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. Jahrestagung der Dt. Ges. f. Pathologie e.V.

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Supplement 1

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Berlin, 1. Mai – . Juni 212

Editorial Gustavo B . Baretton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Eingeladene Referate und Keynote Lectures Kolorektales Karzinom 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-001 – VO-003 . . . . . . . . . . . 6

. Jahrestagung der Deutschen Gesellschaft für Pathologie e.V.

Kolorektales Karzinom 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-005 . . . . . . . . . . . . . . . . . . . . 7 Keynote Lecture – Deep Sequencing - new frontiers in GI-tumor pathology

Schwerpunkt der Jahrestagung

N. Papadopoulos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-007 . . . . . . . . . . . . . . . . . . . . 7

F Gastrointestinale Pathologie F Translationale Forschung in der Pathologie

Primäre Entzündungen im GI-Trakt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-008 – VO-013 . . . . . . . . . . . 7 Keynote Lecture –

Vorsitzender der Gesellschaft Prof . Dr . med . Manfred Dietel

Genetic determinants for cancer progression and individual therapy selection

Tagungspräsident Prof . Dr . med . Gustavo Baretton

A. Ullrich . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-014 . . . . . . . . . . . . . . . . . . . . . 9 Translationale Forschung und Diagnostik –

Herausgeber im Auftrag der Gesellschaft Holger Moch, Zürich Kongressorganisation und Industrieausstellung Elisabeth Jacob Project Manager MCI Berlin Office Elisabeth .Jacob@mci-group .com

Lunge, Sarkome, GIST . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-015 – VO-017 . . . . . . . . . . . 9 Translationale Forschung und Diagnostik – Niere, abl . Harnwege, Prostata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-018 – VO-020 . . . . . . . . . .10 Gastric cancer – English . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-024 – VO-026 . . . . . . . . . .11 Keynote Lecture – Mechanisms of androgen resistance in prostate cancer D.J. Tindall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-027 . . . . . . . . . . . . . . . . . . . .12 Pankreaskarzinom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-029 – VO-031 . . . . . . . . . .12 Keynote Lecture The epithelial-mesenchymal transition and cancer stem cells R.A. Weinberg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-032 . . . . . . . . . . . . . . . . . . .13 Mechanisms of Progression and Therapy resistance of Cancer I – English . . . . . . . . . . . . . . . . . . . . . . . . . .VO-033 – VO-035 . . . . . . . . . .13 Mechanisms of Progression and Therapy Resistance of Cancer II – English . . . . . . . . . . . . . . . . . . . . . . . .VO-036 – VO-038 . . . . . . . . . .14 Translationale Forschung und Diagnostik – Mamma/Schilddrüse/Melanom . . . . . . . . . . . . . . . . . . . .VO-039 – VO-040 . . . . . . . . . .15

Titelbild:  © W .M .

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Ausgewählte Vorträge aus den Einsendungen (Hauptprogramm   und Arbeitsgemeinschaften) AG Gastroenterologische Pathologie I – Leber . . . . . . . . . . . . . . . . . . .DO-001 – DO-007 . . . . . . . . .15 AG Gastroenterologische Pathologie IV – Oberer GI-Trakt . . . . . . . .DO-015 – DO-018 . . . . . . . . .18 AG Gastroenterologische Pathologie VI – Unterer GI-Trakt . . . . . . . .DO-020 – DO-024 . . . . . . . . .19 AG Pneumopathologie I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DO-025 – DO-029 . . . . . . . . .21 AG Pneumopathologie III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DO-032 – DO-036 . . . . . . . . .23 AG Hämatopathologie I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DO-043 – DO-054 . . . . . . . . .24 AG Hämatopathologie II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DO-055 – DO-066 . . . . . . . . .28 AG Orthopädische Pathologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DO-079 – DO-086 . . . . . . . . .32 AG Oralpathologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DO-087 – DO-098 . . . . . . . . .35 AG Herz- und Gefäßpathologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DO-100 – DO-110 . . . . . . . . . .38 AG Molekularpathologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DO-111 – DO-125 . . . . . . . . . .42 Workshop Informatik – Strukturierte Befunde . . . . . . . . . . . . . . . . . . .DO-001 – DO-002 . . . . . . . . .46 AG Dermatopathologie und AG Zytopathologie I – Endokrine Themen I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-001 – FR-008 . . . . . . . . . . .47 AG Dermatopathologie und AG Zytopathologie II – Endokrine Themen II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-009 – FR-015 . . . . . . . . . . .49 Aktuelle Habilitationen I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-016 – FR-020 . . . . . . . . . . .51 Aktuelle Entwicklungen in der Forschung mit Vortrag des Promotionspreisträgers . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-021 – FR-024 . . . . . . . . . . .53 Promotionspreis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-026 . . . . . . . . . . . . . . . . . . . .54 Translationale Forschung und AG Molekularpathologie . . . . . . . . . .FR-027 – FR-035 . . . . . . . . . . .55 Aktuelle Habilitationen II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SA-001 – SA-004 . . . . . . . . . .58 Aktuelle Entwicklungen in der Forschung II – Gastrointestinaltrakt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SO-001 – SO-009 . . . . . . . . . .59 Aktuelle Entwicklungen in der Forschung III – Translationale Forschung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SO-010 – SO-018 . . . . . . . . . .62 AG Gynäkopathologie und Mammapathologie I . . . . . . . . . . . . . . . . .SO-019 – SO-027 . . . . . . . . . .65 AG Gynäkopathologie und Mammapathologie II . . . . . . . . . . . . . . . . .SO-029 – SO-037 . . . . . . . . . .69 AG Paidopathologie I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SO-038 – SO-045 . . . . . . . . . .72 AG Paidopathologie II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SO-046 – SO-058 . . . . . . . . . .75 AG Urologische Pathologie I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SO-061 – SO-69 . . . . . . . . . . .78 AG Urologische Pathologie II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SO-071 – SO-074 . . . . . . . . . .81

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Deutsch-Chinesisches Symposium Colorectal Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SG-129 – SG-136 . . . . . . . . . . .83 Pancreatic Adenocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SG-137– SG-140 . . . . . . . . . . .85 Mammary Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SG-141 – SG-145 . . . . . . . . . . .86 Malignant Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SG-146 – SG-147 . . . . . . . . . . .87

Poster Poster: Deutsch-Chinesisches Symposium . . . . . . . . . . . . . . . . . . . . . . .SG-P-112 – SG-P-133 . . . . . . .88 Poster: GI-Trakt: Ösophagus, Magen . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-P-036 – FR-P-055 . . . . . . .94 Poster: GI-Trakt: GIST, Dünndarm, Kolorektum . . . . . . . . . . . . . . . . . . .FR-P-056 – FR-P-074 . . . . . .101 Poster: GI-Trakt: Kolorektum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-P-075 – FR-P-093 . . . . . .107 Poster: GI-Trakt: Hepatobiliäres System und Pankreas . . . . . . . . . . . .FR-P-094 – FR-P-111 . . . . . .113 Poster: Herz- und Gefäßpathologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-P-112 – FR-P-129 . . . . . . .119 Poster: Pneumopathologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-P-130 – FR-P-146 . . . . . 125 Poster: Hämatopathologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-P-147 . . . . . . . . . . . . . . . . .130 Poster: Autopsie/Fallstudien/Sonstiges . . . . . . . . . . . . . . . . . . . . . . . . . .FR-P-161 – FR-P-180 . . . . . 134 Poster: Gynäkopathologie und Mammapathologie I . . . . . . . . . . . . .SA-P-005 – SA-P-019 . . . . .141 Poster: Gynäkopathologie und Mammapathologie II . . . . . . . . . . . . .SA-P-020 . . . . . . . . . . . . . . . . .145 Poster: Molekularpathologie I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SA-P-035 – SA-P-053 . . . . .150 Poster: Molekularpathologie II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SA-P-054 – SA-P-068 . . . . .156 Poster: Paidopathologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SA-P-069 – SA-P-077 . . . . 160 Poster: Uropathologie I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SA-P-078 – SA-P-089 . . . . 163 Poster: Uropathologie II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SA-P-090 – SA-P-100 . . . . 166 Poster: Informatik . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SA-P-101 – SA-P-111 . . . . . .170

Ergänzung: AG Informatik in der Pathologie . . . . . . . . . . . . . . . . . . . .DO .067 – DO .078 . . . . . . . . .177

ige e z n A e ein t h e t s r Hie nt e m e s i t r ve d a n a s i This

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H e rau sg e ber

Der Pathologe

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Supplement 1

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Mai 2012

Organ der Deutschen Gesellschaft für Pathologie Organ der Deutschen Abteilung der Internationalen Akademie für Pathologie Organ der Österreichischen Gesellschaft für Pathologie Organ der Schweizerischen Gesellschaft für Pathologie Organ des Bundesverbandes Deutscher Pathologen Federführende Schriftleitung / Editor-in-Chief

Schriftleitung / Editors

Univ.-Prof. Dr. K.W. Schmid, Institut für Pathologie und Neuropathologie, Universitätsklinikum Essen

Prof. Dr. L. Bubendorf, Institut für Pathologie, Universitätsspital Basel, Schweiz Prof. Dr. W. Feiden, MVZ für Histologie, Zytologie und Molekulare Diagnostik, Trier Prof. Dr. C. Kuhnen, Institut für Pathologie am Clemenshospital Münster Univ.-Prof. Dr. S. Lax, Institut für Pathologie, LKH Graz West, Österrreich Prof. Dr. T. Mentzel, Dermatopathologische Gemeinschaftspraxis, Friedrichshafen Prof. Dr. W. Saeger, Institut für Pathologie des Marienkrankenhauses Hamburg Prof. Dr. D. Schmidt, Institut für Pathologie, Referenzzentrum für Gynäkopathologie, Mannheim Prof. Dr. Annette Schmitt-Gräff, Abt. Allgemeine Pathologie und Pathologische Anatomie, Institut für Pathologie, Universitätsklinikum Freiburg PD Dr. M. Vieth, Institut für Pathologie, Klinikum Bayreuth GmbH PD Dr. M. Werner, Institut für Pathologie, HELIOS Klinikum Emil von Behring, Berlin

In Zusammenarbeit mit / In cooperation with Prof. Dr. G.B. Baretton, Institut für Pathologie, Universitätsklinikum „Carl Gustav Carus“, TU Dresden Prof. Dr. R. Büttner, Institut für Pathologie, Universitätsklinikum Köln Prof. Dr. H.H. Kreipe, Institut für Pathologie, Medizinische Hochschule Hannover Prof. Dr. H. Moch, Institut für Klinische Pathologie, UniversitätsSpital Zürich, Schweiz Prof. Dr. P. Schirmacher, Pathologisches Institut, Universität Heidelberg

Rubrikherausgeber / Section editors Pitfalls / Pitfalls Prof. Dr. C. Kuhnen, Münster

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Editorial

96. Jahrestagung   der Deutschen Gesellschaft  für Pathologie e. V. Berlin, 31. Mai – 3. Juni 2012

Liebe Kolleginnen und Kollegen, sehr geehrte Damen und Herren, ich freue mich, Sie bei der 96. Jahrestagung der Deutschen Gesellschaft für Pathologie e.V. in Berlin begrüßen zu dürfen, die zum vierten Mal gemeinsam mit der Tagung des Bundes­ verbandes Deutscher Pathologen e.V. und mit Beteiligung der Internationalen Akademie für Pathologie, Deutsche Abteilung e.V., als „Berliner Woche der Pathologie“ stattfindet. Mit den diesjährigen Hauptthemen „Gastrointestinale Pathologie“ und „Transla­ tionale Forschung in der Pathologie/Prädik­ tive Diagnostik“ werden hoch relevante und aktuelle Aspekte der gewebsbasierten Forschung und Diagnostik in den Fokus der Tagung gestellt. Für beide Themen­ bereiche ist es gelungen, national und inter­ national renommierte Experten aus dem Fach Pathologie und aus anderen Feldern der Biomedizin und Grundlagenforschung zu gewinnen. Die Beiträge der eingeladenen Referenten sollen einen umfassenden Über­ blick über den aktuellen Stand der Forschung bieten und aufzeigen, in welche Richtung sich das Fach Pathologie weiterentwickelt. Mit der Verbindung von wissenschaftlichen Keynote­Vorträgen und der Präsentation von wichtigen diagnostischen Themen (teil­ weise im Dialog mit Klinikern) soll sowohl für junge Pathologinnen und Pathologen als auch für erfahrene Kolleginnen und Kollegen ein interessanter Bogen geschlagen werden. Die Sitzungen der Arbeitsgemeinschaften, welche das Hauptprogramm umrahmen, runden das Programm ab und bieten Ein­ blicke in neueste wissenschaftliche Erkennt­ nisse in den verschiedenen Organsystemen. Gerade der Nachwuchsförderung soll auch auf der 96. Jahrestagung wieder große Beach­ tung geschenkt werden. Dem erfolgreichen

Format der im letzten Jahr in Leipzig erst­ mals durchgeführten Nachwuchslounge wird diesmal noch mehr Zeit eingeräumt. Es soll damit ein möglichst großer Kreis von inter­ essierten jungen Nachwuchswissenschaftlern angesprochen und motiviert werden, sich weiter für die Forschung in der Pathologie zu begeistern und Kontakte zu knüpfen. Erfreulich ist außerdem das große Interesse der Industrie an unserem Fach, was durch die große Zahl der Aussteller und zahlreiche Satelliten­Symposien zum Ausdruck kommt. Dabei weitet sich das Spektrum von den Anbietern labortechnischer und analytisch­ apparativer Verfahren zunehmend auch auf die pharmazeutische Industrie aus. Dahin­ ter steht die Intention der Hersteller, eine flächendeckende qualitätsgesicherte prädik­ tive Diagnostik für die zielgerichteten Thera­ peutika sicherzustellen, die eine zunehmende Bedeutung in der Onkologie spielen. Die Bündelung der Veranstaltungen der 96. Jahrestagung der Deutschen Gesellschaft für Pathologie e.V., des Bundesverbandes Deutscher Pathologen e.V. und der Inter­ nationalen Akademie für Pathologie, Deut­ sche Abteilung e.V., als „4. Berliner Woche der Pathologie“ soll nachdrücklich die Gewebekompetenz der Pathologie als Al­ leinstellungsmerkmal unseres Fachs doku­ mentieren. Es soll gezeigt werden, dass die Grenzen zwischen akademischer Pathologie und praktischer pathodiagnostischer Tätig­ keit offen sind und offen bleiben müssen; denn gerade die zeitnahe Integration trans­ lationaler Forschungsergebnisse in die Praxis bringt – wie in der Vergangenheit immer wieder eindrucksvoll dokumentiert – unser Fach weiter voran und sichert seine Zukunft. Die erfolgreiche Zusammenarbeit mit der Internationalen Akademie für Pathologie/ IAP wird in bewährter Weise fortgesetzt,

indem die IAP dankenswerterweise wieder 4 Tutorials im Rahmen der Tagung anbietet. Eine Besonderheit der diesjährigen Tagung stellt das angeschlossene „Chinesisch­ deutsche Symposium“ dar, welches mit Un­ terstützung durch die Deutsche Forschungs­ gemeinschaft/DFG die Zusammenarbeit in der wissenschaftlichen Pathologie zwischen der deutschen und der chinesischen Fach­ gesellschaft weiter voranbringen soll. Ins­ besondere junge Nachwuchswissenschaftler/­ innen aus beiden Ländern sollen so die Möglichkeit erhalten, persönlich in Kontakt zu kommen und bilaterale Kooperationen in der Forschung einzugehen. Ich wünsche Ihnen eine interessante Tagung mit vielen neuen Erkenntnissen sowie der Möglichkeit zum kollegialen Dia­ log und regen wissenschaftlichen Austausch. Prof. Dr. Gustavo B. Baretton

Tagungspräsident der 96. Jahrestagung der DGP e.V.

Korrespondenzadresse Prof. Dr. G. B. Baretton Institut für Pathologie Universitätsklinikum Dresden Fetscherstr . 74 01307 Dresden gustavo .baretton@uniklinikum-dresden .de Der Pathologe Suppl 1 · 2012  | 

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Abstracts Pathologe 2012 [Suppl 1] · 33:6–180 DOI 10 .1007/s00292-012-1584-x © Springer-Verlag 2012

96. Jahrestagung   der Deutschen Gesellschaft  für Pathologie e. V. Berlin, 31. Mai – 3. Juni 2012

Eingeladene Referate und Keynote Lectures Kolorektales Karzinom 1 VO-001 The natural history of colorectal adenomas and early colorectal cancer M . Risio1 1 Institute for Cancer Research and Treatment (IRCC), Candiolo – Torino, Italy It is well known that adenomas represent the morphologically categorized precursor of the vast majority of colorectal cancers. In accordance with the stochastic modeling of colorectal tumor progression, each step of the adenoma-carcinoma sequence, from single-crypt dysplasia to cancerised adenoma, can be probabilistically profiled in terms of evolutive pathways: although every adenoma has the capacity of malignant evolution, most adenomas stabilize their progression or even regress. Easily identifiable pathological features (size, type, architectural growth, grade, and gross organization of dysplasia) are predictive of the natural history of adenomas in terms of potential and times of cancerisation. Interestingly, the link between size and adenoma malignancy is greater than the one linking dysplasia and malignancy, suggesting the need to perfect the histological criteria now used for grading dysplasia in order to improve the detection rates of faster-progressing precursors. Regression of both micro- and gross adenomas is histologically well established, but it is thought to be a dynamic process, with cycling fluctuation of phases of regression and growth of adenomatous tissue. Colorectal carcinoma invading the submucosa but not the muscular layer (pT1, early invasive cancer) represents the earliest form of clinically relevant colorectal cancer. Neoplastic invasion of the submucosa, in fact, opens the way to metastasis and the choice between surveillance and major surgery will turn on its metastatic potential. Grade of differentiation of carcinoma, lymphovascular invasion and state of the resection margin predict the risk of metastasis and the different clinical outcomes: a positive resection margin is predictive of local disease, vascular invasion of lymph node metastasis, poorly differentiated carcinoma of hematogenous metastasis and cancer-related mortality. Microstaging of invasive cancer, namely the width and the depth of submucosal invasion, together with tumor budding at the advancing edge allow the metastatic risk to be further stratified in minimal, low, and high. There evidence exists that cancerised adenomas represent the end point of two different,

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although morphologically undistinguishable, tumorigenic pathways, the former blocking the growth of early cancer, the latter allowing its fast progression towards advanced cancer: new biomarkers are needed to distinguish progressive from non-progressive pT1 neoplasia.

VO-003 The mismatch repair deficient crypt focus – Der mismatch repair defiziente Kryptenfokus H . Bläker1, M . Kloor2 1 Institut für Pathologie, Campus Charite Mitte, Universitätsmedizin Berlin, Institute of Pathology, Campus Mitte, Berlin, 2Heidelberg, Institute of Pathology Keimbahnmutationen in einem der Mismatch-Repair(MMR)-Gene, zumeist MLH1 und MSH2, sind ursächlich für das Lynch-Syndrom, häufig auch als „hereditary non-polyposis colorectal cancer“-Syndrom (HNPCC) bezeichnet. Anders als bei den Polyposis-Syndromen, insbesondere der familiären Adenomatosis polyposis coli, fehlt bei HNPCC die deutlich erhöhte Zahl der adenomatösen Karzinom-Vorläufer. Der MMR-defiziente Kryptenfokus, der hier vorgestellt wird, ist eine neu entdeckte, nichtpolypöse Läsion, die in hoher Zahl, etwa einmal pro cm2, in der nichttumorös veränderten Dünn- und Dickdarmschleimhaut von HNPCC-Patienten auftritt. Der MMR-defiziente Kryptenfokus zeigt die typischen molekularen Veränderungen HNPCC-assoziierter Tumoren. Während kleine, nur eine bis wenige Krypten umfassende Läsionen histologisch unauffällig sind, finden sich mit zunehmender Größe architekturelle und zytologische Veränderungen, die sich keiner Adenomform zuweisen lassen. Das Transformationspotential der MMR-defizienten Kyrptenfoci muss in Anbetracht der erheblichen Diskrepanz zwischen Menge an MMRdefizienten Foci und Anzahl von Karzinom bei HNPCC ausgesprochen gering sein. Möglicherweise sind diese Läsionen selbstlimitierend, wobei die Mechanismen der Elimination noch offen sind. Zusammenfassend erklärt die Assoziation des HNPCC-Syndroms mit dem MMR-defizienten Kryptenfokus das Fehlen einer Polyposis bei diesem Syndrom. Die weitere Charakterisierung der MMR-defizienten Kryptenfoci und ihr Verlauf versprechen neue Erkenntnisse über initialisierende und limitierende Mechanismen des autonomen Wachstums.

Kolorektales Karzinom 2 VO-005 Translating biology of colorectal cancer into clinical applications G .A . Meijer1 1 VU University Medical Center, VU University Medical Center, Amsterdam, Netherlands Colorectal cancer (CRC) is one of the most common malignancies and represents a substantial burden for society, in terms of patient suffering as well as economically. As cancer is an evolutionary process in which genotype drives phenotype, knowledge of the biological mechanisms underlying CRC can help to improve patient outcome. Translational research in CRC mainly focuses on unmet clinical needs in three stages of the disease, i.e. early detection or screening, prognostication in primary CRC and prediction of response to drug therapy in metastatic CRC. As CRC develops over a number of years from a detectable precursor (adenoma), there is a window of opportunity for early detection and curative intervention. Our increased understanding of CRC biology, and in particular adenoma to carcinoma progression, has yielded a number of markers based on e.g. DNA or proteins that hold great promise for the next generation CRC screening tests. In primary CRC, standard UICC staging is still the most important method for stratifying patients by risk of recurrence. Yet, the substantial number of stage II patients that do develop recurrences, and the still small proportion of stage III patients that actually benefit from adjuvant therapy, underline the need for additional diagnostic arsenal. Since CRC biologically is a heterogeneous disease, it does not come as a surprise that there is a large number of markers for which some level of evidence exists that they could have additional prognostic value. The main challenge here is to make the next step to get these markers validated and implemented in routine diagnostic practice. In a similar way, much translational research has successfully translated biology of CRC into diagnostic tests that more rapidly have been implemented, like KRAS testing for anti-EGFR therapy. The fact that these predictive markers have entered the field much more rapidly than the prognostic markers most likely is associated with more efficient business development, stimulated by the companion diagnostic concept. Yet, also here challenges remain, as because of the complexity of CRC biology it is much more likely that we will have complex decision trees rather than single parameter tests to stratify patients for drug therapy. The exciting developments that will bring whole cancer genome sequencing within the reach of pathologists will eliminate current barriers for the full exploration of tumor biology for the purpose of diagnostic pathology.

Keynote Lecture VO-007 Deep Sequencing - new frontiers in GI-tumor pathology N . Papadopoulos Johns Hopkins University, Baltimore, USA Inflammatory bowel diseases represent a chronic disorder accompanying mostly young people throughout their life. Thus therapeutic strategies allowing for a normal life are mandatory. Although the incidence is increasing and the research of the last decades provides a detailed view of the pathogenesis, the available therapeutic strategies are still symptomatic. The primary aim is to induce a stable remission. Depending on disease localization as well as associated complications such as fistulas, abscesses and malnutrition, the therapeutic strategies range from local applications up to systemic immunosuppressive medications. Despite the availability of highly potent agents including azathioprine, calci-

neurin inhibitors as well as anti-TNF antibodies, there is still a subset of patients that remains with active disease. Thus it is crucial to predict the disease course early on, in order to decide whether early aggressive therapy is required or a less aggressive treatment is sufficient. Some factors helping with this decision have already been identified. Novel data suggest that the expression profile of CD8+ cells may help in predicting the disease course. However, these data will have to be confirmed in prospective studies. Equally important is the question when immunosuppressive therapies can be paused with low risk. Do we need clinical, endoscopic or even histological remission? On a long-term view mucosal healing gains impact since it reduces the risk of developing colorectal cancer. The discussed points emphasize that therapeutic strategies in inflammatory bowel diseases represent an increasingly individualized therapy in order to allow for a high life quality of our patients.

Primäre Entzündungen im GI-Trakt VO-008 Clinical view and novel therapeutic strategies in inflammatory bowel diseases B . Siegmund1 1 Charité – Universitätsmedizin Berlin, Klinik für Gastroenterologie, Infektiologie und Rheumatologie der Charite Campus Benjamin Franklin, Berlin Inflammatory bowel diseases represent a chronic disorder accompanying mostly young people throughout their life. Thus therapeutic strategies allowing for a normal life are mandatory. Although the incidence is increasing and the research of the last decades provides a detailed view of the pathogenesis, the available therapeutic strategies are still symptomatic. The primary aim is to induce a stable remission. Depending on disease localization as well as associated complications such as fistulas, abscesses and malnutrition, the therapeutic strategies range from local applications up to systemic immunosuppressive medications. Despite the availability of highly potent agents including azathioprine, calcineurin inhibitors as well as anti-TNF antibodies, there is still a subset of patients that remains with active disease. Thus it is crucial to predict the disease course early on, in order to decide whether early aggressive therapy is required or a less aggressive treatment is sufficient. Some factors helping with this decision have already been identified. Novel data suggest that the expression profile of CD8+ cells may help in predicting the disease course. However, these data will have to be confirmed in prospective studies. Equally important is the question when immunosuppressive therapies can be paused with low risk. Do we need clinical, endoscopic or even histological remission? On a long-term view mucosal healing gains impact since it reduces the risk of developing colorectal cancer. The discussed points emphasize that therapeutic strategies in inflammatory bowel diseases represent an increasingly individualized therapy in order to allow for a high life quality of our patients.

VO-010 Microscopic colitis: clinical appearance and therapy S . Miehlke1 1 Magen-Darm-Zentrum, Hamburg Microscopic colitis (MC) is a chronic inflammatory bowel disease which is increasingly recognized as a common cause of chronic, non-bloody diarrhoea. Besides watery diarrhea, many patients also suffer from abdominal pain, weight loss and fecal incontinence which severely deteriorate their quality of life. There is a female predominance with an average age at diagnosis around 60 years. Smoking appears to be a relevant risk factor. Epidemiological studies have shown a rising incidence in the last

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Abstracts decade and meanwhile it appears that MC is almost as common as classic IBD, i.e. Crohn‘s disease and ulcerative colitis. The endoscopic appearance of the colon is usually normal or may show only subtile alterations. The diagnosis can be made only by histology and the specific findings reveal also the subtypes of MC, lymphocytic (LC) or collagenous colitis (CC). The key histological feature is a thickened subepithelial collagenous band >10 µm in CC, and an increase number of surface intraepithelial lymphocytes >20 IEL/100 epithelial cells) in LC. Patients with chronic diarrhea not completely fulfilling the histological CC/LC criteria may have incomplete MC. The primary aim of medical therapy is to achieve and maintain clinical remission and to improve patient‘s quality of life. The strongest evidence is currently available for budesonide, a locally acting corticosteroid with an extensive first-pass metabolism in the liver. Three randomized controlled trials in CC and two in LC have proven budesonide 9 mg per day effective for induction of clinical remission with a pooled response rate of 81%, and a NNT of 2 patients. The majority of patients response rapidly and experience a substantial improvement in their quality of life. After cessation of budesonide, symptomatic relapse may occur in 60– 80% of patients. Two randomized controlled trials have now shown that clinical remission and histological response can be maintained in the majority of patients with budesonide 6 mg per day for 6 months with a pooled response rate of 83% and a NNT of 2 patients. Other drugs such as mesalazine, bismuth or loperamide are occasionally used; however, the benefit is unclear due to lack of adequate clinical trials. There is currently also no evidence to recommend immunosuppressives. However, recent case reports suggest that azathioprin or anti-TNF natibodies might be an option in individual refractory cases.

VO-011 Histopathology of microscopic colitis D. Aust1 Institute for Pathology TU Dresden, Dresden

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Microscopic colitis (MC) is recognized to be a common cause of chronic, non-bloody diarrhea. Numerous epidemiological studies, mainly in the USA and Sweden, have shown a rising incidence in the last decade. The diagnosis can only be made by histology and the specific histological findings define the subtypes of MC, lymphocytic (LC) or collagenous colitis (CC). As LC and CC share clinical similarities and histopathological features, and many patients with CC also fulfil the histological criteria for LC, it has been discussed whether the two are in fact different stages of disease development. Conversion of LC to CC or vice versa is infrequent, and at present LC and CC is considered two separate but related entities. In MC, the lamina propria shows increased numbers of plasma cells and lymphocytes with loss of the normal gradient, even eosinophilic and neutrophilic granulocytes may be present. But these histological features do not warrant the diagnosis of MC even though they may be responsible for the clinical symptoms. The key histological feature of LC is an increased number of surface intraepithelial lymphocytes (IEL). Usually >20 IELs/100 epithelial cells are requested to warrant the diagnosis of LC. IELs are mostly cytotoxic CD8+ T-lymphocytes. The epithelium itself can show regressive changes with focal or diffuse flattening of the columnar cells, loss of mucin, decreased goblet cells and signs of degeneration such as cytoplasmic vacuoles and pycnotic nuclei. The key histological criterion for CC is a continuous subepithelial fibrous band underneath the surface epithelium (>10 µm). Other hallmarks of CC are chronic mucosal inflammation, the collagen band contains entrapped capillaries, red blood cells and inflammatory cells. Damaged epithelial cells appear flattened, mucin depleted and irregularly oriented. Focally, small strips of surface epithelium may lift off from their basement membrane. The terms MC not otherwise specified (MCnos) or MCi (microscopic colitis incomplete) was suggested for a subgroup of patients with diarrhea

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and an increase in cellular infiltrate in the colonic lamina propria and either an abnormal collagenous layer and/or intraepithelial lymphocytes coming short of fulfilling the criteria for CC and LC. The histological features of MC, diagnostic algorithms and possible differential diagnoses of MC will be discussed in this talk.

VO-012 Eosinophilic esophagitis: role of the gastroenterologist A. Schöpfer1 1 University of Lausanne, Department of Gastroenterology and Hepatology, CHUV, Lausanne, Switzerland Eosinophilic esophagitis (EoE), first described in the early 1990’s, has rapidly evolved as distinctive chronic inflammatory oesophageal disease with increasing incidence and prevalence in the westernized countries (prevalence of about 1/2000). Currently, EoE represents the main cause of dysphagia in adult patients. EoE is defined as chronic, immune/antigen-mediated esophageal disease characterized clinically by symptoms related to esophageal dysfunction and histologically by eosinophil-predominant inflammation. The presence of at least 15 eosinphils per high power field is considered a minimum threshold for EoE diagnosis. The disease is isolated to the esophagus, and other causes of esophageal eosinophilia should be excluded. Other diseases associated with esophageal eosinophilia are e.g. gastroesophageal reflux disease (GERD), eosinophilic gastrointestinal disorders, celiac disease, Crohn’s disease, invasive parasites, achalasia, or drug hypersensitivity. EoE is more prevalent in males and is frequently associated with allergies. It is currently under discussion to what extent and by which methods allergic testing should be performed. Therapeutic strategies for EoE can be summarized by the “3 D’s”: drugs, diet, dilation. Topical corticosteroids lead to a rapid improvement of active EoE clinically and histologically. Especially in children, elimination diets can have similar efficacy as topical corticosteroids. Oesophageal dilation of EoE-induced oesophageal strictures can also be effective in improving symptoms, but this therapy has no effect on the underlying inflammation. Neither the diagnostic nor long-term therapeutic strategies are yet defined.

VO-013 The role of the pathologist in the diagnosis of eosinophilic ­esophagitis (EoE) C. Bussmann1 1 Pathology Cantonal Hospital Lucerne, Luzern, Switzerland EoE is a chronic, immune-mediated esophageal disease with clinical symptoms and eosinophil-predominant inflammation. The diagnosis of EoE is therefore complex. It is not reflux or infectious or drug-induced. Histological cases with a high number of eosinophils are not a diagnostic problem. However, limits are of necessity in borderline cases. These must be based on the high power field (HPF) size and the percentage of squamous epithelium covering an HPF, which may greatly vary. Also, it must be considered that EoE is patchy in nature. In order to establish a reliable diagnosis a minimal number of biopsies are essential. Alongside the number of eosinophils further histological features associated with EoE, such as abscesses of eosinophils or basal layer enlargement, may be of diagnostic help in borderline cases.

Keynote Lecture VO-014 Genetic determinants for cancer progression and individual therapy selection A . Ullrich1 1 Department of Molecular Biology, Max Planck Institute of Biochemistry, Martinsried For the past years we have investigated various aspects of signaling systems in tumor cells in order to identify critical switch points in the patho-physiological process that results in malignancy. These efforts aim at the selective blockade of abnormal, disease-promoting signaling mechanisms by monoclonal antibodies, or small molecule kinase inhibitors. This strategic approach began with the cloning of the EGF receptor cDNA and the related receptor HER-2/neu and translated the animal oncogene concept into target-directed personalized therapy of human cancer. This work yielded the first specific oncogene target-based, FDAapproved (1998) therapeutic agent, “Herceptin”, for the treatment of metastatic breast cancer. Earlier and subsequent “target-driven drug development” efforts that employed various genomic analysis strategies led to the cancer therapies that are based on EGFR, HER3, FGFR4, Axl/Ufo and Flk-1/VEGFR2 as critical signaling elements in tumor progression. The latter served, in cooperation with SUGEN Inc./Pharmacia/Pfizer, as basis for the development of SU11248. The drug discovery process that led to SU11248 represents a prototypical example for the adaptation of cancer therapeutics from highly specific to multi-targeted drugs. While all novel cancer therapies target genetic alterations in tumor tissues innovative strategies are aimed at investigating the contribution of germ line determinants of the patient to disease progression and therapy response. One example is the common polymorphism at codon position 388 in the human FGFR4 gene of which the Arg388 allele represents a target for the development of individual genotype-dependent cancer therapy development. Current findings and their consequences for patient-specific cancer therapy will be discussed.

Translationale Forschung und Diagnostik – Lunge, Sarkome, GIST VO-015 Translational lung cancer research S . Perner1 University Hospital of Bonn, Institute of Pathology, Bonn

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Lung cancer is the most common malignant disease leading to death worldwide. Histologically, it is broadly subcategorized into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with the latter mainly consisting of the three major entities – adenocarcinoma, squamous cell carcinomas and large cell carcinomas. In the recent past, surgical resection and chemotherapy were the only therapeutic options available. However, genetic profiling of various lung cancer entities have revealed major genetic differences within distinct histological tumor entities, enabling specific diagnosis, individual prognosis and rational treatment for the disease. Mutation of the Epidermal Growth Factor Receptor (EGFR) in lung cancer of non-smoking patients was the first major discovery leading to novel therapeutic strategies, which included treatment with tyrosin kinase inhibitors (TKI) gefitinib and erlotinib. EGFR mutated cases are more sensitive to TKI treatment, whereas cases harboring KRAS mutations are associated with a resistance to TKI. Moreover, the occurrence of KRAS and EFGR mutations are mutually exclusive and are correlated with poor prognosis. In an effort to further subclassify lung cancer at the molecular level, large lung cancer cohorts were characterized using high-throughput technologies. The lineage survival oncogene TTF1 is found to be the most common amplification occurring in pulmonary adenocarcinomas. In squamous cell lung cancer, SOX2 was identified as the most frequently amplified lineage survival oncogene. Amplification of either gene proved to be associated with better overall survival rates. In 2010, Weiss et al. described Fibroblast Growth Factor Receptor 1 (FGFR1) amplification in 20% of squamous cell lung cancer. FGFR1 amplified tumors were shown to be sensitive to FGFR1 small molecule inhibitors in cell lines and murine xenograft models. This finding paved the way to the first rational therapy in a significant subset of molecularly defined squamous cell lung cancers. Moreover, our discovery of FGFR1 amplification in squamous cell cancers of the head and neck area might broaden the therapeutic spectrum of the FGFR1 inhibitors. These findings, among others, can only estimate the genetic complexity of lung tumors. Large-scale molecular profiling has the potential to identify novel diagnostic, prognostic and predictive markers as well as therapeutic targets.

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Abstracts VO-017 Translational research and diagnostics – GIST E . Wardelmann1 1 University of Cologne, Institute of Pathology, Köln Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors in the digestive tract. In up to 90% of cases, they are characterized by activating mutations in the KIT or the PDGFRA gene. GIST turn out to be a paradigm for successful targeted treatment with tyrosine kinase inhibitors (TKI). Since the approval of the TKI imatinib in 2002 the survival of patients with metastatic GIST has been tripled. The next logical step was the concept to use imatinib in an adjuvant approach which recently showed to increase the overall survival significantly. In both settings, the mutational status has high predictive implications. In detail, GIST with KIT exon 11 mutations show the best response rates with partial remission rates of up to 80%. In KIT exon 9 mutations, a doubled daily dose of 800 mg imatinib is now the standard. The PDGFRA exon 18 mutation D842V has been shown to lead to a primary resistance. Conclusively, the treatment strategy in GIST is driven by their molecular characterisation. Further research has increased the knowledge about resistance mechanisms in solid tumors against TKI. The number of patients with secondary resistance due to acquired KIT mutations is increasing with treatment duration. Strategies to address this situation are the introduction of novel pathway inhibitors targeting different levels of signal transduction pathways such as the mTOR/Akt pathway, the RAS/RAF pathway, histone deacetylation, and others. Among the GIST without mutations in the common hot spot regions of KIT and PDGFRA, so-called wild type GIST, further genomic subgroups have been identified. One such subgroup carries inactivating germline mutations in the genes encoding succinate dehydrogenase B, C, or D. They are associated with the occurrence of paragangliomas, the so-called Carney-Stratakis syndrome. Most frequently, GIST are located in the stomach and show an epithelioid phenotype and a multinodular growth pattern. They preferentially occur in young females and often show lymph node metastases. In Carney’s triad additional pulmonary chondromas are observed. Another small subgroup of sporadic GIST present with BRAF mutations as an alternative genomic event. Finally, very rare kindreds with germline mutations in either KIT or PDGFRA have been described. In summary, the molecular characterisation of GIST has revolutionized their treatment because of increasing knowledge about the high relevance of predictive molecular typing in solid tumors.

Translationale Forschung und Diagnostik – Niere, abl. Harnwege, Prostata VO-018 Biomarker for diagnosis, prognosis and prediction in renal cancer H . Moch1 University Zurich, Institute for Pathology, Zürich, Switzerland

Vancouver 2012. The Working Group discussed the use of immunohistochemical markers as well as the use of cytogenetics or other molecular technologies in the characterization of renal neoplasms. In this presentation, the results of the Working Group Meeting are summarized. In detail, the value of immunohistochemistry for the differential diagnosis in different diagnostic situations, e.g. in renal tumors with clear or granular cytoplasm, diagnosis of unclassified renal cancer is commented. Further, the view of the Working Group regarding use of predictive or prognostic biomarkers in routine pathology is provided.

VO-019 Translational research and diagnostics: urinary tract R . Knüchel-Clarke1, N .T . Gaisa2, M . Rose2, C . Henkel3, E . Dahl2 1 Universitätsklinikum Aachen, Institut für Pathologie, Aachen, 2University Hospital, RWTH Aachen, Institut für Pathologie, Aachen, 3Ruhr-University Bochum, Medical Proteomics Center, Bochum In urothelial cancer as a frequent tumor entity two main fields of the clinical situation deserve special attention and need support from basic research. 1. Amongst the most frequent non-invasive papillary low grade tumors morphology alone is insufficient to predict recurrence rate or even more importantly progression. 2. Amongst the already muscle invasive tumors which mostly undergo cystectomy, neoadjuvant and adjuvant chemotherapy concepts still have a limited supportive role, and more effective individualized treatment concepts are desirable. Ad 1. While the WHO classification of tumors has integrated data from genetic analysis of tumors to diagnose genetically stable (low grade) and unstable (high grade) tumors, FGFR3 mutations and additional molecular alterations may help to define the non-progressing, i.e. nearly benign tumors within the group of low grade tumors. This could support therapy decision favoring avoidance of intravesical chemotherapy and allowing less follow up. A multiparametric approach will be necessary and becomes increasingly feasible due to e.g. epigenetic or proteomic marker sets. Ideally these marker sets are not only valid for tissue analysis but also sufficiently sensitive to predict benign disease course in cytology specimens. Ad 2. Within the group of genetically instable tumors it seems necessary to define tumors different from mere urothelial differentiation as squamous and glandular as well as variants of urothelial carcinoma as micropapillary or small cell carcinoma in a first step. All these tumor variants are found in other organs as well. Consequently the multicentric collection of cases and the molecular analysis of pathways of growth signalling is an apt approach for target identification. Data should be compared to already established data in other cancers as colon and lung cancer and will allow the inclusion of cases in prospective studies using e.g. small molecule- like tyrosine kinase inhibitors. Own research efforts and recent data from the literature that intend to improve the two clinical settings in urothelial cancer will be presented. With support of a START grant RWTH Aachen (MR, NTG and ED) and a DFG grant GA 1384/2-1 (NTG).

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Biomarkers are frequently used in aiding diagnosis, and to predict prognosis or response to therapy in neoplasms. In renal cancer, immunohistochemistry is commonly used in the routine for the classification of renal tumors. Conventional karyotyping, fluorescence in situ hybridization and molecular cytogenetics are less commonly used. Expression profiling, and mutational analysis are currently performed to identify specific molecular pathways in renal cancer, mainly for research purposes. In this presentation, we document results of a Working Group Meeting conducted by the International Society of Urological Pathology (ISUP) in

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VO-020 Translational research and diagnosis of prostate cancer G . Kristiansen1 Institut für Pathologie der Universitätsklinik Bonn, Bonn

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Prostate cancer is the most common non-cutaneous malignant tumour in men and is a major research focus of pathologists, urologists and urooncologists alike. Due to PSA-screening and risen patient awareness, the practising pathologist is confronted with a steadily increasing number of prostate biopsies, necessitating ancillary tests in morphologically challenging cases. Next to basal cell markers, additional positive markers

(AMACR, FASN, GOLM1, GSP-pi, ERG) that aid in the differential diagnosis are presented and discussed here. The clinical decision of urologists or radiooncologists, whom and how to treat these men, still rests predominantly on histological parameters. This urges us to increase standardization of the way we handle and diagnose our specimens. Processing and diagnosing of prostatectomy specimens has been the topic of the 2009 consensus conference of the International Society of Urologic Pathology (ISUP) and the most important recommendations of this conference will be discussed in comparison to the current S3-guidelines. As Gleason Score is one of the strongest prognostic parameters in prostate cancer, standardization is particularly important. The long overdue update on Gleason scoring by the ISUP 2005 recommendations has generally increased awareness, but has also led to some confusion among pathologists and clinicians and has possibly even induced a systematic over-grading of biopsies. A recent study conducted by the European Network of Urinary Pathologists focussed on particularly challenging cases to discriminate problematic areas in the differentiation of Gleason patterns 3 and 4 in order to establish helpful diagnostic guidelines. Despite the clinical need to identify insignificant and lethal prostate cancer at the biopsy stage, this estimation is still left exclusively to conventional clinical and histological parameters and no molecular biomarker has entered clinical practice yet. A critical overview of recent developments of prognostic biomarkers in prostate cancer is given. Finally, a brief outline of the PREFERE study, a prospective therapy study beginning in late 2012 in Germany, that aims to compare the outcomes of 7600 patients over a period of nearly 20 years, will be presented.

Gastric Cancer – English VO-024 Hereditary gastric cancer F . Carneiro1 Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) and Medical Faculty of Porto/Centro Hospitalar S . João, Porto, Portugal, Porto, Portugal 1

Familial aggregation of gastric cancer (GC), both of the diffuse and of the intestinal type, occurs in a variable proportion of cases, pointing to genetic predisposition in these settings. In 1998, Guilford et al identified the first inherited gastric cancer syndrome, designated as Hereditary Diffuse Gastric Cancer (HDGC), caused by germline alterations at the CDH1 (E-cadherin) gene. In 1999, the International Gastric Cancer Linkage Consortium (IGCLC) defined the criteria for the different types of familial gastric cancer syndromes: 1) two GC cases in a family, one confirmed DGC <50 years; 2) three confirmed DGC cases in 1st or 2nd degree relatives independent of age. In HDGC families, penetrance of gastric cancer is >80% at the age of 80 in both genders, and lobular breast cancer is 60% in women by age 80. About one third of families fulfilling the criteria for HDGC carry germline alterations of the CDH1 gene. To date, about 100 different germline CDH1 alterations have been identified in HDGC families, mainly point mutations and large deletions. In 2010, the IGCLC updated the recommendations for CDH1 testing, including: 1) histological confirmation of DGC only required for one family member; 2) DGC <40 years of age; 3) individuals and families with diagnoses of both diffuse gastric cancer and lobular breast cancer (one diagnosis <50 years). In mutation positive individuals prophylactic total gastrectomy at a centre of excellence is recommended. Protocolised endoscopic surveillance in centres with experienced endoscopists and pathologists is recommended for: those opting not to have gastrectomy; those with mutations of undetermined significance; those families for whom no germline mutation is yet identified. The systematic histological study of prophylactic gastrectomies shows pre-invasive lesions including in situ signet ring carcinoma with

pagetoid spread of signet ring cells. Expert histopathological confirmation of these early lesions is recommended. In 2011, a new hereditary gastric cancer syndrome was identified: Gastric Adenocarcinoma and Proximal Polyposis of the Stomach (GAPPS). GAPPS is a unique gastric polyposis syndrome with a significant risk of gastric adenocarcinoma, characterized by the autosomal dominant transmission of fundic gland polyposis, with areas of dysplasia or intestinal-type GC, restricted to the proximal stomach, with no evidence of colorectal or duodenal polyposis or other heritable gastrointestinal cancer syndromes.

VO-025 Micro RNA and novel biomarkers in stomach cancer W . Yasui1 Department of Molecular Pathology, Hiroshima University Institute of Biomedical & Health Sciences, Hiroshima, Japan

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Stomach cancer is one of the most common malignancies worldwide and is the second most frequent cause of cancer-related death. Although recent advances in diagnosis and treatment have offered excellent survival for early stomach cancer, prognosis of patients with advanced cancer still remains poor. A better understanding of molecular bases may lead to new approach to diagnosis, treatment and prevention of stomach cancer. MicroRNAs have been focused on the role in cancer development and progression through epigenetic gene regulation. By miRNA microarray analysis using 182 stomach cancer samples, several miRNAs were upregulated or down-regulated in stomach cancer, many of those were associated with histology, progression and patient’s prognosis. MiR125b, miR-199a, and miR-433 are important miRNAs involved in cancer progression, while low expressions of let-7g and miR-433 and high expression of miR-214 are independent unfavorable prognostic markers. MiR-148a is frequently down-regulated in stomach cancer by DNA methylation, and modulates cell invasion through regulation of MMP-7 and EGFR expression. MiR-143/145, whose expression is higher in diffuse type stomach cancer than intestinal type, is mainly produced by caner-associated fibroblasts and may mediate cancer-stromal interaction. MiRNAs are stable in human circulation in a cell-free form and may be a powerful new class of blood-based biomarkers for stomach cancer. The levels of the certain miRNAs in serum assessed by quantitative RT-PCR are significantly higher in stomach cancer patients than in non-cancerous controls, and correlate with tumor stage. Transcriptome dissection by using SAGE (serial analysis of gene expression) and CAST (Escherichia coli ampicillin trap) methods, we have identified several genes including Reg IV, OLFM4, SPC18, TSPAN8, TM9SF3 and ZDHHC14 as candidate diagnostic and therapeutic targets of stomach cancer. Reg IV activates EGFR and participates in 5-fluorouracil resistance and peritoneal metastasis. Combination of Reg IV and OLFM4 serves as highly sensitive serum tumor marker. SPC18 stimulates tumor growth and invasion through TGF-alpha up-regulation. TSPAN8 participates in with progression of gastric mucin phenotype of stomach cancer, while TM9SF3 is associated with poor prognosis of diffuse type. Information obtained from “Omics” studies greatly contributes to new developments for diagnosis and treatment of stomach cancer.

VO-026 The molecular biology of gastric cancer C . Röcken1 Christian-Albrechts-University, Department of Pathology, Kiel

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Gastric cancer is one of the most common cancers worldwide, ranking fourth in overall frequency, and accounting for over 870,000 new cases and over 650,000 deaths annually. While the incidence of gastric cancer has decreased, the absolute number of gastric cancer patients has increased due to an aging population. Mortality from gastric cancer is Der Pathologe Suppl 1 · 2012  | 

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Abstracts second only to lung cancer. In recent decades we witnessed major advancements in the understanding of the epidemiology, pathology and pathogenesis of gastric cancer. Infection with H. pylori or Epstein-Barr virus, dietary and lifestyle factors contribute to the risk of developing gastric cancer. With regard to pathogenesis, at least three distinct types of gastric cancer exist, i.e. (1) proximal, (2) distal diffuse, and (3) distal non-diffuse type. Genetic and epigenetic alterations are related to oncogene mutations and tumor suppressor gene inactivations, e.g. by loss of heterozygosity or methylation. Canonical oncogenic pathways such as E2F, KRAS, p53, and WNT/β-catenin signaling are de-regulated in gastric cancer. Microsatellite instability is observed in approximately 10–15% of the cases. Hereditary and familial type gastric cancers are currently linked to 122 different CDH1-mutations (25–30% of the cases with Hereditary Diffuse Gastric Cancer) and various gene polymorphisms determining disease susceptibility. Molecular subtypes of gastric cancer were identified, which separate diffuse from intestinal type gastric cancer and are not entirely congruent with the histopathological phenotype according to Laurén, but may influence chemosensitivity. Putative cancer stem cell markers of gastric cancer were found (e.g. ADAM17, CD133, FZD7, LGR5), and correlate with patient prognosis. Perioperative chemotherapy has improved patient survival and targeted therapy is applied in patients overexpressing Her2/neu. With regard to patient prognosis, complete surgical resection is still the most important predictor of patient outcome, followed by tumor stage, lymph node ratio, and mucin phenotype (Muc2). Among the diverse anxillary biomarkers that have been sought and identified, including class I histone deacetylases, none has reached a broader clinical application. Thus, molecular phenotyping of gastric cancer is still in its infancies and the search continues for novel diagnostic, prognostic and predictive biomarkers.

Keynote Lecture VO-027 Mechanisms of androgen resistance in prostate cancer D .J . Tindall1 1 Department of Urology, Mayo Clinic Foundation, Rochester, United States The androgen receptor (AR) signaling axis plays a critical role in the development, function and homeostasis of the prostate. The classical action of AR is to regulate gene transcriptional processes via AR nuclear translocation, binding to androgen response elements on target genes and recruitment of, or crosstalk with, transcription factors. Prostate cancer initiation and progression is also uniquely dependent on AR. Androgen deprivation therapy remains the standard of care for treatment of advanced prostate cancer. Despite an initial favorable response, almost all patients invariably progress to a more aggressive, castrate-resistant phenotype. Considerable evidence now supports the concept that development of castrate-resistant prostate cancer (CRPC) is causally related to continued transactivation of AR. Understanding the critical events and complexities of AR signaling in the progression to CRPC is essential in developing successful future therapies. This talk provides a synopsis of AR structure and signaling in prostate cancer in progression, with a special focus on recent findings on the role of AR in CRPC. Clinical implications of these findings and potential directions for future research are also outlined.

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Pankreaskarzinom VO-029 Molecular alterations in pancreatic ductal adenocarcinoma B . Sipos1 1 University of Tübingen, Department of Pathology, Tübingen In the last decade significant results have been achieved in understanding the molecular pathogenesis of pancreatic ductal adenocarcinoma (PDAC). Holistic approaches searching for genetic abnormalities indicate that PDACs harbor one or more genetic alterations in the majority of core pathways. These pathways include apoptosis, DNA damage and G1-S phase transition control; homophilic cell adhesion and integrin signaling; c-Jun, K-ras and other small GTPases associated pathways; TGF-beta, hedgehog and Wnt/notch signalling and finally the regulation of invasion. In the initiation of human PDACs K-ras, p53, DPC4/ Smad4 and p16/CDKN2a play a pivotal role, which is demonstrated by the increasing rate of alterations of these genes during progression of pancreatic intraepithelial neoplasia. Beyond these descriptive data from human studies on pancreatic intraepithelial neoplasia, experiments using genetically engineered mouse models (GEMM) provide functional evidence that K-ras, p53, p16/ CDKN2a alterations are key factors in emerging PDACs. GEMM that express mutant oncogenes and/or tumor suppressor genes under the control of pancreas specific promoters such as elastase, Pdx-1 and p48/ Ptf1a recapitulate the progression of pancreatic intraepithelial neoplasia to PDAC, following a characteristic acinar-ductal metaplasia in the pancreatic parenchyma. These GEMM give rise to PDAC, but also to other types of cancer. Targeting K-ras and p53 in GEMM results in well-reproducible tumor development, which allows reliable pre-clinical in vivo experiments in conjunction with sophisticated small animal imaging. High stroma content and paucity of intratumoral vessels are hallmarks of human PDAC. The stroma contains stellate cells, immune cells and large amounts of extracellular matrix, which all contribute to the malignant traits of PDACs and may even exert a selective pressure on tumor cells. Desmoplasia probably contributes to the innate chemoresistance of PDACs by hindering drug delivery. To date, there is no efficient targeted therapeutics for human PDAC. Targeted treatment may fail due to numerous affected pathways that facilitate evasion of tumor cells from the pressure of selective blocking agents. In the next decade the big challenge is to find the Achilles’ heel of PDAC, probably using intelligent combination of smart molecules and targeting of the stroma.

VO-030 The CRM concept of pancreatic cancer – a proposal for the new S3-Guideline A . Tannapfel1 1 Institut für Pathologie, Bochum Pancreatic ductal adenocarcinoma is diagnosed in about 13,000 patients each year in Germany being the fourth leading cause of cancer mortality. The 5-year survival rate remains less than 5% because of metastatic disease at time of initial diagnosis. It becomes evident, that ductal pancreatic cancer develops in a sequential process from lesions, named as Pancreatic Intraepithelial Neoplasia (PanIn). The curative removal of the tumor (R0 resection) may improve survival, but survival remains poor even in optimally resected patients. Loco regional and metastatic recurrence is frequent. The rate of microscopic margin involvement (R1) varies markedly in the current literature (from 5 to 85%). The rate of R1 resections is frequently underreported. One possible reason is the lack of the uniform use of R classification, followed by the lack of quality assessment for pathological examination of pancreaticoduodenectomy specimens. A standardized protocol for pathological examination should be used

to assess the correct rate of R1 resected patients and also the correlation between R1 resections and clinical outcome. The optimal standardized protocol for Whipple specimens, involving multicolor margin staining, axial slicing and extensive tissue sampling, has to be applied. The R classification is defined for any given tumor as R0, no residual tumor; R1, microscopic residual tumor; R2, macroscopic residual tumor. Since these definitions are internationally accepted, the concept of „circumferential resection margins“ is introduced for pancreatic cancer, describing the relation of the tumor edge to the circumferential margin (CRM). The pathologist has to measure the exact distance of the tumor to the definite resection margin. Tumors with a minimal distance from the CRM of < or=1 mm are categorized as CRM-positive, tumor with a distance of >1 mm are CRM-negative – in both cases a curative resection occurred. Only in the case of tumor cells visible directly at the resection margin, a R1 resection is diagnosed. The different use of both definitions of resection margin involvement improves valid comparisons between reports on treatment results. The CRM concept of pancreatic carcinoma will be introduced in the revised version of the S3 guideline.

VO-031 Molecular pathology of pancreatic adenocarcinoma P . Michl1 1 Klinik für Gastroenterologie, Endokrinologie und Stoffwechsel, PhilippsUniversität, Marburg Pancreatic cancer carries the most dismal prognosis of all solid tumors and is associated with a 5-year survival rate of less than 5%. Identification of novel, urgently required therapeutic modalities depends on detailed knowledge of the underlying molecular pathology. During recent years, progress has been made in deciphering the complex network of signaling cascades involved in cancerogenesis and tumor progression in pancreatic cancer. Furthermore, genetically engineered mouse models have been developed, including a model with conditionally activating mutation of K-Ras and inactivating mutation of p53, both of which are frequently found in the human disease. The murine tumors closely recapitulate histological and clinical presentation of human pancreatic tumor development and progression and are suitable for studying the impact of genetic manipulation of specific genes as well as for testing novel pharmacological inhibitors. In addition, accumulating evidence suggests a dominant role for the desmoplastic stromal reaction that comprises up to 90% of the tumor volume in mediating tumor progression. Signaling events within the tumor stroma such as activation of the hedgehog pathway have been shown to influence tumor growth, angiogenesis and resistance to chemotherapy. Moreover, infiltrating inflammatory cells within the stromal compartment have been shown to contribute to the resistant phenotype of this tumor entity. Numerous preclinical and clinical trials targeting tumor cell autonomous and non-autonomous stromal signaling cascades are currently underway to overcome the resistance to chemotherapy and to improve the appalling prognosis of pancreatic cancer.

Keynote Lecture VO-032 The epithelial-mesenchymal transition and cancer stem cells R .A . Weinberg1 1 Whitehead Institute, Massachusetts Institute of Technology, Cambridge, United States

vasion-metastasis cascade”, which includes local invasion by primary tumor cells, intravasation, passage through the systemic circulation, extravasation, formation of micrometastatic colonies, and the outgrowth of the latter in macroscopic growths, often termed colonization. In recent years, a cell-biological program termed the epithelial-mesenchymal transition (EMT) has been studied in great depth because it holds the promise of explaining many of the steps of this complex cascade. Thus, by passing through this program, a carcinoma cell acquires the attributes required to complete most of the steps of the invasion-metastasis cascade, quite possibly all of them except for the last step, colonization. This represents an enormous simplification of our conceptualization of this process, as it may represent nothing more than the activation of an otherwise-latent cell-biological program that is normally operative during embryogenesis and, in adults, wound-healing. Over the past several years, an additional aspect of the EMT program has come to light, as it has been found to be intertwined with the epithelial stem cell program. Thus, cells that have passed through the EMT program approach the stem-cell state. This holds important implications for both normal epithelial and neoplastic epithelial cells, as they both exploit the same program to organize themselves. In the context of metastasis, this implies that a cell that has pass through an EMT also acquires the self-renewal capability needed to seed a new colony of metastatic cells, an important prerequisite to successful colonization

Mechanisms of Progression and Therapy Resistance of Cancer I – English VO-033 Programmed necrosis W . Roth1 Institute of Pathology, University Hospital Heidelberg, and German Cancer Research Center, Heidelberg

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The research on cell death regulation has become one of the most important areas in tumor biology. Due to the central role of cell death regulation in tumor development and therapy resistance, a detailed knowledge of the molecular mechanisms of cell death opens up the possibility to develop novel rational therapeutic approaches for cancer. During the last decades the research on cell death was dominated by an oversimplified dual concept of apoptosis versus necrosis: Apoptosis was defined as an active, molecularly determined signaling cascade leading to “programmed cell death”, whereas necrosis was conceived as a passive process resulting from unspecific cellular damage. However, in the last few years this dogmatic conception has dramatically changed. Several studies clearly demonstrate that certain forms of necrotic cell death are executed in a strictly regulated and ordered fashion. The best known type of programmed necrosis is “necroptosis” which describes a signaling cascade mediated by TNF receptor 1 and RIP1 leading to cell death. Necroptosis plays an important role in the embryonic development, but also in the pathophysiology of certain diseases such as chronic inflammatory bowel disease. Yet another necrosis-like type of cell death can be induced by the HMGB1 protein. The HMGB1-induced cell death is morphologically characterized by the formation of giant mitochondria and metabolically by a severe deregulation of mitochondrial oxidative phosphorylation. The elucidation of the molecular mechanisms of necrotic cell death is an important step to develop novel strategies to overcome the classical resistance to apoptotic cell death which is one of the main reasons for therapy resistance in many types of cancer.

The molecular and the cellular mechanism of tumor progression have been elusive until recently. In particular, the mechanisms of invasion and metastasis have proven difficult to elucidate. These last steps of malignant progression involve a succession of steps often termed the “inDer Pathologe Suppl 1 · 2012  | 

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Abstracts VO-034 Interplay of cadherins in breast cancer progression M . Rezaei1, K . Friedrich1, A . Kettelhake1, B . Wielockx1, G . Baretton1, G . Breier1 University Hospital Carl Gustav Carus, TU Dresden, Institute of Pathology, Dresden

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Introduction. Deregulation of cadherin expression, such as the loss of epithelial (E-)cadherin and gain of neural (N-)cadherin, has been implicated in carcinoma progression. We have previously shown that vascular endothelial (VE-)cadherin can be expressed on human breast cancer cells, in addition to tumor endothelial cells. This aberrant expression pattern was recapitulated in a mouse mammary carcinoma model, and functional studies showed that VE-cadherin promotes experimental tumor growth by stimulating transforming growth factor (TGF)-beta signaling in cancer cells. Here, we have investigated the functional interplay between N-cadherin and VE-cadherin in breast cancer. Methods. The expression of N-cadherin and VE-cadherin was evaluated by immunohistochemistry in a tissue micro-array with 84 invasive human breast carcinomas. VE-cadherin and N-cadherin expression in mouse breast cancer cells was manipulated by RNA-interference or overexpression and analysed by immunofluorescence, reverse transcriptasepolymerase chain reaction, and western blot. Experimental tumors were generated by transplantation of the modified mouse breast cancer cells into immunocompetent mice. Tumor growth was monitored, and tumor tissue was subjected to histological analysis. Results. VE-cadherin and N-cadherin were largely co-expressed in invasive human breast cancers. Silencing of N-cadherin in mouse mammary carcinoma cells led to decreased VE-cadherin expression and induced changes indicative of mesenchymal-epithelial reverting transition (MET), as indicated by re-induction of E-cadherin, localisation of β-catenin at the cell membrane, decreased expression of vimentin and SIP1, and gain of epithelial morphology. Suppression of N-cadherin expression in mammary carcinoma cells inhibited tumor growth in vivo even with forced expression of VE-cadherin. Conclusions. The results underline the critical role of N-cadherin in breast cancer progression and show that N-cadherin is involved in the maintenance of the malignant fibroblastoid tumor cell phenotype. Ncadherin prevents the re-expression of E-cadherin and the localisation of β-catenin at the plasma membrane; consequently, β-catenin can exert its known protumorigenic activity in the cell nucleus. N-cadherin is also required to maintain the expression and protumorigenic activity of VEcadherin in malignant tumor cells but not vice versa. Thus, N-cadherin acts in concert with VE-cadherin to promote tumor growth.

VO-035 Cancer stem cells: targets and potential biomarkers for radiotherapy M . Krause1 Dept . of Radiation Oncology, OncoRay Center for Radiation Research in Oncology

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Radiotherapy has a curative potential in solid human tumours. Even in locally advanced, inoperable tumours, many patients can still be cured by radiotherapy or radiochemotherapy, e.g. up to 40% in advanced head and neck cancer. The current understanding of cancer stem cells (CSC) defines a CSC as a tumour cell that has the unique potential to self-renew and to regenerate a complete tumour with all its sublines of tumour cells. This definition implies that all CSC need to be inactivated to reach a permanent local tumour control, or, that a single surviving CSC after treatment will cause a recurrence. Thus, CSC should be ideal biomarkers and targets for radiotherapy. Today, there some evidence for a higher radioresistance of CSC measured by surface markers that are higher expressed in CSC versus nonCSC. If such biological differences hold true, CSC need to be included

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into the development of new predictive biomarkers. Recently, for the first time a systematic clinical study has shown a predictive value for the expression of the surface marker CD44 for local tumour control after primary radiotherapy of early laryngeal cancer. However, it has to be expected that in other tumour entities, the heterogeneity will be larger due to confounding factors of radiation resistance, e.g. tumour size or tumour micromilieu. The talk will give an overview on the current knowledge of the potential value of CSC for prediction of tumour control after radiotherapy. First attempts of specific targeting approaches will be discussed.

Mechanisms of Progression and Therapy Resistance of Cancer II – English VO-036 The role of HIF-prolyl hydroxylase-2 (PHD2) during physiological and pathological processes in mice B . Wielockx1 TUDresden – Pathology, Dresden

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Hypoxia is a prominent feature during development and physiological as well as pathological conditions in adults. An oxygen-sensing machinery is therefore very important to help the cells adapt instantaneously to any unacceptable O2 level. Such a system relies on the oxygen dependent HIF-prolyl hydroxylases (PHD1–3), enzymes that can inactivate the alpha subunit of the hypoxia inducible transcription factor (HIF). HIF1α is ubiquitously expressed in all tissues, whereas HIF2α is restricted to certain cell types. In case of low oxygen availability, PHDs lose their functionality and allow the HIF complex, composed of HIFα and a constitutive HIFβ subunit, to promote biochemical and physiological changes including anaerobic glycolysis, angiogenesis and hematopoiesis. We produced a mouse line that lacks HIF prolyl hydroxylase2 (PHD2) in different cell types (e.g. hematopoietic cells, epithelial cells). Moreover, these conditional PHD2-deficient mice display strongly elevated hematocrit levels (up to 85%) together with high EPO concentrations in the blood produced by kidney and brain. Remarkably, these mice show no premature lethality. In addition, we observed an enlargement of the spleen which we showed to be the major organ responsible for the enormous overproduction of RBCs. Double cKO mice revealed that the erythrocytosis phenotype is exclusively driven by HIF2 α whereas HIF1 α is responsible for the survival of cKO mice. Next, we found that the hematopoietic stem cell (HSC) compartment in the bone marrow was significantly altered. Detailed FACS analyses demonstrated that cKO mice contain much more proliferating multipotent progenitors (MPPs) under steady state conditions; an effect induced by HIF1  α . On the other hand, severe stress situations pushed quiescent cKO CD34neg HSCs to self-renewal. In addition, we subjected these cKO mice to different in vivo models highlighting the central role of PHD2 during inflammatory related disorders.

VO-037 Role of autophagy in cancer K . Datta1 1 Department of Biochemistry, University of Nebraska Medical Center, Nebraska, United States Autophagy is a regulated catabolic pathway that promotes lysosomal degradation of damaged proteins, cellular organelles, and other macromolecules. This self-digestion process, which facilitates the recycling of bioenergetic components, is activated by a number of stimuli, including the presence of reactive oxygen species, deprivation of growth factors,

DNA damage, and cytotoxic drugs. Autophagy dysregulation is associated with a number of disease states, including cancer. Autophagy plays different roles during the initiation and progression of cancer. While autophagy acts as a tumor suppressor during the initiation phase of cancer, it promotes tumor progression and metastasis in established cancers. Metastatic cancer cells that usually grow in a nutrient-poor microenvironment utilize autophagy to fulfil their high metabolic demand. Autophagy can facilitate survival during anchorage-independent growth or anoikis, and promotes therapeutic resistance. Furthermore, recent studies indicated that genetic or pharmacologic inhibition of autophagy sensitized tumor cells to anti-cancer treatment. It is therefore important to study the role of autophagy and its regulations in cancer cells, which will help defining optimal strategies to modulate autophagy for therapeutic advantage.

VO-038 Markers of autophagy in cancer M . Muders1 1 University Hospital Carl Gustav Carus at the University of Dresden, Institute of Pathology, Dresden Autophagy has been implicated in cancer progression and therapy resistance. Accordingly, different methods to identify and quantify autophagy in tissue samples and cell culture models will be applied more frequently. The traditional way to visualize autophagy at the ultrastructural level is electron microscopy. Electron microscopy has the ability to detect important structures that are involved during lysosomal degradation of organelles like autophagosomes. Easier and more cost effective is the immunohistochemical detection of autophagy substrates like LC3 or p62. In addition, proteins involved in autophagy like Beclin-1 could serve as an indicator of autophagy. In cell culture models, functional studies like detection of autophagic flux as well as long-lived protein degredation are used to monitor autophagic activity. However, all methods have their limitations and should be applied only after careful consideration of their strengths and weaknesses.

Translationale Forschung und Diagnostik – Mamma/Schilddrüse/Melanom VO-039 Translationale Forschung und Diagnostik: Karzinome der Schilddrüse A . Perren1, A .M . Schmitt1, M . Dettmer2 Institut für Pathologie, Bern, Switzerland, 2University of Pittsburgh, Department of Pathology and Laboratory Medicine, Pittsburgh, United States

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Histopathology and treatment of thyroid carcinomas poses several challenges: On a diagnostic level there is a group of tumors difficult (or impossible) to classify as benign or malignant, more importantly, the 10% of patients that cannot be cured by surgery and radio-iodine treatment is difficult to predict. The grey zone of follicular thyroid tumors of unknown malignant potential and morphological criteria of an adverse outcome (poorly differentiated thyroid carcinomas and tall-cell papillary thyroid carcinomas) will be discussed. On a molecular level, well differentiated thyroid carcinomas are well classified and the genetic basis is well known, however genetic events during carcinoma progression are less well understood. The most important genetic changes helping diagnosis, determination of prognosis will be discussed. Their role in guiding future targeted therapy will have to be shown in clinical trials.

VO-040 Translational research and diagnostics: Melanoma J . Rüschoff and Panel Members of DGP/BDP BRAF Testing Ring Study 1 Pathologie Nordhessen

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Most recently the first molecularly defined targeted therapy in metastatic and/or irresectable melanoma has been approved by EMA (20.2.2012). Vemurafinib (Zelburaf) is a small molecule that selectively inhibits BRAF kinase in its mutated form. About 50% of metastatic melanoma exhibit mutations within the BRAF oncogene almost exclusively at codon V600 activating the RAS-RAF-MEK-ERK signal transduction pathway. About 90% of mutations lead to an exchange of valin and glutamate (V600E). Within a large phase III clinical trial (BRIM3) median survival of Vemurafinib treated patients was 5.3 months instead of 1.6 months after chemotherapy with response rates of 48.4% versus 5.5%, respectively (Chapman PB et al. NEJM 2011; Sosman JA et al. NEJM 2012). In light of these data mutation testing of BRAF is becoming standard of care in malignant melanoma. This raises the question about testing methods and quality assurance. A working group of the DGP and BDP has addressed these aspects by performing a ring study where 9 Institutes of Pathology together with their clinical colleagues participated. Recommendations of testing and evaluation have been determined and a QUIP-based approach of quality assurance will be available in 04/2012 headed by the Universities of Heidelberg and Berlin [1]. Sensitivity and specificity of testing platforms (Sanger-, Pyro-, 454-Sequencing, real-time-PCR-based cobas® 4800) will be discussed together with the need of a strict tissue based approach making use of tumor cell enrichment, e.g. by microdissection. In addition to BRAF testing in melanomas further mutation analyses will be needed, e.g. of NRAS and CKIT, where targeted drugs are either available (CKIT) or under development (NRAS). Potential impact of new targeted drugs on testing probably in specific tumor subtypes such as acral or mucosal melanoma will be discussed. References

1 . Panel Members: M . Dietel (Berlin), A . Enk (Heidelberg), A . Lehmann (Berlin), J .N . Bauer (Tübingen), C . Garbe (Tübingen), U . Kellner (Minden), T . Kirchner (München), A . Jung (München), H . Kreipe (Hannover), S . Merkelbach-Bruse (Köln), R . Büttner (Köln), W . Schlake (Gelsenkirchen), P . Schirmacher (Heidelberg), R . Stadler (Minden) als Verfasser entsprechender Ankündigungspublikationen in JDDG und Der Pathologe, eingereicht 2012 .

Ausgewählte Vorträge aus den Einsendungen (Hauptprogramm und Arbeitsgemeinschaften) AG Gastroenteropathologie I – Leber DO-001a miR-101 is involved in steatosis and steatohepatitis of Non- Alcoholic Fatty Liver Disease (NAFLD) K .S . Ommer1, N . Elfimova1, A . Noetel1, H .-P . Dienes1, M . Odenthal1, N . Winkler2, M . Quasdorff2, I . Strack1, J . Riemer1 1 University Hospital of Cologne, Institute for Pathology, Köln, 2University Hospital of Cologne, Department of Gastroenterology and Hepatology, Köln Aims. The Non-Alcoholic Fatty Liver Disease (NAFLD) is a rising widespread disease. Frequently, steatosis results in steatohepatitis (NASH), however the factors, responsible for inflammatory progression, are yet unknown. Since previous profiling studies have pointed to miR-101 as a candidate involved in steatohepatitis, we have studied the role of miR101. Der Pathologe Suppl 1 · 2012  | 

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Abstracts Methods. LDL-receptor knockout mice were fed with a fatty diet. Subsequently, the miR-101 level was detected by Real-Time PCR. Primary hepatocytes of mice as well as human hepatoma cells were stimulated with free fatty acids or with proinflammatory factors TNF-a or IL-6. Following, the cellular and extra-cellular miR-101 levels were analyzed by PCR. Additionally, miR-101 was quantified in histological characterized biopsies (NASH-score 1–8) and serum samples of 55 patients with NAFLD (Ntissue, Nserum=55) and correlated to clinical data as well as liver apoptosis determined by M30/M65 measurement. Results. The expression of miR-101 was increased in fatty livers of LDLreceptor mouse model as well as in human patients with steatosis. In contrast, the miRNA level was diminished in livers with inflammatory changes. Both, the miR-101 enhancement in fatty liver as well as repression of miR-101 upon inflammation could be mimicked in vitro in a hepatoma cells stimulated with free fatty acids or proinflammatory mediators, respectively. Interestingly, miR-101 was also detected in human serum. These circulating miR-101 levels were associated with the M30 apoptosis values and with grades of steatosis and inflammation. In addition, circulating miR-101 levels inversely correlated to the expression of miR-101 in liver tissues. Conclusions. In NAFLD, the expression of miR-101 in liver tissues is contrarily influenced by free fatty acids and proinflammatory mediators. Alterations of miR-101 serum levels are suggested to indicate NAFLD progression into steatohepatitis.

DO-002a Regulation and function of the nuclear transport factor CAS in hepatocarcinogenesis J. Winkler1, J. Samarin1, V. Ehemann1, K. Breuhahn1, P. Schirmacher1, S. Singer1 1 Institute of Pathology/University Hospital Heidelberg, Heidelberg Aims. There is rising evidence that deregulation of nuclear transport factors contributes to cancer formation. An important member of the nucleocytoplasmic transport machinery is the RanGTPase dependent exporter CAS (Cellular Apoptosis Susceptibility) that recycles importinalpha from the nucleus to the cytoplasm. CAS was also shown to bind to p53 target gene promoters (e.g. PIG-3) and to be involved in apoptosome formation. Considering these pro-apoptotic properties high expression levels of CAS observed in different malignant tumors suggest additional protumorigenic functions. Here, we analyzed the expression, function, and regulation of CAS in hepatocarcinogenesis. Methods. CAS expression analyses in 188 HCCs, 9 dysplastic nodules and 20 normal liver samples were performed by using immunhistochemistry and in a subset of samples by semiquantitative real-time PCR (qRT-PCR). The impact of siRNA mediated CAS knockdown on cell viability, cell cycle, and apoptosis was analyzed in different HCC cell lines by using MTT-assays and FACS. The role of p53 and mTOR (the mammalian target of rapamycin) in regulating CAS expression in HCC cell lines was investigated by Westernblot (WB) and qRT-PCR upon treatment with appropriate compounds. Results. CAS was overexpressed on mRNA and protein level in up to ~70% of the HCCs analyzed and its expression was positively correlated with tumor dedifferentiation, proliferation (Ki-67) and nuclear accumulation of p53. A significantly decreased cell viability and increased apoptosis was observed upon CAS knockdown. Induction of wild-type p53 by Nutlin-3 led to reduced CAS protein and mRNA expression levels. Lowered levels of CAS protein were also observed after Rapamycin (mTOR inhibitor) treatment. Conclusions. Our data suggest a protumorigenic role of CAS in hepatocarcinogenesis apparently linked to a pro-survival function. We also conclude that CAS is a target of p53 mediated repression and identified mTOR as a positive regulator of CAS expression. Future studies in vitro and in vivo are required to gain further mechanistic insights into CAS dependent functions and to examine if CAS is a potential therapeutic target in HCC.

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DO-003 Molecular and functional analysis of long non-coding RNAs in hepatocellular carcinoma M. Hämmerle1, T. Gutschner1, M. Polycarpou-Schwarz1, C. Hildenbrand1, K. Breuhahn2, T. Longerich2, P. Schirmacher2, S. Diederichs1 1 Institute of Pathology, University Hospital & German Cancer Research Center (DKFZ), Heidelberg, 2Institute of Pathology, University Hospital, Heidelberg Aims. The vast majority of the human genome is represented by nonprotein-coding RNAs (ncRNAs), which are ribonucleic acids of different lengths without an open reading frame. Recently, different functions have been attributed to the few well-characterized ncRNAs, e.g. in epigenetics and cancer. However, the function of most of the newly discovered long ncRNAs is still unknown and a detailed analysis is lacking. Since cancer research has focused on protein-coding genes for the last decades, the potential of involvement of ncRNAs in the pathogenesis and prognosis of hepatocellular carcinoma (HCC) is not known so far. Therefore, our study aimed at identifying differentially expressed ncRNAs in HCC compared to control liver samples and at elucidating their role on the cellular and molecular level in order to draw conclusions about their contribution to the development of HCC. Methods. We screened for the expression of 17,000 ncRNAs in 32 cases of HCC and 7 control tissue samples. After identifying tumor-specific candidates, their expression was validated in HepG2 and Huh7 cells. Their impact on cell viability was uncovered after siRNA-mediated ncRNA knockdown in liver cancer cell lines. By using RNA affinity purification (RNA-AP), protein interaction partners were identified. Results. Statistical analysis unravelled 187 upregulated and 278 downregulated ncRNAs in HCC. One ncRNA, LOHC (Long non-coding RNA Overexpressed in Hepatocellular Carcinoma), was highly expressed in liver cancer compared to normal liver patient samples. Knockdown of LOHC expression significantly reduced cell viability and influenced cell cycle progression of HepG2 and Huh7 cells. Using RNA-AP, IGF2 mRNA binding proteins (IMPs) were identified as LOHC interaction partners. Moreover, interaction of LOHC and IMPs was largely different in diverse stages of cell cycle, which additionally influenced LOHC expression and stability over time. Conclusions. LOHC is an important ncRNA in HCC, which regulates cell viability and cell cycle. It was found to be an interaction partner of IMPs, which can regulate LOHC stability and have a major role in the pathogenesis of liver cancer. These data show that besides protein-coding genes, the expression of ncRNAs could be highly and specifically regulated in HCC, which will allow conclusions about the use of ncRNAs as potential diagnostic and prognostic markers. Most importantly, ncRNA expression profiling in cancer has identified functionally important players in liver tumorigenesis.

DO-004 miR-125b regulates the lin28/IGF-II axis during hepatocellular carcinogenesis N. Elfimova1, K.S. Ommer1, N. Winkler2, M. Quasdorff2, I. Strack1, J. Riemer1, A. Noetel1, H.-P. Dienes1, M. Odenthal1 1 University Hospital of Cologne, Institute for Pathology, Köln, 2University Hospital of Cologne, Department of Gastroenterology and Hepatology, Köln Aims. MicroRNA (miRNA), involved in posttranscriptional regulation of gene expression, play an important role in cell proliferation and differentiation. miR-125b expression was shown to be divergently expressed in liver carcinogenesis. Here, we focused on the role of miR-125b in development of hepatocellular carcinoma (HCC). Methods. A Cre-expressing adenoviral vector was applied to Alb-SV40 T-Ag transgenic mice in order to induce liver carcinogenesis. Expression levels of miR-125b were determined at different time points of tumorgenesis and in human hepatoma cell lines. Additionally, from 52 human

HCV-positive formalin-fixed and paraffin-embedded biopsies, sections were prepared and HCC were macrodissected. Subsequently, miR-125b was analyzed by real-time PCR. Putative miR-125b binding sites were fused to the luciferase reporter and reporter assays were carried out with miR-125b treated hepatoma cells. Results. During development of mouse HCC, the expression of miR-125b progressively decreased. In agreement miR-125b was reduced in human hepatoma cells in comparison to normal liver. Furthermore, miR-125b decrease depending on the progression of hepatocarcinogenesis was confirmed in human samples, showing significant lower levels in HCC high grades than in dysplastic foci or cirrhosis. Overexpression of miR125b in Hep3B and Pop10 cells resulted in a pronounced reduction of cell growth. Screening of putative miR-125b target transcripts by various algorithm calculations identified various pathways involved in proliferation and apoptosis. Reporter assays of 3’-UTR-regions of the putative targets identified miR-125b binding sites in lin-28 mRNA. Since lin28 is known to effect synthesis of the mitogen IGF-II, the miR-125b/lin28 axis is suggested to be involved in HCC pathogenesis by IGF-II mediated regulation of cell growth. Conclusions. Expression of miR-125b is down-regulated during progression of hepatocarcinogenesis leading to up-regulation of lin-28 that in turn triggers enhanced cell growth and proliferation.

DO-005 The PI3K-AKT-mTOR axis contributes to the functional inactivation of p53 through stabilization of MDM4 in human hepato­cellular carcinoma R. Pellegrino1, O. Neumann1, P. Schirmacher1, T. Longerich1 University Hospital Heidelberg/Institute of Pathology, Heidelberg

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Aims. Mutational inactivation of p53 gene is rare in Western hepatocellular carcinoma (HCC). MDM4, one of the main p53-regulating factors, is frequently upregulated in human HCC. This overexpression can be in part explained by chromosomal gains at 1q34.1. Here we investigated the role of the PI3K-AKT axis in the stabilization of the MDM4 protein. Methods. All experiments were performed in human HCC cell lines with different p53 gene status. PI3K and mTOR were specifically inhibited using chemical compounds in vitro; specific siRNAs were transiently transfected to target AKT1 and ATK2 and to validate the results from drug treatment. Realtime RT-PCR analysis was used to check for restored p53 transcriptional activity. In addition, combined cycloheximide and siRNAs treatment was performed to study the protein stability of MDM4 under these experimental conditions. Results. Using a specific PI3K inhibitor or siRNAs targeting AKT1/2 in HCC cell lines, we observed a strong decrease of MDM4 protein levels, which resulted in the activation of p53 target genes (e.g. PUMA, BAX and p21) indicating restored p53 gene function in these lines. Cycloheximide treatment combined with siRNA-mediated AKT inhibition indicated that MDM4 is phosphorylated by AKT2 and that this phosphorylation is responsible for the stabilization of the protein via the protection from proteasomal degradation. This effect was independent from both MDM2 and p53 gene status. Furthermore, we showed that the Eukaryotic translational Elongation Factor 1 alpha 2 (EEF1A2), which we reported upregulated in human HCC, is involved in the activation of the PI3K-AKT axis. Specific siRNA inhibition of EEF1A2 resulted in decreased pAKT and MDM4 protein levels in HCC cell lines. Moreover, treatment with the mTOR inhibitors, Rapamycin and PI-103, decreased MDM4 protein levels indicating that the PI3K-AKT-mTOR axis is involved in the MDM4 regulation in vitro. Conclusions. Our data demonstrate that the EEF1A2-PI3K-AKT-mTOR axis is involved in maintaining protumorigenic MDM4 levels in human HCC cell lines, which in turn promotes functional inactivation of p53. Moreover, we showed that the AKT-mediated phosphorylation of MDM4 is the crucial mechanism to prevent its proteasomal degradation.

DO-006 HSF1 is a downstream effector of Ras and AKT protooncogenes and contributes to hepatocellular carcinoma development and progression D.F. Calvisi1, S. Mattu1, S. Delogu1, V. De Murtas1, G. Gasparetti1, G. Destefanis1, X. Chen2, F. Dombrowski1, M. Evert1 1 University Medicine Greifswald, Institute for Pathology, Greifswald, 2 University of San Francisco, Liver Center, San Francisco, United States Aims. Recent evidence suggests an oncogenic role of heat shock transcription factor 1 (HSF1) in cancer, but its functional relevance in hepatocellular carcinoma (HCC) remains poorly delineated. Methods. We have investigated HSF1 function both via in vitro and in vivo approaches as well as in a collection of human HCC. Results. In human liver specimens, we found that HSF1 was progressively induced from non-tumorous surrounding livers to HCC, reaching the highest levels in tumors with a poorer outcome (as defined by the length of patient’s survival). In HCC cell lines, overexpression of HSF1 resulted in increased activity of MAPK and AKT/mTOR pathways and suppression of JNK cascade, leading to augmented proliferation and angiogenesis and reduced apoptosis in vitro. Conversely, suppression of HSF1 in HCC cell lines decreased MAPK and AKT/mTOR activity, and induced JNK-dependent apoptosis. Forced overexpression of either Ras or AKT protooncogenes triggered upregulation of HSF1 in HCC cell lines via the small RalA GTPase. Of note, HSF1-overexpressing cells were specifically sensitive to growth inhibition and induction of apoptosis following the treatment with either AMPK activators or hexokinase inhibitors. Finally, overexpression of a HSF1 dominant negative form by hydrodynamic gene delivery strongly reduced the oncogenic potential of activated Ras and AKT in a mouse model of aggressive liver cancer. Conclusions. Altogether, the present data indicate that activation of HSF1 plays a major role in hepatocarcinogenesis by enhancing the activity of Ras and AKT, and might represent a valuable candidate for innovative targeted therapies against human HCC.

DO-007 Perturbation of hepatocytes metabolism by AKT contributes to growth in insulin-induced hepatocarcinogenesis and is reverted by the PI3K/mTOR dual inhibitor NVP-BEZ235 M. Evert1, D.F. Calvisi1, K. Evert1, V. De Murtas1, G. Gasparetti1, S. Mattu1, G. Destefanis1, S. Thiel1, A. Thiele1, S. Ribback1, F. Dombrowski1 1 University Medicine Greifswald, Institute for Pathology, Greifswald Aims. Mounting evidence supports a role of insulin signaling deregulation and diabetes mellitus in human hepatocarcinogenesis. To study the oncogenic effect of chronically elevated secretion of insulin on hepatocytes in the presence of mild hyperglycemia, we developed a model of pancreatic islet transplantation into the liver. Methods. In this model, islets of a donor rat are transplanted into the liver of a recipient diabetic rat, with resulting local hyperinsulinism that leads to the development of preneoplastic lesions and hepatocellular carcinoma (HCC). Here, we investigated the metabolic and growth properties of the AKT pathway in this model of insulin-induced hepatocarcinogenesis. These findings were recapitulated in HCC cell lines in vitro. Results. We found that activation of insulin signaling triggers a strong induction of the AKT cascade that is paralleled by increased synthesis of fatty acids, cholesterol, and triglycerides, induction of glycolysis and decrease of fatty acid oxidation and gluconeogenesis in rat preneoplastic and neoplastic liver lesions when compared with normal liver. AKT-dependent metabolic effects of insulin on hepatocytes were recapitulated in vitro using human HCC cell lines. In these cells, suppression of lipogenesis, glycolysis, and the pentose phosphate pathway triggered a strong growth restraint despite insulin administration. Of note, metabolic abnormalities and proliferation driven by insulin were effectively reverted

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Abstracts using the dual PI3K/mTOR inhibitor, NVP-BEZ235, both in vitro and in vivo. Conclusions. Thus, AKT activation by unconstrained insulin signaling induces a defined module of metabolic alterations in hepatocytes contributing to aberrant cell growth. The inhibition of AKT and related metabolic changes might represent a novel preventive and therapeutic approach to effectively inhibit insulin-induced hepatocarcinogenesis.

AG Gastroenteropathologie IV – Oberer GI-Trakt DO-015 STAT3 activation and Mcl-1 and MMP9 target gene expression is preferentially seen in esophageal squamous cell carcinomas, but not Barrett‘s adenocarcinomas S . Timme1, K . Atanasov1, C .D . Fichter1, A . Schoepflin1, L . Bogatyreva2, D . Hauschke2, L . Tang3, H . Geddert4, G . Faller4, D . Klimstra3, O . Opitz5, M . Werner1, S . Lassmann1 1 Institute of Pathology, University Medical Center, Freiburg, 2Institute of Medical Biometry and Medical Informatics, University Medical Center, Freiburg, 3Dept . of Pathology, Memorial Sloan Kettering Cancer Center, New York, United States, 4Institute of Pathology, St-Vincentius-Kliniken, Karlsruhe, 5 Tumorzentrum Ludwig Heilmeyer – CCCF, Freiburg Aims. Active (phosphorylated) signal transducer and activator of transcription 3 (P-STAT3) is translocated to the nucleus. By this, (P-)STAT3 suppresses apoptosis or induces cell migration via Mcl-1, Bcl-xl and Survivin or matrix metalloproteinases (e.g. MMP9) expression, respectively. This STAT3 activity can be triggered by an active EGFR. To complement our data on P-STAT3 expression in esophageal squamous cell carcinomas (ESCC) and Barrett’s adenocarcinomas (BAC), we investigated EGF-mediated STAT3 activity in ESCC and BAC cell lines as well as inactive STAT3 expression in ESCCs and BACs. Methods. Serial sections of 105 esophageal carcinomas (n=60 BAC; n=45 ESCC) were evaluated for STAT3 expression by semi-quantitative immunohistochemistry. Data of nuclear P-STAT3 expression was already available. Statistical analysis was performed using Mann-Whitney-U tests at p<0.05 (SPSS v 18). ESCC (OE21) and BAC (OE33) cell lines were subjected to 1) EGF stimulation and 2) STAT3 inhibition (siRNA), with subsequent analysis (q-RT-PCR, Western blot) of STAT3 activation and STAT3 targets (Cyclin-D1, Survivin, Bcl-xl, Mcl-1, MMP9). Results. In tissue specimens, STAT3 was similarly expressed in ESCCs and BACs (p=0.358), but preferentially activated (P-STAT3) in ESCCs (p=0.013). In vitro, EGF treatment resulted in a dramatic activation and phosphorylation of STAT3 in ESCC (OE21), but not BAC (OE33) cells. This was accompanied by marked up-regulation of Mcl-1 and MMP9 mRNA expression in OE21 cells. Cyclin-D1, Survivin and Bcl-xl mRNA expression showed no changes. STAT3-specific siRNA downregulated STAT3 expression and activation in both OE21 and OE33 cells. This was accompanied by marked apoptosis in OE21, but not OE33 cells. Conclusions. Our data demonstrate that STAT3 signalling differs in tissue specimens and cell lines of ESCC and BAC. EGF may activate STAT3 and induce MMP9 in OE21 cells, but not in BAC cells. Therefore, STAT3 signalling may contribute to stromal remodelling in ESCCs. Finally, also potential therapeutic STAT3 inhibition may be more effective in ESCC (OE21).

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DO-016 Alterations of PIK3CA/PTEN and KRAS/NRAS/BRAF in primary resected esophageal adenocarcinomas: loss of PTEN is associated with worse clinical outcome S . Berezowska1, M . Bettstetter2, G . Keller3, J . Slotta-Huspenina3, E . Drecoll3, M . Feith4, H . Höfler3, R . Langer5 1 University of Bern, Institute of Pathology, Bern, Switzerland, 2Teilgemeinschaftspraxis Molekularpathologie Südbayern, Munich, 3Technische Universität München, Munich, Institute of Pathology, 4Technische Universität München, Munich, Department of Surgery, 5Technische Universität München and University of Bern, Institutes of Pathology, Switzerland Aims. Targeted therapy against EGFR is currently under investigation to become an interesting approach for the treatment of highly malignant esophageal adenocarcinoma. In colorectal carcinomas, mutations of KRAS, NRAS, BRAF, PTEN and PIK3CA, have been shown to influence the responsiveness of tumors against anti-EGFR therapy in a large number of studies. In contrast, there are only few data about the prevalence of alterations of these molecules in esophageal adenocarcinomas. We therefore aimed to investigate the prevalence of mutations of KRAS, NRAS, BRAF, PTEN and PIK3 in a large collective of esophageal adenocarcinomas and to determine their clinicopathologic impact. Methods. A total of 144 primary resected tumors were investigated. Mutation analysis for KRAS (Exons  2, 3), NRAS (Exons  2, 3), BRAF and PIK3CA (Exons 9, 20) was performed after DNA extraction using realtime PCR with high resolution melting analysis (HRMA), followed by sequencing of positive cases. For assessment of PTEN status, immunohistochemistry was performed. Results were correlated with pathological findings (UICC pTNM category, tumor differentiation) and survival. Results. In total, 3/134 tumors (2%) had a KRAS G12D mutation, one tumor had a KRAS G12V mutation and one tumor had an E545K mutation of PIK3CA. No NRAS or BRAF mutation was detected. 16/117 cases (14%) were immunohistochemically negative for PTEN. There was no correlation between mutational status of the tumors and histopathologic features. However, loss of PTEN was associated with shorter overall and disease free survival (p<0.001 each) and also an independent prognostic factor in multivariate analysis (HR=2.1; p=0.044 and HR=2.1; p=0.039, respectively). Conclusions. Loss of PTEN can be detected in a significant subset of esophageal adenocarcinomas and is associated with an aggressive phenotype. Therefore PTEN may be useful as a prognostic biomarker. In contrast, mutations of KRAS, NRAS, BRAF and PIK3CA appear only very rarely if at all in esophageal adenocarcinomas. The role of PTEN for response to anti EGFR treatment may be part of further investigations, while determination of RAS/RAF/PIK3 mutations may only have impact for very few patients in this context.

DO-017 Relevance of MET activation and genetic alterations of KRAS and E-cadherin for cetuximab sensitivity of gastric cancer cell lines S . Heindl1, E . Eggenstein1, S . Keller1, J . Kneißl1, G . Keller1, K . Mutze1, S . Rauser2, G . Gasteiger1, I . Drexler1, A . Hapfelmeier1, H . Höfler1, B . Luber1 1 TU München, München, 2Helmholtz Zentrum München, Neuherberg Aims. The therapeutic activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab in gastric cancer is currently being investigated. Reliable biomarkers for the identification of patients who are likely to benefit from the treatment are not available. The aim of the study was to examine the drug sensitivity of five gastric cancer cell lines towards cetuximab as a single agent and to establish predictive markers for chemosensitivity in this cell culture model. The effect of a combination of cetuximab with chemotherapy was compared between a sensitive and a non-sensitive cell line. Methods. EGFR expression, activation and localisation, the presence and subcellular localisation of the cell adhesion molecule E-cadherin as

well as MET activation were examined by Western blot analysis, flow cytometry and immunofluorescence staining. Cells were treated with varying concentrations of cetuximab and cisplatin and 5-fluorouracil in tumor relevant concentrations. The biological end point was cell viability, which was measured by XTT cell proliferation assay. Response to treatment was evaluated using statistical methods. Results. We assessed the activity of cetuximab in five gastric cancer cell lines (AGS, KATOIII, MKN1, MKN28 and MKN45). The viability of two cell lines, MKN1 and MKN28, was significantly reduced by cetuximab treatment. High EGFR expression and low levels of receptor activation were associated with cetuximab responsiveness. MET activation as well as mutations of KRAS and E-cadherin were associated with cetuximab resistance. Conclusions. These data indicate that our examinations may be clinically relevant and the candidate markers should therefore be tested in clinical studies.

DO-018 Notch2 expression and chemoresistance in neoadjuvant treated gastric cancer L . Bauer1, R . Langer1, M . Mandl1, K . Becker1, J . Slotta-Huspenina1, A . Novotny2, A . Hapfelmeier3, H . Höfler1, G . Keller1 1 Technische Universität München, Department of Pathology, München, 2 Technische Universität München, Department of Surgery, München, 3 Technische Universität München, Department of Medical Statistics and Epidemiology, München Aims. In a recent study analyzing the prognostic significance of the expression of cancer stem cell (CSC) related genes in residual gastric tumor cells after neoadjuvant chemotherapy, Wnt and Notch signaling genes, among others, showed a prominent association with survival. The aim of this study was to assess selected genes for differential expression between pretherapeutic biopsies and resected specimens. In vitro we investigated the impact of Notch activity on chemosensitivity in gastric cancer cell lines. Methods. Expression of 12 genes was compared between corresponding biopsies and resected specimens from patients treated with neoadjuvant chemotherapy (CTx) demonstrating partial (n=22) or minimal/ no tumor regression (n=22). mRNA was isolated from macrodissected FFPE tissues and gene expression was quantified by real time PCR using TaqMan® low density arrays. Immunohistochemical staining (IHC) for Notch2 was performed on biopsy/resected-specimen pairs from patients with sub-total, partial and minimal/no tumor regression (n=22, each) and from patients not treated by CTx. (n=16) and evaluated by a semiquantitative scoring system. Chemosensitivity of three gastric cancer cell lines to the gamma-secretase inhibitor DAPT alone or in combination with cisplatin was determind by XTT or colony formation assays. Results. Differential expression analysis revealed an increase of Notch2 and POU5F1 from biopsies to resected tumors in tumors with partial response (p=0.002 and 0.028) and minimal/non responding tumors (p=0.062 and 0.002). In contrast a decrease in expression was observed in both tumor groups for Notch1 (p=0.072 and 0.001). Immunhistochemical analysis of Notch2 revealed that cytoplasmic staining intensities of tumor cells decreased significantly in all groups of CTx-treated patients (p=0.016, .001 and 0.017) but not in non-CTx patients. IHC analysis of Notch1 is in progress. Treatment of gastric cancer cell lines with 10 µM DAPT and 2 µM cisplatin led to a synergistic reduction of metabolic activity in comparison to the single drugs. Conclusions. The comparison of mRNA expression between corresponding biopsies and resected specimens revealed alterations consistent with an enrichment of chemotherapy resistant residual tumor cells. Results of Notch2 IHC are in line with the mRNA data. The synergistic effect of cisplatin and the gamma-secretase inhibitor DAPT in vitro suggests that Notch signaling might be involved in chemoresistance of gastric cancers.

AG Gastroenteropathologie VI – Unterer GI-Trakt DO-020 Aurora-A protein expression is associated with multipolar mitoses independent of molecular class of colorectal carcinomas* D . Batarello1, A . Schoepflin1, D . Hauschke2, M . Werner3, S . Lassmann1 1 Institute of Pathology, University Medical Center Freiburg, Freiburg, 2 Institute of Medical Biometry and Medical Informatics, University Medical Center Freiburg, Freiburg, 3Institute of Pathology, University Medical Center Freiburg Aims. Aurora-A overexpression may induce supernumerary centrosomes, respective multipolar mitoses, and aneuploidy in model systems. Here, we examined the occurrence of Aurora-A positive multipolar mitoses in aneuploid (microsatellite-stable, CIN-type) versus near-diploid (microsatellite-instable, MIN-type) colorectal carcinomas (CRC). Methods. Three-dimensional immunofluorescence (3D-IF) of Aurora-A was performed on 8µm thick FFPE tissue sections of 18 previously characterized colorectal carcinomas. Stained sections were screened by a x63/1.3 oil objective at 0.7µm image stacks (one x63 field = high power field/HPF; total of 374 HPFs, range 10–46 HPF per case). Total numbers of mitoses (n=476, range 11–57 per case) and numbers of bipolar (2 Aurora-A positive centrosomes/spindle poles) and aberrant multipolar (>2 Aurora-A positive centrosomes/spindle poles) mitoses were counted. For differences of mitotic counts and frequencies between CIN-type and MIN-type CRCs, the Wilcoxon Test (exact, two-sided; with p<0.05) was applied. Results. In situ, three-dimensional immunofluorescence (3D-IF) revealed similar numbers of mitoses in CIN- and MIN-type CRCs (CIN=31±17; MIN=25±12, p=0.463). Both bipolar and multipolar Aurora-A positive mitoses were detected within the same case of CIN- or MIN-type CRCs. Multipolar mitoses also included pro-/metaphase mitoses with multiple Aurora-A signals, respective supernumerary centrosomes in both CIN(23±13%) and MIN- (30±14%) type CRCs. Bipolar mitoses (CIN=73±14%; MIN=66±14%) and multipolar mitoses (CIN=27±14%; MIN=34±14%) were statistically equally distributed between CIN- and MIN-type CRCs (p=0.224). Conclusions. Aurora-A positive multipolar mitoses, respective mitotic cells with supernumerary centrosomes, occur similarly frequent in CINand MIN-type colorectal carcinomas. The occurrence of multipolar mitoses in colorectal carcinomas is hence not exclusively linked to increased Aurora-A gene copy numbers and/or high Aurora A (over)expression. Aurora-A overexpression may play a more prominent role for establishment of aneuploidy in earlier steps of colorectal carcinogenesis. *Study supported by DFG (LA1290/3-1), associated publication: Herz et al . Mol Carcinog . 2011 Jul 7 . doi: 10 .1002/mc .20823 .

DO-021 MiR-214-induced chemoresistance in CIMP+ colorectal adenocarcinomas J . Munding1, S .-T . Liffers1, S . Ladigan1, A . Tannapfel1, A . Mirmohammadsadegh1 1 Ruhr University Bochum, Institute of Pathology, Bochum Aims. Colorectal adenocarcinomas can be divided in subgroups according to their CPG-island methylation (CIMP) status. Previous analysis has shown differences in miRNA expression patterns according to the CIMP status. MiR-214 turned out to be more frequently up-regulated in CIMP+ compared to CIMP− tumors. Therefore we aimed to characterize its functional impact in a model system consisting of a CIMP+ and a CIMP− colorectal adenocarcinoma cell line. Methods. MiR-214 expression was analyzed in micro-dissected CIMP+ (n=30) and CIMP− (n=35) tumors by qRT-PCR compared to normal muDer Pathologe Suppl 1 · 2012  | 

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Abstracts cosa. MiR-214 expression was stably reconstituted in the colorectal cancer cell lines RKO (CIMP+; MSI) and SW480 (CIMP−; MSS) via retroviral gene transfer. Its impact on tumor growth and response to oxaliplatin and 5-FU treatment was analyzed by MTT assays, Annexin V staining and cell cycle analysis by flow cytometry. Results. Up-regulation of miR-214 in CIMP+ colorectal adenocarcinomas could be demonstrated in more than 45% of the tumor specimens analyzed. MiR-214 overexpressing cells showed an increased proliferation regardless of the CIMP background in vitro. Further, we observed that only the CIMP+ colorectal adenocarcinoma cell line RKO overexpressing miR-214 was more resistant to oxaliplatin as well as 5-FU treatment. Conclusions. Up-regulation of miR-214 expression is frequently observed in CIMP+ tumors. Its increased expression is linked to a higher growth rate. Additionally, we were able to show that the overexpression of miR214 in a cell line with a CIMP+ phenotype leads to a 5-FU and oxaliplatin chemoresistance.

DO-022 EWSR1: Identification and functional characterization of a novel target gene locus in Lynch syndrome S. Piscuoglio1, S. Kishore2, V. Mele3, F. Trapani3, M. Zavolan2, M. Kovac1, L. Terracciano3, K. Heinimann1 1 University of Basel, Department of Biomedicine, Basel, Switzerland, 2University of Basel, Biozentrum and Swiss Institute of Bioinformatics, Basel, Switzerland, 3University of Basel, Institute of Pathology, Basel, Switzerland Aims. Lynch syndrome represents the most common, autosomal dominantly inherited cancer predisposition worldwide and accounts for 3-5% of the total colorectal cancer (CRC) burden. It is caused by germline mutations in DNA mismatch repair (MMR) genes. MMR deficiency results in microsatellite instability (MSI). MSI, used as a diagnostic tool to identify HNPCC-related CRCs, can also affect repeat tracts within or immediately adjacent to the coding sequence of so-called target genes which are thought to fuel carcinogenesis in HNPCC. In search for novel target gene loci we identified a large poly-T tract, (T)16, in the 3’ untranslated region of the Ewing sarcoma breakpoint region 1 (EWSR1) gene. Our aims are: to determine 1) type and frequency of instability at the EWSR1 (T)16 in 78 HNPCC and 123 sporadic colorectal cancers and 2) its possible effect on EWS expression. Methods. We determined the length of the EWSR1 3’UTR tract motif, (T)16, by PCR amplification and fragment analysis of 78 CRCs from HNPCC patients with identified germline mutation, 123 sporadic microsatellite-stable CRCs as well as 5 CRC cell lines (2 MMR proficient, 3 MMR deficient). EWS protein expression was assessed by immunohistochemistry (IHC) on a tissue microarray and immunoreactivity scored semi-quantitatively. Statistical comparisons were performed using Chi-square or Student’s t test where appropriated (two-tailed p-values, considering p<0.05 to be statistically significant). Results. Novel alleles at the EWSR1 3’UTR (T)16 tract were observed in all (n=78 ) HNPCC but none (0/123) of the sporadic CRCs. The type (insertion/deletion) and frequency of somatic alterations were: ins1-2 (2.5%), del3 (22.6%), del4 (34.4%), del5 (28.54%) and del>6 (11.92%). Similar observations were made for the MMR-deficient cell lines whereas the MMR-proficient ones were stable. RNA secondary structure prediction suggested gross structural alterations for deletions >4 Ts. IHC showed significant downregulation of EWS expression in sporadic CRCs (p<0.005), but no difference in HNPCC CRCs (p=0.7). Since the (T)16 tract may represent a binding site for AU-rich element binding proteins it could have an effect on EWSR1 mRNA stability/translation. Conclusions. The (T)16 tract in the 3’UTR of the EWSR1 gene represents a novel target gene locus in Lynch syndrome allowing for highly sensitive and specific identification of MMR-deficient CRCs. Moreover, IHC analysis suggests a regulatory role of this locus on EWS protein expression in HNPCC CRC cancers.

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DO-023 Molecular basis of serrated lesions of the colon T. Gaiser1, S. Meinhardt1, D. Hirsch1, J.K. Killian2, J. Gaedcke3, P. Jo3, I. Ponsa4, J. Rueschoff5, G. Seitz6, Y. Hu1, J. Camps1, T. Ried1 1 Section of Cancer Genomics, Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, United States, 2Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, United States, 3GeorgAugust-University, Department of General and Visceral Surgery, Göttingen, 4 Universitat Autònoma de Barcelona, Universitat Autònoma de Barcelona, Barcelona, Spain, 5Institute of Pathology Nordhessen, Kassel, 6Hospital of the Sozialstiftung Bamberg, Department of Pathology, Bamberg Aims. Colorectal cancer (CRC) develops from a variety of polyps and is mainly forced by three molecular pathways: chromosomal instability (CIN), microsatellite instability (MSI) and CpG island methylation (CIMP). Polyps are classified histologically as conventional adenoma, hyperplastic polyp, sessile serrated adenoma/polyp (SSA/P) and traditional serrated adenoma (TSA). However it is unclear which lesion follows which pathway. DNA was extracted from colon tumors and mucosa samples and analyzed by array comparative genomic hybridization (n=125), by sequence analysis of the oncogenes BRAF, KRAS, PI3K (n=96), and by the GoldenGate™ Methylation Cancer Array (n=136). Methods. Microsatellite instability (MSI) was tested indirectly by immunohistochemistry (IHC). A loss of MLH1, MSH2, MSH6 or PMS2 (n=130) was assigned as high microsatellite instability (MSI-H) while low microsatellite instability (MSI-L) showed only mgMT IHC negativity (n=10). Results. Chromosomal imbalances were detected in 78% of all MSI-H CRCs, most commonly as a gain of chromosome 8. On average, MSI-H CRCs showed 3 imbalances per sample, while microsatellite stable (MSS) CRCs showed 7  imbalances per sample (MSI-L CRCs showed an average of 5). Methylation data analyses allowed classification of samples into four groups and detected similar methylation profiles of SSA/Ps and MSI-H CRCs. TSAs also revealed aberrant methylation pattern, but clustered more heterogeneously and closer to MSS CRCs. Conclusions. Chromosomal instability, in contrast to the established doctrine, is a common phenomenon in MSI CRCs yet to a lower extent and at later stages compared to MSS CRCs. Methylation analyses suggest that SSAs are precursors for MSI-H CRCs and follow the CIMP pathway.

DO-024 Upregulation of c-MYC and SIRT1 expression correlates with the step-wise malignant transformation in the serrated route to colorectal cancer L. Kriegl1, M. Vieth2, T. Kirchner3, A. Menssen3 1 Ludwig-Maximillians-Universtiät, Institute for Pathology, München, 2 Klinikum Bayreuth, Institute of Pathology, Bayreuth, 3Ludwig-MaximilliansUniverstiät, Institute for Pathology, München Aims. Approximately 7.5% of all colorectal cancers are considered to originate from the alternative, serrated route. Here, we investigate the expression of the c-MYC oncogene and the SIRT1 protein deacetylase in subgroups of colorectal serrated lesions that were characterized by different molecular alterations. Methods. A collection of 121 serrated lesions was classified with regard to BRAF(V600E) exon 15 and KRAS exon 2 codon 12/13 mutational status. Immunohistochemical staining of C-MYC, SIRT1 and β-catenin was done. c-MYC and SIRT1 expression was evaluated regarding nuclear expression pattern ranging from 0–3, for negative, low, moderate and strong staining. Regarding β-catenin staining, only nuclear β-catenin expression was taken into account. Results. We demonstrate a correlation of the expression of c-MYC and SIRT1 with mutant KRAS and BRAF. Increased expression of c-MYC and SIRT1 was strongly associated with higher grades of malignancy in this group. In contrast, in the majority of serrated lesions without KRAS

or BRAF mutation, c-MYC and SIRT1 expression was not found increased. In addition, within the group of serrated lesions with wild type KRAS and BRAF, a subgroup was characterized by elevated c-MYC and SIRT1 expression. In these lesions with mostly high grade intraepithelial neoplasia and carcinomas, nuclear localization of β-catenin suggested that activation the wnt signalling pathway may mediate the induction of c-MYC at the transcriptional level. Conclusions. In summary, we established a link of oncogenic K-Ras and B-Raf as well as wnt signalling to activation of the c-MYC oncogene and SIRT1 in the serrated route to colorectal cancer. The elevated expression levels observed within higher grades of malignancy point to a crucial function of c-MYC and SIRT1 in the malignant transformation.

AG Pneumopathologie I DO-025 HOPE-technique improves diagnostics of Bronchoalveolar Lavage (BAL) E . Vollmer1, S . Marwitz1, M . Abdullah1, C . Vock1, J .S . Fine2, S . Visvanathan2, K . Gaede1, H .-P . Hauber1, P . Zabel1, T . Goldmann1 1 Research Center Borstel, Borstel, 2Roche, Inflammation Discovery, United States Aims. Besides its application in pulmonary routine diagnostics BAL is a useful tool for scientific investigations. Because of its limitations in long term storage we explored in this study the utility of a novel fixative (HOPE, Hepes glutamic acid buffer mediated Organic solvent Protection Effect) for retrospective and standardized cell analysis also in regard of immunological and molecular techniques. This study has been performed in accordance with the 1964 Declaration of Helsinki and its later amendments. Methods. BAL samples, obtained by flexible bronchoscopy from patients with different diseases, were diluted to a standard cell number of one million cells and incubated in HOPE-fixative as well as in neutral buffered 4% formalin with a subsequent paraffin-bloc-embedding. In addition, for addressing RNA preservation, fresh frozen samples were included. For enhanced/expanded high-throughput analyses of multiple BAL samples tissue microarrays of paraffin embedded HOPE-BAL were also produced. Results. We have shown that HOPE-BAL cells have an excellent morphology; besides that this technique allows archiving of BAL cells. By preservation of proteins and nucleic acids it allows the application of immunocytochemistry as well as a plethora of molecular techniques like in situ hybridization, quantitative PCR, transcription microarray analysis, 2-D-Gel-Electrophoresis etc. We showed by targeting some exemplary molecules the power of screening and validating HOPE-BAL for new biomarkers. Conclusions. The HOPE-BAL-technique allows long term storage of BAL cells and is a unique and novel tool for various molecular based applications in pulmonary medicine. It combines easy handling in the form of paraffin blocks with almost no limitations in readout techniques thus being a step forward into enhanced molecular diagnostics and biobanking.

DO-026 Tissue sparing application of the newly proposed IASLC/ATS/ ERS classification of non-small cell lung cancer shows practical diagnostic and prognostic impact W . Sterlacci1, S . Savic2, T . Schmid3, M . Fiegl4, A . Tzankov2 Academic Teaching Hospital Feldkirch, Feldkirch, Austria, 2University Hospital Basel, Switzerland, 3Medical University Innsbruck, Center of Operative Medicine, Austria, 4Medical University Innsbruck, Department of Internal Medicine, Austria

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Aims. The histologic subtype of non-small cell lung cancer (NSCLC) determines treatment strategies and the need for genetic analyses. Since most NSCLC are diagnosed on small biopsies or cytology specimens, an accurate but also tissue sparing approach is necessary. To date, consensus for a general diagnostic algorithm is lacking. Methods. To test the diagnostic and clinical relevance of the recently published multidisciplinary guidelines by the International Association for the Study of Lung Cancer, American Thoracic Society and European Respiratory Society, we examined 371 surgically resected NSCLC brought into tissue microarray format as a surrogate for small biopsies. Adenocarcinomas (ACA) were graded according to architecture considering lepidic as well, acinary and papillary as moderately and micropapillary and solid as poorly differentiated. Results. The antibody panel TTF-1, p63, CK5/6 and CK7 proved diagnostic for most cases. The positive predictive value (PPV) of p63 and CK5/6 for squamous cell carcinoma (SCC), and of CK7 and TTF1 for ACA was 88.9%, 84.9%, 88.4% and 97.7%, respectively. The negative predictive value (NPV) of p63 and CK5/6 for SCC, and of CK7 and TTF1 for ACA was 99.5%, 99.5%, 93.6% and 76.9%, respectively. Faint/focal staining for CK7 is negligible for classificatory purposes and focal expression of TTF-1 with variable staining intensity is a feature compatible with SCC (approximately 3% of cases). Overall survival in months for ACA according to architecture-based tumor grade was 72.5 for well, 71.0 for moderate and 35.7 for poor differentiation (p=0.039). Conclusions. We propose double stains combining an above mentioned nuclear and membranous marker, which is highly diagnostic for NSCLC on small biopsies while conserving tumor tissue for subsequent analyses. No recommendations using less than 2 sections exist, however a panel consisting of TTF-1 in combination with CK5/6 may be feasible, since TTF-1 appears to be the most specific discriminating marker between ACA and SCC (best PPV for ACA) and the only unequivocally evaluable staining combination is with cytoplasmic staining for CK5/6, which also achieved the best NPV for ACA. When grading ACA, the histologic tumor architecture should be the determining factor. This approach primarily has prognostic implications but will also result in easier comparisons of future studies.

DO-027 Multi-immunassay with concurrent staining of 6 antibodies allows tissue-sparing diagnosis on small tissue samples on nonsmall cell lung carcinomas with high diagnostic accuracy G . Kayser1, A . Csanadi1, C . Otto1, S . Dango2, B . Passlick2, M . Werner1 Institute of Pathology, University Hospital Freiburg, Freiburg, 2Department of Thoracic Surgery, University Hospital Freiburg, Freiburg 1

Aims. Today the histological differentiation of non-small cell lung carcinomas NSCLC into adenocarcinomas (LAC), squamous cell carcinomas (SCC), and large cell neuroendocrine carcinomas (LCNEC) is not only of prognostic relevance but far more of predictive value for different therapeutic regimes. As these decisions are of utmost relevance in advanced cancer stages, pathologists are asked to perform a highly accurate diagnosis on small tissue samples. We here investigated the possibility of simultaneous staining of widely agreed upon markers for the histological classification of NSCLC. Der Pathologe Suppl 1 · 2012  | 

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Abstracts Methods. Of 261 NSCLC patients tissue multi arrays (TMA) with a core diameter of 2  mm were composed which served as a simulation model for endobronchial biopsies. The TMAs were stained with TTF1 (8G7G3/1) and Vimentin (VIM3B4) visualized by amino-ethylcarbazol first. Second staining was performed with p63 (4A4) and a neuroendocrine (NE) cocktail (CD56 (NCL-L-CD56-1B6), synaptophysin (SPII), chromogranin (DAK-A3)) visualized by diaminobenzidine. For histological classification hematoxilin-eosin (HE) stained TMA-slides were evaluated. Independently from HE-classification immunohistochemical (IHC) classification was performed by a stepwise decision tree: 1) morphologically clear adenoid or squamous growth patterns: LAC or SCC; 2) TTF1 positive: LAC; 3) TTF1 negative and p63 positive: SCC; 4) TTF1 and p63 negative: large cell carcinoma; 5) TTF1 negative, p63 negative, NE positive: LCNEC. Statistical analyses included kappa-values and Kaplan Meier survival curves. Results. Evaluation of specific staining of the different antibodies was easy to perform and compared to a set of carcinomas which were stained with the multi-IHC protocol and each antibody separate did not show any different staining patterns. Analyzing the inter-core variability, IHC classification was superior to HE-diagnosis. Furthermore, a better separation of the Kaplan-Meier survival curves could be achieved by IHC classification as compared to HE classification alone. Conclusions. Multi-immune assays for classification of NSCLC are feasible and deliver more accurate results than HE-diagnosis alone. IHC classification shows higher intra-tumor homogeneity. As different entities are most probable to show different biological behavior IHC classification delivers the best separation of survival curves and should thus be applied to all lung cancer specimens for accurate pathological classification on small biopsies.

DO-028 The novel IASLC/ATS/ERS classification is a stage-independent predictor of survival and correlates with the response to adjuvant therapies A. Warth1, T. Muley2, M. Meister3, A. Stenzinger1, J. Cortis1, M. Thomas4, P. Schirmacher1, P.A. Schnabel1, J. Budczies5, H. Hoffmann6, W. Weichert1 1 University Hospital Heidelberg, Institute for Pathology, Heidelberg, 2Thoraxklinik Heidelberg, Translational Research Unit, 3Heidelberg University Hospital, Translational Research Unit, 4Thoraxklinik Heidelberg, Oncology, 5 Charité University Hospital Berlin, Institute for Pathology, 6Thoraxklinik Heidelberg, Thoracic Surgery Aims. Our aim was to analyze and to validate the prognostic impact of the novel IASLC/ATS/ERS proposal for an architectural classification of invasive pulmonary adenocarcinomas (ADC) across all tumor stages. Methods. The architectural pattern of a large cohort of 500 resected ADC (stages I–IV) was retrospectively analyzed in 5% increments and classified according to their predominant architecture (lepidic, acinar, solid, papillary, micropapillary), as proposed by the IASLC/ATS/ERS. Subsequently, histomorphological data were correlated with clinical data, adjuvant therapy and patient outcome. Results. Overall survival differed significantly between lepidic (78.5  months), acinar (67.3  months), solid (58.1  months), papillary (48.9  months), and micropapillary (44.9  months) predominant ADC (p=0.007). When patterns were lumped into groups this resulted in even more pronounced differences in survival (pattern group 1: 78.5 months, group 2: 67.3 months, group 3: 57.2 months, p=0.001). Comparable differences were observed for overall, disease specific and disease free survival. Pattern and pattern groups were stage- and therapy-independent prognosticators for all three survival parameters. Survival differences according to patterns were influenced by adjuvant radiochemotherapy, especially solid predominant tumors had an improved prognosis under adjuvant radiotherapy. The predominant pattern was tightly linked to the risk of developing nodal metastases (p<0.001).

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Conclusions. Besides all recent molecular progress, architectural grading of pulmonary ADC according to the novel IASLC/ATS/ERS scheme is a rapid, straightforward and efficient discriminator for patient prognosis and may support patient stratification for adjuvant radiochemotherapy. It should be part of an integrated clinical, morphological and molecular subtyping to further improve ADC treatment.

DO-029 Radiomorphological characteristics for histotype prediction of invasive pulmonary adenocarcinoma A. Warth1, M. Lederlin2, M. Puderbach2, P.A. Schnabel1, J. Cortis1, H.-U. Kauczor2, C.-P. Heussel3, T. Muley4, H. Hoffmann5, W. Weichert1 1 University Hospital Heidelberg, Institute for Pathology, Heidelberg, 2University Hospital Heidelberg, Radiology, 3Thoraxklinik Heidelberg, Radiology, 4 Thoraxklinik Heidelberg, Translational Research Unit, 5Thoraxklinik Heidelberg, Thoracic Surgery Aims. Recently a novel staging system including a novel grading scheme with respect to tumor growth patterns have been proposed for pulmonary adenocarcinomas (ADC). It has been confirmed to have high impact on survival and may also allow patient stratification for adjuvant therapies. Radiological imaging, together with histological evaluation, is the basis of evaluating pulmonary neoplasms. For imaging-based prediction of ADC subtypes and thus prognosis it is of paramount importance to investigate the correlations of parameters of these two important evaluation modalities. Methods. The association between staging and growth patterns as evaluated by histology and data from preoperative assessment of the same lesions by thin-section computed tomography (CT) imaging of 174 resected pulmonary ADC was analyzed. Results. Configuration as well as solidity of the tumors as determined by CT were associated with distinct growth patterns. Histomorphological solid predominant ADC preferably had smooth margins and were also solid in CT scans, while lepidic predominant ADC had mixed margins and were associated with a high percentage of ground glass opacity (GGO) elements. Agreement of preoperative stage assessment with final pathological stage was fair. CT estimation of tumor size was quite accurate. Non-spherical tumor growth was a negative predictor of overall and disease specific but not of disease free patient survival. Conclusions. CT morphologic image modalities are associated with histomorphologic growth patterns of pulmonary ADC. This may allow for the development of algorithms for accurate growth pattern estimation of non-resectable cases on the basis of biopsy data and radiological imaging.

AG Pneumopathologie III DO-032 Remodelling-related molecular profiles in interstitial pulmonary fibrosis D . Jonigk1, J . Rische1, L . Maegel1, H . Golpon2, N . Izykowski1, C . Bockmeyer1, T . Welte2, S . Janciauskiene3, J . Gottlieb2, G . Warnecke4, A . Haverich5, H . Kreipe1, F . Laenger1 1 Hannover Medical School (MHH), Institute of Pathology, Hannover, 2 Hannover Medical School (MHH), Department of Pneumology, Hannover, 3 Hannover Medical School (MHH), Department of Respiratory Medicine, Hannover, 4Hannover Medical School (MHH), Department of Throracic Surgery, Hannover, 5Hannover Medical School (MHH), Department of Thoracic Surgery, Hannover Aims. Idiopathic pulmonary fibrosis (IPF) is the most important representative of the idiopathic interstitial pneumonia group (IIP) and is a disease characterized by an overall poor prognosis and unresponsiveness to currently available therapies. Thus elucidation of molecular pathways to gain better insight into the pathogenesis and identify potential therapeutic targets is warranted. Methods. We performed compartment-specific analyses using laser microdissection, RT-PCR based microarray techniques and immunohistochemistry in lung samples from well defined patients with UIP, nonspecific interstitial pneumonia (NSIP), organizing pneumonia (OP) patterns and controls (n=5 of each group). Results. Notably, we identified cardinal regulatory genes that were differentially up-regulated in UIP (BMP 4 and MMP13), NSIP (BMP6 and CXCR4) and OP (BMP1, IL-13 and TGFB3), respectively. In UIP, remodelled areas showed a prominent up-regulation of fibrosis-associated genes like BMP7, MMP2 and TIMP2, while non-remodelled zones were characterized by a significantly higher expression of BMP6 and pro-inflammatory mediators IL-8 and IL-17A. Conclusions. Our findings show that distinct, morphologically defined IPF subgroups show specific cytokine expression patterns. Moreover, BMPR2 and MMP13 up-regulation correlates significantly with the absence of interstitial scarring in UIP pattern lungs. These results are promising regarding their potential as diagnostic adjunct and therapeutic targets.

DO-033 MALAT1 is essential for lung cancer metastasis in a novel human knockout model T . Gutschner1, M . Eißmann2, M . Hämmerle1, M . Baas1, C . Hildenbrand1, M . Groß1, M . Zörnig2, S . Diederichs1 1 Heidelberg University Hospital & German Cancer Research Center (DKFZ), Institute of Pathology, Heidelberg, 2Georg-Speyer-Haus, Frankfurt Aims. The highly conserved long non-coding RNA MALAT-1 (Metastasis-Associated in Lung Adenocarcinoma Transcript 1) had been discovered as a prognostic marker associated with poor survival and development of distant metastasis in lung adenocarcinoma. Since then, it has been found to be deregulated in numerous tumor entities and has been linked to splicing. However, its functional relevance in tumor cells remains to be elucidated. Knockdown models for MALAT1 have been described but suffer from insufficient silencing efficiency of the highly abundant, nuclear non-coding RNA (ncRNA). Methods. In this project, we have developed a novel strategy to create ncRNA knockouts in human cancer cell lines. Results. We have successfully used a synthetic Zinc Finger Nuclease engineered to target the 5’-region of MALAT1 to stably and biallelically integrate RNA-destabilizing elements into the genome of human lung cancer cells (A549). This approach resulted in a specific and more than

1000-fold silencing of MALAT1 in individual clones compared to a less than 5-fold silencing using siRNAs. Thus, this approach can be used to create functional knockouts of coding as well as non-coding genes also in human tumor cell lines allowing loss-of-function studies also of nonconserved ncRNAs in the future. Phenotypically, the MALAT1-Knockout cells (KO) greatly differ from their parental cell line and wild type clones (WT): Next to morphological changes, the migration of the KO cells is largely impaired as shown in scratch assays. In xenograft assays after i.v. injection, the KO cells form significantly fewer and smaller lung metastases than their WT counterparts. Since no large difference was observed after subcutaneous injection of the WT and the KO cells, this indicates a specific, active and essential function of MALAT1 in metastasis. Conclusions. Taken together, we have developed a novel, highly effective approach for the knockout of genes that can be used for non-coding as well as coding RNAs in human tumor cells. Knockout of MALAT1 in human lung cancer cells revealed its essential function in migration and metastasis.

DO-034 Rationale for treatment of metastatic squamous cell carcinoma of the lung using FGFR inhibitors A . Franzen1, F . Göke1, R . Menon1, D . Goltz1, R . Kirsten1, D . Böhm1, W . Vogel1, A . Göke1, V . Scheble2, J . Ellinger3, U . Gerigk4, F . Fend5, P . Wagner6, A . Schröck7, S . Perner1 1 University Hospital of Bonn, Institute of Pathology, Bonn, 2University of Tübingen, Department of Hematology and Oncology, Tübingen, 3University Hospital of Bonn, Department of Urology, Bonn, 4Malteser Hospital Bonn, Department of Thorax Surgery, Bonn, 5University Hospital of Tübingen, Institute of Pathology, Tübingen, 6University of Pittsburgh Medical Center, Division of Surgical Oncology, Pittsburgh, United States, 7University Hospital of Bonn, Department of Head and Neck Surgery, Bonn Aims. We previously identified amplification of the fibroblast growth factor receptor 1 (FGFR1) gene as a potential therapeutic target for a small molecule inhibitor therapy in squamous cell lung cancer (L-SCC). Currently, clinical phase 1 trials are underway to examine whether patients with FGFR1-amplified L-SCC benefit from a targeted therapy approach using small molecule inhibitors of FGFR. As most lung cancer patients present with metastatic disease, we investigated whether lymph node metastases in L-SCC share the FGFR1 amplification status of their corresponding primary tumor. Methods. Our study cohort consisted of 72 patients with L-SCC, 39 of whom presented with regional lymph node metastases. Tissue microarrays were constructed from formalin-fixed, paraffin-embedded tissue of the primary tumors and, where present, of the corresponding lymph node metastasis. A biotin-labeled target probe spanning the FGFR1 locus (8p11.22-23) was used to determine the FGFR1 amplification status by fluorescence in-situ hybridization (FISH). Results. FGFR1 amplification was detected in 16% (12/72) of all primary lung SCC. Among patients with metastatic L-SCC, 18% (7/39) of the lymph node metastases displayed a FGFR1 amplification, and an exact correlation between the FGFR1 amplification status was observed between the primary tumor and metastatic tissue. Conclusions. FGFR1 amplification is a common genetic event in squamous cell carcinomas of the lung. Moreover, lymph node metastases derived from FGFR1-amplified L-SCCs also exhibit FGFR1 amplification. Therefore, we suggest that the FGFR1 amplification is a clonal event in tumor progression. Beyond this biologically relevant observation, our findings carry therapeutic implications, in that small-molecule inhibitors may be applicable to the treatment of squamous cell carcinomas of metastatic squamous cell carcinoma of the lung.

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Abstracts DO-035 FISH assays for the detection of FGFR1 amplifications and EML4ALK translocations in NSCLC H .-U . Schildhaus1, K . Schmitz1, L . Heukamp1, S . Merkelbach-Bruse1, R . Büttner1 1 University of Cologne, Institute of Pathology, Köln Aims. Easy, reliable and standardized tests for predictive diagnoses and targeted therapies are needed. A small subset of pulmonary adenocarcinomas (AC) harbor therapeutically relevant EML4-ALK translocations. The incidence of squamous cell carcinomas (SC) of the lung increases dramatically, but targeted therapeutic options are not well established for this subgroup of NSCLC. Recently, Fibroblast Growth Factor Receptor 1 (FGFR1) amplification in SC was described to be associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients. Methods. A total of 400 NSCLC samples were included in this study. For detection of EML4-ALK translocations a triple color FISH assay was used: two probes labeled orange and green flank the breakpoint region of ALK in telomeric and centromeric direction, and a third probe, labeled in blue, spans the entire EML4 gene. A dual color FGFR1/CEN8 probe was used to evaluate the prevalence of FGFR1 amplification in SC and to develop an evaluation strategy for FGFR1 FISH assays. Results. Using the triple color EML4-ALK probe specific signal patterns were found for different types of ALK rearrangements. An inversion of chromosome 2p results in (1) split (separated) orange and green signals, (2) a split (doubled) blue signal and (3) a colocalisation (fusion) of the separated orange and green signals with blue signals. Additional specific signals patterns were seen in cases with inversions/deletions and interstitial deletions alone. Investigating a subset of 254 SC with a two colour FISH assay we found FGFR1 amplifications in 20% of SCC but not in AC. Low amplification levels (as defined by ≥5 FGFR1 signals in ≥50% of tumor cells) were rare with a frequency of 6.3% while high level amplifications (as defined by a FGFR1/CEN8 ≥2.0, or average number of FGFR1 signals per tumor cell nucleus ≥6, or the percentage of tumor cells containing ≥15 FGFR1signals or large clusters ≥10%) occurred in 15% of all SC. Conclusions. The novel triple color split/fusion approach decreases the number of questionable or borderline cases at high sensitivity and specificity and allows the diagnosis of specific types of ALK translocations. FGFR1 amplification is one of the most frequent therapeutically tractable genetic lesion in NSCLC. Standardized reporting of FGFR1 amplification in SCC will become increasingly important to correlate therapeutic responses to FGFR1 inhibitors in clinical studies.

DO-036 Mutation analysis from the REASON study, a Registry for the Epidemiologic and Scientific evaluation of EGFR mutation status in newly diagnosed NSCLC patients stage IIIB/IV P . Schirmacher1, W . Schütte2, M . Thomas3, W . Eberhardt4, J .-M . Graf von der Schulenburg5, S . Zaun6, M . Dietel7 1 University of Heidelberg, Institute of Pathology, Heidelberg, 2Städtisches Krankenhaus Martha Maria, Halle-Dölau, 3Heidelberg University Hospital, Thoraxklinik, Heidelberg, 4Universitätsklinikum der GSH Essen, Essen, 5 Leipniz University of Hannover, Hannover, 6AstraZeneca GmbH, Wedel, 7 Humbold University Berlin, Institute of Pathology, Berlin Aims. Somatic mutations in the EGFR gene predict for sensitivity to EGFR tyrosine kinase inhibitors (TKI) in patients with advanced NSCLC, however, little is known about the prevalence of such mutations in the German population. The REASON study aims to generate key data on the EGFR mutation status and its association with major clinicopathological parameters derived from a sufficiently large sample of stage IIIB/IV NSCLC patients with a predominantly Caucasian ethnic background in Germany.

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Methods. REASON is an AstraZeneca sponsored German registry. 4255 subjects with stage  IIIB/IV NSCLC for whom EGFR mutation testing was planned were enrolled at 151 participating sites throughout Germany (129 hospital-based, 22 office-based). EGFR mutation testing was done at 56 QuIP certified and 11 additional pathology institutions. While analysis of exons 19 and 21 was obligatory, analysis of exons 18 and 20 was not routinely done at all labs. The primary aim was to collect epidemiological data on EGFR mutation status (M+, M−) in the German population and to correlate EGFR mutation status with clinicopathological characteristics (e.g. smoking status, gender, histology, etc.). As secondary objectives, real-life clinical outcome data of all EGFR M+ patients (PFS, OS,DCR), clinical management and pharmaco-economic data (resource use) associated with diagnosis and treatment of EGFR M+ patients will be collected. Results. Interim analysis data was available for 3612 patients (Caucasian patients only, 99.4% of all patients). 62% of patients are male and 38% female; with 81% being ever-smokers vs. 19% non-smokers. Adenocarcinoma histology is most frequent (68%), followed by squamous epithelial carcinoma subtype (20%). 358 EGFR mutations were found, the rate of EGFR mutations predicting TKI sensitivity was 9.5%, with 0.4% resistant mutations. Most common mutations were exon 19 deletions (49.6%; predominantly Del E746-A750) followed by exon 21 mutations [38%, mostly L858R (93 or 26.8% of all mutations)]. Exon 18 and 20 mutations (incl. 3 T790M) were found in 6.3% and 9.2% of patients, respectively. The differentiated panel of identified mutations will be shown. Conclusions. REASON provides the largest data base to date on EGFR mutations status of patients with newly diagnosed stage IIIB/IV NSCLC in Germany. In addition, data on baseline epidemiological and further clinicopathological characteristics will enable us to better understand the association of these factors with different mutations and clinical outcomes.

AG Hämatopathologie I DO-043 ID2 and ID3 protein expression differs between hematopoietic cell lineages and acute leukemias of distinct clinicopathological entities A .M . May1, A .-V . Pfister1, L . Bogatyreva2, M . Benkisser3, D . Hauschke2, M .  Lübbert4, M . Werner1, J . Hasskarl4, S . Lassmann1 1 University Freiburg Medical Center, Institute of Pathology, Freiburg, 2University Freiburg Medical Center, Institute of Medical Biometry und Medical Informatics, Freiburg, 3University Freiburg, Freiburg, 4University Freiburg Medical Center, Department of Hematology and Oncology, Freiburg Aims. Inhibitors of DNA binding (ID) proteins regulate cellular differentiation and proliferation through formation of heterodimers with basic helix-loop-helix transcription factors. To elucidate the role of ID proteins in hematopoiesis and in acute leukemia, we analysed ID2 and ID3 protein expression in hematopoietic cells and leukemic blasts. Methods. Primary bone marrow biopsies (BMB) of patients with AML with myelodysplasia related changes (AML-MD) (n=19), de novo AML (n=20), cALL (n=23) and T-ALL (n=19) as well as BMB of healthy bone marrow stem cell donors (n=19) were stained for ID2 and ID3 protein expression by immunohistochemistry (IHC). In healthy BMB, each 200 cells/hematopoietic lineage were evaluated, differentiating between mature and immature granulopoiesis. In acute leukemias, the staining pattern and intensity of 200  blast cells/BMB were assessed. Statistical analyses were done by the nonparametric Kruskal-Wallis test (two-sided, significance level 0.05), and in case of a significant result by an additional pairwise Wilcoxon test. Results. In normal BMB hematopoietic cells and maturation stages displayed significant differences in ID2/3 protein expression. While the

immature granulopoiesis showed a strong ID3 protein expression, the more mature granulocytes only displayed a minimal reactivity. In contrast, erythropoiesis remained negative for ID2/3 (p<0.001). The ID2/3 protein expression of immature granulopoiesis differed significantly from the ID2/3 expression of AML blast cells (p<0.001). Moreover, a significant differential expression of ID proteins was seen between the different acute leukemia subgroups: AML, especially AML-MD had more ID2- (p<0.001) and ID3- (p<0.001) positive blasts than ALL. Conclusions. In the morphological context of the bone marrow, this study shows loss of ID2/ID3-protein expression during maturation of granulopoietic cells. Erythropoieses, however, seems to be regulated via ID2/3-independent mechanisms. Furthermore, the significantly different ID2/3 protein expression in acute leukemias indicates a specific role of ID2/3 proteins in de novo AML and AML-MD, as well as in T- and BALL. Whether or not ID proteins are of therapeutic relevance in patients with acute leukemias has to be clarified in further studies. Grant support: Deutsche José Carreras Leukämie Stifung (R09/26).

DO-044 Immunogenetic polymorphisms are associated with the occurrence of cmL but not with interferon-α responsiveness E. Riedl1, A. Becker1, D. Belharazem1, M. Körner1, S. Rothkegel1, C. Sauer1, S. Küffer1, P. Ströbel1, A. Marx1 1 University of Heidelberg, Faculty of Clinical Medicine Mannheim, University Medicine Mannheim, Mannheim Aims. Chronic myeloid leukemia (CML) is a clonal stem-cell disease. For yet unknown reasons, 8–30% of cmL patients respond to interferon-α (IFN-α ) therapy with a cytogenetic remission. T-cell mediated immunity to cmL-specific self-antigens is important in the pathogenesis of cmL and could explain the response to IFN-α . In the present study, we investigated the impact of functional immunogenetic polymorphisms involved in T-cell reactivity on the development of cmL and IFN-α responsiveness. Methods. 250 BCR-ABL-positive cmL patients on IFN-α were divided into two groups: responders (n=70) with a major/complete cytogenetic response and non-responders (n=180). 7 functional single nucleotide polymorphisms (SNPs) associated with autoimmune disease or tumor were tested: CTLA4 rs231775, PTPN22 rs2476601, PDCD1 rs11568821, RUNX1 rs2268277, FCLR3 rs7528684, SLC9A3R1 rs734232 and cFLIP rs10190751. Patients were genotyped using TaqMan® SNP Genotyping Assays (Applied Biosystems). Allelic frequencies were compared to distribution in a healthy population. Fisher‘s exact or χ2 tests were performed to evaluate the impact of the SNPs. Results. A significant association between cmL and cFLIP rs10190751 was found (CML vs. healthy control; p=0.00001; odds ratio=2.25; 95% CI=1.15–3.31). Moreover, PDCD1 rs11568821 was significantly less frequent in cmL patients (Allelic frequency of rs11568821 cmL vs. healthy control; 13 vs. 22%; p=0.0001). SNP frequencies did not significantly differ between IFN-α responder and non-responder (responder vs. non-responder, p>0.05 for all tested SNPs). Conclusions. The significant association between cFLIP/PDCD1 and the occurrence of cmL suggests that immune-mechanisms could play a role in the pathogenesis of cmL. By contrast, cFLIP- and PDCD1-dependent immune-mechanisms are apparently not involved in IFN-α responsiveness of a subgroup of patients.

DO-045 Mesenchymal stem cells – key players in myelofibrosis? I. Leisten1, A. Schumacher2, S. Ziegler2, D. Fahrenkamp3, G. Müller-Newen3, T.H. Brümmendorf2, E. Jost2, R. Knüchel1, P. Ziegler2, R.K. Schneider1 1 RWTH Aachen University, Institute of Pathology, Aachen, 2RWTH Aachen University, Department of Hematology, Oncology and Stem Cell Transplantation, Aachen, 3RWTH Aachen University, Institute of Biochemistry, Aachen Aims. Myeloproliferative neoplasms (MPN) are clonal disorders characterized by excessive production of mature blood cells. Pathogenetic mechanisms in MPN include secondary stromal changes in the bone marrow leading to myelofibrosis. Stroma cells of the bone marrow (e.g. mesenchymal stem cells, MSC) might be conditioned by abundantly released growth factors from the malignant hematopoietic clone and in turn contribute to a modified, fibrotic niche environment. The aim of the study is the comparative analysis of MSC from MPN patients and healthy donors regarding their phenotype and extracellular matrix (ECM) remodelling capacities. Methods. MSC isolated from bone marrow of MPN patients (all myelofibrosis grade 0–2) and healthy subject were cultured after isolation and characterization in 3D collagen gels (Schneider et al. Biomaterials 2010) for studying the ECM remodelling. ECM remodelling potential of MSC in the 3D culture system was determined by immunohistochemistry and qtRT-PCR. Results were correlated to immunohistochemistry of corresponding bone marrow punches. Results. When activated through contact with the collagenous matrix, MSC from MPN-patients extensively remodelled and contracted the collagenous matrix compared to healthy donors. The intensive ECM remodelling was proven by qtRT-PCR and immunohistochemistry for collagen I, IV, fibronectin, laminin and osteopontin. In particular, the fibronectin production was strongly up-regulated. The matrix remodelling observed in vitro was comparable to the corresponding bone marrow punches. Immunofluorescence double stainings confirmed the co-localization of fibronectin accumulations and MSC (CD271+) in the bone marrow. Compared to bone marrow of healthy donors, MSC (CD271+, CD146+) seem to be mobilized from their perivascular and endosteal niche and localized diffusely in the bone marrow, surrounded by intense matrix production and dysplastic megakaryocytes. Conclusions. In conclusion, MSC from MPN patients induce extensive matrix remodelling and seem to actively participate in the process of myelofibrosis. MSC might be attracted and recruited from the niche by the malignant hematopoietic clone. Further analysis will show if epigenetic chances are responsible for this fibrotic MCS phenotype.

DO-046 In vivo expression profile of the antiviral restriction factor and tumor-targeting antigen CD317/BST-2/HM1.24/tetherin in humans F. Lasitschka1, E. Eriksson2, T. Adam2, S. Schmidt2, J. Lehmann-Koch1, B. Over2, C. Goffinet2, C. Harter3, I. Bekeridjian-Ding2, S. Sertel4, P. Schirmacher1, O. Keppler2 1 University Hospital Heidelberg, Institute of Pathology, Heidelberg, 2 ­ University Hospital Heidelberg, Department of Infectiology, Heidelberg, 3 University Hospital Heidelberg, Department of Internal Medicine, Heidelberg, 4University Hospital Heidelberg, Department of Otolaryngology, Head and Neck Surgery, Heidelberg Aims. Human CD317 is an intrinsic immunity factor that restricts the release of enveloped viruses, including the major pathogens HIV and Lassa virus, from infected cells in culture. Its importance for infection control in humans is unclear, due in part to its incompletely defined in vivo expression pattern. CD317 also has been proposed as a selective target for immunotherapy of multiple myeloma. Methods. To provide a framework for studies of the biological functions, regulation, and therapeutic potential of CD317, we performed microar-

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Abstracts ray-based expression profiling in 468 tissue samples from 25 healthy organs from more than 210 patients. Results. We found that CD317 protein was expressed to varying degrees in all organs tested and detected in a number of specialized cell types, including hepatocytes, pneumocytes, ducts of major salivary glands, pancreas and kidney, Paneth cells, epithelia, Leydig cells, plasma cells, bone marrow stromal cells, monocytes, and vascular endothelium. Although many of these cell types are in vivo targets for pathogenic viruses, restriction by CD317 or virus-encoded antagonists has been documented in only some of them. Limited cell type-dependent co-expression of CD317 with the IFN biomarker MxA in vivo and lack of responsive stimulation in organ explants suggest that interferons may only partially regulate CD317. Conclusions. This in vivo expression profiling sheds light on the biology and species-specificity of CD317, identifies multiple thus far unknown interaction sites of viruses with this restriction factor, and refutes the concept of its restricted constitutive expression and primary IFN inducibility. CD317’s widespread expression calls into question its suitability as a target for immunotherapy.

DO-047 Frequent detection of the Merkel cell polyomavirus in B-lymphocytes: implications for non-Hodgkin lymphomagenesis? D. Rennspiess1, A. Haugg1, J. Beckervordersandforth1, A.K. Kurz2, R. Plusquin1, G. Cathomas3, C. Wendtner4, E.-J. Speel1, H. Kvasnicka5, A. zur Hausen1 1 Maastricht University Medical Center, Department of Pathology, Maastricht, Netherlands, 2University Hospital Aachen, Department of Internal Medicine IV, Aachen, 3Kantonsspital Liestal, Institute of Pathology, Switzerland, 4University Hospital Cologne, Department I of Internal Medicine, Köln, 5 University Hospital Frankfurt, Institute of Pathology, Frankfurt Aims. The recent discovery of the Merkel cell polyomavirus (MCPyV) in Merkel cell carcinomas (MCC) also had an important impact on the well established epidemiological association of CLL/SLL with MCC. We have recently demonstrated the presence of MCPyV DNA in highly purified CD5+/CD19+ CLL cells. Meanwhile, the presence of MCPyV DNA in CLL/SLL cells has been independently demonstrated by two other groups reporting MCPyV-DNA in approx. 21–33%. In addition, we were able to demonstrate MCPyV integration by fluorescence in situ hybridisation (FISH). Here we extended our analyses to different types of non Hodgkin lymphomas (NHL) as well as to non neoplastic reactive follicular lymph nodes for the presence of MCPyV by FISH and/or MCPyV DNA PCR. Methods. DNA PCR was carried out on 8 formalin-fixed and paraffin embedded SLL cases and 1 DLBCL case and on 39 reactive lymph nodes as previously described. On these and on a tissue microarray (TMA) containing CLL/SLL (n=43), MZL (n=44), FL (n=40), MALT (n=47), DLBCL (n=32), and T cell lymphomas (n=19) MCPyV FISH was performed. Results. MCPyV DNA was detected by PCR in 6 of the 8 SLL cases and in 13 of the 39 reactive lymph nodes. By MCPyV FISH sharply punctate dots – compatible with viral integration – were identified in 29% (n=15/51) of CLLs, in 9% (n=4/45) of MZL, in 35% (n=14/40) of FL, in 15% (n=7/47) of MALT, and in 18% (n=6/32) of DLBCL. All T cell lymphomas were MCPyV negative. Of interest, small clusters of MCPyV DNA positive B cells were detected in the follicle centres of the reactive lymph nodes. These hybridization signals were punctate and multiple, and some of them revealed a diffuse hybridization pattern. Conclusions. MCPyV FISH confirms our previous data on the integrated presence of MCPyV in CLL. Other B-cell NHL might be associated with the presence of integrated MCPyV. The finding of small clusters of follicular B-cells harbouring integrated MCPyV DNA is suggestive for an early and rather common MCPyV infection of premature B-cells which normally – during the process of follicular B-cell maturation – are eliminated. MCPyV-positive follicular B-cells which are not eliminated, thus not recognised as “foreign”, are likely to be the reservoir of cells

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which during a long-term transformation process by an accumulation of oncogenic mutations or deletions turn into a NHL cell. Functional studies concerning MCPyV and lymphomagenesis are ongoing in order to elucidate a possibly underlying etiopathogenic role for MCPyV in NHL lymphomagenesis.

DO-048 The majority of immunohistochemically BCL2 negative FL grade I/ II carry a t(14;18) with mutations in exon 1 of the BCL2 gene and can be identified with the BCL2 E17 antibody I. Bonzheim1, R. Baumann1, P. Adam1, F. Fend1, L. Quintanilla-Martinez1 1 University Hospital Tübingen, Institute of Pathology and Neuropathology, Tübingen Aims. Follicular lymphoma (FL) is characterized by the translocation t(14;18)(q32;q21) resulting in constitutional overexpression of the antiapoptotic protein BCL2. However, 10–15% of FL grade I/II remain negative in the immunohistochemical (IHC) staining for BCL2. The aims of this study were: 1) to investigate the incidence of IHC BCL2 negative FL grade I/II diagnosed at our institution, 2) to analyze BCL2 IHC negative FL with the alternative BCL2 antibody (clone E17) and perform FISH analysis for the t(14;18), and 3) to elucidate the molecular mechanism of immunohistochemical BCL2 negativity. Methods. FL grade I/II diagnosed between 01/2005 and 08/2011 at the Institute of Pathology of the University of Tübingen were included in the study. All cases were stained with the standard BCL2 antibody (clone 100D5; DCS). BCL2 negative cases were subsequently stained with the BCL2 antibody, clone E17 (Zytomed) and analyzed by FISH using a BCL2 break-apart probe (LSI BCL2 BAP, Vysis). Exon 1 of the BCL2 gene, where the epitope of the standard BCL2 antibody resides, was amplified and sequenced. Results. 23 (9.6%) of the 240  identified cases of FL grade  I/II were negative with the standard BCL2 antibody. Of these, 13 cases (57%) were positive for the E17 antibody and 10 cases (43%) remained negative. All E17 positive cases showed a BCL2 break by FISH analysis, indicative of a t(14;18), whereas the E17 negative cases lacked BCL2 alterations. Two of the E17 negative cases carried a BCL6/IGH translocation. Sequencing of BCL2 exon 1 revealed missense point mutations resulting in amino acid substitutions in all 9 analyzable E17-positive cases, with a hot spot around codon 144. Conclusions. The incidence of immunohistochemically BCL2-negative FL grade I/II in our series is comparable to published data. The E17 antibody reveals the presence of BCL2 protein undetectable with the standard antibody due to exon 1 missense mutations in the majority of BCL2 “negative” FL grade I/II and correlates with the presence of the t(14;18). The molecular pathogenesis of the BCL2 (E17)- and t(14;18) negative FL grade I/II remains to be determined.

DO-049 Follicular lymphoma with prominent mantle zones – a FICTION analysis P. Kosmidis1, P. Adam1, I. Bonzheim1, L. Quintanilla-Fend1, P. Bauer2, M. Scharpf1, T. Henopp1, F. Fend1 1 Eberhard-Karls-University, Institute of Pathology and Neuropathology, Tübingen, 2Eberhard-Karls-University, Institute of Human Genetics, Tübingen Aims. Follicular lymphoma (FL) is characterized by the recurrent translocation t(14;18), resulting in BCL2 protein overexpression. Most cases show an indolent clinical course, which in part may be a result of the influence of the complex tumor microenvironment of FL. Involvement of different lymph node compartments may indicate a biologically more advanced lymphoma, whereas restriction of neoplastic cells to germinal centers might represent earlier disease stages. We have therefore exami-

ned, if well-defined mantle zones, which can be identified in a subset of FL including FL “in situ”, are part of the malignant clone. Methods. FL cases with morphologically detectable mantle zone structures and a case of follicular lymphoma “in situ” were selected from a series of 240 FL grade  1/2. Fluorescence in situ hybridization (FISH) for the detection of the chromosomal translocation t(14;18)(q32;q21) was combined with a simultaneous immunofluorescence staining for IgD for the identification of mantle zone cells (FICTION). Results. 10 of 17 (59%) FL cases with morphological detectable mantle zone structures lacked a t(14;18) by FISH. In the remaining 7 t(14;18) positive FL cases and the FLIS case, the IgD+ mantle zone cells did not show a chromosomal break in the BCL2 gene locus. Conclusions. 1) A significant part of FL cases with prominent mantle zone structures are t(14;18) negative. Further investigations are necessary to elucidate the molecular background of this subgroup and to define the border with nodal marginal zone lymphoma with follicular colonization. 2) The mantle zone cells in t(14;18)+ FL with prominent mantle zones in their majority are not part of the malignant clone and therefore most likely represent either pre-existent or newly recruited reactive cells.

DO-050 Reactive tumor infiltrating T-cells predict survival in mantle cell lymphoma: an immunohistochemical study of 81 patients C. Schrader1, Ö. Akalthun1, P. Meusers2, G. Brittinger2, J. Claasen1, W. Klapper3 University Hospital of Kiel, 2nd Department of Medicine, Kiel, 2University Essen, Department of Hämatology, 3UKSH, Campus Kiel, Department of Pathology

1

Aims. The role of tumor infiltrating T-Cells in malignant B-Cell lymphomas is discussed controversial. There are only limited data on CD 8 and FOXP3 positive cells in mantle cell lymphoma. Methods. 81 biopsy specimens of patients (64 men and 17 women) with mantle cell lymphoma and a median age of 64 years (range: 41 to 86 years) were included in this study. The slides were stained immunohistochemically with CD3, CD8 and FOXP3. Positive T-cells of 10 High power fields (HPF) were counted and the average value was calculated. Results. The CD 8 staining showed a range of 0 to 138 positive cells per HPF with a mean value of 19.4/HPF. A high account of CD 8 positive cells was associated with a significantly longer overall survival time (42 months) compared to MCL with a low account of CD 8 positive cells (28.8  months, p=0.029). FOXP3 staining had a range of 0 to 104/HPF with a mean value of 28. Patients with MCL and a high number (>25/ HPF) of FOXP3 positive cells had a median survival time of 38.2 months compared with the group with low account (<20/HPF) of FOXP3 positive cells (23 months). Kaplan Meier analysis revealed a significant difference (p=0.015) in overall survival time. Conclusions. High number of CD 8 and FOXP 3 T-Cells predicts a superior clinical outcome in patients with mantle cell lymphoma.

DO-051 Incidence of preclinical manifestations of mantle cell lymphoma and mantle cell lymphoma in situ in reactive lymph nodes – a comparative analysis P. Adam1, A.-I. Schiefer2, S. Prill1, T. Henopp1, L. Quintanilla-Fend1, H.-C. Bösmüller3, A. Chott2, F. Fend1 1 Eberhard Karls University, Institute of Pathology, Tübingen, 2Medical University Vienna, Clinical Institute of Pathology, Wien, Austria, 3Krankenhaus Barmherzige Schwestern Linz, Department of Pathology, Linz, Austria

in MCL patients, and their biological and clinical significance are still not clear. Methods. A total of 1294 reactive lymph nodes from unselected consecutive surgical specimens of 132 patients without a history of lymphoma obtained over a 3 months’ period were stained for Cyclin D1. Additionally, a search was performed to identify MCL patients, of whom morphologically reactive lymphoid tissues and extranodal reactive lymphoid infiltrates predating the lymphoma diagnosis were available. These were also stained for Cyclin D1. Results. No case of MCLIS was identified among the 1294 consecutively analyzed reactive lymph nodes of the control population. For 36 of 422 (8.5%) MCL patients diagnosed from 2000–2011 in three different institutions, additional morphologically uninvolved lymphoid tissues or extranodal tissues with reactive lymphoid infiltrates obtained more than 2 months before the diagnosis of MCL (3–86 months, median 23 months) were available. None of these showed a MCLIS. In 3 (8%) patients, early MCL involvement with a mantle zone growth pattern not identifiable on routine stains was detected retrospectively. In 7 (19%) patients, small accumulations of cyclin D1 positive cells were detected in morphologically reactive extranodal infiltrates. Conclusions. MCL “in situ” is an extremely rare phenomenon in individuals without a history of MCL, in line with the low prevalence of t(11;14) positive cells described in the peripheral blood of healthy adults. In contrast, in a significant minority (19%) of MCL patients a preclinical MCL manifestation antedating the lymphoma diagnosis can be detected. In our series these either represent small groups of MCL cells in extranodal reactive lymphoid infiltrates or early MCL with mantle zone pattern, rather than MCLIS.

DO-052 Cell cycle dynamics of mantle cell lymphoma N. Vogt1, K. Koch1, D. Abramov1, W. Klapper1 1 University of Kiel, Department of Pathology, Kiel Aims. Previous studies have identified the Ki67-index (percentage of Ki67 positive cells) to be the most important prognostic marker in Mantle Cell Lymphoma (MCL). The translocation (11;14) places the CCND1 locus under the control of the immunoglobulin heavy chain gene promoter leading to an overexpression of Cyclin D1. Cyclin D1 promotes the transition from G1 to S-phase in the cell cycle. Our goal was to determine the characteristics of all cell cycle dysregulation in MCL. Methods. We analyzed 18 cases of MCL. All lymphomas overexpressed Cyclin D1 in immunohistochemical stainings. Immunofluorescence double stainings were performed and analyzed by digital image analysis using TissueQuest 2.2. Two combinations of antibodies were used: Ki67 (positive in G1, S, G2, M-phase) + Survivin (positive in S, G2, M-phase) and Ki67 + Phospho-Histone H3 (PH3, positive in M-phase). Results. The percentage of Survivin positive cells among all Ki67 positive cells ranged between 45.47% and 73.13%. The percentage of PH3 positive cells among all Ki67 positive cells ranged between 2.06% and 3.84%. Interestingly, the percentage of Survivin or PH3 positive cells among the Ki67 positive cells was constant over a wide range of the Ki67-index. Conclusions. In MCL the speed of the cell cycle is independent of the number of cells in the cell cycle. Thus the Ki67-index reflects the absolute tumor duplication rate. Since the level of Cyclin D1 expression is reported to be associated with the Ki67 value, the role of Cyclin D1 in cell cycle dysregulation in MCL remains still uncertain.

Aims. “In situ” mantle cell lymphoma (MCLIS) is defined as the occurrence of cyclin D1 positive B-cells with MCL phenotype and genotype in the inner mantle zones of morphologically inconspicuous lymph nodes. Prevalence of this and similar early lesions in the general population and Der Pathologe Suppl 1 · 2012 

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Abstracts DO-053 Account of tumor infiltrating macrophages is a prognostic factor for patients with mantle cell lymphoma

AG Hämatopathologie II

C . Schrader1, F . Sirin1, P . Meusers2, G . Brittinger2, J . Claasen1, W . Klapper3 University Hospital of Kiel, 2nd Department of Medicine, Kiel, 2University Essen, Department of Hämatology, 3UKSH, Campus Kiel, Department of Pathology

DO-055 Bcl6 expression is not limited to germinal center B-cells and is associated with progression of extranodal marginal zone B-cell lymphomas of the gastrointestinal tract

Aims. Mantle cell lymphoma (MCL) is a malignant lymphoma associated with a relatively aggressive clinical course and a median overall survival time of 3–4 years. Only limited data about tumor associated macrophages and their influence on survival in MCL exists. Methods. We analyzed the amount of CD68 macrophages in relation to the clinical outcome in patients with MCL. Lymph node biopsies of 77 untreated patients (17 women and 60 men) enrolled in two multicenter trials (1975–1985) with a median age of 66 years (range 41–86 years) were included in this study. Biopsy specimens were investigated immunohistochemically with monoclonal antibodies against CD68 (Ki-M1P). 10 High power fields (HPF) were evaluated by random. Results. Patients with low account (less than 10/HPF) of CD 68 positive macrophages had a median overall survival time of 38.2 months, compared to 24.2 months for patients with high (more 10/HPF) CD 68 positive macrophages. The Kaplan-Meier analysis showed a significant difference in the overall survival time (p=0.0027). Conclusions. Patients with mantle cell lymphoma and a low number of CD 68 positive macrophages have a better prognosis and can predict outcome.

U . Boruschek1, L . Floßbach1, M . Buck1, S . Brüderlein1, P . Möller1, T .F .E . Barth1 1 Ulm University, Pathology, Ulm

1

DO-054 Transformation of gastritis to gastric marginal zone lymphoma is associated with deregulated expression of microRNAs C . Thorns1, J . Kuba1, A .C . Feller1, V . Bernard1, A . Senft2, S . Szymczak2, H .-W . Bernd1 1 UKSH, Campus Lübeck, Pathology, 2University Lübeck, Institute for medical bioinformatics and statistics Aims. Gastric extranodal marginal zone lymphoma (MALT lymphoma) generally evolve from a chronic Helicobacter pylori-positive gastritis. The mechanisms that promote the malignant transformation from gastritis to lymphoma are not well understood. This study aims to identify microRNAs that might be involved in the process of neoplastic transformation. Methods. Gastric biopsies were scored as 0 (normal), 1 (gastritis), 2 (follicular gastritis), 3 (suspicious, probably reactive), 4 (suspicious, probably lymphoma) and 5 (MALT lymphoma) (Wotherspoon scores). Groups 3, 4, and 5 were further evaluated for monoclonality by immunohistochemistry for immunoglobulin light chains and/or by polymerase-chainreaction for the immunoglobulin heavy chain locus (IgH). MicroRNAsignatures of 68 cases were generated by RT-PCR for 376 miRNAs. Results. MicroRNA signatures revealed a total of 41 miRNAs that were significantly upregulated (n=33) or down regulated (n=8) in succession from normal mucosa to gastritis and to MALT-lymphoma. Some of these reflect the normal expression in lymhocytes (e.g. miR-566 and -212), while others are known to be the effect of Helicobacter pylori infection (e.g. miR-155 and let7f). A group of five miRNAs (miR-150, -550, -124a, -518b and -539) were differentially expressed in gastritis (Wotherspoon scores 1, 2 and polyclonal 3 and 4) and lymphomas (monoclonal scores 3, 4, and 5). These are likely to be involved in the malignant transformation of gastritis into MALT lymphoma. Conclusions. The development of gastric MALT-lymphoma out of chronic gastritis is paralleled by the deregulation of distinct microRNAs which might thus be centrally involved in the process of malignant transformation.

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Aims. We have previously shown that progression in gastrointestinal B-cell lymphomas is associated with changes in the BCL6 locus and with an increased expression of Bcl6 [Flossbach et al. Int J Cancer 2011; 129(1):70–7]. Bcl6 is a well known germinal center marker; the extranodal marginal zone B-cell lymphomas (MALT lymphomas) are supposed to stem from cells of the extrafollicular space, therefore the expression of Bcl6 in these lymphomas during progression seems conflicting. We have analyzed the detailed expression of Bcl6 in lymph nodes with toxoplasmosis since one of the cells discussed to be the potential progenitor of MALT lymphomas is the monocytoid B-cell. Methods. We studied lymph node paraffin sections of four patients with toxoplasmosis by double immunofluorescence staining using a broad panel of antibodies. For this purpose we first established a new technique based on sequential heating of the samples that now even allows the application of antibodies from the same species or the simultaneous use of monoclonal and polyclonal antibodies. Further we performed stimulation experiments with lymphoblastoid B-cells. Results. We found a strong expression of Bcl6 in the germinal center. However, we also detected some scattered Bcl6 positive cells in the extrafollicular space. These extrafollicular Bcl6-positive cells were characterized as: CD19+,CD75+, AID+, IgA+, IgG+/−, CD30−, CD10−, CD38−, Bcl2−, ZAP70−, cRel−, IgM−, IgD−, CD11c−. Monocytoid B-cells expressed Bcl6 in a subfraction of less than 1%. The profile of the Bcl6+ extrafollicular B-cells corresponds to an activated post germinal center B-cell differing from monocytoid B-cells. Expression of Bcl6 was partially inducible in lymphoblastoid EBV transformed B-cells by TNFα, TPA, and LPS. Conclusions. We conclude that Bcl6 expression is not limited to germinal B-cells but is also found in extrafollicular B-cells. We suggest that the blastic variant of MALT lymphoma may have preserved the potential of up-regulating Bcl6 as an antiapoptotic mechanism.

DO-056 Characterization of gastrointestinal marginal zone B-cell lymphoma and large cell variants using high-resolution SNP-arrays L . Floßbach1, K . Holzmann2, T . Mattfeldt1, P . Möller1, T .F .E . Barth1 1 Ulm University, Pathology, Ulm, 2Ulm University, IZKF, Ulm Aims. Gastrointestinal marginal zone B-cell lymphomas (MALT Lymphomas) are a model for tumor progression since we and others have shown that the frequently coexisting more aggressive large cell component is clonally related to the small cell lymphoma. We used SNP analysis to further characterize these lymphomas. Methods. We extracted genomic DNA from frozen tissue samples of 28 gastrointestinal marginal zone B-cell lymphomas (n=7) and large cell variants (n=21). We performed SNP analysis using the Affymetrix HGW SNP array 6.0 platform. Results were correlated with FISH and IHC analyses. Results. While small cell lymphomas have on the average 8 aberrations longer than 0.2MB each case, large cell variants have more than 14. Both small and large cell lymphomas have losses in regions 1p13 and 6q15 as well as gains on 1p36 and 17q21. Gains on 9q12i-j (2/7) are restricted to small cell lymphomas. Losses on 6q24 (5/21) and gains on 11q23 (8/21) are restricted to the large cell lymphomas. Most frequent losses or deletions concern region 6q14.1a-c containing HTR1B, IRAK1BP1, PHIP, HMGN3, LCA5 and SH3BGRL2 (5/21 large cell and 1/7 small cell lym-

phomas). Most prominent gains or amplifications (6/21 large cell lymphomas) concern region 2p16.1a-15d containing PAPOLG, REL, PUS10 and PEX13. Amplification of REL was confirmed by FISH in these cases. In all these lymphomas, immunohistochemical staining for c-Rel was positive in at least 30% of the tumor cells. Regions with putative acquired uniparental disomies (aUPD) are more present in the large cell lymphomas: on the average 40 regions vs. 9 regions in the small cell lymphomas. Comparing the SNP profiles of two areas of the same tumor both with a t(11;18) Api2/Malt1 but with slightly different morphology, the analysis revealed additional gains in the more blastic part. Investigating two lymphoma samples from the same patient with an interval of two years, FISH analysis showed a signal pattern pointing to a large deletion in the IGH locus exclusively in the later sample. SNP analysis confirmed the FISH result and revealed about ten additional aberrations illustrating increasing genomic complexity during lymphoma progression. Conclusions. Small and large cell variants of gastrointestinal marginal zone B-cell lymphomas have distinct patterns of genomic aberrations but share some overlapping features. REL is frequently amplified in the large cell variants. In general, during lymphoma progression, the SNP data correlate with a more complex pattern of aberrations.

DO-057 Comparative analysis of gene expression profiles defines large cell variants of gastric marginal zone B-cell lymphoma as a distinct subgroup L. Floßbach1, J. Kraus2, K. Holzmann3, P. Möller1, H.A. Kestler2, T.F.E. Barth1 Ulm University, Pathology, Ulm, 2Ulm University, Neural Information Processing, Ulm, 3Ulm University, IZKF, Ulm 1

Aims. Gastrointestinal marginal zone B-cell lymphomas (MALT Lymphomas) often collocalize with a more aggressive, large cell component. The WHO classifies these lymphomas as “extranodal DLBCL with or without residing MALT component”. We have shown that the small and the large cell components of these composite lymphomas (ComL) are mostly clonal and, in addition to that, that the gene expression profiles of small cell MALT lymphomas and lymphoma components are similar to those of the large cell components or lymphomas. This suggests that most of the gastrointestinal DLBCL are indeed blastic variants of marginal zone B-cell lymphomas (MZBL) [Barth et al., J Pathol 2007; 211(3):305; Flossbach et al. Int J Cancer 2011 129(1):70]. To further distinguish these large cell variants of MZBL from nodal and extranodal DLBCL we performed a comparative analysis of gene expression profiles from B-cell lymphomas. Methods. We extracted RNA from frozen tissue samples of 28 gastrointestinal marginal zone B-cell lymphomas (n=7), large cell components of ComL (n=8) and large cell variants (n=13). Gene expression profiling was performed using the Affymetrix U 133 plus 2.0 array. Additional datasets created with the same chip from DLBCL (n=119), PMBL (n=20), BL (n=33), FL (n=38), MCL (n=7), pulmonary MALT lymphomas (n=35) and normal B-cells (n=20) were obtained from the Gene Expression Omnibus (GEO) database. After normalization and based on a subset of NF-κB target genes [Compagno et al., Nature, 459, 717, 2009], we performed hierarchical clustering analysis. Additionally, we performed cluster robustness analysis using the k-means algorithm. Results. Cluster number estimation was robust for k=8. These eight clusters consisted mostly of: 1. naïve B-cells and memory B-cells, 2. centroblasts and centrocytes, 3. Burkitt’s lymphoma, 4. pulmonary MALT lymphoma, 5. follicular lymphoma, mantle cell lymphoma, and gastrointestinal MALT lymphoma, 6. PMBL and DLBCL, 7. DLBCL, 8. blastic MZBL and DLBCL. The dendrogramm, generated by the hierarchical average linkage clustering process, was consistent with this classification. In comparison to all DLBCLs and PMBLs, the blastic MZBLs had relatively underexpressed PTPN3 and relatively overexpressed BANK, CD44, CD63, and FAS.

Conclusions. These data confirm our view of blastic marginal zone B-cell lymphomas as a distinct group of extranodal diffuse large B-cell lymphomas.

DO-058 Prognostic phenotypic and genotypic in situ biomarkers in diffuse large B-cell lymphomas: preliminary translational report of the prospective SAKK 38/07 trial A. Tzankov1, N. Leu1, S. Muenst1, D. Klingbiel2, C. Mamot3, S. Dirnhofer1 University Hospital Basel, Pathology, Basel, Switzerland, 2SAKK Coordinating Center, Swiss Group for Clinical Cancer Research, Bern, Switzerland, 3 Cantonal Hospital Aarau, Aarau, Switzerland 1

Aims. Diffuse large-B cell lymphoma (DLBCL) exhibits variable outcomes and risk assessment is based on the international prognostic index (IPI), which takes into account primarily patient-related parameters. The prognostic role of tumor-related parameters is a matter of controversy. Methods. We prospectively analyzed the prognostic value of phenotypic and genotypic profiles suggested to play a role in DLBCL on a clinical trial collective of 124 DLBCL patients homogenously treated with six cycles of rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone, followed by 2 cycles rituximab (R-CHOP). Evaluation of the role of positron emission tomography was a main objective, and was performed before, after 2 cycles of therapy and at the end of treatment. Immunohistochemical (BCL2, BCL6, CD5, CD10, CD20, CD95, CD168, Cyclin E, FOXP1, GCET, Ki-67, MUM1p, pSTAT3) and in situ hybridization analyses [BCL2 break apart probe (BAP), C-MYC BAP and C-MYC/ IgH double-fusion probe (DFP) and Epstein-Barr virus probe (EBER)] were performed and correlated with clinicopathological parameters and outcome. Results. The median patients’ age was 59 years; 68 were males, 56 females. BCL2 gene breaks were observed in 11% of cases, and those cases also expressed BCL2 in a mean of 95% of tumor cells, compared to 42% in nonrearranged instances; 85% of the rearranged cases were of the germinal center (GC) phenotype according to the Tally algorithm. 3% of cases (all of the non-GC phenotype) showed BCL2 amplifications. C-MYC breaks were observed in 10% of cases; 66% were of the GC phenotype. Of the CMYC rearranged cases only a third displayed C-MYC/IgH fusions corresponding to t(8;14), the others being assumed to have alternative C-MYC rearrangement partners. Cases with rearranged C-MYC showed as high Ki-67  labeling as non-rearranged. Cases with both BCL2 and C-MYC rearrangements were not observed. A complete response (CR) defined by Cheson’s criteria was achieved in 90 out of 117 patients, for 7 there were no data. Factors that were linked to failure to achieve CR were CD5 positivity (11% compared to 2%, p=0.051), EBER positivity (4% of cases compared to 0% of those with CR, p=0.072) and presence of either BCL2 or C-MYC gene rearrangements (46% compared to 18%, p=0.132), but not IPI or Tally phenotype. Conclusions. Phenotypic and genotypic studies with carefully selected biomarkers like CD5, EBER and BCL2- as well C-MYC BAP might be of prognostic value in DLBCL patients treated by R-CHOP.

DO-059 Phenotype of primary testicular diffuse large B-cell lymphomas T. Menter1, M. Ernst1, S. Dirnhofer1, A. Braghorn2, P. Went1, A. Tzankov1 1 University of Basel, Institute of Pathology, Basel, Switzerland, 2 Medica Zürich, Switzerland Aims. Primary testicular diffuse large B-cell lymphomas (tDLBCL) are rare neoplasms with few comprehensive studies conducted so far. We therefore aimed to systematically analyze the morphology and phenotype of tDLBCL.

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Abstracts Methods. Forty-three patients from 3 different Swiss hospitals were included in the study. The tumors were diagnosed between 1972 and 2009. The protein expression profile was assessed by immunohistochemistry. Results. 39 of the tumors showed centroblastic and 1 immunoblastic morphology, three were not classifiable. All cases were positive for CD79a, followed by CD20 and PAX5 (95% of cases) and CD19 (93%). Most cases (68%) expressed the post-germinal center (GC) marker FOXP1 and 21% expressed MUM1, while the GC markers CD10, LMO2, BCL6 and GCET1 were expressed in 27, 16, 8 and 6%, respectively (cut-off levels for the respective markers were determined by ROC). BCL2 was expressed on >50% of the tumor cells in 69% of cases. 83% of cases were phenotypically classifiable as non-GC DLBCL according to the Tally algorithm. There was no evidence for EBV- or HHV8-association. 70% of the tumors showed active STAT signaling by expression of either pSTAT1 or pSTAT3, but not pSTAT5. p53 was expressed in 12% of cases, but p21 staining (p21/ p53) did not suggest presence of TP53 mutations.. Mean mitotic index was 18/mm2, median MIB1 labeling index was 40% (±25%). Tumors with lymphoepithelial lesions in seminiferious tubules showed a lower mitotic activity, although the association was weak. Interestingly, one tumor was positive for OCT4. All 43 cases were negative for NUT1 and PLAP. Only limited clinical data were available: mean age at diagnosis was 69 years (range: 43–87 years, n=41). There was no side predilection of the tumors. One tumor was bilateral at diagnosis, one tumor presented simultaneously in the testis and the CNS. Of ten tumors, five did not relapse (mean follow up time 48 months). Five tDLBCL relapsed, thereof two in the contralateral testis, two in the CNS and one in the skin. Conclusions. We conclude that tDLBCL are predominantly centroblastic and of non-GC phenotype. Since occasionally tDLBCL can express germ cell markers or be CD20-negative, multimarker phenotyping is important for lineage determination. There was no shift of morphology or protein expression profile over time. tDLBCL have active STAT signalling mediated through pSTAT1 and pSTAT3. tDLBCL are of non-/post-GC origin and not hyper-proliferative. TP53 mutations are unlikely.

DO-060 C-MYC aberrations characterize a subset of patients with diffuse large B-cell lymphoma with poor outcome when treated with rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone A. Tzankov1, M. Gerhard1, S. Dirnhofer1, C. Visco2, K. Young3 1 University Hospital Basel, Pathology, Basel, Switzerland, 2San Bortolo Hospital, Internal Medicine, Vicenza, Italy, 3The University of Texas MD Anderson Cancer Center, Pathology, Houston, United States Aims. Diffuse large-B cell lymphoma (DLBCL) exhibits variable outcomes and risk assessment is based on the international prognostic index (IPI), which takes into account primarily patient-related parameters. Gene expression profiling (GEP) can stratify patients with different prognoses into germinal center B-cell (GCB) and activated B-cell subtype (ABC). These groups remain of prognostic importance with the addition of rituximab (R) to chemotherapy. The prognostic role of C-MYC gene status in the era of modern treatment is still debatable. Methods. To address this question, we analyzed C-MYC gene abnormalities by interphase fluorescence in situ hybridization (FISH) utilizing break-apart- (BAP) and a IgH/C-MYC double-fusion (DFP) probes in 601  patients with de novo DLBCL treated with R, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) and 332 patients treated with CHOP; all patients had clinical follow-up and GEP data. Results. C-MYC gene abnormalities were detected in 67 of 672 evaluable cases (10%), including 7 amplifications (2 ABC, 5 GCB), 4 rearrangements, detectable only with the DFP (2 ABC, 2 GCB), 23 rearrangements, detectable only with the BAP (5 ABC, 18 GCB) and 33 rearrangements, detectable with both the BAP and DFP (12 ABC, 21 GCB; p=0.032 for the differences between ABC and GCB). In multivariable models the last two C-MYC aberration types represented IPI- and GEP-independent

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prognostic factors for event-free survival in R-CHOP treated patients (p=0.003; relative risk 1.46) and in patients with GCB (p=0.014; relative risk 1.29), but (though being of prognostic significance in univariable models) not in CHOP treated ones and in ABC, where IPI was the sole prognosticator. Conclusions. C-MYC aberrations are observed in 10% of DLBCL, more commonly in GCB cases. Alternative C-MYC rearrangements with nonIgH partners, which are detectable only with BAP, account for a third of all C-MYC structural genetic abnormalities. C-MYC aberrations detected by BAP add IPI independent prognostic information for individual DLBCL risk estimation in R-CHOP treated cases.

DO-061 MYC-binding sites in Burkitt lymphoma identified by deep sequencing V. Seitz1, P. Butzhammer2, B. Hirsch1, J. Hecht3, I. Gütgemann4, A. Ehlers1, D. Lenze1, E. Oker1, A. Sommerfeld1, E. von der Wall1, C. König5, C. Zinser6, R.  Spang2, M. Hummel1 1 Institute of Pathology, Charité – University Medicine, Berlin, 2University of Regensburg, Institute for Functional Genomics, Regensburg, 3Charité – University Medicine, Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, 4University Hospital of Bonn, Department of Pathology, Bonn, 5 imaGenes GmbH, Source BioScience, Berlin, 6Genomatix Software GmbH, Genomatix Personalized Medicine, München Aims. MYC is a key transcription factor involved in central cellular processes such as regulation of the cell cycle, histone acetylation and ribosomal biogenesis. It is overexpressed in the majority of human tumors including aggressive B-cell lymphoma. Especially Burkitt lymphoma (BL) is a highlight example for MYC overexpression due to a chromosomal translocation involving the c-MYC gene. However, so far genomewide analysis of MYC-binding sites by chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-Seq) has not been conducted in BL. Therefore, our objective was the precise and representative consideration of the MYC landscape in BL due to a much higher coverage of MYC-binding sites employing ChIP-Seq. Methods. We carried out ChIP in 5 human BL cell lines, employing a MYC-specific antibody followed by next generation sequencing of the precipitated DNA fragments. To assess the functional consequences of MYC binding, the ChIP-Seq data were supplemented with siRNA-mediated knock-downs of MYC in BL cell lines followed by gene expression profiling. Results. Our ChIP-Seq analysis gave rise to 7,054 MYC-binding sites after bioinformatics analysis of a total of approx. 19 million sequence reads. In line with previous findings, binding sites accumulate in genes known to be involved in the cell cycle, ribosomal biogenesis, histone acetylation and DNA-methylation demonstrating a regulatory role of MYC in these processes. Unexpectedly, MYC-binding sites also accumulate in many B-cell relevant genes and expression analysis after knock-down of MYC by siRNA identified genes involved in the B-cell function which are upregulated in response to MYC silencing. Conclusions. The 7,054 MYC-binding sites identified by our ChIP-Seq approach greatly extend the knowledge regarding MYC binding in BL and shed further light on the enormous complexity of the MYC regulatory network. Especially our observations that (i) many B-cell relevant genes are targeted by MYC and (ii) that MYC down-regulation leads to an upregulation of B-cell genes highlight an interesting aspect of BL biology.

DO-062 Tumor-associated macrophages in pediatric classical Hodgkin lymphoma: associations with Epstein-Barr virus, lymphocyte subsets and prognostic impact M. Barros1, R. Hassan2, G. Niedobitek1 Unfallkrankenhaus Berlin, Institut für Pathologie, Berlin, 2Brazilian National Cancer Institute, Bone Marrow Transplantation Center, Rio de Janeiro, Brazil

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Aims. The number and type of tumor-associated macrophages are implicated in the biology of classical Hodgkin lymphoma (cHL) in adults. Due to the immunological differences between children and adults, and to the cross-talk between innate and adaptive immune system, it is hypothesized that number, function and prognostic impact of macrophages in pediatric cHL may be different from adult cases. Methods. We analyzed the number of macrophages and dendritic cells (DCs) in the tumor microenvironment of pediatric cHL by immunohistochemistry (IHC) and image analysis system. Epstein-Barr virus (EBV) status was determined by EBER-specific in situ hybridization and IHC. Results were analyzed in the context of age group, histological characteristics and clinical follow-up. Results. The younger ages were characterized by a more intense CD14+ and CD163+ cell infiltrate (p=0.01 and p=0.025, respectively). In relation to nodular sclerosis, mixed cellularity subtype was characterized by high number of CD14+, (p=0.003) and CD163+ cells (p=0.027). EBV+ cases exhibited higher numbers of CD14+ (p<0.0005), CD68+ (p=0.005) and CD163+ cells (p=0.02). CD14+ cells were directly correlated with the number of TBET+ (p<0.0005), CD8+ (p=0.019), TIA1+ (p=0.024) and Granzyme B+ cells (p=0.009). CD68+ cells were directly correlated with the number of FOXP3+ (p<0.001), TIA1+ (p=0.001) and Granzyme B+ cells (p< 0.001). CD163+ cells were directly correlated with the number of TBET+ (p=0.001), CD8+ (p=0.025), TIA1+ (p=0.001) and Granzyme B (p<0.0005) cells. CD83+ cells were directly correlated with the number of CD4+ cells (p=0.01), FOXP3+ cells (p=0.003) and CD20+ lymphocytes (p=0.018). A worse overall survival (OS) was observed in cases with CD163/CD8 ratio >1 (p=0.007). High number of CD163+ cells was associated with worse progression-free survival (PFS; p=0.015) in univariate analysis. However, an independent prognostic impact was observed only when the innate and adaptive immune variables were analyzed together (high numbers of CD163+ cells and Granzyme B+ cells, respectively; p=0.038). Conclusions. Our results suggests that in pediatric cHL the macrophage composition is numerically and functionally distinct from adults; EBVstatus influences the composition of macrophages in the tumor microenvironment and the variables from innate and adaptive immune system may have to be considered together when the prognostic impact of the tumor microenvironment is analyzed.

DO-063 Three different expression patterns of T-bet in angioimmunoblastic T-cell lymphoma K. Jöhrens1, M. Dietel1, I. Anagnostopoulos1 Charité Medical University Berlin, Institute of Pathology, Charité Campus Mitte, Berlin

pattern II showed a T-bet expression pattern B (6/8 cases), while the majority of those with pattern III exhibited the T-bet pattern A (11/21 cases). Conclusions. We propose that T-bet expression by B-cells represents a T-cell independent immune response trying to cope with opportunistic infections, while T-bet expression by neoplastic T-cells is linked to the introduction of a Th17 response responsible for the immunologic derangements characteristic of AITL.

DO-064 Molecular characterization of paediatric T-cell lymphoblastic lymphoma using gene expression profiling E. Niendorf1, M. Szczepanowski1, I. Salaverria2, B. Burkhardt3, W. Klapper1 Christian Albrecht University of Kiel, Department of Pathology, Kiel, 2Christian Albrecht University of Kiel, Institute of Human Genetics, Kiel, 3University Hospital Münster, Department of Pediatric Hematology and Oncology

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Aims. T-cell lymphoblastic lymphoma (T-LBL) is an aggressive T-cell lymphoma predominantly affecting children. Relapse of the disease is virtually always fatal. Therefore, identification of T-LBL at risk for a later relapse is important for therapy stratification. The aim was to characterize paediatric T-LBL by gene expression profiling to identify differences between lymphomas with and without later relapse and differences between assumed histological subtypes. Methods. Biopsies of 28 paediatric T-LBL were characterized by immunohistochemistry and gene expression profiling (GEP) using Affymetrix GeneChip U133A arrays. Data were analyzed using Partek® Genomics Suite software. Immunohistochemistry was performed on Tissue Micro Arrays (TMA) using an independent set of T-LBL. Results. GEP did show significant differences between T-LBL with and without a later relapse. Among the most differentially expressed genes with a P value up to 0.002, some are known to be involved in cell cycle regulation and the pathogenesis of different malignancies such as HIPK2, SSX1 or CTAGE5. Surprisingly, the subgroup of cortical T-LBL defined by CD1a expression did show only minor differences in gene expression profiles compared to the non-cortical T-LBL. Using immunohistochemistry for one of the differentially expressed genes, MLC1, we were not able to reproduce the differential mRNA expression between cortical and non-cortical T-LBL in staining. Conclusions. Prognostic bio-marker identifying paediatric T-LBL at risk for a later relapse are urgently needed. The differences in GEP between T-LBL with and without a later relapse reported herein, might present a starting point to develop such markers for routine use e.g. by the use of immunohistochemistry or quantitative RT-PCR. Furthermore, a detailed study of the differentially expressed genes might provide insight into the pathomechanisms that render the lymphoma prone to relapse after therapy. Currently, T-LBL are classified by immunohistochemistry into a cortical and a non-cortical subtype by the use of CD1a protein expression assuming that this classification reflects differences in the maturation stage of the “cell of origin”. The very limited number of differentially expressed genes between both groups however suggests, that this classification does not reflect biologically relevant subgroups.

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Aims. Angioimmunoblastic T-cell lymphoma (AITL) exhibits a multifaceted clinical picture and distinct architectural patterns that correlate with disease progression and the number of neoplastic cells. In this study we investigated the expression of the transcription factor T-bet and correlated it with the architectural patterns (I, II or III) of AITL. Methods. 29 cases of AITL were investigated by double immunolabelings for T-bet and for CD20, CD3 or PD1. Results. Following patterns were revealed: predominant T-bet expression by neoplastic T-cells (pattern A), by aggregates of small B-cells (pattern B) or by B-immunoblasts (pattern C). The majority of cases of AITL

DO-065 Identification of miRNAs characteristic of ALK+ ALCL J. Steinhilber1, I. Bonzheim1, S. Schäfer1, F. Fend1, L. Quintanilla-Martinez1 1 University Hospital Tübingen, Eberhard-Karls-University, Institute of Pathology and Neuropathology, Tübingen Aims. ALK+ anaplastic large cell lymphomas (ALCL) overexpress C/ EBPβ, as a consequence of NPM-ALK kinase activity. The aim of this study was to identify miRNAs, which are differentially regulated by C/ EBPβ in ALK+ ALCLs and to find differentially expressed miRNAs between ALK+ ALCLs, ALK− ALCLs and T-cells.

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Abstracts Methods. Three ALK+ ALCL cell lines were transduced with C/EBPβ shRNA by lentiviral gene transfer. The C/EBPβ knockdown was quantified by RT-qPCR and Western Blot. MiRNA expression in ALK+ ALCL cell lines with and without C/EBPβ knockdown, ALK− ALCL cells and T-cells were analyzed by deep-sequencing, to determine differentially regulated and expressed miRNAs in ALK+ ALCL. The influence of C/ EBPβ on the expression of miRNA candidates and the differential expression in ALK+ ALCL was validated by RT-qPCR in cell lines and in primary tumors. Results. Next-generation sequencing analysis resulted in 80 significantly regulated miRNAs after C/EBPβ knockdown in the three ALK+ ALCL cell lines. Three of these miRNAs (miR-181a*, miR-146b-5p, miR-203) were significantly regulated in all three cell lines. The C/EBPβ dependent regulation of these miRNAs was confirmed in two cell lines by RTqPCR. Comparison of the results of the ALK+ ALCL cell line SUDHL-1 and T-cells or SUDHL-1 and the ALK− ALCL cell line revealed 379 or 301 significantly regulated miRNAs respectively. ALK− ALCL cells and T-cells showed a significant difference in the expression levels of 366 miRNAs. A hundredfold change in expression levels was observed for a few interesting miRNAs of the different signatures. The differential expression of some of the most remarkable miRNAs in ALK+ ALCL was validated in primary human ALK+ and ALK− ALCLs by RT-qPCR. Several of these miRNAs play important roles in diverse cancers with tumor-suppressing or oncogenic functions. Conclusions. Three miRNAs were found to be regulated by C/EBPβ in two ALK+ ALCL cell lines. Numerous miRNAs are differentially expressed in ALK+ or ALK− ALCL cells and T-cells. Several miRNAs which are significant differentially expressed in ALK+ and ALK− ALCLs separate ALCLs depending on their ALK status. We identified a miRNA profile specific to ALK+ ALCLs.

DO-066 The role of C/EBPβ in the phenotype of ALK+ anaplastic large cell lymphoma J .-A . Schmidt1, I . Bonzheim1, S . Schäfer1, F . Fend1, L . Quintanilla-Martinez1 1 University Hospital Tübingen, Institute of Pathology and Neuropathology, Tübingen Aims. ALK+ anaplastic large cell lymphoma is characterized by the loss of pan T-cell antigens and the unusual expression of myelomonocytic surface markers. The forced overexpression of the transcription factor C/ EBPβ in B and T-cells has been shown to induce transdifferentiation into macrophages. Since C/EBPβ has been shown to play a central role in the pathogenesis of ALK+ ALCL, the aim of the study was to investigate its influence on the unusual phenotype of ALK+ ALCL. Methods. The expression of 242 surface antigens was investigated in ALK+ ALCL cell lines before and after the specific knockdown of C/ EBPβ using the BD Lyoplate Human Cell Surface Marker Screening Panel by flow cytometry. A highly specific C/EBPβ-shRNA was transduced by lentiviral infection. The expression of significant surface markers was validated by immunohistochemistry and Western blot in primary ALK+ and ALK− ALCL cases. Results. The surface marker screening confirmed the loss of T-cell specific markers, such as TCR chains and CD3, and overexpression of EMA and activation markers CD25 and CD30 but did not support the unusual expression of myeloid markers as CD13 or CD33, as previously described. Activation surface markers, including CD25, CD30, CD97 and CD98, showed downregulation after C/EBPβ knockdown indicating a role for C/EBPβ in the activation of ALK+ ALCL cells. CD147 (EMMPRIN) was strongly expressed in ALK+ ALCL cells, and was downregulated after C/ EBPβ knockdown. Interestingly, CD147 was differentially expressed in ALK+ ALCL when compared to ALK− ALCL primary cases. Conclusions. Surface marker screening in ALK+ ALCL revealed an influence of C/EBPβ on the activation phenotype of the neoplastic T-cells and expression of CD147, an inductor of matrix metalloproteinases implica-

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ted in tumor progression and metastasis in solid tumors. The differential expression of CD147 in ALK+ ALCL suggests its involvement in the pathogenesis of ALK+ ALCL.

AG Orthopädische Pathologie DO-079 Histomorphological and clinical characterisation of Epstein-Barr virus-associated post-transplant smooth muscle tumours L . Maegel1, J . Rische1, C . Tiede1, J . Salem1, F . Laenger1, H . Kreipe1, K . Hussein1, D . Jonigk1 1 Hannover Medical School, Institute of Pathology, Hannover Aims. Histomorphological and clinical characterisation of Epstein-Barr virus-associated post-transplant smooth muscle tumours. Methods. Own cohort: Five PTSMT were examined by histology, immunohistochemistry and molecular methods [mean age of patients 10 years; PTSMT developed after a mean interval of 44 months following liver (n=2), heart (n=2) or bone marrow (n=1) transplantation]. Metadata analysis: Data of 64 PTSMT cases (including our cohort) of the last 20 years were re-evaluated by applying survival analysis. Molecular analyses: All specimen of our cohort underwent compartment-specific laser microdissection and processed for further quantitative real-time PCR analysis. Gene expression of transcripts for a set of 20 EBV-associated endogenous human genes were analysed by qPCR: transcription factors MYC, TP53, NFKB1; apoptosis factors such as BAX; JAK3/STAT signal factors; cytokines such as VEGF; miR-155 and miR-146a. To evaluate the origin of PTSMT – either from the recipient or from the donor – short tandem repeat (STR)-PCR was performed (“molecular fingerprinting”). Results. Histomorphology and immunoprofile (total cohort, n=64): – Spindle shaped, leiomyogenous cells (actin+/desmin+/EBER+), local invasion. – Most PTSMT show no marked cellular atypia, no tumour necrosis, <1/10HPF and <5 cm diameter. – PTSMT are not restricted to a specific organ (58% PTSMT manifested in the liver/transplant liver, 42% in other organs such as, respiratory tract, spleen, gastrointestinal tract, brain). – PTSMT can present metachronous, synchronous and metastatic. Risk profils (total cohort, n=64): – Most patients were kidney transplanted (58%). – Tumours occurred after a median interval of 48 months (range 5–348 months) after transplantation. – PTLD preceded PTSMT in a subfraction of cases (19%). Survival-related factors (total cohort, n=64): – Surgery and reduced immunosuppression have the same benefit. – PTSMT manifestation in the brain has a poorer out-come than multiple tumour localisation and/or transplant organ involvement. Molecular characteristics of PTSMT (own cohort, n=5): – STR-PCR demonstrate the donor origin of 1/5 PTSMT. – MYC is overexpressed in PTSMT. Conclusions. PTSMT represent an entity with unknown malignant potential and shows no features of typical sarcomas in immunocompetent patients. The major prognostic factor is brain involvement.

DO-080 DOG1: an immunohistochemical marker for neoplastic chondroblasts in chondroblastoma H. Akpalo1, C. Lange2, J. Zustin1 University Medical Center Hamburg-Eppendorf, Institute of Pathology, Hamburg, 2University Medical Center Hamburg-Eppendorf, Clinic for Stem Cell Transplantation, Hamburg

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Aims. Chondroblastoma represents less than 1% of all primary bone tumors and typically presents in the epiphysis of long bones of young patients. The tumor is composed of cartilaginous and osseous matrix along with cellular portions with polygonal chondroblasts with indented nuclei and scattered osteoclast-type multinucleated cells. In the current study, we wished to investigate the expression of several established immunohistochemical markers in the cellular portions of chondroblastomas. Methods. Nine chondroblastomas, seven chondromyxoid fibromas, five giant cell tumors of bone and tissues from five fetal femoral bone endings were analyzed using immunohistochemical antibodies (CD34, SMA, DOG1, CD117, and CD163). Results. The cellular areas of chondroblastoma cases contained nests of DOG1+ SMA+ CD117− CD34− chondroblasts, a phenotype that was not detected in chondromyxoid fibroma cases or in giant cell tumors. The remaining chondroblasts showed only low expression of DOG1 or negative reaction. Furthermore, numerous CD163+ macrophages were detected in all tumors which were analyzed in our study: chondroblastomas, chondromyxoid fibromas and giant cell tumors. Conclusions. We described membranous DOG1+ chondroblasts located within cellular portions of chondroblastoma along with CD163+ macrophages and multinucleated osteoclastic giant cells. Therefore, chondroblastoma can be added to the tumors that are usually positive for DOG1, alongside GIST, rare solid-pseudopapillary neoplasms of the pancreas as well as exceptional mesenchymal tumors including peritoneal leiomyomatosis, uterine type retroperitneal leiomyoma, and synovial sarcoma.

DO-081 Bone erosion and inflammation – polymorphonuclear neutro­ phils promote generation of osteoclasts in osteomyelitis patients M.M. Gaida1, B. Mayer2, S. Stegmaier2, P. Schirmacher1, C. Wagner3, G.M. Hänsch2 1 University of Heidelberg, Institute of Pathology, Heidelberg, 2University of Heidelberg, Institute of Immunology, Heidelberg, 3BG Trauma Center Ludwigshafen, Ludwigshafen Aims. Chronic and persistent inflammatory processes in the proximity of bones may lead to severe bone erosion, requiring the amputation of the respective limb. Aim of the present study was to elucidate the process of bone erosion in the context of inflammation. Methods. To explore the relationship between inflammation and bone erosion, biopsies of patients with osteomyelitis due to arterial occlusive disease or to diabetes mellitus were examined (n=31) and the inflammatory infiltrate, bone erosion and infiltration of osteoclasts were quantified. In parallel, interleukin (IL)-8-induced differentiation of CD14+ monocytes derived from the peripheral blood of healthy individuals to osteoclasts was tested in vitro. The cells were cultivated with monocyte colony stimulating factor (M-CSF) and IL-8, and for comparison with the well established osteoclast-inducing receptor activator of Nf κ B ligand (RANKL). To verify the activity of newly generated osteoclasts the ability to degrade ivory slices was tested. The classical pathway of the osteoclastogenesis (NFATc1 and c-fos) was explored by Western blot analysis of isolated cytoplasmic and nuclear proteins after stimulating the isolated monocytes with IL-8. Results. In tissue sections of osteomyelitis patients, in areas of bone destruction, the number of osteoclasts correlated significantly with the extent of the leukocytic infiltrate, particularly with the number of polymorphonuclear neutrophils (PMN). PMN recovered from the infec-

ted sites showed characteristics of activation and produced interleukin (IL)-8. CD14+ monocytes derived from the peripheral blood of healthy individuals were cultivated with (M-CSF) and IL-8, and within 3 days, a translocation of the transcription factor NFATc1 into the nucleus was seen, as begin of the differentiation into osteoclasts. By 10 to 20 days, multinucleated cells with the typical osteoclast morphology appeared which expressed tartrate-resistant acid phosphatase (TRAP) and cathepsin K. Moreover, these cells were able to resorb bone. Conclusions. In patients with persistent inflammatory disease and loss of bone, the abundance of PMN in areas of bone resorption correlated with the number of osteoclasts. Since activated PMN are known to produce IL-8, which is able to induce osteoclast formation, we propose that PMN promote bone destruction by local generation of osteoclasts and thus provide a link between the inflammation and bone erosion.

DO-082 High IGF2 and FGFR3 are associated with tumor progression in pleomorphic undifferentiated sarcomas, but EGFR and FGFR3 mutations are a rare event K. Rüping1, D. Katenkamp1, Y. Chen1, A. Altendorf Hofmann2, U. Settmacher2, I. Petersen1, T. Knösel1 1 Friedrich-Schiller University, Institute of Pathology, Jena, 2Friedrich-Schiller University, Department of General, Visceral und Vascular Surgery, Jena Aims. Pleomorphic undifferentiated sarcoma (formerly known as malignant fibrous histiocytoma, MFH) is meanwhile recognized as a morphological growth pattern shared by a wide variety of poorly differentiated malignant neoplasms, which include specific subtypes of pleomorphic sarcomas. Nevertheless prognostic and therapeutic options in these tumors are urgently needed. Methods. 327 fibroblastic/myofibroblastic differentiated tumors consisted of 203 pleomorphic undifferentiated sarcomas, 42 low grade sarcomas (10 low grade fibromyxoid sarcoma, 32 low grade myofibroblastic sarcomas) and 82 pseudosarcomatous tumors of the fasciitis family were analyzed immunohistochemically and correlated with clinicopathological parameters. Additionally mutational analysis was performed on high expressed specimens of EGFR and FGFR3. Results. High expression was found in PDGFRA (45%), PDGFRB (35%), EGFR (3.4%), TFE (30%), KDR (1.5%), IGF2 (68%), FGFR1 (6.5%) and FGFR3 (52%). High expression of IGF2 and FGFR3 was significantly correlated with higher tumor grading (low versus high, p<0.001), higher tumor size (5 versus >5 cm, p<0.05) and higher Ki67 Index (20 versus 20, p<0.001). Mutational analysis of high expressed cases of EGFR and FGFR3 showed no mutations on the hotspots (EGFR: hotspots exon 19, 20 and 21. FGFR3: hotspots exon 7, 10 and 15). Conclusions. High expression of IGF2 and FGFR3 is significantly associated with tumor progression in fibroblastic/myofibroblastic sarcomas including pleomorphic undifferentiated sarcomas and classify these tumors in prognostic and therapeutic subgroups. These biomarkers might guide targeted therapies in these neoplasms.

DO-083 Analysis of potential therapeutic targets in synovial sarcomas M. Straub1, M. Bettstetter2, G. Keller1, R. Gradinger3, H. Höfler1, K. Specht1 Technical University Munich, Institute of Pathology, München, 2Molecular Pathology Southern Bavaria, 3Technical University Munich, Department of Orthopedics 1

Aims. Synovial sarcomas are mesenchymal tumors of unknown histogenesis accounting for 5–10% of all soft tissue sarcomas. Their molecular signature is a specific t(X; 18)(p11.2;q11.2) translocation which fuses SS18 (SYT) with either SSX1, SSX2 or SSX4. Clinical management consists primarily of surgery and radiotherapy or cytotoxic chemotherapy and no effective targeted therapies are currently available. The aim of this Der Pathologe Suppl 1 · 2012 

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Abstracts study was to evaluate the expression and mutational status of potential molecular therapeutic targets in synovial sarcomas. Methods. 38 well characterized and molecularly confirmed cases of synovial sarcomas were included in this study. Immunohistochemical stainings of tyrosine kinase receptors of the EGF-R family (EGF-R, HER2/ neu, HER3, HER4), the hepatocyte growth factor c-met, and signaling molecules implicated in the mTOR pathway (AKT, mTOR, PTEN), as well as E-Cadherin and snail was performed. In addition, cases were screened for mutations in the EGFR, PIK3C, B-RAF, K-RAS and N-RAS genes. Results. Members oft the EGF-receptor family of kinases as well as ECadherin and snail are important for defining the tumor phenotype by determining epithelial-mesenchymal transition of synovial sarcomas. Activation of c-met and signaling molecules oft the mTOR pathway are seen in a significant number of cases. Mutations of the genes studied (EGFR, PIK3C, B-RAF, K-RAS, N-RAS) are an overall rare event in synovial sarcomas. Conclusions. EGF-R expression is found in many synovial sarcomas, however, activating mutations in the tyrosine kinase domain or downstream signaling molecules appear to be a rare event or are absent. Activation of c-met and molecules oft the mTOR pathway is frequently seen in synovial sarcomas. The benefit of targeted therapy against these genes in synovial sarcomas remains to be determined.

DO-084 Allergy to metal implants: Immunological und histological analysis of patients with intolerance reaction after knee arthroplasty J. Schneider1, M. Flaig1, B. Summer1, C. von der Helm1, C. Schopf1, V. Krenn2, M. Thomsen3, L. Frommelt4, P. Thomas1 1 Ludwig-Maximilians-University, Munich, Dermatology, München, 2Zentrum für Histologie, Zytologie und Molekulare Diagnostik Trier, 3OrthopädischeDRK-Klinik, Baden-Baden, 4Endoklinik, Hamburg Aims. Allergic reaction to metal implants as a reason for implant loosening or other complications is controversially discussed. Therefore we analysed 10 patients with metal allergy but no infection or mechanical problems in knee arthroplasty who needed revision surgery. Methods. In periprosthetic tissue of the 10  patients with metal hypersensitivity (patch testing and/or lymphocyte transformation test (LTT) positive) and CoCrMo based arthroplasty, histological classification (according to consensus classification of periprosthetic interface membranes) and molecular cytokine analysis (Realtime-PCR) was performed. The results were compared with a control group of 5  patients without metal hypersensitivity. After implant replacement to titanium or oxinium based arthroplasty the symptoms were monitored with the WOMAC-Score. Results. Patch testing: 4/10 reactivity to nickel, 3/10 to cobalt, 1/10 to chromium. Enhanced LTT reactivity: 8/10 to nickel, 1/10 to cobalt. Cytokine expression: IFNy 4/10 vs. 0/5; TGFβ 8/10 vs. 5/5; IL-8 8/10 vs. 0/5; IL-6 6/10 vs. 1/5, IL10 7/10 vs. 5/5. In histopathology primarily the indeterminate type of periprosthetic tissue (type IV) or arthrofibrosis and a varying degree of lymphocytic infiltration were detected. The WOMAC-Score increased from 40.4±20.58 to 55.59±20.14. Conclusions. The combination of allergological, immunological and histopathological diagnostic steps helps to identify patients with implant intolerance reaction and may support the decision to a replacement with alternative material, such as titanium based arthroplasty.

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DO-085 Expression patterns of microRNA in SFT – prediction of malignancy? C. Poremba1, C. Altmann1, N. Arens1, J. Kriegsmann1, P. Knöß2 1 Research Park Trier, Center of Histopathology, Cytology and Molecular Diagnostics (CHCMD) Trier, Trier, 2Center of Histopathology, Cytology and Molecular Diagnostics Trier Aims. The clinical and biologic behavior of solitary fibrous tumor (SFT) has been a problematic issue, mainly because of inconclusional criteria. SFT harboring “malignant” features such as high cellularity, >4 mitotic figures/10HPF and necrosis/hemorrhage are at risk for metastasis/recurrence. However, in biopsies those criteria may not be evident. In our study, we analyze if expression patterns of microRNA, a class of posttranscriptional regulators, differs between localized, relapsed and metastatic SFT, and may help to predict clinical and biologic behavior in this tumor entity. Methods. In this pilot study, tumor tissues from 6 patients suffering from SFT (3 localized without recurrence/metastasis, 3 with recurrence/metastasis) are analyzed by microRNA assay (Applied Biosystems, Carlsbad, CA USA). Clinical follow-up is available up to 10 years after initial diagnosis. Expression patterns of microRNAs are compared between localized SFT versus relapsed/metastatic SFT and within this group between the initial tumor (areas of different cellularity) and its respective recurrence/metastasis after microdissection. Results. In ongoing analyses, expression patterns of microRNAs are compared between localized vs. relapsed/metastatic SFT and are correlated to clinical outcome. Conclusions. We investigate if different expression patterns of microRNAs between localized vs. metastatic/relapsed SFT may help to identify patients with higher risk for unfavourable clinical outcome based on the initial biopsy of SFT. Statistical analyses are ongoing.

DO-086 Defining arthrofibrosis in histopathological specimens: an evolving concept P. Knöß1, C. Dierkes1, M. Ruppert2, T. Gehrke3, D. Kendoff3, C. Theiß4, V. Krenn1 Medical health center for histology, cytology and molecular diagnostics Trier, 2Brothers of mercy hospital Trier, 3ENDO Clinic Hamburg, 4Ruhr University Bochum

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Aims. Arthrofibrosis is the most severe complication in endoprothetic surgery leading to a complete loss of joint function. In the past we reported a proposal for a histopathological grading system for arthrofibrosis (grade 1–3). This time we wanted to verify our results immunohistochemically using β-catenin, a marker known for his association with fibromatosis. Methods. 262 specimens of patients with clinical evidence of arthrofibrosis were graded semiquantitatively for fibroblast density using our proposed grading system, stained with antibodies against β-Catenin and compared with a reference group of 29 neosynovialitis type 4 specimens. Results. In 85.1% of the specimens the histopathologic diagnosis was arthrofibrosis. The distribution for the fibroblast density was 28.8% grade 1, 47.7% grade 2 and 23.4% grade 3. The cellularity was significantly higher in every arthrofibrosis grade than in the reference group (p<0.05 grade 1, p<0.001 grade 2, p<0.001 grade 3). 20 β-catenin positive fibroblasts per high power field were established as a cut off for distinguishing arthrofibrosis from neosynovialitis type 4 with a sensitivity of 73% and a specifity of 87%. Conclusions. The histopathological diagnosis of arthrofibrosis can be defined as fibrotic synovial tissue with an increased cellularity of β-catenin staining fibroblasts. An easily measurable cut off (20 β-catenin positive fibroblasts per high power field) distinguishes arthrofibrosis from neosynovialitis type 4 with a sensitivity of 73% and a specificity of 87%.

AG Oralpathologie DO-087 Detection of human papillomavirus infection in head and neck squamous cell carcinoma J . Dreyer1, M . Barros1, G . Niedobitek1 1 Unfallkrankenhaus Berlin, Institute for Pathology, Berlin Aims. A sub-group of head and neck squamous cell carcinoma (HNSCC) is associated with HPV infection, mainly with HPV16. The incidence of this subset has increased and there is evidence to suggest that HPV-positive HNSCC show a more favourable prognosis. There is, however, controversy regarding the most suitable method for the detection of HPV infection in this setting. We have compared HPV DNA in situ hybridisation (ISH) with p16 immunohistochemistry (IHC). Methods. 141  cases of HNSCC diagnosed between 1997 and 2010 were identified. 119  patients (84.4%) were male and 22 (15.6%) were female. Primary site and pTNM stage as well as all other relevant clinicopathological data were extracted from the clinical and pathological files. Primary sites were oropharynx in 65 cases, hypopharynx in 21 cases, floor of mouth in 31 cases, tongue in 20 cases and other sites in the oral cavity in 4 cases. Tissue-micro-arrays (TMA) with three 2 mm cores per case were constructed. IHC for p16 (mtm laboratories) and ISH for high-risk and low-risk HPV-DNA (Ventana) were carried out. Results. 23  cases (16.3%) were positive for p16. Of these, 17  cases (12.1% overall) also showed a nuclear signal for high-risk HPV-DNA by ISH. No p16- cases were positive by ISH and no infection with low-risk HPV types was detected. Thus, there was a statistically significant association of p16 expression and high-risk HPV infection in our series (p<0.0005, Fisher’s test). The HPV ISH+ cases were localised preferentially in the oropharynx (15/17, 88.2%) and to a minor extent at the floor of mouth (2/17, 11.8%). No HPV DNA+ cases were seen in the hypopharynx or at the tongue (p=0.006). Five of 6 p16+/HPV DNA- cases (83.3%) were from the oropharynx. There was no association of HPV infection with other clinicopathological parameters (stage, grade, gender). Conclusions. Using ISH, we confirm that HPV infection is preferentially detected in HNSCC arising in the oropharynx. Our results furthermore support the notion that p16 is a suitable surrogate marker for HPV infection in HNSCC. This conclusion is also supported by the observation that 5 of 6 p16+/HPV DNA- HNSCC were localised in the oropharynx, the preferred site for HPV+ HNSCC. The observation of six p16+/HPV DNA- cases may be due to limited sensitivity of the ISH assay. Alternatively, HNSCC may be infected with HPV types not included in the probe cocktail. The suitability of a new system for the in situ detection of HPV E6 and E7 RNA transcripts is currently under investigation.

DO-088 HPV status predicts therapy response in head and neck squamous cell cancer patients receiving radiotherapy independent of the accompanying treatment modality A . Stenzinger1, A . Jensen2, J . Debus2, A . Muckenhuber1, A . Warth1, B . Sinn3, F . Klauschen3, J . Cortis1, W . Weichert1 1 University Hospital Heidelberg, Institute of Pathology, Heidelberg, 2 Department of Radiation Oncology, University Hospital Heidelberg, 3 Institute of Pathology, University Medicine Charité, Berlin Aims. HPV− HNSCC (head and neck squamous cell cancers) differ from HPV+ HNSCC in their biological behavior. Patients with HPV+ HNSCC show better responses to radiotherapy treatment and improved survival when compared to patients with HPV− HNSCC. However, until now, only sparse data exist regarding tumoral HPV status and the respective associations in comparison between differing accompanying treatment

modalities, especially induction chemotherapy, conventional chemotherapy and immuntherapy. Methods. We retrospectively analyzed the HPV status of a well defined cohort of 73  patients with locally advanced HNSCC by both, p16 immunostaining and PCR-based chip analysis for the HPV subtype. The patients were stratified according to the combined modality approach used for therapy: radiochemotherapy (RChT; 19 patients), radioimmunotherapy with cetuximab (RIT; 42 patients), or induction chemotherapy (IN) combined with either RChT or RIT (12 patients). The outcome was estimated using the Kaplan-Meier analysis and correlated with the HPV status and the therapy regimen respectively. Results. Dependent on the scoring algorithm and method used for detection between 15% and 37% of cases were p16/HPV positive. HPV status as determined by p16 immunoreactivity was a significant predictor of overall survival as well as of local and distant disease free survival. This effect was independent of the combination treatment modality (RIT, RChT, IN) used. Hazard ratios for HPV+ tumors in multivariate survival analysis were well below 0.5 for all modalities. In addition, the association of p16 positivity with survival was independent of the respective scoring algorithm used for the discrimination between positive and negative cases. Conclusions. To our knowledge, this is the first study showing that the predictive and prognostic effect of HPV in HNSCC patients receiving radiotherapy is independent of the accompanying treatment regimen. Patients with HPV+ HNSCC benefit from the addition of EGFR inhibition to radiotherapy and have a significantly improved survival compared to patients with HPV− HNSCC. Larger and prospective studies are needed to confirm these results.

DO-089 EBV- and HPV-associated carcinoma of the head and neck: Why is the topographical manifestation so different? M . Mollenhauer1, P . Zengel2, G . Assmann3, O . Guntinas-Lichius4, S . Ihrler5 Technische Universität München, Institute of Pathology, München, 2Ludwig Maximilian University Munich, Department of Otorhinolaryngology, Head and Neck Surgery, München, 3Ludwig Maximilian University Munich, Institute of Pathology, München, 4University of Jena, Department of Otorhinolaryngology, Jena, 5Laboratory for Dermatohistology und Oral Pathology, München 1

Aims. In the head and neck region EBV-associated carcinomas are limited to the nasopharynx, while HPV-associated carcinomas are predominantly localized in the palatinal tonsils and base of tongue (developing from cryptal epithelium) with rare cases in the nasopharynx. The reason for this diverse manifestation is unclear, especially since these three regions are regarded as equivalent lymphoepithelial tissue of Waldeyer’s ring. Methods. Comparison of microscopic anatomy of Waldeyer’s ring and determination of topographical development of nasopharyngeal carcinoma in situ (CIS). Results. Microscopic anatomy is not identical: All three regions show reticulated lymphoepithelial epithelium in the crypts, while on the surface palatinal tonsils and base of tongue show conventional squamous epithelium and nasopharygeal tonsils show respiratory epithelium. We demonstrate a case of EBV-associated nasopharyngeal CIS, which is a very rare occurrence, topographically located to respiratory surface epithelium. In a literature search we found a study, locating nasopharyngeal CIS to the surface epithelium in 82% and to the cryptal epithelium in 18%. Conclusions. While the mode of EBV-infection in the nasopharynx remains obscure, our data and literature support a correlation of precursor EBV-associated nasopharyngeal CIS to the respiratory surface epithelium. Hence, the divergent microscopic anatomy might be responsible for the striking different topographical manifestation of both carcinomas. While the occurrence of HPV-associated carcinomas in all regions obviously relates to the overall presence of lymphoepithelial cryptal epithelium in the three organs, the restriction of EBV-associated carcinomas to Der Pathologe Suppl 1 · 2012  | 

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Abstracts the nasopharyngeal tonsil might relate to the anatomical restriction of respiratory surface epithelium to this type of tonsil and might thereby give a clue of the primary site of malignant transformation by EBV.

DO-090 Analysis of human papilloma virus (HPV) and Epstein-Barr virus (EBV) in salivary gland adenocarcinomas E. Senft , H. Kreipe , K. Hussein Hannover Medical School, Institute of Pathology, Hannover 1

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Aims. Viruses are known to be associated with neoplastic proliferation, e.g. epitheliotropic human papilloma virus (HPV) can be detected in squamous neoplasms such as cervix carcinoma and oropharynx carcinoma while Epstein-Barr virus (EBV) infects B cells and can induce malignant lymphomas. Furthermore, EBV persists in the ductal epithelial cells of salivary glands and can be associated with solid neoplasms such as benign Warthin tumour. Systematic analyses have been performed in squamous carcinomas of the head and neck but not in salivary gland adenocarcinomas. Methods. Histological re-evaluation and selection of samples: adenoidcyctic carcinomas (n=20), adenocarcinomas, NOS (n=17), mucoepidermoid carcinomas (n=11), pleomorphic adenoma (n=4), carcinoma ex pleomorphic adenoma (n=3), non-neoplastic salivary glands (n=65). Analysis of multi-blocks with a total of 120 formalin-fixed and paraffinembedded (FFPE) tissue samples by HPV immunhistochemistry and EBER in situ hybridisation. DNA extraction from FFPE tumour samples and evaluation of HPV by multiplex PCR. Results. HPV and EBV were not detectable in salivary gland carcinomas. Conclusions. This is the first systematic analysis which demonstrates that the two human pathogenic viruses HPV and EBV are not involved in the pathobiology of salivary gland adenocarcinomas.

DO-091 SOX2 amplification is a common event in sinunasal squamous cell and undifferentiated carcinomas F. Göke1, A. Franzen2, R. Menon2, S. Huss3, D. Boehm2, W. Vogel2, F. Bootz4, S. Ihrler5, A. Schroeck4, S. Perner1 1 University Hospital Bonn, Pathology, 2University Hospital Bonn, 3University Hospital Cologne, Institute of Pathology, 4University Hospital Bonn, Head and neck department, 5Laboratory for Dermatohistology and Oral Pathology Aims. Although carcinomas of the nasal cavities are known to differ significantly from other cancers of the head and neck, regarding causing noxa, clinical behavior, and treatment, they share histological appearance. SOX2, a transcription factor-coding gene located at 3q26.33, is known to be recurrently amplified in squamous cell carcinomas (SCCs) of the lung, esophagus, skin, penis, cervix uteri and oral cavity. The aim of our study was to assess if SOX2 amplifications also occur in different tumor entities of the paranasal sinuses. Methods. Using fluorescence in-situ hybridization, we assessed for SOX2 amplification status in a cohort consisting of sinonasal SCCs (n=65), sinonasal undifferentiated carcinomas (SNUC, n=18), adenocarcinomas (n=25), and adenoid cystic carcinomas (n=18). Furthermore, we performed SOX2 immunohistochemical staining to quantify protein expression. Results. We detected SOX2 amplifications in 36% of sinunasal SCCs, 35% of SNUCs, 9% of adenocarcinomas, but none of the adenoid cystic carcinomas. Moreover, we found that the SOX2 amplification is associated with a SOX2 protein overexpression in SCCs and SNUCs. Conclusions. SOX2 amplification is not an organ site specific event, but is a frequent genetic alteration occurring in SCCs of various organs. Since SNUCs also harbor SOX2 amplifications in similar frequencies, we

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hypothesize that SNUCs may be undifferentiated SCCs of the sinunasal cavity.

DO-092 Expression of differentiation factor caspase 14 in oral squamous carcinomas C. Scharenberg1, H. Kreipe1, K. Hussein1 1 Hannover Medical School, Institute of Pathology, Hannover Aims. Caspase 14 is not involved in apoptosis (in contrast to all other caspase family members) but in differentiation of squamous epithelia. Caspase 14 is expressed mainly in the suprabasal layers, particularly the Str. intermedium/spinosum. Systematic analyses have been performed in cervix carcinomas and skin cancer but not oral cavity squamous carcinomas. Methods. Histological re-evaluation and selection of samples: squamous carcinomas of the oral cavity (n=30) and oral leukoplakia (n=10). Caspase 14 expression analysis by immunhistochemical evaluation of formalin fixed and paraffin embedded (FFPE) tissue samples. Results. Nuclear and cytoplasmatic caspase 14 expression is evident in non-neoplastic epithelial cells of the Str. intermedium but absent or weak in the basal and superficial layers. In leukoplakia the protein expression is increased in cells with keratinisation. In invasive lesions, caspase 14 is mainly expressed in the cells with keratinisation but absent or weak in neoplastic cells without keratinisation. Conclusions. This is the first experimental evidence that differentiation factor caspase 14 is expressed in oral cavity squamous carcinomas. ­Similar to leukoplakia expression of caspase 14 is increased in carcinoma cells with keratinisation.

DO-093 FGFR1 amplification in metastatic squamous cell carcinoma of the head and neck – a potential target for a rational therapy? F. Göke1, A. Franzen2, R. Menon2, R. Kirsten2, D. Boehm2, W. Vogel2, F. Bootz3, A. Schroeck3, S. Perner1 1 University Hospital Bonn, Pathology, 2University Hospital Bonn, 3University Hospital Bonn, Head and neck department Aims. Currently, patients with FGFR1 amplified squamous cell lung cancers (L-SCC) are treated in phase I clinical trials using small molecule inhibitors. Of interest, SCC of the lung share common molecular alterations with squamous cell head and neck cancers (HN-SCC). Aim of our study is to assess if HN-SCCs also harbor FGFR1 amplifications. Furthermore, we aim to identify a HN-SCC cell line harbouring FGFR1 amplification and inhibit cell proliferation using a small molecule inhibitor. Methods. We put together a cohort of 227 patients suffering from HNSCC, with 97 of these suffering from metastatic disease. Primary tumors and, where available, metastatic tumors were assessed for FGFR1 copy number status using fluorescence in-situ hybridization (FISH). We tested different cell lines for FGFR1 amplification status and inhibited these with small molecule inhibitors. Results. 20.3% of primary HN-SCC displayed FGFR1 amplifications. Of interest, almost all metastatic tumor samples revealed a FGFR1 amplification if the corresponding primary tumor harbored the amplification. The cell lines HN and SCC-25 harboured FGFR1 amplifications. HN cell proliferation was inhibitable with small molecule inhibitors. Conclusions. FGFR1 amplification frequently occurs in primary and metastatic HN-SCC and proves as a potential target for small molecule therapy in non-operable or metastatic disease. Furthermore, cell growth of FGFR1 amplified cell lines is inhibitable with small molecule inhibitors. Additional functional studies and subsequent clinical trials are needed for further validation of our findings.

DO-094 Do activated fibroblasts influence epidermal growth factor receptor (EGFR) inhibitor sensitivity in oral squamous cell carcinoma cells (OSCC)? P. Richter1, N. Neumann2, J. Schulte3, K. Schult1, S. Weisheit4, M. Franz5, O. Guntinas-Lichius6, C. Liebmann4, I. Petersen1, A. Berndt1 1 Jena University Hospital, Institute of Pathology, Jena, 2University Hospital Zurich, Institute of Surgical Pathology, Zurich, Switzerland, 3Ludwig-Maximilian-University, Munich, Großhadern Medical Center/Department of Cardiac Surgery, München, 4Friedrich Schiller University Jena, Institute of Biochemistry and Biophysics, Jena, 5Jena University Hospital, Clinic for Internal Medicine I, Jena, 6Jena University Hospital, Department of Otorhinolaryngology, Jena Aims. Although EGFR is involved in development of OSCC and EGFRinhibitor sensitivity could be shown in OSCC cell lines, a therapeutic benefit of an EGFR-inhibitor therapy can be observed only in a minority of patients. It was suggested that the carcinoma microenvironment have modulating effects on inhibitor sensitivity in vivo. One possible hypothesis is that carcinoma associated fibroblasts induce phenotype changes such as epithelial-mesenchymal transition (EMT) which are accompanied by alterations in EGFR signalling. Thus, the study was aimed at investigating the influence of growth factor activated fibroblasts on EGFRinhibitor sensitivity of OSCC in vitro applying Gefitinib. Methods. “Activated” fibroblasts were generated by stimulation of hTERT-BJ1 fibroblasts with TGFbeta1, PDGFAB, aFGF, and TGFbeta1/ aFGF, respectively. They were characterized with regard to proliferation, expression of fibroblast markers, activation of EGFR signalling, and the capability to induce OSCC cell invasion as well as their Gefitinib sensitivity using immunohistochemistry, western blotting, rtRT-PCR, MTT test and Boyden chamber assay. Furthermore, Gefitinib sensitivity was evaluated in different OSCC cell lines by MTT test. The impact of TGFbeta1 and TGFbeta1/aFGF activated fibroblasts on EGFR inhibitor sensitivity was assessed by pre-culturing of OSCC cells in fibroblast conditioned media. Results. In vitro activated fibroblasts showed a strong upregulation of ASMA and fibronectin due to stimulation with TGFbeta1. PDGFAB stimulation induced proliferation with up-regulation of EGFR and pAKT. aFGF and TGFbeta/aFGF stimulation resulted in an intermediate phenotype. TGFbeta1 stimulated fibroblasts exhibited the highest capability to induce invasion in the OSCC cell line PE/CA-PJ15 with upregulation of N-cadherin. Interestingly, activated fibroblasts differed in their ­Gefitinib sensitivity (TGFbeta-stim.=”low” and PDGFAB stim.=”high”). OSCC cell lines tested so far show a different Gefitinib sensitivity. Furthermore, pre-culturing in fibroblast conditioned ­medium leads to a partial reversibility of the Gefitinib effect. Conclusions. Results indicate that: 1) EGFR inhibition by Gefitinib ­affects OSCC cells as well as activated stromal fibroblasts, 2) the different Gefitinib sensitivity of fibroblast phenotypes may lead to a selective ­accumulation of myofibroblasts during treatment, and 3) OSCC cell sensitivity is modulated by stromal fibroblasts.

DO-095 Evaluation of post-transplant lymphoproliferative diseases (PTLD) with manifestation in the oral cavity C. Tiede1, B. Maecker-Kolhoff2, H. Kreipe1, K. Hussein1 1 Hannover Medical School, Institute of Pathology, Hannover, 2Hannover Medical School, Hannover

Methods. Evaluation of histomorphology and clinical data on early lesion, polymorphic, and monomorphic PTLD. Total cohort of 212 patients (median age 9  years, range 0.5–70  years, 57% males/43%females, 75.5% children/24.5% adults). Results. Oral cavity PTLD manifestation was found in 61/212  patients (29%): 42/61 early lesion PTLD (20%), 12/61 polymorphic PTLD (6%) and 7/61 monomorphic PTLD (3%). Tonsils were the most frequent site of manifestation (n=57/61, 93%) including early lesion PTLD (n=42/57, 74%), polymorphic PTLD (n=12/57, 21%) and monomorphic B cell PTLD (n=3/57, 5%). Other localisations were gingiva (n=2/61, 3%; EBV+ plasmocytomas), maxillary bone (n=1/61, 1.5%; EBV+ plasmocytomas) and larynx (n=1/61, 1.5%; EBV-T-cell PTLD). Conclusions. Tonsils are the main site of PTLD manifestation in the oral cavity and comprise mainly benign early lesion PTLD and polymorphic PTLD. Oral cavity monomorphic PTLD is rare and is located outside of the tonsils in a considerable proportion (n=4/7, 57%) of cases.

DO-096 Non-sebaceous lymphadenoma of salivary glands: proposed development from intraparotid lymph nodes and risk of misdiagnosis C. Weiler1, A. Agaimy2, P. Zengel3, J. Zenk4, T. Kirchner1, S. Ihrler5 1 Ludwig Maximilian University, Institute of Pathology, München, 2University Hospital Erlangen, Institute of Pathology, Erlangen, 3Ludwig Maximilian University, Head and Neck Surgery, München, 4University Hospital Erlangen, Head and Neck Surgery, 5Laboratory for Dermatohistology and Oral Pathology, München Aims. Non-sebaceous lymphadenoma (NSLA) is a rare benign salivary gland tumour composed of lymphoid and epithelial components. Definitionally, the epithelial component lacks sebaceous differentiation and, instead, displays a wide range of histological differentiation. In this study, we have collected 9 cases of NSLA to characterize their histological and immunohistochemical profile. Methods. The samples were histologically reviewed, and immunohistochemical stains for CK5/6, CK7, CK14, CK18, p63, and Ki67 performed. Patients were 6 males and 3 females (mean age, 50 years). Results. All tumours were located in the parotid gland and showed intimate intermingling of lymphoid tissue with islands or strands of epithelium with a wide spectrum of histological differentiation. The immunohistochemical profiles mirrored the epithelial differentiation; hence, areas with basaloid or lymphoepithelial differentiation strongly expressed CK5/6, CK14, and p63, while areas with ductal differentiation showed strong positivity for CK18/CK7 and CK5/6/CK14/p63 in luminal and basal cell layers, respectively. A hilus structure with salivary inclusions or D2-40 (podoplanin) positive marginal sinus were identifiable in 4 and 9 of the cases, respectively, confirming origin within intra-/periparotid lymph nodes. Six cases were initially misdiagnosed as other benign (n=4) or malignant tumours (n=2). Conclusions. Our study on the largest series of NSLA reported to date provides strong evidence that NSLA belongs to the group of salivary gland tumours that pathogenetically develop from embryonic salivary gland inclusions in intra-/periparotid lymph nodes. Knowledge of the wide histological spectrum of this rare and presumably underreported tumour is important in order to avoid misdiagnosis, particularly as malignant tumour.

Aims. Inspection of the oral cavity can be easily performed in transplanted patients with an oral tumour mass and suspected Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD). We aimed to evaluate the main sites of manifestation and the morphological subtypes of PTLD in the oral cavity.

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Abstracts DO-097 Cytokeratin-positive epithelioid angiosarcoma presenting in the tonsil: a diagnostic challenge A . Agaimy1, H . Kirsche2, S . Semrau3, H . Iro2, A . Hartmann1 Friedrich-Alexander University of Erlangen, Institute of Pathology, Erlangen, 2 Friedrich-Alexander University of Erlangen, Department of Otorhinolaryngology, Head and Neck Surgery, Erlangen, 3Friedrich-Alexander University of Erlangen, Department of Radiation oncology, Erlangen 1

Aims. The majority of malignant neoplasms of the head and neck represent squamous cell carcinomas while sarcomas are rare in this anatomic region. Primary oral cavity sarcomas are exceedingly rare and may pose a great diagnostic challenge. Methods. A 71-year-old woman without previous history of malignancy or radiation to the head and neck presented with an antibiotic-refractory diffuse painful swelling of the right tonsil necessitating tonsillectomy. Within months the patient underwent surgical resection of multiple bleeding intraoral and gastrointestinal metastases. She is currently alive with disease 9 months from diagnosis. Results. Histological evaluation revealed subtotal replacement of the right tonsil by a high-grade epithelioid neoplasm displaying extensive ulceration, necrosis and primitive vasoformation. Immunohistochemistry showed strong/diffuse expression of pancytokeratin (CK) antibodies KL-1 and Lu5, CK8, CK18, CK19, vimentin, CD31, ERG and FLI-1. High molecular weight cytokeratins (CK5, 34ß12), CK7, CK13 and CK20 were not expressed. Conclusions. To our knowledge, this case represents the first well documented primary epithelioid angiosarcoma of the tonsil. The strong cytokeratin expression in epithelioid angiosarcomas represents a diagnostic pitfall. Thus, awareness of this rare and highly aggressive neoplasm is necessary for distinguishing it from poorly differentiated and acantholytic squamous cell carcinoma and diffuse large cell lymphoma.

DO-098 Lipomatous neoplasms of the salivary glands. A series of 23 cases with emphasis on oncocytic lipoadenoma and sebaceous differentiation A . Agaimy1, B . Märkl2, H . Arnholdt2, J . Zenk3, V . Bonkowsky4, M . Michal5, A . Skalova5, A . Hartmann1, S . Ihrler6 1 Friedrich-Alexander University of Erlangen, Institute of Pathology, Erlangen, 2Augsburg Clinic Center, Augsburg, 3Friedrich-Alexander University of Erlangen, Department of Otorhinolaryngology, Head and Neck Surgery, Erlangen, 4Nürnberg Clinic Center, Department of Otorhinolaryngology, Head and Neck Surgery, Nürnberg, 5Charles University, Medical Faculty Hospital, Department of Pathology, Pilsen, Czech Republic, 6Ludwig Maximilian University, Munich, Department of Pathology, München Aims. Lipomatous tumors of salivary glands are rare and have been the subject of rare case reports in the literature. Accordingly, their morphologic spectrum and clinicopathological features from a consecutive case series have not been studied. Methods. We collected 23 fatty tumors from consecutive surgical pathology files at four large hospitals (n=17) and from consultation files of two centers (n=6). Fat-containing pleomorphic adenoma and lipomatous myoepitheliomas were excluded. Results. There were 15 males and 8 females aged 18–89 yrs (mean, 55 yrs). Most tumors (n=21) originated in the parotid gland. Two affected the submandibular gland. Histologically, the tumors could be categorized into three groups: ordinary lipoma (n=16; 70%), oncocytic lipoadenoma (n=4) and non-oncocytic adenolipoma/sialolipoma (n=2). Ordinary lipomas were either completely intraglandular or they have been submitted as a lipomatous nodule containing minor foci of residual atrophic serous acini at the periphery of the lipoma beneath the capsule. None of the lipomas contained sebaceous elements or oncocytic cells. The less

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common oncocytic lipoadenoma (synonym: oncocytic sialolipoma) had a fatty component ranging from 10% to 95% of the lesion that was usually interspersed between the oncocytic acini. Sebaceous islands were found in three of the four cases. The oncocytes showed either diffuse solid sheets with a lobular architecture interrupted by scattered adipocytes or were scattered between plentiful fatty tissues. The two non-oncocytic adenolipoma (synonym: non-oncocytic sialolipoma) were predominantly fatty (70–80%) and prominently lobulated. They displayed a biphasic pattern with serous tissue diffusely distributed between the fatty components. One lesion showed foci of sebaceous metaplasia. Conclusions. Lipomatous tumors of the salivary glands are rare and most represent intraglandular ordinary lipomas that are otherwise similar to their soft tissue and cutaneous counterparts. While fairly absent in ordinary salivary lipomas, sebaceous differentiation seems to be a common feature of oncocytic lipoadenoma and non-oncocytic sialolipoma. Lipomatous lesions of the salivary glands do not seem to be association with other significant salivary gland pathology or to carry a risk of malignant degeneration. Although lipomatous components in these lesions might derive from intraglandular adipose tissue, the pathogenesis of the oncocytic and sebaceous elements (metaplastic vs. neoplastic) remains unclear.

AG Herz- und Gefäßpathologie DO-100 microRNA-143 is essential in arteriogenesis K . Troidl1, G . Jung1, C . Troidl2, W . Schaper1, T . Schmitz-Rixen3 1 Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, 2 Kerckhoff Heart Centre, Bad Nauheim, 3Goethe University, Frankfurt Aims. Arteriogenesis – the growth of pre-existing collateral arterioles to functional arteries – is triggered by increased fluid shear stress (FSS). MicroRNAs (miRNA) are implicated in post-transcriptional regulation of gene expression. A FSS-induced signature pattern of miRNAs during collateral growth might influence signal transduction of the physical stimulus into a cellular response. We investigated the involvement of miRNAs in arteriogenesis in a rat model of chronically elevated FSS in collateral arteries. Methods. 6 sprague dawley rats were subjected to femoral artery ligature (FAL). A side-to-side anastomosis distal to the ligature was created between the femoral artery and the accompanying vein, which leads to chronically elevated FSS inside the collaterals. Following dissection of collateral tissue 7 d after surgery, miRNA was isolated and an expression profile was generated by microarray analysis. Differential expression was confirmed by qRT-PCR. Cellular localization of selected miRNAs was assessed by in situ hybridisation combined with immunostaining. By local blockage of specific miRNAs in mouse collaterals we analyzed their functional involvement in arteriogenesis. Results. Growing collaterals showed a significant up-regulation of 6 miRNAs when compared to sham operated controls. miR-143, miR-195, and miR-24 were localized in the media of growing collaterals. miR-24 is also expressed in the FSS-stimulated endothelium. Blockage of miR-143 led to severe impairment of arteriogenesis. Conclusions. These data indicate that miRNas are involved in arteriogenesis. miR-143 belongs to a cluster which has been assigned to the phenotypic switch of smooth muscle cells. We identified a functional implication during vascular remodeling. Targeted modulation of this miRNA in vivo suggests new treatment options in improvement of collateral growth.

DO-101 Histopathological analysis of proteases in abdominal aortic aneurysm wall J. Pelisek1, M. Rudelius2, C. Reeps1, F. Lohoefer1, C. Lipp1, H.-H. Eckstein1 1 Klinikum rechts der Isar der TU München, Clinic of Vascular Surgery, München, 2Klinikum rechts der Isar der TU München, Institute of Pathology, München Aims. Abdominal aortic aneurysm (AAA) wall is characterised by degradation of extracellular matrix through plethora of proteases. In contrast to the already well explored matrix metalloproteinases (MMPs), little is known about other groups, such as ADAM family of metalloproteases (a disintegrin and metalloprotease) or cathepsins. The aim of the study was therefore a detailed analysis of expression of selected ADAMs and cathepsins with known proteolytic activity of relevant extracellular components of the vessel wall and their inhibitors in specimens of patients with AAA. Methods. Tissue samples of vessel wall of 35 AAA patients and 10 organ donors were analysed by immunohistochemistry for expression of MMP-1, -2, -3, -7, -8, -9, -12, -13, ADAM 8, 9, 10, 12, 15, 17, and cathepsin B, D, K, L, S in all cells located within AAA. In addition, the known inhibitors of these proteases TIMP-1, -3, and cystatin C were analysed. Results. Endothelial cells (ECs) were positive for MMP-1, -3, -9, neovessels expressed all MMPs tested except for MMP-13. Aortic medial smooth muscle cells (SMCs) expressed MMP-1,-2,-3,-9. Inflammatory infiltrates expressed all MMPs tested except for MMP-2, macrophages expressed all MMPs. ADAMs were expressed in both AAA and control aorta without any significant differences between the groups. SMCs, neovessels, and macrophages were positive for all ADAMs tested. ECs of AAA were positive for cathepsin D and partially for cathepsin B, K, and S. Macrophages, neovessels and SMCs were positive for all cathepsins tested. Inflammatory infiltrates expressed all cathepsins in the following manner: D>B=S>K
DO-102 Histopathologic evaluation of cryopreserved arterial allografts L. Morawietz1, M. Poetzsch2, R. Eisele3, O. Guckelberger3 1 Klinikum Stuttgart – Katharinenhospital, Institute of Pathology, Stuttgart, 2 Charite University Hospital, Department of Anesthesiology and Operative Intensive Care Medicine, Berlin, 3Charite University Hospital, Center for General, Visceral and Transplantation Surgery, Berlin Aims. Cryopreserved arterial allografts from organ donors are used for reconstructive purposes in tumor surgery and in the treatment of aortic aneurysms. Before these grafts are allowed for allograft tissue banks, a histopathologic evaluation needs to be performed. To date no standard exists which features should be evaluated, and it is not known if there are histologic criteria that define the suitability of the grafts for implantation. Therefore, this study aims at establishing criteria for the standardized evaluation of arterial allografts. Methods. 40 consecutive patients received cryopreserved arterial allografts in a total of 45 surgical procedures. The grafts had been examined histopathologically before cryoconservation and after thawing before implantation. Using standard stains (H&E, EvG, Alcian blue, CD34 im-

munohistochemistry), the following criteria were evaluated: Atherosclerosis, medial degeneration, endothelial injury, necrosis, inflammatory infiltrate. Also, in T1 samples attention was paid to alterations that might have been caused by cryoconservation. Furthermore, the postoperative clinical course was recorded for a mean time of 576 days. Results. No atherosclerosis or only initial changes were found in about 60% of samples, while more advanced lesions were observed in as many as 40%. However, the degree of atherosclerosis had no impact on the postoperative outcome. Signs of medial degeneration were significantly more pronounced after cryoconservation than before. The degree of medial generation did not influence the postoperative clinical course, either. A desquamation of the endothelium could be observed both before and after cryoconservation, with no significant differences. The degree of desquamation did not correlate with the postoperative outcome. Damages, which could unequivocally be attributed to the cryoconservation procedure, could not be observed. Conclusions. Apart from degenerative changes in the media, which were significantly more pronounced after cryoconservation, no specific histopathologic damages could be observed after cryoconservation. In this patient collective, no features with significant impact on the postoperative outcome could be established. Somewhat surprisingly, advanced atherosclerotic lesions were observed in 40% of the grafts, but these did not impair the outcome, either. Still, it seems advisable to perform standardized examination of vessel allografts and to evaluate the clinical outcome in larger patient collectives.

DO-103 Expression pattern of the DNA sensor AIM2 in vascular cells and atherosclerotic lesions suggests a role in vascular pathogenesis S. Dihlmann1, M. Hakimi1, A. Peters1, D. Böckler1 1 University of Heidelberg, Department of Vascular Surgery, Heidelberg Aims. Absent in melanoma (AIM2), a cytosolic DNA sensor in innate immunity, is associated with both, infection defence and tumor pathology. In addition to its activation by cytosolic DNA, AIM2 is inducible by type I and II interferons in different cell types. Based on our previous studies on tumor cells, we hypothesized that AIM2 may act as a danger signal that is activated in response to cellular stress likewise in vascular cells. We thus addressed AIM2 expression in different vascular lesions with respect to intra-plaque localization, cellular origin, and expression intensity. Moreover, we aimed to characterize AIM2 expression in cultured human vascular endothelial cells (HAoEC) and smooth muscle cells (HAoSMC) in response to different stimuli. Methods. Carotid lesions from patients who underwent endartectomy and healthy carotid specimen from autopsies were formalin-fixed, paraffin-embedded and analyzed by immunohistochemistry using antibodies specific for AIM2 and different cellular marker proteins (CD68, VSMC, CD31), respectively. Commercially purchased HAoEC and HAoSMC were treated with different cytokines (IFN-gamma, TNF-alpha) to induce an inflammatory response. RNA extracts and cell lysates derived from treated and untreated cell cultures were analyzed for expression of AIM2 and several AIM2-responsive target genes by real time RT-PCR and Western blotting. Results. Endogenous AIM2 expression was detected in endothelial cells of the intima and vasa vasorum of healthy arteria carotis interna (ACI). Within the media, AIM2 was highly expressed in SMC of healthy ACI, whereas it was absent or expressed at a lower level in SMC of atherosclerotic ACI-lesions. In addition, AIM2 was highly expressed in infiltrating inflammatory cells accumulating around calcification and cholesterol plaques in atherosclerotic ACI-lesions. No AIM2 expression was detected in cultured untreated and TNF-alpha treated HAoEC and HAoSMC, whereas it was strongly induced upon treatment of the cells with IFNgamma. In addition, AIM2 expression correlated with induction of several AIM2-responsive target genes and appears to be associated with stress-induced cellular senescence. Der Pathologe Suppl 1 · 2012 

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Abstracts Conclusions. The AIM2 expression and induction patterns suggest a role in vascular pathogenesis. AIM2 might act as a danger signal in vascular EC, SMC and infiltrating inflammatory cells.

DO-104 Carbamylated EPO-fusion protein and recombinant human EPO during porcine kidney I/R injury F. Simon1, M. Gröger2, O. McCook2, E. Calcia2, P. Radermacher2, H. Schelzig1 University of Düsseldorf, 2University of Ulm, Ulm

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Aims. A newly carbamylated erythropoietin-fusion protein (cEPO-FC) and recombinant human EPO (rhEPO) equally protected against spinal cord I/R injury in young, healthy swine [1]. In a recent clinical trial, however, rh-EPO did not affect acute kidney injury in ICU patients [2]. Since patients often present with vascular disease and consecutive organ dysfunction, we compared cEPO-FC and rh-EPO in swine with ubiquitous atherosclerosis [3]. Methods. Pigs randomly received either of cEPO-FC (50 μg/kg), rh-EPO (5000 IU/kg) or vehicle twice over 30 min before and during the first 4 h of reperfusion after 120 min of aortic occlusion using inflatable balloons. We assessed creatinine-clearance, fractional Na+ excretion, blood NGAL, cytokine and NO levels together with tissue histology and immune-histochemistry and -blotting for iNOS, HO-1, HIF-1α , NF-κB, and markers of apoptosis. Results. All pigs presented with reduced glomerular filtration (creatinine-clearance 74±24 vs. 90–140 mL/min normal value) and pre-existing histological organ damage. Neither cEPO-FC nor rh-EPO beneficially influenced the I/R-induced kidney dysfunction, histological organ damage nor tissue inflammation and apoptosis. Conclusions. Pre-existing atherosclerosis-induced kidney dysfunction and tissue damage may reduce the efficacy of cEPO-FC and rh-EPO to prevent I/R-induced kidney damage. References

1. Simon F et al (2011). Intensive Care Med, in press 2. Thim T et al (2010). EuroIntervention 6:261–8 3. Endre Z et al (2010). Kidney Int 77:1020–30

DO-106 Antibody-mediated rejection in cardiac transplant recipients is a seasonal disease K. Wassilew1, N.E. Hiemann1, D. Kemper1, R. Hetzer1 Deutsches Herzzentrum Berlin, Berlin

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Aims. Diagnosis of antibody-mediated rejection (AMR) is still a matter of controversial discussion. We suggested that staining for immunoglobulins might improve the diagnostic spectrum of AMR because of seasonal effects in complement deposition. Methods. We studied prospectively all endomyocardial biopsies harvested since 01/2011 (n=205) for acute cellular rejection, activated endothelial-cells and deposition of C4d, C3d and IgA/M/G in interstitial capillaries by paraffin immunohistochemistry. Histologic and immunohistochemical parameters of AMR were classified according to the ISHLT and studied for seasonal effects in an ordinal (Jan–Mar vs. Apr– Jun vs. Jul–Sept vs. Oct–Dec) and nominal model (Oct–Mar vs. Apr– Sept). Results. Overall, 16% of biopsies showed signs of acute cellular rejection of any grade and 46% of samples showed evidence of endothelial-cell swelling. In the ordinal model (Jan–Mar vs. Apr–Jun vs. Jul–Sep vs. Oct–Dec), seasonal effects were found for endothelial cell swelling (83% vs. 14% vs. 49% vs. 40%; p<0.001), IgA deposition (62% vs. 71% vs. 86% vs. 92%; p=0.046), C4d deposition (36% vs. 9% vs. 20% vs. 15%; p=0.003) and C4d deposition in combination with endothelial cell swelling (35% vs. 5% vs. 9% vs. 4%; p<0.001). In the nominal model (Oct–Mar vs. Apr–Sep) we

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found similar results showing seasonal effects for endothelial cell swelling (70% vs. 30%; p<0.001), C4d deposition (29% vs. 14%; p=0.012) and C4d deposition in combination with endothelial cell swelling (25% vs. 7%; p<0.001). No other combinations of histopathological features, immunohistochemical findings or acute cellular rejection showed seasonal behavior. AMR according to the ISHLT was not associated with impaired graft function in echocardiography. Conclusions. Complement deposition seems to be more pronounced in autumn and winter. The finding of capillary IgA deposition might add important information about coincident alloimmune responses derived from mucosal surfaces and might be considered to complement current diagnostic parameters.

DO-107 Systematic histological analysis of thrombotic microangiopathy in a porcine xeno-kidney-transplant-model C. Bockmeyer1, W. Ramackers2, J. Wittig1, P.A. Agustian1, M.E. Dämmrich1, H. Kreipe1, V. Broecker1, M. Winkler2, J.U. Becker1 1 Medical School Hannover, Institute for Pathology, Hannover, 2Medical School Hannover, Clinic for General, Abdominal and Transplant Surgery, Hannover Aims. Despite different pharmacologic and genetic interventions thrombotic microangiopathy is still a big challenge in porcine xenotransplantion. A detailed molecular analysis of histological patterns in xeno-kidney-transplant-models is necessary for a targeted anticoagulant therapy and to better understand the pathophysiology of human transplant associated thrombotic microangiopathy. Methods. Porcine kidneys were perfused with human whole blood (n=5). Xenotransplants were compared to different intervention groups [application of antithrombin and activated Protein C (aPC), each n=5] and controls (perfusion with porcine whole blood, n=4). Microthrombi positive for fibrin (Fib+MT) and activated platelets (CD61+MT) were defined as lumen occlusion of more than 50% with fibrin and activated platelets. 100 glomeruli und preglomerular vessels per sample were analyzed. Results. Preglomerular CD61+MT were diminished in xenotransplants after application of aPC and antithrombin. Glomerular CD61+MT were increased in xenotransplants with and without intervention compared to controls. Only aPC, but not antithrombin was able to diminish significantly glomerular CD61+MT in xenotransplants. There were no differences in glomerular and preglomerular Fib+MT between both intervention groups. In total CD61+MT were more frequently observed than Fib+MT in xenotransplants. Conclusions. Microthrombi in xenotransplants seem to be composed predominantly of platelets. In dependence on different antithrombotic interventions the number of microthrombi is diminished and the ratio of Fib+MT compared to CD61+MT is altered. Underlying mechanisms and the role of the vessel wall will be further investigated by gene expression analysis of pro- and antithrombotic factors. These data will be correlated with histological and laboratory data. These data will hopefully guide targeted antithrombotic interventions.

DO-108 Identification of potential criteria for a successful Rituximab salvage therapy in kidney allograft rejection M. Dämmrich1, C. Blume2, C. Bockmeyer1, S. Immenschuh3, A. Schwarz2, D. Agustian1, V. Broecker1, H. Kreipe1, J. Becker1 1 Hannover Medical School, Institute for Pathology, Hannover, 2 Hannover Medical School, Centre for Internal Medicine, Hannover, 3 Hannover Medical School, Institute for Transfusion Medicine, Hannover Aims. Rituximab (anti-CD-20 antibody) is often used in kidney transplantation to treat rejection refractory to standard treatment. Rituximab therapy carries a risk for serious infectious complications and is not effective in all cases. Therefore clinica, serological or histopathological criteria to predict Rituximab response are most desirable. As a first step for the identification of useful criteria we did a retrospective exploratory analysis to identify criteria that were different between Rituximab responders and non-responders. Methods. 18 renal transplant recipients who received Rituximab (375 g/ m2 body surface, 1–2 courses after steroid bolus therapy, 15× combined with up to 5 courses of plasmapheresis) for standard-therapy resistant rejection were included in the study with their last biopsy before therapy. 10 were identified by terminal loss of transplant function as nonresponders, 8 as responders. Clinical, serological and histopathological parameters were compared between both cohorts by Wilcoxon- or χ2 tests. P-values were regarded as descriptive in this retrospective analysis. Results. At time of biopsy more of the Rituximab non-responders had panel-reactive antibodies (>85%), and their serum creatinine before therapy was higher. Among the histopathological criteria tubulitis was less severe in responders. No significant differences were found for any of the other clinical, serological or histopathological criteria. Conclusions. In this retrospective exploratory study we identified lower serum creatinine before therapy, the presence of panel- reactive antibodies in more than 85% at time of biopsy, and a less severe Banff t-score as possible criteria to predict responsiveness to Rituximab therapy. These criteria need validation in future prospective randomized studies.

DO-109 Splenectomy and postconditioning with the sphingosine-1-phosphate agonist FTY720 protect the myocardium against ischemia reperfusion injury D. Goltz1, S. Huss2, E. Ramadori1, L. Diehl3, R. Büttner2, R. Meyer4 1 University of Bonn, Dept. of Pathology, Bonn, 2University of Cologne, Dept.  of Pathology, Köln, 3University of Bonn, Institute of Molecular Medicine, Bonn, 4University of Bonn, Physiology II, Bonn Aims. The pathogenesis of myocardial ischemia reperfusion injury (MI/R) involves the inflammatory response of the innate immune system. A modulation of this response could be a potential future target in the management of the acute coronary syndrome. Recently, the spleen has been proved an important origin of a Ly6-Cpos monocyte subset that readily invades the injured myocardium upon ischemic damage. We operated a murine myocardial ischemia reperfusion model in splenectomised animals to reduce the invasion of phagocytic active immune cells. In a second pharmacologic approach we applied the sphinosine-1phosphate analogue FTY720 with reperfusion to interfere with the maturation of monocytic derived macrophages. The aim of this study was to evaluate the short and long term outcome of MI/R after modulation the immune response. Methods. In a murine closed-chest ischemia-reperfusion model, myocardial infarct volume was assessed by TTC staining after 24  h of reperfusion and by planimetric quantification of fibrosis on the Masson stained specimen after 21 days of reperfusion in control animals, splenectomised animals and FTY720 treated mice. Within the same interval, cardiac function was evaluated by catheterisation using a Millar cathe-

ter. The immune response was characterised by FACS analysis in whole heart specimens after 24 h. Results. After 24 h of reperfusion, splenectomised animals and FTY720 treated animals revealed a significant reduction of infarct volume. The invasion of monocytes, especially of Ly6-Cpos monocytes was significantly reduced in splenectomised animals compared to the control group. Moreover, the ratio of inflammatory macrophages to resident macrophages was significantly smaller in the splenectomised group. FTY720 treated animals showed an almost exclusive invasion by monocytes within the remote myocardium. Functional data show a significantly improved systolic cardiac function in both, the splenectomised and the FTY720 treated groups compared to the control animal after 21  days of reperfusion. Quantification of the area of fibrosis, however, revealed a trend towards reduced myocardial scarring in both target groups, but the decline failed to reach a level of significance. Conclusions. Both, splenectomy and postconditioning with FTY720 are cardioprotective within 3 weeks after MI/R. In splenectomised animals, this effect is due to a reduction of early invading monocytes. The mechanism of FTY720 efficiency still warrants further investigation.

DO-110 Nestin expression in fetal and adult lungs vessels as well as vascular tumors S. Gerlach1, G. Kristiansen1, A.M. Müller2 University Bonn Medical Center, Institute of Pathology, Bonn, 2 University Bonn, Department of Pediatric Pathology, Bonn

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Aims. The neural stem/progenitor cell marker Nestin is a class VI intermediate filament protein. It is not only expressed by undifferentiated central nervous system (CNS) cells during development and adult CNS cells but various tumour cells as well as in injured and regenerating tissues, indicating nestin as a marker for activated, migrating, proliferating cells. Recently it has been described in proliferating endothelial cells (EC). We were interested in any differences Nestin expression in fetal and adult vessels as well as vascular tumors. Methods. Endothelial Nestin and D2-40 expression was studied by immunolocalisation in chorionic tissue from early abortions (n=5), fetal (n=21) and adult (n=3) lungs, infantile hemangiomas (n=5) and angiosarcomas (n=5). Results. Although Nestin was expressed by EC of all blood vessels but not lymphatic vessels there were differences concerning expression intensity. In the first trimester, EC in chorionic and villous tissue as well as EC of pulmonary veins, arteries and capillaries showed the same strong Nestin-positivity. Starting in the second half of the second trimester Nestin expression in venous EC began to fade, while arterial EC still showed a strong staining. In lungs of neonates and adults the Nestin expression was even weaker, although in arteries it was still stronger expressed than in veins. In angiosarcomas Nestin was strongly expressed by the tumour cells. EC of all infantile hemangiomas were clearly but in comparison to angiosarcomas weaker stained. Conclusions. The intermediate filament Nestin can be regarded as endothelial marker expressed by vascular EC already during the first weeks of life. As it is not expressed by lymphatic EC it can help discriminating blood vessel endothelium from lymphatic endothelium. In accordance with other endothelial markers like Angiotensin I converting enzyme it is weaker expressed in adult pulmonary veins than adult pulmonary arteries. The strong Nestin expression in fetal vessels – when compared to EC of mature vessels or hemangiomas – corresponds to a strong Nestinpositivity in malignant endothelial tumor cells. At present we analyse the role of Nestin in colorectal cancer resp. its tumour vessels concerning expression patterns in different stages of cancer.

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Abstracts AG Molekularpathologie DO-111 Improved method of detecting the ERG gene rearrangement in prostate cancer using combined dual-color chromogenic and silver in-situ hybridization M . Braun1, J . Stomper1, D . Böhm1, W . Vogel1, V . Scheble2, N . Wernert1, Z . Shaikhibrahim1, F . Fend3, G . Kristiansen1, S . Perner1 1 University Hospital Bonn, Institute of Pathology, Bonn, 2University Hospital Tübingen, Division of Hematology and Oncology, Tübingen, 3University Hospital Tübingen, Institute of Pathology, Tübingen Aims. The recently detected TMPRSS2-ERG fusion revealed as a recurrent and prevalent prostate cancer (PCa) specific event, which potentially qualifies it for clinical utilizations. To detect this alteration, fluorescence in-situ hybridization (FISH) is the method of choice. However, FISH harbors some disadvantages for widespread adoption in clinical practice. Subsequently, the chromogenic in-situ hybridization (CISH), that uses organic chromogens, and the enzymatic metallography silver in-situ hybridization (SISH) emerged as promising bright-field alternatives. Compared to CISH, SISH signals are very distinct and superior with regard to signal clarity and resolution, but rule out a multi-color protocol. However, the precise localization of genomic targets using a dual-color approach is indispensable for gene break-apart and fusion assays. In order to bridge this gap, we aimed to develop a dual-colour combined CISH and SISH (CS-ISH) gene break-apart assay on the example of the ERG gene commonly rearranged in PCa. Methods. On the basis of the ERG break-apart FISH assay, we established a dual-colour ERG break-apart CS-ISH assay and compared these results with those obtained by FISH. We assessed 178 PCa and 10 benign specimens for their ERG rearrangement status applying a dual-colour FISH and CS-ISH ERG break-apart assay on consecutive sections. Results. We observed a highly significant concordance (97.7%) between FISH-based and CS-ISH-based results (Pearson’s correlation coefficient 0.955, p<0.001). Conclusions. Our findings demonstrate that the ERG rearrangement status can reliably be assessed by CS-ISH. Further, we confirm that the CS-ISH technique combines the accuracy and precision of FISH with the ease of bright field microscopy. We developed a tool which allows a much broader spectrum of applicants to study the biological role and clinical utilization of ERG rearrangements in PCa. Moreover, our study is the first proof-of-principle for bright-field CS-ISH gene fusion or break-apart assays.

DO-112 Evaluation of different molecular methods for BRAF mutation analysis M . Kleine1, H . Künstlinger1, J . Fassunke1, H .-U . Schildhaus1, R . Büttner1, S . Merkelbach-Bruse1 1 University Hospital Cologne, Institute of Pathology, Köln Aims. Metastatic melanoma is an aggressive disease with only few therapeutic options. The approval of Zelboraf (Vemurafenib) in the US by the FDA in 2011 improved the therapy of unresectable or metastatic melanoma. But only mutation-positive patients show complete or partial response. These patients carry a substitution of valine to glutamic acid at codon 600 (p.V600E) and rarely a substitution of valine to leucine (p.V600K) of the serine-threonine protein kinase BRAF. Therefore, the precise identification of these somatic mutations is essential. The aim of this study was to evaluate the sensitivity and feasibility of the detection of mutations in the BRAF gene by four different molecular methods. Methods. A collective of samples harbouring p.V600E mutations as well as rare mutations like p.V600K or p.V600R in exon 15 of the BRAF gene

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was set up and compared to wild type samples. DNA for mutational analysis was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automatic extraction using the BioRobot M48 (Qiagen). BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, Pyrosequencing (therascreen® BRAF Pyro Kit) and by the cobas® BRAF V600 test (Roche). All mutations were independently reassessed by Sanger sequencing. Sequencing analysis was carried out in duplicates. Results. Using high resolution melting (HRM) analysis, wild-type samples could be distinguished from mutated samples. Furthermore, based on differential melting, the specific mutations could be further distinguished and characterised. The spectrum of mutations was likewise detected using Pyrosequencing. The cobas® BRAF V600 test is specific for the p.V600E mutation. All mutations found by the three different methods could be confirmed by Sanger sequencing. Conclusions. A comparison of 4 different methods showed no significant difference in sensitivity. The high resolution melting (HRM) analysis was not more sensitive than sequencing by Sanger. But interestingly, this method can be used not only as a pre-screening method, but also to distinguish different mutations of the BRAF gene by showing distinct melting curves. Concerning the analysis of the results, pyrosequencing requires many experiences for a correct interpretation of the results. The cobas® BRAF V600 test has limited utilisation as it can only detect p.V600E, p.V600K and p.V600D mutations but is unable to distinguish between p.V600X mutations.

DO-113 Perspectives of the 454 parallel sequencing approach in the EGFR mutation analysis of routinely processed lung cancer biopsies U . Koitzsch1, S . Merkelbach-Bruse1, N . Helmhold1, J . Fassunke1, C . Vollbrecht1, L . Heukamp1, R . Büttner1, M . Odenthal1 1 University Hospital Cologne, Institute for Pathology, Köln Aims. Prognosis and therapeutic sensitivity of lung cancer is associated to somatic mutations in the kinase domain of the EGFR gene (exon 18– 21) affecting cell growth, proliferation and survival. Thus, EGFR gene mutations are important targets of molecular diagnostic approaches. In the present study we evaluated the feasibility of 454 next-generation sequencing for mutation analysis in formalin-fixed, paraffin-embedded (FFPE) biopsies and lung cancer cell preparations, routinely processed in pathology. Methods. Quantities of DNA, extracted from 60 FFPE biopsies or cell preparations by means of the M48 platform (Qiagen, Hilden), were determined. A multiplexed amplicon library of the EGFR exon 18–21 was produced and taken for emulsion PCR and parallel 454-sequencing using the GS Junior equipment. All steps were performed in agreement and according to the recommendations of Roche Diagnostics and Roche Pharma. In order to evaluate sensitivity of mutation detection, EGFR exon 19 and 21, carrying a deletion or a point mutation, respectively, were cloned into TOPO plasmids. 102 to 105 copies of formalin treated plasmids were mixed to 100 ng human DNA representing 1.5×105 wild type diploid genomes, and then applied to the parallel sequencing assay. Results. Multiplexing of 12 tumor samples for EGFR exon 18, 19, 20, 21 parallel sequencing resulted in 500 to 1000 reads of each exon. Mutations were detected in a rate of 10% to 50% depending on histology and cell compostion of the lung tumors, when 50–100 ng DNA were applied. In order to determine the cut off of the occurring variations, normal, tumor-free FFPE biopsies were analysed. Variant frequency in all positions was less than 1%. Next, we investigated the sensitivity of the mutation detection using the dilution series of cloned EGFR exons 19 or 21, carrying a codon 746–751 deletion (exon 19) or a point mutation (exon 21), respectively. Sequencing revealed that both mutations were clearly defined when constituting more than 10% of the applied DNA, but when only 100 molecules were mixed in 1.5×105 human DNA copies (<1%), mutations could not be differentiated from the background variance.

Conclusions. In conclusion, 454 parallel sequencing is a very useful and economic approach for molecular pathology due to sample multiplexing and simultaneous target analyses. Both, FFPE extracted DNA and DNA from cell preparations may be applied to the approach. If the mutation rate is higher than 10% in the FFPE sample, the mutation is easily detected.

DO-114 IMDA: a methodical approach enhancing molecular diagnostic of microcarcinomas and small biopsies F. Mairinger1, K. Worm1, W. Grüning2, T. Mairinger3, K.W. Schmid1 1 University Hospital Essen, Department of Pathology und Neuropathology, Essen, 2Helios Klinikum Emil von Behring, Department of Pneumology, Berlin, 3Helios Klinikum Emil von Behring, Department of Pathology, Berlin Aims. The isothermal multiple displacement amplification (IMDA) would be a powerful tool in molecular routine diagnostics for preamplification of extraordinary small tumor samples (biopsies containing small amount of tumor, microcarcinomas) but is not banked in pathological laboratories. We designed a study to check the feasibility and convenience of these methods for routine diagnostics on LCM microdissected FFPE samples. Methods. For validation of the IMDA assay, different benign FFPE tissue samples were microdissected using LCM technology (areas ca. 50×50 µm up to 150×100 µm) and afterwards preamplified by an commercial IMDA-kit (Qiagen REPLI-g FFPE assay). Chromosomal representation was tested using qPCR of genes spanning regions on different chromosomes. Afterwards, patient samples from the Helios Klinikum Emil von Behring with a too small amount of material for conventional molecular analysis were pre-amplified by IMDA and further processed with the “normal” routine samples for EGFR analysis. Results. With an amplification time duration of 3h and a starting material of 50×50 µm extension a yield of total 250 µg DNA (concentration of 5 µg/µl) could be generated. Preliminary results show an acceptable relative chromosomal representation, the presence of diagnosis relevant genes in clinical samples could be proven. Mutational analysis of clinical samples was accomplishable and shows concordance with earlier diagnostically findings. Conclusions. We could proof the diagnostic feasibility and convenience of IMDA for routine diagnostics. Also small amount samples, until now not analyzable with molecular methods, will be sufficient for all-embracing molecular routine diagnostics.

DO-115 miRNA 26b stabilizes the pro-apoptotic DAP Kinase by inhibiting its E3 ubiquitin ligase DIP1 S. Knaup1, S. Wach2, A. Agaimy1, J. Schulze-Luehrmann1, M. Hugele1, S. Chakilam1, R. Atreya3, R. Wirtz4, T.T. Rau1, R. Schneider-Stock1 1 University of Erlangen-Nuremberg, Institute of Pathology, Erlangen, 2 University of Erlangen-Nuremberg, Institute of Urology, Erlangen, 3 University of Erlangen-Nuremberg, Department of Medicine I, Erlangen, 4 STRATIFYER Molecular Pathology GmbH, Cologne Aims. Tumor necrosis factor α (TNF) is a pro-inflammatory cytokine involved in the inflammatory reaction of the intestinal mucosa, but also mediates tumor eliminating effect. We have shown recently that treatment of HCT116 colorectal tumor cells with TNF led to a higher expression of death-associated protein kinase (DAPK) and induced caspase-dependent apoptosis. It is also known, that DAPK can be found in a complex together with DAPK-inter-acting protein (DIP1) that antagonizes the pro-apoptotic function of DAPK by ubiquitination. Upon TNF treatment a decrease of the DIP1 protein level could be detected, while there were no matching changes in the DIP1 mRNA levels. This

raised the question of a potential involvement of a miRNA binding to the 3‘UTR of DIP1 regulating its degradation or translational inhibition. A miRNA microarray analysis of TNF-treated HCT116 cells revealed a significant up-regulation of miRNA 26b. Methods. The potential of miRNA 26b to target DIP1 and thereby influencing apoptosis through regulation of DAPK had to be verified. This was achieved by a luciferase reporter assay and overexpression of miRNA 26b as well as knock-down of the miRNA and DIP1, followed by westernblot and Real-Time PCR analysis. To assess the in vitro findings in vivo, in situ hybridization was performed on tissue microarrays of normal, inflamed (ulcerative colitis) and inflammation-associated colorectal tumor samples to check the localization and abundance of miRNA 26b. Results. In the luciferase reporter assay, miRNA 26b binds to DIP1, confirming it as a miRNA 26b target. This was further verified by overexpression, as well as a knock-down of miRNA 26b. More miRNA led to a decrease of DIP1 protein levels and in return to a stabilization and increase of DAPK protein levels. Vice versa, reduction of miRNA 26b resulted in higher DIP1 protein levels and less DAPK protein. Knockdown of DIP1 by siRNA showed an increase of apoptosis via caspase 3 cleavage. In human tissues of ulcerative colitis patients, in situ hybridization verified a higher level of miRNA 26b in the inflamed colon crypts, consistent with the grade of inflammation. Conclusions. miRNA 26b promotes apoptosis in human colon cancer cells by targeting the E3 ubiquitin ligase DIP1 and thereby stabilizing the pro-apoptotic DAPK. miRNA 26b is strongly expressed in the inflamed tissue of ulcerative colitis patients, suggesting a possible role in the regulation of the inflammatory process.

DO-116 MRNA and microRNA stability in surgical tissue: an issue for biobanking and biomarker identification C. Schuster1, W.E. Thasler2, K.-F. Becker3, T. Kirchner1, F. Hlubek1 1 Ludwig-Maximilians-University München, Institute of Pathology, München, 2Ludwig-Maximilians-University München, Department of Surgery, Grosshadern Hospital, München, 3Technical University Munich, Institute of Pathology Aims. Human frozen tissues are one of the best sources for molecular analyses such as microarrays, qPCR or Next-Generation-Sequencing. In addition, frozen tissues are particularly valuable for biomarker identification. Several biobanks comprising non-fixed frozen tissues have been established alongside with corresponding clinical data repositories to facilitate biomarker studies relevant for clinical diagnostics. Apart from proteomic approaches, mRNA- and microRNA-expression profiles have shown to be highly valuable for biomarker studies. Since RNA is generally a fragile molecule, RNA integrity in tissue specimens has tremendous impact on gene expression analyses, requiring a rigorous quality assessment of biobank tissue samples. Methods. To address this issue, we established a tissue quality test system based on RNA integrity and differential gene expression. We used normal and cancerous surgical tissue that was stored under various conditions and for different time periods of ischemia prior to being snap frozen. Results. The RNA was isolated and the quality was assessed in four steps: First, the RNA was quantitated by spectrophotometry (NanoDrop) and total RNA quality was determined by on-chip electrophoresis (Experion). Second, the degree of RNA degradation and the maximum length of RNA molecules available for downstream applications were determined by amplicon length analysis of housekeeping genes using PCR amplification. Third, the RNA expression level of selected genes were determined and correlated to the time and condition of ischemia. The genes analysed comprised highly regulated genes, signaling pathway genes and genes induced by hypoxia or apoptosis. Fourth, the expression of selected miRNAs representing different expression levels was determined by quantitative PCR.

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Abstracts Conclusions. Taken together we analysed mRNA and miRNA quality in correlation to the time and condition of warm and cold ischemia of the same tissue samples. The results enabled us to establish a RNA-based quality assessment procedure of tissue specimen for frozen tissue biobanks. This study may facilitate and optimise the logistics of biobanking processes.

DO-117 Quantitative genome-wide methylation profiling of human breast cancer reveals subtype-specific patterns of epigenetic instability U. Lehmann1, J. Rößler1, O. Ammerpohl2, J. Gutwein2, R. Geffers3, W. Hofmann4, F. Länger1, H. Kreipe1 1 Medical School Hannover, Institute of Pathology, Hannover, 2University Hospital Schleswig-Holstein, Institute for Human Genetics, Kiel, 3Helmholtz Centre for Infection Research, Genom analysis/Gen Regulation and Differentation, Braunschweig, 4Medical School Hannover, Institute of Cell and Molecular Pathology Aims. This project addresses the question whether a subgroup of human breast cancer is characterized by widespread epigenetic instability, in contrast to genetic instability which is typical for e.g., familial breast cancer. Methods. High molecular weight DNA was isolated from 28 histologically examined fresh-frozen human breast cancer specimens and 4 normal mammary epithelial fractions using standard procedures. DNA methylation patterns were analyzed using the newly developed 450k methylation array from Illumina as well as methyl binding domain (MBD)-based affinity enrichment and subsequent hybridization to a CpG-island and promotor array from Agilent. For data analysis software provided by the manufacturers of the arrays were employed. These analyses were complemented and extended by employing commercially as well as freely available software packages (Omics Explorer from Qlucore and the R package IMA). The results for individual loci were validated using conventional pyrosequencing and independent breast cancer specimens. Results. In comparison to normal mammary epithelial cell fractions all breast cancer specimens display several thousand statistically significant aberrations in DNA methylation (number of CpG sites for p<0.001: 3,000–11,000). However, the number of affected loci and the extent of hyper- and hypomethylation are not uniformly distributed. Also, individual loci show characteristic differences between histological subtypes. Early onset familial breast cancer displayed the lowest number of aberrantly methylated loci. The two array formats used in this project show different strengths and weaknesses and represent rather complementary than competitive approaches for a genome-wide study of DNA methylation in human specimens. Conclusions. A subgroup of human breast cancer is characterized by high frequency of DNA methylation aberrations indicating the presence of widespread epigenetic instability, which is not uniformly distributed across human breast cancer subtypes. The development of familial breast cancer seems to be dominated by the genetic lesion.

DO-118 Inhibition of mTOR and insulin receptor in hormone receptor positive and Tamoxifen resistant breast cancer cells F. Mietzsch1, H.P. Sinn1, P. Schirmacher1, S. Aulmann1 University Hospital Heidelberg, Institute of Pathology, Heidelberg

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Aims. The mutation of PI3KCA is frequently observed in hormone-receptor positive breast carcinomas and sensitizes the tumour cells to therapeutic mTOR inhibition. Our aim was to investigate the effects of mTOR inhibition as well as possible interactions with insulin receptor signalling in Tamoxifen-resistant breast cancer cells.

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Methods. MCF7 breast cancer cells, harbouring an activating PIK3Ca mutation (Exon  9 1633G>A), and tamoxifen-resistant MCF7 cells (TMCF7) were treated with the allosteric mTOR complex  1 (mTORC1) inhibitor Everolimus and the active-site mTORC1/mTORC2 kinase inhibitor PP242. In this setting, the effects of insulin receptor signalling on cell growth, motility and viability were investigated by stimulation with insulin or IGF1 and in the presence of siRNA inhibition of the insulin receptor (IR) and insulin like growth factor 1 receptor (IGF-1R). Results. T-MCF7 showed elevated level of IR/IGFR expression as well as an activated (phosphorylated) ERK1/2 in contrast to the untreated MCF7. The addition of insulin resulted in an increased signal transduction via AKT and ERK1/2. Simultaneous inhibition of mTORC1/2 through PP242 abolished AKT-phosphorylation and led to a complete cell cycle arrest in G0/G1 as well as a substantial decrease of cell viability in MCF7 and T-MCF7. However, mTORC1-inhibition alone using Everolimus resulted only in a partial G0/G1-arrest which could be reversed by addition of insulin. siRNA inhibition of IR demonstrated an effective reduction of MAPK-signalling in both MCF7 and T-MCF7 while siRNAs against IR or IGF1R resulted in an additional decrease of cell viability. Conclusions. Inhibition of mTOR-signalling reduced cell viability and proliferation in PIK3CA-mutated breast cancer cells independent of an acquired Tamoxifen resistance. However, our data indicate that IR and IGF1R-conferred cell growth may reduce the effects of isolated mTOR inhibition in Tamoxifen-resistant breast cancer cells and that additional targeting of the insulin receptor pathway may prove useful in this setting.

DO-120 Strong negative feedback from Erk to Raf confers robustness to MAPK signaling R. Fritsche1, F. Witzel2, A. Sieber1, R. Herr3, N. Schmidt1, S. Braun3, T. Brummer3, C. Sers1, N. Blüthgen1 1 Charité University Hospital Berlin, Pathology, Berlin, 2Charité University Hospital Berlin, Pathology, 3ZBSA, Albert Ludwigs-University, Freiburg Aims. Protein levels within signal transduction pathways vary strongly from cell to cell. For example, it has been reported that concentrations of the last kinase within the MAPK signalling module, Erk, varies about 4-fold between clonal cells under the same conditions. In the present study, we analysed how signalling pathways can still process information quantitatively despite strong heterogeneity in protein levels. Methods. Mathematical analysis of isolated de- and phosphorylation cycles predicts that phosphorylation of a signalling molecule is proportional to the protein concentration. We combined mathematical modelling and experimental analysis and systematically perturbed the protein levels of Erk by siRNA. Our experiments also included the analysis of Erk phosphorylation under Mek overexpression, measuring transcript levels of negative feedback regulators, and the application of generic inhibitors. Results. We found that the steady-state phosphorylation of Erk is very robust against perturbations of Erk protein level, suggesting that there are mechanisms that provide robustness to the pathway against protein fluctuations. Using mathematical modelling, we identified three potential mechanisms that may provide robustness: 1. kinetic effects, 2. transcriptional negative feedbacks, 3. negative feedbacks on the post-translational level. By experimental analysis of the systems we could exclude kinetic effects and transcriptional negative feedback as mechanisms of robustness. By analysing a panel of cell lines we found that cells are robust as long as the signal passes through Raf-1. In contrast, cells where the pathway is activated by a mutation in B-Raf loose robustness. Therefore, once the feedback is broken, the system loses robustness and can be readily modulated by low concentrations of targeted inhibitors. In contrast, if the feedback is intact, inhibition of the pathway is inefficient. Conclusions. This finding explains why Mek inhibition has shown little success in the past in cancer treatment. However, it also shows that a

subgroup of patients with B-Raf mutation will likely benefit, and that due to the robustness of the healthy cells that have no B-Raf mutation side effects might be minimal. We believe that analysing robustness of other signalling pathways in a similar way will be the key to devise efficient targeted interventions for these, and will unveil which mutations in the pathway will break robustness and thereby open the door for efficient intervention.

DO-121 The nuclear localization and transcriptional activation of β-catenin are independent of each other S. Ormanns1, T. Kirchner1, A. Jung1 1 Ludwig-Maximilians-University, Institute of Pathology, München Aims. Mutations in components of the Wnt signaling pathway are the drivers of carcinogenesis in the majority of colorectal cancers. They result in the accumulation of the transcription factor β-catenin which exerts its function by the induction of the hallmarks of cancer like epithelio-mesenchymal transition, stemness, chemoresistance, proliferation, invasion or apoptosis besides others. The transcriptional activity of β-catenin depends on its nuclear localization and posttranslational modifications. As it is unknown how both processes are regulated, we asked if the nuclear accumulation of β-catenin and its activation induced via the PI3K-AKT signaling pathway were independent of each other. Methods. To discriminate between the nuclear localization and additional activation steps of β-catenin an experimental cell culture system was designed that allowed the forced nuclear translocation of β-catenin independent of additional activation steps. The subcellular localization of β-catenin was assessed by immunofluorescence. The transcriptional activity of β-catenin was determined by TOP-flash luciferase reporter gene assays. The activity of PI3K-AKT was interfered by the specific inhibitor LY294.002. Results. Inhibiting PI3K/AKT lead to a significant dose-dependent reduction of endogenous β-catenin activity in the cell lines SW480 and RWP-1 harbouring inactivating mutations in the APC gene. Conversely, stimulating the Wnt-signaling pathway or adding degradation resistant β-catenin both resulted in the activation of β-catenin in the cell lines 293T, CHO or HeLa. Here, the nuclear translocation of β-catenin also resulted in an activation of its transcriptional activity which could be blocked by inhibiting PI3K. In contrast the nuclear translocation of β-catenin did not result in transcriptional activity in A431 cells. Conclusions. We provide experimental evidence that the nuclear transport and the transcriptional transactivation of β-catenin are independent processes. Thus, β-catenin signaling depends at least on active PI3K/ AKT signaling. Taken together, the transcriptional activity of β-catenin is regulated at least by two signals which might open the opportunity for clinically interfering with the hallmark of CRC, the activation of the β-catenin pathway.

DO-122 Activation of the EGFR-MAPK signaling pathway is dependent on FAM125 proteins S. Müller1, G. Baretton2, G. Fitze1, M. Haase3 1 TU Dresden, Pediatric Surgery, Dresden, 2TU Dresden, Pathology, Dresden, 3 TU Dresden, Pediatric Surgery and Pathology, Dresden Aims. Ionizing radiation leads to complex changes in tissues such as changes in cell survival, cell differentiation and loss of function. In order to find proteins that are overexpressed in irradiated tissue, we constructed a differential cDNA library. Among other proteins, we found FAM125A (family 125A), a protein component of transport vesicles that has been reported to play a role in the internalization of epidermal growth factor receptor (EGFR). The aim of the study was to get further insights into the function of FAM125 proteins.

Methods. A differential cDNA library was constructed from cDNA obtained from radiated lung tissue of the rat. mRNA expression analysis was done by quantitative RT-PCR. Protein expression was quantified by western blot analysis. Tissue distribution was analyzed by immunohistochemistry on tissue microarrays (TMAs). Down-regulation of mRNA/proteins was achieved by stable transfection of sh-RNA vectors into HELA cells. Protein extracts from these cells were prepared from the membrane, cytoplasmic and nuclear fractions. Results. FAM125A protein is expressed in most cell types. A very high expression is seen in tissues with very active membrane transport processes such as renal tubule cells and glandular cells. FAM125A mRNA and protein are overexpressed in irradiated tissue. Down-regulation of FAM125A and B leads to a decrease of total EGFR and phosphorylated EGFR (Y1045 and Y1068) in the membrane fraction. In addition, it leads to an accumulation of phosphorylated Akt (S473) and c-Src (T416) in the membrane fraction whereas phosphorylation of p42/44 MAPK (T202-Y204) is decreased. Conclusions. FAM125 proteins seem to play a role in transport processes of molecules including signaling proteins. Down-regulation of EGFRactivity correlates with decreased activity of the p42/44 MAPK pathway whereas Akt and c-Src activity are not affected. This suggests that the EGFR-MAPK pathway is dependent on FAM proteins whereas the Akt and c-Src pathways act independently. Further research should clarify the association of various signaling molecules to transport vesicles and should provide an insight into signaling processes in radiation-damaged cells.

DO-123 DUSP4 expression increases cell proliferation in colorectal cancer (CRC) cells and is associated with microsatellite instability in CRC B. Gröschl1, M. Bettstetter1, W. Dietmaier1 University of Regensburg, Institute of Pathology, Regensburg

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Aims. DUSP4, a member of the mitogen-activated protein kinase phosphatase (MKP) family and a potential tumor suppressor, negatively regulates the MAPKs (Mitogen-activated protein kinases) ERK, p38 and JNK which play a crucial role in cancer development and progression. Our aim was to investigate DUSP4 expression in high frequent microsatellite unstable (MSI-H) and microsatellite stable (MSS) colorectal cancers (CRC) as well as its influence on potential MAPK downstream targets and its effect on the proliferation in CRC cells. Methods. We studied DUSP4 mRNA levels in 19 MSI-H and 19 MSS CRC compared to matched normal tissue as well as in CRC cell lines by RTqPCR. Promotor methylation of the DUSP4 gene was analyzed using Methy-QESD (Quantification of Endonuclease-Resistent DNA) and coding regions were assessed for mutations through Sanger sequencing. We overexpressed DUSP4 in CRC cell lines and analyzed expression of potential downstream target genes as well as cell growth by Real-Time Cell Analysis (RTCA). Results. DUSP4 mRNA was elevated in all 19  MSI-H tumors and in 14 MSS tumors. Median expression levels in MSI-H tumors were significantly higher than in MSS-tumors (p<0.001). Consistently, MSI-H CRC cell lines showed higher DUSP4 mRNA levels than MSS cell lines. Neither DUSP4 promoter methylation nor genomic mutations in the DUSP4 gene could be detected. DUSP4 overexpression in CRC cell lines caused upregulated expression of MAPK targets CDC25A, CCND1, EGR1, MYC and CDKN1A in HCT116 as well as downregulation of MSH2 in SW480. Furthermore DUSP4 overexpression led to increased proliferation in CRC cells. Conclusions. Our findings suggest that DUSP4 acts as an important regulator of cell growth within the MAPK pathway and causes enhanced cell growth in MSI-H CRC.

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Abstracts DO-124 Gastrointestinal stromal tumors of the stomach rarely harbour KIT exon 9 mutations and are mostly associated with a low or no malignant potential H . Löser1, S . Huss1, W . Jeske1, M . Fielenbach1, P . Hohenberger2, P . Reichardt3, H .-U . Schildhaus1, R . Büttner1, E . Wardelmann1 1 University of Cologne, Institute of Pathology, Köln, 2University of Heidelberg, Department of Surgery, Mannheim, 3Helios Klinikum, Hematology/ Oncology, Bad Saarow Aims. Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the gastrointestinal (GI) tract. Up to 90% of them carry an activating mutation in the KIT or the PDGFRA (platelet-derived growth factor alpha) gene both encoding type III receptor tyrosine kinases. In both genes, hot spot regions have been identified, i.e. exons 9, 11, 13, and 17 in KIT and exons 12, 14 and 18 in PDGFRA. The distribution among these different exons is not balanced. More than 60% of cases carry KIT exon 11 mutations followed by 10 to 15% of tumors carrying either a KIT exon 9 or a PDGFRA exon 18 mutation. All other locations are very rare (less than 2% for each exon). The different mutational subtypes in KIT and PDGFRA are found in variable frequences in different parts of the GI tract. In detail, KIT exon 9 mutations are nearly always found in the small bowel and rectum and but only rarely in gastral GISTs. In contrast, PDGFRA mutations are nearly always restricted to gastric GISTs. We were interested to know how often stomach tumors carry KIT exon 9 mutations and whether there is an association with an aggressive behavior as demonstrated for GISTs in a non-gastric location. Methods. We evaluated more than 2000 cases in our GIST and Sarcoma Registry Cologne/Bonn (GSRCB) for gastric GISTs with KIT exon 9 mutations. Sequences were analysed by direct Sanger Sequencing. We evaluated pathomorphological and clinical data and compared them to GISTs in other primary locations. Results. We could identify 19 gastric cases carrying a KIT exon 9 mutation. The average tumor diameter was 4.1 cm. According to the AFIP classification (Miettinen 2006), 15 tumors belonged to the groups of no or low aggressive behavior. Three GISTs were classified as high risk lesions with a mitotic count of 13, 25 and 132/50 HPFs, resp. one other belonged to the intermediate risk group. 18 tumors carried the classical 6 base pairs insertion in KIT exon 9 (p.A502_Y503dup). One low-risk tumor showed a novel 12 bp deletion in KIT exon 9 (p.K484_G487del) which has not been described before. Conclusions. KIT exon 9 mutations (typically a 6 bp insertion; p.A502_ Y503dup) occur preferentially in a non-gastric location of GISTs. In these, the mutational subtype frequently implicates an aggressive behavior. In contrast, gastric tumors with exon  9 mutation often are associated with a low or no malignant potential. Conclusively, in the vast majority of these lesions there is no implication for an adjuvant treatment with imatinib.

DO-125 Functional phosphoproteomics for therapy response prediction in malignant thymomas and thymic carcinomas S . Küffer1, A .-L . Bohlender1, C . Sauer1, D . Belharazem1, A . Marx1, P . Ströbel1 1 University Medical Centre Mannheim of the University of Heidelberg/Institute of Pathology, Mannheim Aims. Thymomas (TH) and thymic carcinomas (TC) are rare mediastinal tumor with a high tendency for local therapy failures. Relapsed tumors require first or second line adjuvant treatments, which are not well established. Our group has recently reported clinical response to the multikinase inhibitor sunitinib in a small series of patients with metastatic TC. In an attempt to better understand the underlying molecular conditions and to eventually predict sunitinib response, we investigated TH and TC by different phosphoproteomic approaches.

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Methods. Functional kinomics were carried out by spiking sunitinib into a tumor lysate from one patient with clinical response to sunitinib and from one patient with a resistant tumor and subsequent measurement of 144 receptor tyrosine kinase (RTK) substrates. Snap-frozen tumour tissues of 63 TH and TC samples were analyzed using Phospho-ProteinArrays to detect activation of RTKs and MAPKs. Subsequently, primary cells from 10 tumor samples with known RTK/MAPK activation status were tested with sunitinib, an Akt inhibitor and a JNK inhibitor. Results. Comparing the clinical responder and the non-responder, 44/144 peptide substrates were found a) significantly inhibited by sunitinib and b) significantly different between the two samples. Pathway analysis revealed a prominent role of the EGFR, and to some extent, VEGFR signalling pathways, with involvement of PI3K and ras/raf as downstream targets. Analysis of a large number of TH and TC revealed activation of the EGFR alone or in combination with other RTKs in 40/63  cases (63%). Analysis of MAPK revealed a dichotomic pattern with actvation of the PI3K/AKT pathway in 46% and activation of JNK kinases in 54% of cases. Preliminary data with ex vivo cell cultures from TH and TC treated with AKT and JNK inhibitors suggested better responses to AKT inhibitors in the “AKT group” and vice versa. Conclusions. Our results suggest that the EGFR – the single most frequently activated RTK in TH and TC – as well as its downstream effectors PI3K/AKT and the ERK pathway may be a prominent sunitinib target in these tumors. Our findings are surprising, since small clinical trials using targeted EGFR inhibition (e.g. through gefitinib) were disappointing. Given the possible involvement of the VEGFR, inhibition of multiple kinases may be a preferable therapeutic approach. Our results also indicate that inhibition of specific pathways (such as the AKT) in highly selected patients may further improve therapeutic response rates.

Workshop Informatik – Strukturierte Befunde DO-001b Structured reports in pathology – current status and activities T . Schrader1, F . Oemig2, J . Thümmler3, U . Altmann4, G . Haroske5 University of Applied Sciences Brandenburg, FB Informatics & Media, Brandenburg, 2Agfa Healthcare, Standards and Interoperability, 3Vivantes GmbH, Berlin, Ressort IT/TK, 4Justus-Liebig-Universität Gießen, Institut für Medizinische Informatik, 5Krankenhaus Dresden-Friedrichstadt, Institut für Pathologie 1

Aims. The application of structured reports (SR) is a pestering request of clinicians, tumor centers, tumor registers and pathologists. In various countries (especially France and Spain) SR’s were developed under the auspices of IHE (Integrating the Healthcare Enterprises) which influences the current discussion of standards development. In Germany new efforts were done to promote the adoption of SR in Pathology and to govern the National and International activities. Methods. The current situation in application and development of SR’s were analyzed. Together with the Federal Society of German Pathologist and with experts from the German Society of Tumor Centers HL7 Germany has identified information blocks of a SR which are then restructured into new templates. They are then compared with current IHE Anatomic Pathology Structured Report (APSR). Results. HL7 Germany coordinates together with the German Society of Tumor Centers a comprehensive balloting process in order to establish Pathology reports. As result of this balloting, different templates of Pathology reports will be approved and could be implemented by vendors of Pathology Laboratory Information Systems. Conclusions. A German implementation guide for CDA-based pathology reports based on APSR is in development including a German description on how to use diagnostic terms and classifications, esp. ICD-10 and TNM.

DO-002b Impact of terminologies in tumor pathology structured reports G . Haroske1, T . Schrader2 Dresden-Friedrichstadt General Hospital, Institute of Pathology, Dresden, 2 University of Applied Sciences Brandenburg, Department Informatics and Media, Brandenburg

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Aims. For information exchange and data mining structured reports in tumor pathology have to be based on controlled vocabulary as to get a model of meaning. So far there is no universal terminology for the wide variety of concepts in tumor pathology. SNOMED CT will probably become a global health terminology standard. National and international initiatives are necessary to reach a growing agreement on particular aspects and needs towards it. Interface terminologies are a tool for drawing existing separate terminology systems to a finally global standard. Methods. Controlled vocabularies in guidelines of German pathologists for a series of tumors, in the basic tumor documentation of cancer registries, and in the HL7 Germany have been mapped to PathLex, an interface terminology of IHE. Results. On average a pathology guideline describes 50 terms which have to be registered as to fulfill the minimum documentation requirements. PathLex provides between 30 to 40 terms per tumor entity, only 80% of them are identical with the German guideline vocabulary. The coincidence of PathLex with HL7 Germany vocabulary or the basic data set of cancer registries is still lower. In contrast to PathLex there is no separation between general and organ-specific information in the German guideline vocabulary. Conclusions. Although based on internationally agreed understanding, sharing the same concepts of tumor pathology, the terminology differences among the different sources are quite obvious. They have to be overcome as to ascertain a reliable information exchange between different actors in the care of tumor patients. Terminology mapping is one solution, but not the optimal one. A closer collaboration with international terminology bodies as well as a sharpened realization of the impact of terminology in home made guidelines would contribute to a better standing of German pathology. A SNOMED membership of Germany would be very helpful.

AG Dermatopathologie und AG Zytopathologie I – Endokrine Themen I FR-001 Unusual HBME1-expression in a hyalinizing trabecular tumor of the thyroid gland: a case report D . Lenggenhager1, E . Marques Maggio1, B . Bösch2, A . Elisa2, M . Rössle1 UniversityHospital Zurich, Institute of Clinical Pathology, Zürich, Switzerland, 2Stadtspital Triemli, Institute of Pathology, Zürich, Switzerland 1

HBME1, but negative for calcitonin, ki67 and CK19. The intertrabecular hyalinized material was positive for diastase-resistant PAS, Collagen IV, and HBME1, exhibiting a filiform and stellate staining pattern. Mutational analysis showed a BRAF wild type. Conclusions. This case shows that HBME1-positivity may occur in HTT, and therefore should be interpreted with caution in differentiating HTT from PTC.

FR-002 Secondary tumours to the thyroid an uncommon but potentially challenging entity: the experience of a single general hospital C . Cacchi1, H . Jähnig1, G . Schenkirsch2, M . Füller3, H . Arnholdt1, B . Märkl1 1 Klinikum Ausburg Insitute for Pathology, Augsburg, 2Klinikum Augsburg, 3 Klinikum Augsburg, Oncology and Hematology Unit Aims. Despite its rich vascular supply, thyroid is a very uncommon location of metastasis. It has been reported that secondary malignancies representing less of 2% of thyroid tumours; the aim of these study is to present experience of a single institution focusing on the differential histological diagnosis. Methods. A total of 13 (8 male and 5 female patients) cases with metastatic disease to the thyroid have been retrieved from the archive of our tumour-registry between 1985 and 2011. All patients have documented histology for both primary and secondary tumour. Patient age, sex, survival, outcome were reordered. Results. The median age of the patients is 69.8 years, actually 4 patients are still alive. Among the other patients only four died as consequence of progression of the primary tumour. The operable cases (9) received in four cases a simple lobectomy, in five cases a total thyroidectomy. A primary tumour was identified in 11 cases: of clear cell renal carcinoma in 6 cases, squamous cell carcinoma in 4 cases (two lung , one larynx and one oesophagus respectively) and one small cell carcinoma of the lung . In two cases the patients have had other malignancies (melanoma, colorectal carcinomas and bile-pancreatic adenocarcinoma). The time between the diagnosis of primary tumours and metastasis is interestingly longer in case of renal carcinoma (144 months) respect to an average value for all cases (77.5 months). Five cases have been studied pre-operative with a fine needle aspiration (FNA), in two cases this procedure were diagnostic. Conclusions. Patients with metastases to the thyroid gland are an uncommon but clinically pathologically relevant issue, particularly when the tumor mass is evident as a single nodule (in our experience in 3 of 13 cases); for example a clear cell renal carcinoma could close mimic a clear cell thyroid adenoma or follicular carcinoma. Moreover, neuroendocrine tumor can metastasise to the thyroid gland simulating a primary thyroid tumour (particularly a medullary carcinoma): a rare but treacherous possibility. To avoid misdiagnosis and to ensure an optimal follow-up for these patients clinical data and a comprehensive immunohistochemical panel are mandatory.

Aims. Hyalinizing trabecular tumour (HTT) is a rare thyroid neoplasm of follicular cell origin with a trabecular growth pattern, marked intratrabecular hyalinization and nuclear features, which are typically found in papillary thyroid carcinoma (PTC). The role of HBME1 in the diagnostic process of PTC has been demonstrated in several studies. We present a case of HTT with patchy, but abundant, hitherto not reported membranous and intrabecular HBME1-positivity. Methods. A 70-year-old woman underwent total thyroidectomy because of cytological diagnosis of PTC. Pathomorphological investigation of the resected specimen was performed. Results. Histologically, the typical trabecular architecture of HTT with elongated tumor cells, markedly hyalinized intratrabecular stroma and oval shaped nuclei with grooves and inclusions was seen. Immunohistochemically, tumour cells were diffusely and strongly positive for thyreoglobulin and TTF1, focally and weekly positive for Galektin3 and Der Pathologe Suppl 1 · 2012  | 

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Abstracts FR-003 Glucagon cell adenomatosis: a novel multifocal neuroendocrine neoplasia restricted to the dorsal pancreas anlage M. Anlauf1, P. Gerlach1, S. Schinner2, M. Schott2, M. Krausch3, K. Cupisti3, R.S. Lanzmann4, C. Antke5, S. Schulz6, G. Klöppel7, H.E. Gabbert1, W.T. Knoefel3, W.A. Scherbaum2 1 Institute of Pathology, University of Düsseldorf, 2Department of Endocrinology, Diabetes and Rheumatology, University of Düsseldorf, 3Department of General-, Visceral- and Pediatric Surgery, University of Düsseldorf, 4Institute of Diagnostic and Interventional Radiology, University of Düsseldorf, 5 Department of Nuclear Medicine, University of Düsseldorf, 6Institute of Pharmacology and Toxicology, University of Jena, 7Institute of Pathology, Technical University of München Aims. Based on observations in four patients, glucagon cell adenomatosis (GCA) has recently been proposed as a novel multifocal neuroendocrine tumor disease of the pancreas. Methods. We report on a 58-year-old patient treated by duodenopancreatectomy who showed distinct features of GCA. In order to study the tumor development and distribution the entire pancreas was embedded and systematically analyzed. Results. The patient presented with recent onset non-insulin-requiring diabetes and abdominal discomfort. On CT the pancreas showed multiple tumors and diffuse nodular enlargement corresponding to the SPECT scintigraphy. The imaging findings were more pronounced in the pancreatic body and tail than in the head. Pathology analysis revealed 12 neuroendocrine macrotumors and more than 10,000 microadenomas mainly composed of glucagon cells. Metastases were not detected. The nontumorous parenchyma revealed a ubiquitous glucagon cell hyperplasia at the expense of the other endocrine islet cell types. These findings were restricted to the body, tail and the upper portions of the pancreatic head. The lower portion of the head of the pancreas that corresponds to the ventral pancreas anlage had a normal appearance. Conclusions. GCA is a novel multifocal neuroendocrine neoplastic disease that is restricted to the dorsal pancreas anlage.

FR-004 Genetic alterations in glucagon cell adenomatosis T. Henopp1, M. Anlauf2, S. Biskup3, G. Klöppel4, B. Sipos1 University Hospital Tübingen, Institute for Pathology and Neuropathology, Tübingen, 2University Hospital Düsseldorf, Institute for Pathology, Düsseldorf, 3Center for Genomics and Transcriptomics, Tübingen, 4Technische Universität München, Institute of Pathology, München

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Aims. Glucagon cell adenomatosis (GCA) was recently recognized by us as a multifocal neoplastic disease of the endocrine pancreas unrelated to MEN1. Multiple micro- and a few macrotumors are found on the background of a hyperplasia of glucagon cells. The disease may cause unspecific abdominal symptoms and only rarely a glucagonoma syndrome. Recently a mutation in the glucagon receptor (GCGR) gene was described in one GCA patient. The aim is to investigate GCGR gene changes in five patients with GCA. Methods. Paraffin embedded and formalin fixed pancreatic tissues from five patients showing multiple microadenomas and in three cases also macroadenomas of glucagon cells were macro- or microdissected. The extracted DNA was sequenced and the GCGR gene analysed for mutations. Results. Sequencing of the GCGR gene revealed germline mutations in three out of five patients. One patient shows two different heterozygous point mutations in the hyperplastic alpha cells as well as in the non-tumorous tissue leading to two premature stop codons. One patient harbors a homozygous stop mutation. The third patient shows two homozygous missense mutations of the GCGR gene that most likely also led to a dysfunction of the GCGR. In the two other patients no germ line mutati-

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ons of the GCGR gene were detected. These variants were not identified in healthy subjects. Conclusions. The finding of germ line and somatic “loss of function” mutations of the GCGR gene in three of five patients with GCA suggests that a change in the signalling function of the GCGR may cause glucagon cell adenomatosis via glucagon cell hyperplasia.

FR-005 Proliferative activity is not associated with tumor aggressiveness in ileal neuroendocrine tumors T. Henopp1, J. Sperveslage1, M. Anlauf2, G. Klöppel3, B. Neumayer1, C.P. Gerlach2, P. Rexin4, T.M. Gress5, R. Moll4, B. Sipos1 1 University Hospital Tübingen, Institute for Pathology and Neuropathology, Tübingen, 2University Hospital Düsseldorf, Institute for Pathology, Düsseldorf, 3Technische Universität München, Institute of Pathology, München, 4 University Hospital Giessen and Marburg, Institute for Pathology, Marburg, 5 University Hospital Giessen and Marburg, Department of Internal Medicine, Marburg Aims. The accuracy of the new WHO grading system for neuroendocrine neoplasms, which is based on proliferative activity, has not yet been validated for ileal neuroendocrine tumors (iNETs). Aim of this study is to analyse the proliferation rates in primary iNETS, lymph node and distant metastases in order to determine the prognostic power of grade 1 and grade 2 categories in the WHO 2010 classification. Methods. 64 primary iNETs, 35 matched node metastases and 20 distant metastases were analysed for proliferation rate (Mib1, Phospho H3) using automated image analysis assessing the maximum (hot spot) and overall proliferative activity. Proliferation rates were compared in different prognostic relevant cohorts (N0M0, N1M0 and N1M1). Results. The maximum Mib1 proliferative rates were: 0.6% (range 0.27– 2.78) for N0M0, 0.77% (range 0.03–2.94) for N1M0 and 1.02% (range 0.2–12.17) for N1M1 primary iNETs. Corresponding node metastases (0.66%; range 0.02–3.38) and distant metastases (0.83%, range 0.01–10.94) showed comparable proliferative rates like primary iNETs. The number of grade 1 and grade 2 iNETs was not different in the three cohorts. Primary iNETs of N1M0 (2 cm; range 1–4.8) and N1M1 (1.95 cm; range 0.4–5) cohorts were significantly larger than N0M0 tumors (0.2 cm; range 0.2–1.6). Conclusions. Grade 1 and grade 2 categories in the WHO classification 2010 do not have discriminatory power regarding prognosis for iNETs. In fact, tumor spread is independent of proliferative activity in iNETs.

FR-006 Comprehensive assessment of Merkel cell polyomavirus in Merkel cell carcinomas: fluorescence in situ hybridization versus qPCR? A. Haugg1, D. Rennspiess1, A. zur Hausen1, E.-J. Speel1, G. Cathomas2, J. Becker3, D. Schrama3 1 Maastricht University Medical Center, Department of Pathology, Maastricht, Netherlands, 2Kantonsspital Liestal, Institute of Pathology, Switzerland, 3 Medical University of Graz, Division of General Dermatology Department of Dermatology, Graz, Austria Aims. Merkel cell polyoma virus (MCPyV) is detected in 80% of Merkel cell carcinomas (MCC). The clonal integration and tumor specific mutations in the large T Antigen (LTAg) gene identify MCPyV as a novel human tumor virus. To date the relationship between the viral presence and cancer induction, development or clinical prognosis is discussed controversially. Yet almost all studies are based on quantitative virus detection, i.e. PCR or qPCR. Here we aimed to gain information about the quality of the viral presence on the single cell level in the histomorphological context.

Methods. We performed MCPyV-FISH of formalin fixed and paraffin embedded (FFPE) MCCs (n=62) on tissue microarrays (TMAs), determined the hybridization patterns and correlated these with qPCR data on the basis of a determined cut-off. MCPyV-FISH was established using the MKL-1 cell line which harbors integrated copies of MCPyV DNA. For MCPyV-qPCR a LT primer pair (Becker et al., 2009) was used on whole tissue sections. Results. MCPyV-FISH on FFPE MKL-1 cells revealed punctate signals compatible with viral integration. The MCPyV-FISH positive MCC cores (76%) mainly revealed two different signal patterns: a punctate pattern (85%) which correlated with a moderate relative viral abundance and in some areas the punctate pattern was combined with a diffuse pattern (15%) indicating the episomal presence of the virus which is linked to viral replication. A mixed hybridization pattern was associated with very high qPCR values. Comparing MCPyV-FISH and qPCR data the results highly correlated (83%) with the MCPyV positive evaluated group, whereas the negative group showed a concordance of 93%. The mean of the qPCR values of all MCPyV positive cores differed significantly from the negative cores (p=0.0076). In some tumor areas of one and the same patients the FISH signals were heterogeneous in intensity, pattern and nuclear localization. Conclusions. A strong correlation between MCPyV FISH and the relative MCPyV abundance by qPCR was detected. Thus, while presence of MCPyV can be verified by qPCR, the quality of the presence can be visualized by MCPyV specific FISH analysis. In this regard, MCPyV qPCR and MCPyV FISH are important complementary tools to gain maximum biological information of the presence of MCPyV in MCC and thus to further elucidate MCPyV related carcinogenesis.

FR-007 A novel BRAF mutation in a patient with metastatic melanoma R . Schneider-Stock1, L . Heinzerling2, E . Kämpgen2, M . Erdmann2, P . Keikavoussi2, A . Agaimy1, A . Hartmann1, G . Schuler2 1 University of Erlangen-Nuremberg, Institute of Pathology, Erlangen, 2 University of Erlangen-Nuremberg, Department of Dermatology, Erlangen Aims. BRAF mutations in melanoma have been identified as a new target for therapy. The V600E mutation is found in 50–68% of malignant melanomas and can be specifically treated with e.g., the BRAF inhibitor PLX4032 now registered as Vemurafenib. We describe a patient with a metastasized nodular melanoma on the right lumbar region which was excised two years before but had already spread to the inguinal lymph nodes. Subsequently, the patient developed multiple metastases of the brain, metastases of the mediastinal, cervical, retroperitioneal, retrocrural, mesenterial, paraaortal and interaortocaval lymph nodes, in the liver, the stomach and the intestines. Previous therapy included lymphadenectomy, radiation therapy, hyperthermia, radiochemotherapy with temozolomide, sorafenib, fotemustine and paclitaxel/carboplatin. Despite these attempts he showed progressive disease and inclusion into a BRAF inhibitor study was considered. Thus the tumor was sent for mutation analysis. Methods. Formalin-fixed and paraffin-embedded tumor sample was used for the extraction of genomic DNA. Mutational analysis was carried out by Pyrosequencing and for confirmation by ABI capillary sequencing of exon 15 of BRAF. Results. In this melanoma patient a new complex mutation was found in BRAF with base substitution of a valine residue at position 600 for glutamic acid (GTG GAA) and deletion of codon 601 (p.V600EK601del; c.1799_1801del3). With this mutation the patient did not qualify for the BRAF inhibitor study. The clinical course of the patient was rapidly progressive with various acute complications (renal insufficiency, ileus) and the patient deceased only 2 months after analysis of the mutation. Conclusions. The V600E mutation constitutively activates RAF/MEK signaling, a major driver of carcinogenesis in various malignancies. Other rather rare mutations like V600K, V600R or V600D might also

cause this constitutive activation and are discussed to be correlated with a yet more aggressive behaviour. It is unclear whether the new mutation described in this case can be considered for targeted BRAF inhibitor therapy.

FR-008 The aid of immunohistochemistry in differential diagnosis between benign and malignant phenotype of difficult melanocytic lesions T . Papadopoulos1 1 Klinikum Nürnberg, Institute of Pathology, Nürnberg Aims. A panel of biological markers differentially expressed in common nevi, dysplastic nevi, Spitz nevi and malignant melanomas are introduced providing a potential tool to differentiate benign from malignant phenotype in difficult melanocytic lesions. The panel includes Cyclin D1, p16, p21 and p53. Methods. Cyclin D1 is markedly expressed in radial growth phase malignant melanomas but is negative in common nevi and in the deepest part of vertical growth phase melanomas as well. The cell cycle inhibitor p16 is positive in benign nevi and radial growth phase melanomas but becomes often negative as malignant melanoma progresses to the vertical growth phase. The cell cycle inhibitor p21 is positive in radial growth phase melanomas and in thin vertical growth phase melanomas but becomes negative in thick malignant melanomas. p21 is negative in common melanocytic nevi. Both cell cycle inhibitors p16 and p21 are upregulated in Spitz nevi and Spitzoid nevi. Finally, p53 is negative or positive at a very low level in melanocytic nevi. Its expression increases with tumor progression from dysplastic nevus to radial growth phase malignant melanoma and vertical growth phase malignant melanomas. Conclusions. This presentation demonstrates characteristic expression patterns of the above mentioned panel that may enable pathologists to differentiate benign from malignant melanocytic lesions even in cases when morphology by itself may not allow a clear classification of the melanocytic lesion. Notiz an die Gutachter des Abstracts: Der Abstract wurde nach Rücksprache und auf Wunsch von Prof . Meister (München) eingereicht . Vorgesehen ist ein etwa 15- bis 20-minütiger Vortrag, quasi als Review für die Immunhistochemie unklarer melanozytärer Läsionen .

AG Dermatopathologie und AG Zytopathologie II – Endokrine Themen FR-009 Epidermal growth factor receptor mutation analysis in pleural effusions of advanced non-small cell lung cancer patients: When only cells are available A . Zimpfer1, B . Schneider1, A . Polak1, A . Bier2, J . Kölbel1, J .C . Virchow2, A . Erbersdobler1 1 University of Rostock, Institute of Pathology, Rostock, 2University of Rostock, Rostock Aims. Epidermal growth factor receptor (EGFR) mutations are associated with an improved clinical outcome due to response to tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). The overwhelming majority of bronchial cancer is diagnosed on often very limited tumor material which is also requested for secondary mutational analysis. This study assessed the feasibility of using PCR and gene-sequencing to screen for (EGFR) mutations in pleural effusions of advanced NSCLC patients. Der Pathologe Suppl 1 · 2012  | 

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Abstracts Methods. EGFR mutation analysis was performed in 16 cases of malignant pleural effusions in lung cancer patients. 6 patients were male (range 48–77 years, median 60.5 years) and 8 were female (range 66–86 years, median 77.5 years). In 1 case 3 pleural effusion specimens of 1 male patient were examined. Precipitations of fibrin in pleural effusions were subjected to cell block technique. Tumor cell enrichment was performed in 2/16 cases. The DNA was extracted and exons 18–21 of EGFR amplified. Sequence analysis of the PCR products was performed using an Applied Biosystems 3500 system. Results. Molecular analysis of all EGFR target sequences was achieved in 14 of 16 (87.5%) cases. EGFR mutations were identified in 3/14 (21.4%) of fully evaluated cases (1 pleomorphic carcinoma and 2 NSCLC). Two cases carried a deletion in exon 19 (K745-A750) and the third case a point mutation in exon 20 (V769M, G779F). Conclusions. EGFR mutations in pleural effusions from advanced NSCLC patients can be detected and characterised by PCR and gene sequencing. Thus, further and costly procedures for tissue retrieval would be unnecessary in some cases. The availability of EGFR mutation testing of cytological specimen is a valuably tool in the concerted cytologic/histologic diagnosis of NSCLC and help to spare precious tissue material for additional tests.

FR-010 Significant increased detection for cervical dysplasia using the ThinPrep Integrated Imager G. Richter1, U. Hahlbohm1, D. Teschner1 1 Institute of Pathology Dr. Richter, Hameln Aims. The liquid based cytology using the ThinPrep PAP test is a standardised method of smear testing and cell processing that was developed in the USA. The ThinPrep Integrated Imager has been available for computer assisted liquid based cytology since autumn 2009. This presentation compares the results of the ThinPrep Integrated Imager of 2010 with the so called normally produced liquid based cytology in 2007. Methods. As a DIN/ISO17020 accredited Institute we have been carrying out the conventional PAP smear test and since 2000 also the liquid based cytology using the ThinPrep PAP test. Since November 2009 the liquid based cytology is carried out computer assisted. Results. In 2007 we manually evaluated 3582 ThinPrep PAP tests of which 98% were of an inconspicous PAP group I or II; 1.6% of PAP group IIW; 0.03% of PAP group III; 0.5% of PAP group IIID; 0.14% of PAP group IVa; 0.03% of PAP group IVb. In 2010 we evaluated 4408 ThinPrep PAP tests using the Integrated Imager of which 94% were of an inconspicuous PAP group I or II; 3.4% of PAP group IIW, 0.05% of PAP group III; 2.6% of PAP group IIID; 0.2% of PAP group IVa and 0.02% of PAP group IVb (p<0.001). Conclusions. The higher sensitivity of the computer assisted liquid based cytology could potentially reduce cervical cancer considerably. Corresponding long term monitoring would be required, to asses the significance of this observation for gynaecological screening.

FR-011 Predictive value of cervical smears reported as glandular neoplasia: 10 years experience with ThinPrep liquid based cytology S. Brockmoeller1, C. Denkert2, M. Dietel2, R. Lonsdale3 1 Norfolk Norwich University, Pathology Department, Norwich, United Kingdom, 2Charite University, Pathology Department, Berlin, 3Norwich and Norfolk University Hospital, Pathology Department, Norwich, United Kingdom Aims. The aim of the study was to audit all cases of cervical cytology (ThinPrep liquid based) in the last 10 years reported as glandular neoplasia (6) and calculate the positive predictive value (PPV) with respect to final histological outcomes.

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Methods. The design of the study was a retrospective review of 244 women who were identified over a 10-year period from the cytology database at the Norwich and Norfolk University Hospital. Histological outcomes were identified for all cases, and the PPVs for neoplasia (including all invasive cancers, CGIN, CIN 3 and CIN 2), invasive cancer and invasive endocervical adenocarcinoma were calculated. Results. The results showed that of the total number of 244 cases, 93 (38%) cases were invasive cancers, 88 (36%) cases were pre-invasive neoplasia (including CIN2/3 and CGIN) and 63 (26%) cases were CIN 1/HPV infection and benign pathology. The PPV for neoplasia was 74%, invasive cancers 38% and endocervical adenocarcinoma 18%. Conclusions. Our analysis from this retrospective study shows that a diagnosis of glandular neoplasia resulted in the detection of clinically significant lesions in 74% of cases. This is similar to published evidence in the literature. More than one third of these patients already have invasive cervical or non-cervical cancer, justifying the urgent clinical referral of patients with this cervical smear result.

FR-012 Management of Pap-negative, HPV-positive women with p16/Ki67 dual-stained cytology – results from the PALMS Trial H. Ikenberg1, M. Sideri2, C. Bergeron3, M. von Knebel Doeberitz4, H. Griesser5, J. Bogers6, H. Neumann7, D. Schmidt8, R. Ridder for the PALMS Study Group9 1 Cytomol, MVZ for Cytology and Molecular Biology, Frankfurt, 2European Institute of Oncology, Mailand, Italy, 3Laboratoire Cerba, Cergy Pontoise, France, 4University of Heidelberg, Applied Tumor Biology, Heidelberg, 5Center for Pathology and Cytodiagnostics, Köln, 6Labo Lokeren – Campus Riatol, Antwerpen, Belgium, 7Institute for Pathology, Schüttorf/Leer, Schüttorf, 8 Institute of Pathology, Mannheim, 9mtm laboratories, Heidelberg Aims. Adding HPV testing to cytology based cervical cancer screening (co-testing) in women aged over 30 years has the potential to increase sensitivity for the detection of high-grade cervical intraepithelial neoplasia (CIN2+). However, there is a need for an efficient clinical management algorithm for women with discrepant, i.e. Pap-negative/HPV-positive test results. We analyzed the performance of a novel immunocytochemical dual biomarker approach that detects co-localization of p16 and Ki-67 expression within the same cervical epithelial cell as an indicator of cell-cycle deregulation, in triaging all Pap-negative/HPV-positive screening results from the PALMS study, a prospective screening trial comprising more than 27,000 women. Methods. Women prospectively enrolled to the PALMS trial were tested using Pap cytology (LBC or conventional Pap smears), HPV (hc2, Qiagen) testing, and p16/Ki-67 Dual-stained cytology (CINtec PLUS, mtm laboratories) from samples collected at the enrolment visit. Women aged over 30 years with any positive test result were referred to colposcopy. Adjudicated tissue diagnosis on cervical biopsies was used as reference standard. Results. Out of a total of 19,205 women aged 30 and older enrolled to the PALMS trial, 1,023 women (5.3%) were tested Pap-negative, HPV-positive. p16/Ki-67 Dual-stained cytology testing at baseline provided positive test results in 225/1,023 (22.0%) women. Within this Dual-stain positive group, 29 out of a total of 36 (80.6%) biopsy-confirmed CIN2+ cases were found. Specificity of dual-stained cytology at the CIN2+ threshold was 79.1%. The negative predictive value (NPV) of a negative dual-stained cytology result was 98.7%. Conclusions. These results from the prospective PALMS trial confirm the high sensitivity and specificity of dual-stained cytology testing recently reported for triaging Pap-negative/HPV-positive screening results. The detection of co-localization of p16/Ki-67 expression in cervical cells identifies women that may benefit most from immediate colposcopy, whereas negative dual-stain test results may exclude pre-cancerous cervical disease within this cohort with a high NPV.

FR-013 HPV-genotype distribution in cytological screening, histology and impact of vaccination M . de Jonge1, G . Busecke2, A . Heinecke3, O . Bettendorf4 1 Institute for Pathology and Cytology Schüttorf/Leer, 2Medical office of General Medicine, Weener, 3Department of Medical Informatics and Biomathematics, University of Münster, 4Insitute of Pathology and Cytology Schüttorf/Leer, Schüttorf Aims. The objective of the research was to investigate the HPV-type distribution in our screening population as well as its correlation to cytological diagnosis and histological outcome, and to calculate the impact of primary HPV vaccination. Methods. HPV genotyping results of 3381 women who attended to the German cervical screening program were retrospectively analysed. The PapilloCheck®-Test was used for HPV genotyping. The distribution of HPV-types with corresponding cytological diagnosis, and – if available – histological outcome, was statistically analysed (SPSS 17.0, SAS9.3, Pearson’s χ2 test). To estimate the possible impact of HPV vaccination on our cohort we calculated the theoretically minimum impact as well as the maximum impact in the prevention of HPV-positive lesions. Results. The HPV-type-distribution showed marked differences among cervical lesions and age. Although HPV-51 was common in all cervical lesions, it was rarely detected as single-type HPV infection in CIN3-lesions. HPV-16 and/or HPV-18 were found in only 58% of the CIN3-lesions, which was still significantly more than the 22% in CIN2-lesions (p<0.0001). According to the theoretically calculated minimum impact of HPV-vaccination, vaccination could have prevented the following percentages of cytologial and histological lesions: 13.1% ASC-US; 10.0% LSIL; 32.7% ASC-H; 20.7% HSIL; 17.2% CIN1; 11.1% CIN2 and 31.3% CIN3. Conclusions. HPV gentoyping is a useful tool for cytological screening, especially for triage of women with HSIL diagnosis and corresponding CIN2-lesions. Primary cervical HPV-screening for HPV-16 and HPV-18 alone would be insufficient to detect CIN-3 lesions. Vaccination would especially have prevented high-grade cervical lesions in our cohort. The effect on preventing low-grade cervical lesions would be less.

FR-014 Human Polyomavirus 6 in keratoacanthomas and squamous cell carcinomas of the skin J . Beckervordersandforth1, D . Rennspiess1, T . Brinkhuizen2, M . van Steensel2, E .-J . Speel1, A . Haugg1, A . zur Hausen1 1 Maastricht University Medical Center, Department of Pathology, Maastricht, Netherlands, 2Maastricht University Medical Center, Department of Dermatology, Maastricht, Netherlands Aims. The human polyomavirus 6 (HPyV6) was recently detected in forehead swabs of healthy persons by Schowalter et al. An initial seroepidemiological study has shown that HPyV6 has a seroprevalence of approx. 69%. Here we assessed the presence of HPyV6 in keratoacanthomas (KA) and squamous cell carcinomas (SCC) by PCR and fluorescence in situ hybridisation (FISH). Methods. HpyV6 PCR was performed according to Schowalter et al. (2010) on 95 squamous cell carcinomas (SCC) and 29 keratoacanthoma (KA) cases. All PCR products were sequenced. HPyV6-FISH was performed according to Hopman et al. with modifications on the MCPyV positive MKL-1 cell line, 26 KA and on 4 PCR positive BCC cases. All tissues were formalin fixed and paraffin embedded. The FISH results were interpreted according to Hafkamp et al. and Cooper et al. Results. Specific HPyV6 DNA was detected by PCR in 52% (15/29) of KA, and 10.5% (10/95) of SCC. The HPyV6 FISH on the MCPyV positive MKL-1 cells was negative. PCR data were confirmed by HPyV6 FISH revealing a punctate, sometimes diffuse pattern in the epithelial and/or lymphocytic compartment in 31% (8/26) cases of the KA.

Conclusions. The low frequency of HPyV6 in SCC (10.5%) is in line with a recent report on the presence of HPyV6 in SCC. The rather frequent detection of HPyV6 DNA in KA (52%) might indicate a role for HPyV6 in the pathogenesis of KA.

FR-015 Hidradenitis suppurativa/Acne inversa: chronology of main histological features M . von Laffert1, V . Stadie2, J . Wohlrab2, M . Dietel1, W .C . Marsch2 1 Pathology Charité Berlin, Berlin, 2Department of Dermatology and Venereology, Martin-Luther-University Halle-Wittenberg Aims. Hidradenitis suppurativa/Acne inversa (HS/Ai) is a chronic-inflammatory, fistulating and scarring disease with topographical predilection in skin folds (axillary, submammary, inguinal, anogenital). Initially the disease was considered to be caused by inflammation and occlusion of the apocrine glands. Histological investigations in the 90ies showed poral occlusion of the terminal follicle structure to be the “primum movens”. In analogy to the formal pathogenesis of acne vulgaris, Plewig introduced the term “Acne inversa” as more suitable. Complete data of operative specimens are scarce. The following investigation describes the morphology (life of lesions/chronology) of HS/Ai. Methods. A total of 485 operative specimens obtained from 128 patients with the diagnosis HS/Ai (surgically treated in the Department of Dermatology and Venerology, Martin-Luther-University Halle-Wittenberg) were examined histologically. Results. HS/Ai shows a heterogeneous histological pattern: hyperkeratosis of the terminal follicles (89%), hyperplasia of follicular epithelium (80%), pronounced perifolliculitis (68%) and follicle rupture (24%). Perifollicultis, follicular hyperkeratosis and hyperplasia occur prior to the rupture of the follicle (early lesions). Other histological criteria are subepidermal inflammatory infiltrates (82%), epidermal “psoriasiform” hyperplasia (56%), pronounced acute dermal inflammation (28%), pronounced chronic dermal inflammation (49%), as well as involvement of the apocrine glands (52%) and the subcutis (31%). Conclusions. Biopsy/operative tissue of the skin folds showing the described histopathological patterns should rise the differential diagnosis of HS/AI, an interdisciplinary (dermatology, surgery, gynecology, urology, pathology), but underestimated disease (prevalence: 1–3%), clinically often not considered and for that reason leading to long disease durations (~8 years) from the first clinical signs to the correct diagnosis and the adequate surgical treatment.

Aktuelle Habilitationen I FR-016 Initiating and prognosis-associated alterations in urothelial carcinoma of the bladder R . Stöhr1 1 University Hospital Erlangen, Institute of Pathology, Erlangen Aims. A lot of effort was made analyzing the molecular basis of bladder cancer (BC). These efforts led to the identification of two separated pathways in the development of papillary and solid BC. Nevertheless, several points of interest are still not covered adequately: little is known about the molecular alterations in the normal urothelium of BC patients. Reliable prognosis-associated markers are still missing, especially progression-related marker for papillary BC. In the actual WHO classification rare tumor variants were acknowledged as discrete BC entities, but molecular analyses of these variants are still missing. Methods. Whole organ-mapping was used for investigating normal urothelium. LOH,- FISH- and FGFR3-mutation analysis was performed to Der Pathologe Suppl 1 · 2012  | 

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Abstracts detect alterations in histopathological inconspicuous urothelium. Gene expression profiling, TMA technology, promoter methylation analysis, qRT-PCR, aCGH and in vitro studies were performed for the identification and functional characterization of progression-related candidate genes in BC. A comprehensive molecular, immunohistochemical and clinicopathological analysis of a large collection of plasmacytoid BC samples was performed. Results. Deletions on chromosomes 9 and 8p were detected in approx. 10% of normal urothelial samples from BC patients. The most common deleted region was on chromosome 9q33.3. Deletions of chromosome 8p were identified as progression markers for papillary BC. Loss of sFRP1 expression (located on 8p11.21) was found in 35% of BC cases and was caused by gene copy loss and/or promoter methylation. Functional studies revealed an influence of sFRP1 on proliferation, cell viability and migration in papillary BC. Patients with papillary BC and sFRP1 loss showed a significant decreased overall survival which was not relevant in patients with solid tumors. Plasmacytoid BC displayed molecular alterations of advanced, aggressive BC. A complete loss of membranous E-Cadherin underlined the high malignant and metastatic potential of this BC variant. Conclusions. BC is not affecting the complete urothelium. Molecular alterations in the normal urothelium could be origins of multifocal tumor growth and argue for the “field cancerization” theory. Deletions on chromosome 8p and expression loss of sFRP1 might act as entity-specific progression markers for papillary BC. The profile of molecular alterations in plasmacytoid BC might help to find the suitable therapeutic intervention for patients with this highly aggressive BC variant.

FR-017 Novel approaches to progressive renal diseases P. Boor1 1 RWTH Aachen University, Aachen Aims. Essentially all chronic renal diseases but also repeated or serious acute insults inevitably lead to chronic kidney disease and renal fibrosis. We currently lack effective treatment options for renal fibrosis; the development of an effective therapy would be invaluable virtually for all renal patients. Methods. We have used various in vivo and in vitro models of renal fibrosis. Results. We have identified PDGF-CC, PDGF-DD, C5a and PPAR-α as novel treatment targets in renal fibrosis. By identifying these targets as crucial components of renal tubulointerstitial fibrosis we also uncovered the mechanisms relevant for their actions, including mitogenic effect of PDGF-DD and proinflammatory effects of PDGF-CC on interstitial fibroblasts and effects of C5a/C5aR and PPAR-α on tubular epithelial cells resulting in reduced profibrotic paracrine signaling to interstitial fibroblasts. We verified all of the targets using tools used in, tested for or developed for clinical use. These studies lay an important experimental basis for translating these targets to clinical practice. In this regard, we also showed that serum PDGF-DD is specifically increased in patients with mesangioproliferative but not other types of glomerulonephritis. In further studies on renal fibrosis we showed that irrelevant IgG has antifibrotic effects in a model of progressive mesangioproliferative glomerulonephritis resulting in improved survival. We also documented that the renal profibrotic effects of cyclosporine A are mediated by Y-box binding protein-1 (YB-1) and that mesenchymal stem cells might maldifferentiate and induce local fibrotic response in progressive glomerulonephritis in rats. We also pointed out that simple non-pharmacological approaches in diabetic rats, e.g. regular moderate exercise, reduced renal fibrosis in a very early stage when no functional changes were yet observed. The mechanism seemed to be via improving metabolism and interfering with an important pathogenic pathway in diabetes, the advanced glycation. We have also identified environmental factors, which led to renal but also cardiac fibrosis in healthy rats, i.e. passive smoking and industrial dust

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particles amozite. These environmental risk factors are external, modifiable and could lead to better monitoring of patients with such (occupational) risk. Conclusions. Still much is to be learned about renal fibrosis. We hope to have uncovered some pieces of this immense puzzle and surely aim to continue to put it together in the future.

FR-018 Using genome-wide molecular screening for the identification of new marker and target genes of human hepatocellular carcinoma T. Longerich1 1 University Hospital Heidelberg/Institute of Pathology, Heidelberg Aims. To identify new diagnostic and prognostic markers that may be used as therapeutic targets and to develop an oncogenetic progression model of human hepatocellular carcinoma (HCC). Methods. A well-characterized human HCC collection was used for systematic genome-wide screening. Identified potential candidate genes were validated via expression analysis and selected candidates were further characterized in vitro using suitable HCC cell lines. Results. Using a meta-analysis of classical comparative genomic hybridisation (CGH) analysis (24  dysplastic nodules, 871  HCCs) a genomic progression model of tumour dedifferentiation (1q – 8q – 4q – 16q – 13q) was generated. Array-based CGH analysis revealed recurrent chromosomal gains at 1q, 6p, 8q, 17q, 19p, and 20q, while genomic losses were observed at 1p, 4q, 8p, 13q, 16q, and 17p. The mouse double minute homolog 4 (MDM4) that functions as a negative p53 regulator could be identified as a target gene of 1q gains in human HCC. Aetiology-dependent copy number gains and MYC overexpression was detected in viral and alcohol-related HCCs. In contrast, cryptogenic HCCs showed neither 8q24 gains, nor MYC overexpression, nor target gene activation. The role of Polo-like kinase family (PLK) members was analyzed in human HCC. PLK1 levels were upregulated in human HCC, reaching the highest expression in tumours with poorer outcome. PLK1 overexpression resulted from activation of the Ha-Ras/FOXM1/PLK1-axis. In contrast PLK2, PLK3, and PLK4 expression were downregulated in HCC, with the lowest levels being detected in HCC with shorter survival. PLK2-4 down-regulation was paralleled by promoter hypermethylation and/or loss of heterozygosity. Immunohistological analysis revealed that a diffuse sinusoidal expression of Annexin A2 has diagnostic value for the biopsy diagnosis of highly-differentiated HCC and may increase the accuracy of the established diagnostic marker panel (GPC3, GS, HSP70) for HCC detection. Conclusions. Five genomic aberrations allow the generation of a robust progression model of human hepatocarcinogenesis. MDM4 upregulation may result in functional p53-inactivation in HCCs that may allow p53-reactivation as a therapeutic strategy. Differential oncogenic and tumour suppressive roles of Polo-like kinases could be identified in human HCC. Systematic genome-wide screening analysis were used to identify new diagnostic marker and potential oncogenic and tumorsuppressive factors that may be promising future therapeutic targets.

FR-019 In situ hybridization in clinical pathology T. Gaiser1 Philipps-University Marburg, Institute of Pathology, Marburg

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Aims. Over the last decade in situ hybridization (ISH) has emerged as a powerful clinical and research tool for the assessment of target DNA dosages within interphase and metaphase nuclei. HER2 detection is the widest application for ISH in routine pathology but EGFR ISH or the newly introduced melanoma FISH are further possible applications, which can be additionally improved by computer based analysis systems.

Methods. Three different techniques FISH, CISH and SISH for in situ hybridization were evaluated regarding their use in routine pathology. Furthermore, a multi-colour probe in malignant melanoma samples was evaluated as test tool in histological borderline melanoma cases and a computer based algorithm was implemented for simplifying and standardization. Results. EGFR SISH amplification studies in archival paraffin-embedded glioblastoma samples showed that SISH can accelerate the diagnostic process in a cost-effective way. Melanoma FISH probes failed to detect a sufficient amount of chromosomal changes necessary for a clinically useful diagnostic tool. Computer based algorithms helped to standardize ISH evaluation. Conclusions. Specific genetic alterations will define new disease entities, requiring ISH as prerequisite to establish the diagnosis. ISH can help to sub-classify morphologically similar neoplasia in terms of therapeutical response and will help to define genetic subgroups within distinct diagnostic groups for treatment purposes.

FR-020 Epithelial-mesenchymal transition of non-small cell lung cancer A . Soltermann1, V . Tischler1, L . Morra1, W . Weder2, H . Moch1 1 University of Zurich, Institute of Surgical Pathology, Zurich, Switzerland, 2 University Hospital Zurich, Division of Thoracic Surgery, Zurich, Switzerland Aims. Non-small cell lung cancer (NSCLC) is a highly fibrotic malignancy, elaborating a prominent desmoplastic stroma reaction or tumor microenvironment, respectively. Four main modes of carcinoma invasion into its own newly formed stroma are generally recognized: Epithelialmesenchymal transition (EMT), amoeboid, infiltration in cohorts and in collective sheets. We aimed for characterization of the matricellular N-glycoprotein periostin, a major EMT indicator, in the tumor microenvironment of NSCLC. Methods. Malignant pleural effusions from lung adenocarcinoma were screened for N-glycoproteins by shotgun proteomics using liquid chromatography following tandem mass spectrometry (LC-MS/MS). The identified EMT protein periostin was validated on both a tissue microarray of surgically resected patients with NSCLC (n total=532) and on tumor whole sections (n=30) by immunohistochemistry. Isoform-specific PCR following sequencing was performed in frozen specimens. Results. In the pleural effusions, 170 non-redundant N-glycoproteins were identified with high protein probability >0.9, belonging mainly to serum factors and extracellular matrix (ECM) constituents. Periostin was the most robustly identified ECM protein in malignant effusions. On tissue microarrays, strong protein upregulation was predominantly observed at the invasive front in both tumor epithelia and the surrounding extracellular matrix, the so-called matricellular space. In comparison to structural ECM proteins such as collagen, elastin, vimentin and versican, high periostin was found to be most closely associated with clinicopathologic parameters of tumor progression such as higher stage, higher pT and larger size; as well as the squamous cell histotype (all p-values <0.05). Further, high stromal periostin was found to be a prognosticator for decreased progression free survival on univariate survival analyses (p-value 0.007). These results were confirmed on whole sections. Sequence analysis detected eight periostin isoforms in fetal lung, but only five in both NSCLC and matched normal lung tissue, suggesting splice-specific regulation during lung embryogenesis. Conclusions. Tumor enlargement of NSCLC is associated with an increase of desmoplastic stroma and concomitant upregulation of EMT markers at the invasive front. Periostin is one of the most robustly upregulated ECM protein in many cancers and therefore a potential target for stroma-directed therapy, using e.g. sc-Fv antibodies.

Aktuelle Entwicklungen in der Forschung mit Vortrag des Promotionspreisträgers FR-021 Brightfield triple color gene-protein assay for human epidermal growth factor receptor 2 (HER2) protein, HER2 gene, and chromosome 17 centromere (CEN17): Protocol development and application for breast cancer tissues H . Nitta1, B . Kelly1, M . Padilla2, N . Wick1, P . Brunhoeber1, I . Bai1, S . Singh1, J . Ranger-Moore1, C . Bieniarz1, H . Tsuda3, T . Grogan1 1 Ventana Medical Systems, Inc ., Tucson, United States, 2Roche Diagnostics, Ltd ., Barcelona, Spain, 3National Cancer Center Hospital, Tokyo, Japan Aims. There are advantages and disadvantages with in situ hybridization (ISH) and immunohistochemistry (IHC) methods for the assessment of HER2 status of breast cancer patients. The combination of HER2 IHC and brightfield ISH (BISH) detections in a “gene-protein assay” might be able to overcome disadvantages of each technology by correlating HER2 protein, HER2 gene, and CEN17 targets and maintaining tissue morphology. Our objectives were: 1) to develop an automated triple color HER2 gene-protein assay using formalin-fixed, paraffin-embedded (FFPE) xenograft tumors and 2) to study the performance of HER2 gene-protein assay comparing to single HER2 IHC and BISH assays with breast cancer tissue microarray (TMA) slides. Methods. The development of HER2 gene-protein assay was conducted with commercial HER2 IHC and dual color HER2 BISH reagents using an automated slide processing system. Technical issues related to the HER2 gene-protein assay were solved using FFPE xenograft tumors: 1) MCF7 (non-amplified HER2 gene and negative HER2 protein) and 2) Calu-3 (amplified gene and positive protein). The HER2 gene-protein assay performance was compared to single HER2 IHC and BISH assays using breast cancer TMA slides with 183 breast tumor tissue cores scored by three pathologists. Results. HER2 gene-protein assay successfully localized HER2 protein, HER2 gene, and CEN17 signals appropriately on FFPE xenograft sections. A technical issue of silver background staining caused by the application of silver detection after DAB IHC was solved using naphthol phosphate as a blocker. There were significant high concordances of HER2 protein and gene status among HER2 IHC, BISH, and HER2 gene-protein assays. Also, HER2 gene-protein assay allowed the assessment of HER2 status based on simultaneous observations of HER2 protein and gene on breast cancer with tumor heterogeneity. Conclusions. Discovery of naphthol phosphate as a blocker for suppressing the silver background staining was the key for successful HER2 gene-protein assay development. HER2 gene-protein assay is a robust and reliable assay that provides potential advantages over single HER2 IHC and HER2 BISH assays.

FR-022 Cancer genetics 2012 – What comes after CGH and FISH? E . Schröck1, B . Klink1, K . Becker1, K . Hackmann1, A . Rump1 1 Institut für Klinische Genetik, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Germany Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) have been proven to be powerful tools in cancer genetics. Both methods have been used to obtain either high resolution genome-wide data on DNA gains and losses or single cell data on gains, losses and structural changes of very defined regions. Nevertheless, the future is already there, as we have been applying Next Generation Sequencing to analyse the exome, transcriptome, methylome, and much more. The ability to fully sequence hundreds and thousands of genes in a single test, including the detection of known “hot-spot mutations” in cancer-relaDer Pathologe Suppl 1 · 2012  | 

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Abstracts ted genes will in the near future allow for providing helpful information on individual cancer-genomes for individualized tumor therapy. Also, NGS will become applicable to rather small specimen, including biopsies, circulating tumor cells and circulating free DNA in plasma. Even so NGS is already used in the clinical setting, for instance for the detection of disease gene mutations of familial breast and ovarian cancer and for genetic diseases, data analysis and interpretation using biostatistics, bioinformatics, systems biological and mathematical approaches including mathematical modeling is still rather complex. Robust strategies for data analysis are being needed and are being developed. Solving the key problems in cancer biology will therefore only be accomplished by orchestrating a manifold collaboration between different disciplines (life sciences, mathematics, and informatics). Platform comparison will be provided for expression data comparing RNA seq and array based methods. We will glimpse into the future and discuss third generation sequencing and single-molecule sequencing technologies. Important goals are to improve our knowledge on the regulation and function of genes and proteins in single cells, tissue and organs. While NGS is just on its way to be established as a routine diagnostics tool, the next next-gen sequencing techniques are already emerging. Whereas current NGS techniques depend on optics and thus require elaborate signal detection, the third and fourth generation techniques simply measure changes of electric current, either when DNA passes through nanopores or bends nanowires in a sequence dependent manner. Being electronic devices like computer processors, the third and fourth generation sequencing machines will be very fast, very small and rather cheap.

FR-023 Prognostoc biomarkers of gastric cancer V . Warneke1, H .-M . Behrens1, C . Röcken1, J . Haag1, K . Balschun1, C . Böger1, C . Böger1, T . Becker2, J . Hartmann3, M . Ebert4, C . Röcken5 1 Christian-Albrechts-University, Department of Pathology, Kiel, 2ChristianAlbrechts-University, Department of Surgery, Kiel, 3Christian-Albrechts-University, Department of Internal Medicine II, Kiel, 4University of Mannheim, Dept . of Medicine II, 5University Hospital Schleswig-Holstein, Campus Kiel, Department of Pathology, Kiel Aims. Chemotherapy for the treatment of gastric cancer (GC) is evolving rapidly and continues to improve patient survival. We studied phenotypic and genotypic biomarkers for GC, in order to test whether these biomarkers are independent prognosticators of patient survival and whether any of these biomarkers should be considered to tailor patient treatment in the future. Methods. 485 patients (299 men, 186 women; median age 68 years) with GC had undergone either total or partial gastrectomy for adenocarcinomas of the stomach or oesophagogastric junction. Survival data and date of death were available for 469 patients. The pTNM-stage was based on surgical pathological examination. The Laurén and mucin phenotype was assessed. H.  pylori- and Epstein-Barr virus infections were documented. The following markers were studied: BRAF-, KRAS-, NRASand PIK3CA (exon 9 and 20)-genotype, microsatellite instability, Her2/ neu-status, E-cadherin, β-catenin and EpCAM-expression. Results. An intestinal type GC was found in 184 patients, a diffuse type in 224. A persistent H. pylori-infection was found in 64 (15.5%) patients, an EBV-infection in 15 (4.0%). Seventeen (3.6%) GCs showed a KRAS-, 12 (2.5%) a PIK3CA (exon 9)- and 9 (1.8%) a PIK3CA (exon 20)-mutation. No NRAS- and BRAF-mutation was found in our series. 424 (95.1%) GCs were EpCAM- and 46 (10.2%) Her2/neu-positive. 33 (7.3%) GCs were highly microsatellite unstable, 31 (93.9%) of which showed loss of expression of MLH1 and PMS2. Patient survival correlated with Laurén phenotype, MSI-H and BerEP4-expression. Patient age, stage grouping according to the Kiel-proposal, lymph node ratio and Mucin 2 were independent prognosticators of patient survival.

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Conclusions. A thorough staging and surgical pathological examination is the most important tool to assess patient prognosis. KRAS, PIK3CA (exon 9 and 20), BerEP4-, Her2/neu- and MSI-status may be considered to tailor patient treatment in the future.

FR-024 Deep-sequencing: Speed-up diagnostics of colorectal carcinoma M . Rechsteiner1, A . Bohnert1, A . von Teichmann1, S . Schmid-Brun1, P . Schraml1, H . Moch1 1 University Hospital Zurich, Surgical Pathology, Zürich, Switzerland Aims. In colorectal carcinoma, KRAS mutations have emerged as a major predictor of resistance to anti-EGFR antibody treatment. Although the role of BRAF mutations, in predicting the response to anti-EGFR drugs still remains controversial, patients with a mutated BRAF gene exhibit a significant shorter survival than patients without a mutation. In this project we aimed to establish a high-troughput ultra-deep sequencing platform to cope with the increased demand for sequence information at medical institutions. With this platform we intend to unravel low frequency mutations below the detection limit of Sanger sequencing and to elucidate throughput power for diagnostics. Methods. A cohort of 120 patients, diagnosed with colorectal carcinoma, was established. The cohort consisted of 45 patients with KRAS mutations in exons 2 or 3 and 75 patients without a KRAS mutation. This was assessed by Sanger sequencing. The 75 patients with a wild-type KRAS gene were further analysed for BRAF mutations in exon  15 by Sanger sequencing. Results. Fifty ng of genomic DNA isolated from FFPE tissue blocks were found to give reproducible results as input material of the PCR to generate amplicons used for deep-sequencing. The target amplicons were KRAS exons 2 and 3 and BRAF exon 15. The exons 5 to 8 of the p53 gene were also analysed due to high mutation rates in colorectal carcinoma. The amplicons of each patient were labelled with multiplex identifiers (MIDs), and these were shown to be highly specific in the data analysis after deep-sequencing. Seven amplicons of 9 patients were pooled in one single 454 Junior Sequencing run. On average, each amplicon was covered 1000-fold which allowed us to identify mutations at a 4% frequency. Integrated computational down-stream analyses enabled us to speed up the detection and classification of mutations. Results from the first 17 patients yielded 14 mutations located in the p53 gene (82%) and 1 in the BRAF gene (6%). The BRAF mutation was identified as the well known activating mutation V600E. We also included a patient with a known KRAS mutation as control which was successfully verified by deep-sequencing. Conclusions. This newly established method allowed us to analyse 7 amplicons of 9 patients (63 amplicons in total) in one deep-sequencing run within 1 week. The capacity limit of a Junior 454 is 4 runs per week which would allow us to analyse the mutation status for the KRAS, BRAF, and p53 genes of 36 patients with colorectal carcinoma

Promotionspreis FR-026 Colocalization algorithms for conversion of traditional immunohistochemistry into virtual multicolor stains A .-S .K . Meyer1, P . Möller1, J .K . Lennerz1 University Ulm, Institute of Pathology, Ulm

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Aims. Diagnostic immunophenotyping is performed via “mentally” combining single immunohistochemistry (IHC) stains. The discrepancy to research settings, were co-visualization predominates, is due to technical- (i.e. species identity, detection systems) and/or practical hurdles

(i.e. investment in equipment and expertise). Here we present simple pixel-algorithms that allow electronic merging and color-range conversion of traditional IHC stains. Methods. Single-label IHC-stains, performed on subsequent sections, are digitized and used as input data. After manual positioning, a merge algorithm compares the input images and selects pixels with higher values. A re-coloring algorithm reassigns color ranges. Algorithms are achieved via a customized link between software platforms (Aperio ImageScope10.0; Adobe PhotoshopCS3; ImageJ Version10.2) using AutoIT (v3.2.12.0), a freeware scripting language for automating the Microsoft Windows GUI. Results. The merge algorithm emulates double staining, which includes preservation of the traditional IHC-views (operative comfort zone). To allow distinction of similarly labeled stains in the merge, a re-coloring algorithm converts the color range of stained elements from each image (e.g. brown vs. red). As a specific re-coloring mode, stained elements can be extracted and converted into pseudo-immunofluorescence (IF), which includes all downstream assessments of co-localized elements (virtual IF-stain). The algorithms solve the common problem of species identity of primary antibodies because they allow co-visualization of markers without compromising staining specificity, while at the same time employing individual stains that remain available for traditional evaluation. When compared to investments in IF-equipment or validation of physical double-stains, the effort to learn and perform the conversion algorithms is negligible. Conclusions. The presented imaging tools emulate the principal advantages of multi-color fluorescence microscopy as well as multi-color IHC. These techniques represent a powerful expansion of one of the most versatile molecular tools in diagnostic pathology. Thereby, the combination of established methods (IHC) and imaging algorithms also exemplifies the potential of digital pathology.

Translationale Forschung und AG Molekularpathologie FR-027 Delay to preservation does not induce a systematic phosphoprotein response during tissue processing S . Gündisch1, C . Schott1, K . Grundner-Culemann2, M . Machatti2, D . Groelz3, C . Schaab2, A . Tebbe2, K .-F . Becker1 1 Technische Universität München, Institute of Pathology, München, 2 Evotec AG, Martinsried, 3PreAnalytiX GmbH, Hombrechtikon, Switzerland Aims. The quality of tissue samples can have a significant impact on analytical data sets for biomarker research. In particular, posttranslational modifications such as phosphorylation need to be systematically investigated in that phosphorylated protein levels indicate the activation status of signal transduction pathways controlled by kinases. However, little is known about the impact of pre-analytical factors on phosphoprotein stability. The aim of this study was to characterize the potential effects of delayed preservation and different preservation methods on the stability of phosphoproteins using targeted and non-targeted proteomic approaches. Methods. Murine and rat liver samples were exposed to different ischemic conditions before preservation and either cryopreserved, formalinfixed or fixed with the PAXgene Tissue System, a new non-crosslinking formalin-free fixative. The phosphoproteome was analyzed using quantitative tandem mass spectrometry (LC-MS/MS) and reverse phase protein array (RPPA) technology. Results. The phosphoproteomic analysis of ischemic mouse liver tissue samples by LC-MS/MS indicated no significant global alterations of more than 5000 phosphosite ratios analysed during 60 minutes of delayed cryopreservation. The analysis of ischemic rat liver tissue samples

by RPPA revealed similar results as investigated phosphoproteins, including phospho-Akt, phospho-p38 MAPK or phospho-p44/42 MAPK, showed very stable profiles during the time-course experiment, independent of the preservation method applied. Conclusions. Since we could not detect significant global changes of the phosphoprotein profiles, neither with a targeted nor a non-targeted approach, we conclude that the phosphoproteome seems to be more stable than expected with regard to delayed preservation. This allows accurate quantitative measurements of the activation state of signalling pathways of tissue samples which had not been immediately preserved. This result is essential for the development of new targeted therapies involving kinase inhibitors which have recently been a focus in the field of personalized medicine. Studies are ongoing to validate our results in human tissue samples as inter-patient variability may occur which is absent in our well controlled model systems. This work has received funding from the Munich Biotech Cluster m4 (www . m4 .de) and the European project SPIDIA (www .spidia .eu) .

FR-028 Validation of a novel DNA methylation based 2-gene biomarker panel for early detection of bladder cancer using urine samples M . Rose1, D . Fiedler1, N .T . Gaisa1, C . Schubert1, P . Antony1, R . Davtalab1, D . Pfister2, A . Heidenreich2, R . Knüchel1, E . Dahl1 1 RWTH Aachen University/Institute of Pathology, Aachen, 2 RWTH Aachen University/Clinic of Urology, Aachen Aims. The early detection of bladder cancer and its recurrent tumours is currently based on cystoscopy, which is highly sensitive and specific, but also invasive and expensive. Alternative methods still lack suitable sensitivity especially with regard to non-invasive bladder tumours. In the line with accumulating evidence suggesting that DNA methylation pattern could serve as sensitive and specific biomarkers, we aimed to identify novel DNA methylation loci potentially useful for early cancer detection and treatment stratification of bladder cancer patients, respectively. Methods. In order to discover potential biomarkers we analyzed the methylation levels of array-based candidate genes by using MSP in bladder cell lines (n=5), non-cancerous (n=20) and cancerous bladder tissues (n=60). Subsequently, we determined the methylation status of selected genes performing MSP assays on a so called “screening cohort” containing DNA extracted from urine sediments of bladder cancer patients (n=60). Age-matched non-urological-cancer patients (n=30) as well as prostate cancer patients (n=15) were included as controls to ensure specificity. The biomarker potential of the most frequently methylated gene loci were then discriminated by using quantitative pyrosequencing in a “validation urine sample cohort” (currently; n=30) of bladder cancer patients in comparison to controls. The performance of a 2-gene-panel was optimised using receiver operator characteristics (ROC) curve analysis. Results. MSP assays performed on bladder cells lines and bladder cancer tissue revealed novel candidate genes exhibiting frequently cancer specific promoter hypermethylation. In the urine screening samples of bladder cancer patients, these gene loci showed frequent methylation with an overall-panel sensitivity of 81.5%, i.e. 81.5% of samples presented promoter methylation in at least one of the two genes. Importantly, none of the age-matched controls exhibited methylation signals excluding rare and weak age-depended side effects. By using quantitative pyrosequencing technique we were able to increase the sensitivity of the 2-gene panel up to 83.3% (p≤0.001, AUC=0.940) with 100% specificity. Conclusions. We have identified a 2-gene putative biomarker panel based on detection of DNA promoter methylation. Applying this panel in urine sediments using pyrosequencing may provide a highly sensitive and specific, non-invasive approach for early detection of primary bladder tumours.

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Abstracts FR-029 Whole exome sequencing identifies potential therapeutic targets for castration resistant prostate cancer R. Menon1, M. Deng1, D. Boehm1, F. Fend2, D. Boehm3, S. Biskup3, S. Perner1 1 Institute of Prostate Cancer Research, Institute of Pathology, University Hospital of Bonn, Bonn, 2Institute of Pathology, University Hospital Tübingen, Tübingen, 3Center for Genomics and Transcriptomics, Tübingen Aims. Castration resistant prostate cancer (CR-PCa) is the most aggressive form of prostate cancer (PCa) having a poor prognosis, and is a significant therapeutic challenge. The key to the development of novel therapeutic targets for CR-PCa is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to perform whole exome sequencing and gene copy number analysis on 5 CR-PCa/normal paired formalin fixed paraffin embedded (FFPE) samples using the SOLiD4 next generation sequencing platform. Methods. Genomic DNA was extracted from 5 CR-PCa/normal paired FFPE samples. The DNA was subjected to targeted exon capture using the Agilent Sure Select kit. The captured DNA was sequenced using the SOLiD4 next generation sequencing platform. The sequencing output was mapped, sorted, filtered and annotated using well-known human genome databases. The results were further analyzed for SNPs and copy number variations. A set of amplified/deleted genes were validated using fluorescence in-situ hybridization (FISH) assays with a PCa progression cohort. The cohort consisted of 138 cases for localized cancer, 105 patients with primary PCa and corresponding LN metastasis, and 39 samples for castration resistant tumors. Results. Whole exome sequencing analysis identified focal regions of deletion, which included well-known tumor suppressors such as NKX3.1 and PTEN. Focal regions of amplification included well-known genes such as cmYC and AR that are known to play a role in PCa. Furthermore, we identified several amplified genes as druggable targets e.g. HDAC6, NTRK1, PLD1, SPHK1, and SIRT7. NTRK1 is a kinase that plays an active role in cell proliferation. HDAC6, PLD1, SPHK1 and SIRT7 regulate numerous complex cellular processes including signal transduction, transcription and apoptosis. Conclusions. This is the first study to use whole exome sequencing approaches on FFPE CR-PCa material to identity novel therapeutic targets. Validation studies would further shed light into the biological understanding of the disease and its plausible treatment options.

FR-030 Cut-offFinder – a web application for cut-off optimization for molecular markers J. Budczies1, F. Klauschen1, W. Schmitt1, C. Denkert1 Charité Hospital, Institute of Pathology, Berlin

1

Aims. In order to translate a continuous diagnostic variable into a clinical decision, it is necessary to determine a cut-off point and to stratify patients into two groups, each of which requires a different kind of treatment. Methods. Cut-offFinder is implemented as Java Server Pages (JSPs) that connect to the statistical engine R. Using a web browser, the user can upload a molecular data set, assign biomarker and outcome variables and determine an optimal cut-off point for the biomarker. The web application offers three different methods for cut-off determination: The first method fits a mixture model of two Gaussian distribution to the distribution of the variable. The optimal cut-off is determined as the value where both probability density functions coincide. For the two other methods, all possible cut-off points are scanned. The second method correlates the dichotomized biomarker with a binary outcome variable using logistic regression. The optimal cut-off is defined as the point with the most significant (Fisher’s exact test) split. The third method fits Cox proportional hazard models to the dichotomized variable and the sur-

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vival variable. Then, the optimal cut-off is defined as the point with the most significant (log rank test) split. Results. As example, we have analyzed gene expression data of estrogen receptor (ESR1) and progesterone receptor (PGR) from a publicly available microarray data set of 286 breast cancer samples (GSE2034 at www. ncbi.nlm.nih.gov/geo) using Cut-offFinder. Histograms of the distribution of ESR1 and PGR showed a clear bimodal shape. Distribution derived cut-offs were located at 10.6 (ESR1) and 5.0 (PGR). The dependence on the cut-off of the odds ratio (OR) for correlation ERS1 expression with ER status (determined by immunohistochemistry) was analyzed and visualized. The optimal cut-off was determined as 10.1 with OR=67.8 (30.2–152.1). Using ERS1 expression measured by the microarray, determination of ER status was feasible with a sensitivity of 85.7% and a specificity of 91.9%. The dependence on the cut-off of the hazard ratio (HR) for correlation of PGR expression with distance-metastasis-free survival was analyzed and visualized. The optimal cut-off was determined as 2.5 with HR=0.46 (0.30–0.71). Kaplan Meier analysis showed a significantly better outcome for patients with high PGR expression (p=0.00028). Conclusions. In summary, Cut-offFinder is a comprehensive and easyto-use web application for cut-off determination for molecular markers.

FR-031 BRAF-testing with pyrosequencing: a reliable alternative for the analysis of highly pigmented and degraded FFPE material A. Lehmann1, C. Schewe1, K. Jöhrens1, C. Denkert1, J. Budczies1, M. Dietel1 Charité Universitätsmedizin Berlin, Institute of Pathology, Berlin

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Aims. The approval of new BRAF inhibitors for the treatment of metastasized melanoma has led to a great demand for BRAF testing in molecular pathology laboratories. However, molecular analysis of formalin-fixed, paraffin-embedded (FFPE) melanoma tissue is challenging. Sanger sequencing often fails due to high amplification lengths and the influence of melanin. In this study we tested the applicability of a new pyrosequencing assay for BRAF analysis on 118 FFPE melanoma tissues and compared the results with those of Sanger sequencing. Methods. The study comprised 118 formalin-fixed, paraffin-embedded tissues of malignant melanoma which were referred to our department of Molecular Pathology for routine BRAF testing. DNA was extracted (QIAamp® DNA Mini Kit, Qiagen) and samples were subjected to both, Sanger sequencing (in-house method) and pyrosequencing (therascreen BRAF Pyro Kit®, Qiagen) of BRAF exon 15, codons 599 and 600. Results. BRAF sequences of 102 of 118 samples (86.4%) were evaluable by both methods pyrosequencing and Sanger sequencing. Mutational status of these samples was consistent in 98.0% (Cohen’s kappa coefficient  =0.96, p=0.000). Sanger sequencing failed for 15 samples, which were mostly highly pigmented. Interestingly, 11 of these samples were still evaluable with pyrosequencing. With a success rate of 95.8% (CI95 [90.5–98.2%]), significantly more cases could be evaluated by pyrosequencing than by Sanger sequencing [success rate Sanger =87.3%, CI95 (80.1–92.1%); p=0.035]. Conclusions. Pyrosequencing requires comparably short target sequences for amplification which makes this method feasible even for problematic material for which standard methods like Sanger sequencing often fail. Particularly with regard to the high impact of BRAF-testing for therapy decision, pyrosequencing is a fast and reliable alternative for BRAF-testing.

FR-032 COLD-PCR: a powerful tool in routine-diagnostic for cost-neutral detection of minor clones using real-time PCR or pyrosequencing quoted by the example of EGFR mutation analysis F. Mairinger1, A. Streubel2, A. Roth2, O. Landt3, W. Grüning4, J. Kohlmeier4, T. Mairinger2 1 University Hospital Essen, Department of Pathology und Neuropathology, Essen, 2Helios Klinikum Emil von Behring, Department of Pathology, Berlin, 3 TIB MOlBIOL Gmbh, Berlin, 4Helios Klinikum Emil von Behring, Department of Pneumology, Berlin Aims. COLD-PCR (co-amplification at lower denaturation temperature-PCR) is a novel method to enrich minority alleles from mixtures of wild-type (wt) and mutation containing (mt) sequences, irrespective of localization and property within the analyzed sequence. The heteroduplexes generated after an initial denaturation step will be preferentially melted at the critical denaturation temperature resulting in a radical enrichment of minor variants, enabling their detection with conventional methods which are intended to analyze normal bi-allelic variations. Molecular testing of tissues is faced with samples containing mixtures of tissues frequently containing only small fractions of mutations. A study was designed to overcome this problem of the too low sensitivity of nowadays routinely used molecular methods. For this, the effect of COLD-PCR in probe-based real-time PCR analysis or as initiating step for pyrosequencing to the sensitivity was tested. Methods. Different dilution steps of artificial EGFR T790M mutated and wild-type EGFR exon20 DNA were analyzed by a LightCycler assay or amplified by full-COLD-PCR using different protocols and afterwards sequenced by pyrosequencing to determine the detection-limit of these methods. Results. For the LightCycler-assay, the best results are rendered with a combination of 10 cycles conventional PCR followed by 45 cycles COLDPCR using 84°C as denaturation temperature. A dilution of down to 0.125% mt-DNA/total-DNA is still detectable. With COLD-PCR amplified DNA, a dilution of 0.125% mt-DNA/total-DNA is still detectable by pyrosequencing in reproducible results. Conclusions. Our results show the exceeding potential of COLD-PCR in enrichment of DNA of underrepresented clones in clinical samples. Because of this, problems like dilution of potentially mutated tumor cells (showing for example EGFR-resistance mutation T790M) with non-resistant tumor cells or benign cells debt to macrodissection or tumor heterogeneity resulting in failing to detect clinically relevant minor clones could be overcome.

FR-033 Fast and reliable detection of mutations in exon 9 of the KIT gene by high resolution melting analysis H. Künstlinger1, M. Kleine1, J. Fassunke1, E. Wardelmann1, R. Büttner1, S. Merkelbach-Bruse1, H.-U. Schildhaus1 1 University Hospital Cologne, Institute of Pathology, Köln Aims. Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the gastrointestinal tract. They harbour activating mutations in the KIT or platelet-derived growth factor (PDGF) receptor. Imatinib mesylate is a potent inhibitor of KIT signalling and is therefore widely used as targeted therapy for GISTs. The mutational status of KIT exon 9 is of special importance for the therapy with Imatinib, because cases with exon 9 mutations need a higher dose of Imatinib. Therefore, in metastasized or high-risk GISTs as well as in primary notoperable tumours it is necessary to obtain the mutational result as fast as possible after diagnosis. At present, mutational analysis of KIT and PDGFR is routinely carried out by Sanger sequencing. This method has certainly its lasting relevance for DNA sections with high variability of mutations (e.g. KIT exon 11), but is relatively expensive and time consuming. Therefore alternative methods need to be established for other

exons (like KIT exon 9) or other certain mutational types to enable a fast and cost efficient detection. Methods. High Resolution Melting (HRM) is a post-PCR mutation scanning tool that detects the change in fluorescence caused by the progressive release of a saturating intercalating dye from DNA duplexes while they are denatured by slight increases in temperature. HRM assays were developed using specifically designed primers and genomic DNA isolated from formalin-fixed paraffin-embedded GIST samples. Melting curve analyses were performed on the LightCycler 480 platform (Roche) and mutation analyses were additionally confirmed by Sanger Sequencing. Results. Conditions for High Resolution Melting analysis of KIT exon 9 could be established using more than 60 GIST samples with known mutational status of the KIT gene. Sensitivity was determined as a minimal proportion of 12.5% mutated alleles. A prospective screening of more than 100 additional GIST samples showed a complete concordance between HRM assay and traditional Sanger sequencing. Conclusions. The established high resolution melting assay represents a highly reliable method for the detection of mutations in exon 9 of the KIT gene. It allows a faster and more cost-effective mutational analysis of KIT exon 9 in the future, which is especially important for dose finding of Imatinib in GIST therapy. The determined sensitivity is comparable to the sensitivity of currently performed Sanger sequencing.

FR-034 Morphological and clinical characterization of a novel mouse model for mutation-activated JAK1 S. Wagner1, E. Janas2, B. Lorenz-Depiereux3, J. Calzada-Wack2, A. Benet-Pagès3, S. Eck3, J.A. Aguilar Pimentel4, B. Rathkolb4, V. Gailus-Durner4, H. Fuchs4, H. Höfler2, M. Hrabé de Angelis1, T. Strom3, F. Neff2 1 Helmholtz Zentrum München, Institute of Experimental Genetics, Neuherberg, 2Helmholtz Zentrum München, Institute of Pathology, Neuherberg, 3 Helmholtz Zentrum München, Institute of Human Genetics, Neuherberg, 4 Helmholtz Zentrum München, German Mouse Clinic, Neuherberg Aims. The members of the Janus kinase family (JAK1, JAK2, JAK3) play important roles in signalling downstream of cytokine receptor activation and are implicated in various physiological processes including the hematopoietic, immune and neuronal systems. However, the lack of successful mouse models for mutation-activated JAK1-induced diseases hampers the understanding of disease pathology. Here, we have produced a novel mutant mouse line leading to an amino acid substitution in the pseudokinase domain of JAK1 using N-ethyl-N-nitrosourea (ENU) mutagenesis. This mutation corresponds to a JAK1-activating mutation (Ser646Phe) described in humans and associated with acute lymphoblastic leukemia (Mullighan et al. 2009). Methods. The ENU mutagenesis was generated in C3HeB/FeJ genetic background. Mutation screening was performed after linkage analysis using single nucleotide polymorphisms (SNP) and chromosome sorting by next generation sequencing. A total of 64 mice at the age of 18 to 31 weeks were analyzed for clinical and immunological parameters as well as histology. Thirty organs were examined by H&E staining and immunohistochemistry. Results. All mutant mice showed a loss of ear cartilage starting with 4 months of age without signs of inflammation, a significant loss in body weight due to an alternation in body composition. In serum, a significant increase of auto-antibodies was observed. Most strikingly, histopathological analysis revealed a nodular regenerative hyperplasia of the liver with a remarkable increased neovascularisation. This vascularisation was also observed in the skin of ears indicating a systemic vasculitis. Conclusions. A significantly increase in auto-antibodies together with the pathological changes observed implicates that the introduced mutation in the pseudokinase domain of JAK1 induces a systemic autoimmune disease.

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Abstracts FR-035 MAP3K7 deletions have prognostic relevance in prostate cancer M . Kluth1, A . Krohn1, J . Hesse1, R . Simon1, T . Schlomm2, H . Huland2, G . Sauter1, S . Minner1 1 University Medical Center Hamburg-Eppendorf, Hamburg, 2Martini-Clinic, Hamburg Aims. MAP3K7 (mitogen-activated protein kinase kinase kinase 7) gene is a component of the MAPK-pathway and plays a central role in cell growth, differentiation and apoptosis. The MAP3K7 gene is located at 6q15, a region, which is commonly deleted in prostate cancer. The aim of this study was to examine the potential relevance of MAP3K7 (6q15) deletions in prostate cancer with respect to tumor phenotype and clinical outcome. Methods. A prostate tissue microarray (TMA) with clinical follow-up data consisting of over 4500 prostate cancer samples was analyzed for MAP3K7 deletions by fluorescence in situ hybridization (FISH). Results were correlated with tumor phenotype, clinical outcome and ERG expression. In addition, 15  tumors with known MAP3K7 deletion and 14 tumors without MAP3K7 deletions were examined for the presence of MAP3K7 mutations (Exon 1–17). Results. Heterozygous MAP3K7 deletions were found in 18.5% (423/2289) of all analyzable prostate cancers. MAP3K7 deletions were associated with advanced tumor stage (p<0.0001), high Gleason grade (p<0.0001), high preoperative PSA levels (p=0.0017) and early biochemical recurrence (p<0.0001). MAP3K7 deletions were inversely associated with ERG expression (p<0.0001). No mutations were found in 29 sequenced cancers. Conclusions. These data suggest that MAP3K7 represents one target gene of the 6q15 deletion since deletions occur in a fraction of prostate cancers with unfavourable tumor phenotype. While our data suggest a clinical significance for MAP3K7 deletions the mechanism for biallelic inactivation remains unclear.

Aktuelle Habilitationen II SA-001 Pathogenesis of diabetes mellitus and diabetic complications: Studies in diabetic mouse models N . Herbach1 LMU Munich, Institute of Veterinary Pathology, München

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Aims. The pathogenesis of diabetes mellitus and diabetic complications to date is only partially understood. Diabetic mouse models, created via random mutagenesis or genetic modification, are essential tools to unravel the mechanisms involved in the development of diabetes and associated diseases. Methods. Three diabetic mutant mouse lines derived from Munich ENU mouse mutagenesis project and one transgenic mouse line were used for the study. The causative mutation was identified in the mutant mouse lines, the diabetic phenotype was analysed in detail and treatment strategies were tested. Glucose homeostasis, postnatal pancreas development and renal lesions were analysed in transgenic mice, expressing a dominant negative glucose dependent insulinotropic polypeptide receptor (GIPRdn) and the effects of dietetic intervention were investigated. Results. An Ins2 mutation and two Gck mutations were identified as cause of diabetes mellitus in the mutant lines. Male Munich Ins2C95S mutants, a model for permanent neonatal diabetes mellitus (PNDM), developed ER stress in pancreatic islets, leading to early onset insulin deficiency, diabetes mellitus, progressive oxidative stress, insulin resistance, hyperglucagonaemia and beta-cell loss. Treatment with insulin or a sodium-dependent glucose transporter 2 (SGLT2) inhibitor ameliorated the diabetic phenotype, insulin resistance, alpha-cell dysfunction, oxidative stress and beta-cell loss of Ins2 mutants. Heterozygous Gck mu-

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tants, models for maturity onset diabetes of the young type 2, developed clinical features that closely resemble the human disease. Homozygous Gck mutants exhibited PNDM and either showed drastically reduced life-expectancy or no change in survival vs. wild-type mice, depending on the location of mutation. In addition, the development of pancreatic islets was slightly disturbed in heterozygous Gck mutants and homozygous mutants of the surviving line showed a severe reduction of beta-cell mass. GIPRdn transgenic mice exhibited early onset insulin deficiency and diabetes mellitus due to defective postnatal expansion of beta-cell mass. Further, progressive diabetes associated renal lesions were observed in GIPRdn transgenic mice. Diabetes and diabetic kidney disease were successfully treated with a high fibre diet. Conclusions. Mutant and transgenic diabetic mouse models analysed in the study were shown to represent valuable models to study the pathogenesis of monogenic diabetes and to establish novel treatment strategies.

SA-002 Tumor cell plasticity and angiogenesis in human melanomas D . Mihic-Probst1, K . Ikenberg1, P . Schraml1, S . Behnke1, H . Moch1, G . Civenni2, L . Sommer2, B . Seifert3, R . Dummer4 1 University of Zurich/Surgical Pathology, Switzerland, 2University of Zurich/ Anatomy, Switzerland, 3University of Zurich/Biostatistic, Switzerland, 4University of Zurich/Dermatology, Switzerland Aims. Recent molecular studies provide evidence for a significant transcriptional plasticity of tumor cell subpopulations. This feature is accompanied by morphological changes both in vitro and in vivo. We were interested to investigate if melanoma cells are contributing to neovascularisation. Methods. We analyzed the morphological plasticity of tumor cells with special focus on vasculogenic mimicry and neovascularisation in human melanoma and mouse xenografts of human melanoma cell lines. In melanoma xenograft experiments, species-specific vessel markers (mouse MECA32 and human CD31) and green fluorescent protein expression were used to investigate whether melanoma cells are contributing to neovascularization. Additionally, we analyzed neovascularization in 49 primary melanomas and 175 melanoma metastases using immunostaining for blood (CD34) and lymphatic (podoplanin) vessel-specific markers. Results. We demonstrate that melanoma cells are contributing to neovascularisation. In addition we found significantly more lymphatic vessels in primary melanomas than in melanoma metastases (p<0.0001). In contrast to the near absence of lymphatic vessels within metastases we found extensive blood micro-neovascularization. Blood micro-neovascularization was absent in micro metastases (less than 2 mm). A significant inverse correlation between Glut-1 expression (implying local hypoxia) and the presence of microvessels indicate their functional activity as blood vessels (p<0.0001). Conclusions. We suggest that the hypoxic microenvironment in metastases contributes to a phenotype switch allowing melanoma cells to physically contribute to blood vessel formation.

SA-003 Combination of ex vivo sentinel lymph node mapping and methylene blue-assisted lymph node dissection in gastrointestinal cancers B . Märkl1, H . Arnholdt1 1 Klinikum Augsburg, Institute of Pathology, Augsburg Aims. Lymph node staging is of crucial importance in all cancers of the gastrointestinal tract. Minimal numbers of evaluated lymph nodes (LNs) for an adequate staging are still matters of controversial debates. Nevertheless, exact numbers are defined by the UICC for all entities. On the other hand it is well known that these numbers are often not reached in daily practice. We recently introduced methylene blue assisted LN

dissection (MBLND) technique to improve the LN staging in gastrointestinal cancers. Moreover, we combined this technique with an ex-vivo sentinel mapping method to further improve the staging. Methods. The technique has been established in pilot studies and than confirmed in prospective and randomized studies. The injections were performed in fresh state. Subserosal or submucosal injection of black ink was performed for the sentinel mapping. Methylene blue (MB) solution was injected afterwards into the main arteries. Results. LN harvest was highly significant improved in the MB groups in comparison to the control groups (colon cancer: 35±18 vs. 17±10; rectal cancer: 30±14 vs. 17±11). MB technique ensured sufficient LN harvest in almost all cases including cases of neoadjuvantly treated rectal cancer. The evSLN detection rate, sensitivity and accuracy in gastric cancer were 87%, 81% and 93%, respectively. Isolated tumor cells were detected after immunohistochemical staining in 3 of 17 cases (18%). The chance to detect a metastasis in an evSLN was 2 times higher in comparison to conventional LNs. In 8% of all cases only evSLN were affected by metastases. Conclusions. MBLND is a highly effective method of improving the LN harvest in gastric cancer. Further application of evSLN mapping is feasible and has the potential to heighten the sensitivity of metastasis detection.

SA-004 EBV-assoziierte Tumoren in Interaktion mit dem Immunsystem M . Büttner1 University Hospital Erlangen, Institute of Pathology, Erlangen

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Aims. EBV-associated tumours are characterized by a prominent inflammatory infiltrate, which, however, is not able to eliminate the tumour cells. Viral proteins like latent membrane protein 1 (LMP1) can interact with intracellular signalling pathways and thereby modulate the immune reaction. In order to better understand immunological processes in EBV-associated tumours different mechanisms were investigated. Methods. Paraffin embedded material of classical Hodgkin lymphomas (cHL), nasopharyngeal carcinomas (NPC) and gastric carcinomas was investigated by immunohistochemistry and in situ hybridization with regard to the expression of EBV-encoded and cellular genes, which are involved in the regulation of the immune reaction. Cell culture experiments were performed for a better understanding of the mechanisms involved on cellular basis. Results. Hodgkin-Reed-Sternberg (HRS) cells of cHL are B cell derivates with a phenotype which is neither typical for a germinal center B-cell nor of a plasma cell, but rather reminds of an abortive plasma cell phenotype. STAT3 a transcription factor with a central role in the linkage of inflammation and tumorigenesis is expressed in cHL and NPC. LMP1 can induce the chemokines RANTES and MCP-1 in an at least partially NFkappaB- dependent manner. However, those chemokines are predominantly expressed in the inflammatory infiltrate rather than the tumour cells. In EBV-associated cardiac carcinoma an increased number of cytotoxic T cells is found, which might be a sign of an on-going antiviral response. Conclusions. In EBV-associated tumours a complex interaction of cellular and viral factors takes place, which can modulate the intratumoural immune reaction. A better understanding of those processes could help to find potential immune evasive mechanisms applied by the tumours, which could interfere with an effective immunotherapy.

Aktuelle Entwicklungen in der Forschung II – Gastrointestinaltrakt SO-001 TNF induces STAT3-mediated inflammation in normal colon epithelial cells S . Chakilam1, A . Agaimy1, R . Atreya2, M . Gandesiri1, J . Ivanovska1, M . RaveFränk3, H . Christiansen3, T .T . Rau1, R . Schneider-Stock1 1 University of Erlangen-Nuremberg, Institute of Pathology, Erlangen, 2 University of Erlangen-Nuremberg, Department of Medicine I, Erlangen, 3 University Göttingen, Department of Radiation Oncology Aims. Tumor necrosis factor α (TNF) is a pleiotropic cytokine that participates in a wide range of biological activities, including inflammation, growth, differentiation, and apoptosis. TNF is a key molecule in the cytokine network, capable of regulating the expression of other cytokines, such as IL-6 which is known to be a major mediator of inflammation through the activation of the STAT-3 pathway. To simulate the effects of TNF to normal intestine we treated immortalized human colon epithelial cells (HCEC) with 0.66 ng/ml TNF and analyzed the possible TNFmediated functions and signal transduction. Methods. HCEC cells were stimulated for various time points (1, 6, 24, 48 and 72  h). Activation of transcription factors (NF-kB, ATF-2, and STAT3) was assessed by western blotting. Inflammation was detected by real-time RT-PCR and cytokine ELISA. Transcriptional transactivation was measured by EMSA and chromatin immunoprecipitation. Apoptosis was analyzed by Annexin V binding assay and M30 staining. Results. TNF-induced a biphasic pattern of activation of transcription factors NF-kB, ATF-2, and STAT3 with NF-κB and ATF-2 phosphorylation at 1 and 6 h, whereas STAT3 activation (pSTAT3Tyr705) and transcriptional activity was induced later at 48 h. TNF also induced secretion of the pro-inflammatory cytokines IL-6 and IL-8. We assumed that the released IL-6 at early time points causes STAT3 activation. Indeed, IL-6 neutralisation significantly attenuated TNF-induced STAT3 phosphorylation. Moreover, AG490 (JAK inhibitor) pre-treatment decreased TNF-induced STAT3 activation and significantly decreased TNF-augmented IL-6 secretion. As expected, IL-6 stimulation alone also increased pSTAT3Tyr705 levels. In parallel, there was no remarkable apoptosis induction despite an increase in DNA-damage measured by pH2AX foci formation. STAT3 signaling was verified in human tissues from ulcerative colitis patients. Conclusions. These data indicate that HCEC cells seem to develop an inflammatory phenotype upon TNF stress. The TNF-induced inflammation is regulated by IL-6 and STAT3. We hypothesize that the normal mucosa is also actively participating in the development and maintenance of inflammatory conditions in the gut.

SO-002 Establishment of a cell culture model to study inflammation- associated oxidative stress as initiator of colorectal neoplasias A . Poehlmann1, K . Reissig1, P . Schoenfeld2, P . Steinberg3, T . Guenther1, A . Roessner1 1 Otto-von-Guericke University Magdeburg, Department of Pathology, Magdeburg, 2Otto-von-Guericke University Magdeburg, Department of Biochemistry and Cell Biology, Magdeburg, 3Institut of Food Toxicology and Analytical Chemistry, Hannover Aims. Accumulating evidence shows that oxidative stress displayed by reactive oxygen species (ROS) is a major contributor to inflammationassociated cancer. Whether oxidative stress in inflammatory bowel disease (IBD)-associated carcinogenesis is only a promoting factor or linked to tumor initiation is still an open question. To find evidence for the transforming capacity and tumor-initiation effects of ROS, we generated Der Pathologe Suppl 1 · 2012  | 

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Abstracts a cell culture model. We mimicked ROS exposure of the epithelium in human IBD using the non-tumor human colon epithelial cell line HCEC and H2O2 as ROS. Methods. The clinical course of IBD with multiple recurrences was simulated by repeated H2O2 exposure cycles of the HCEC cell cultures. We generated ten cell lines and confirmed neoplastic transformation by analyzing them for clonal growth, enhanced proliferation, Colony Formation, ROS-release, and β-galactosidase activity. The p53, Kras, and APC mutation status, as well as microsatellite instability (MSI) were assigned. Results. After three treatment cycles with H2O2, HCEC lost cell-cell contact and started pilling up. Surviving HCEC showed uncontrolled proliferation. Anchorage-independent growth in soft agar is often predictive of tumorigenicity in vivo and is considered the most stringent assay for malignant cell transformation in vitro. As ROS-injured HCEC form colonies in soft agar, we concluded HCEC transformation. By checking further hallmarks of cancer, we detected (i) self-sufficiency in growth signals presumably via ROS-induced ROS-release, (ii) a limitless replicative potential by overcoming cellular senescence, and (iii) evading apoptosis. There were no alterations in p53, Kras, and APC gene, or MSI. Conclusions. Our HCEC cell model provides novel insights into tumorinitiating molecular events in IBD-associated carcinogenesis as these cells are more likely to represent the potential target for tumor initiation in vivo. Oxidative stress alone is sufficient to initiate HCEC transformation. Transformed HCEC do not show well-known genetic alterations. We therefore suppose early epigenetic changes to play an important role in the initiation of the IBD-carcinoma pathway. This would further provide the possibility of unravelling novel subsequent genetic alterations that account for tumor initiation.

SO-003 Acetone compression and its impact on tumor staging of colorectal cancer J. Kitz1, A. Gehoff2, L.-C. Conradi3, T. Sprenger3, O. Basten4, T. Liersch3, P. Middel2, J. Rüschoff2 1 University Medical Center Göttingen, Department of Pathology, Göttingen, 2 Institute of Pathology Nordhessen, Kassel, 3University Medical Center Göttingen, Department of General and Visceral Surgery, Göttingen, 4Institute of Pathology Marburg, Marburg Aims. Acetone compression (AC; Basten et al. Pathologe 2010) is a method that allows for complete lymph node evaluation in colorectal cancer (CRC), which is particularly important in rectal carcinomas pretreated neoadjuvantly (Gehoff et al. Am J Surg Pathol 2011, accepted). In addition, AC also facilitates detection of any further metastases in mesenteric adipose tissue, e.g. perineural infiltration, which has its own prognostic significance (Poeschl et al. JCO 2010). The present study deals with the significance of AC for tumor staging of colorectal cancer. Not only lymph node status but particularly also the significance of tumor cell deposits and perineural infiltration are discussed. Methods. A total of 245 specimens with CRC were analysed prospectively via AC, half of which were rectal carcinomas. In consecutive cases (10–30 tissue samples each) the following parameters were evaluated: a. AC effect on lymph node size and distribution of metastases in comparison to preceding manual dissection on the same sample; b. The incidence of metastatic spread to lymph nodes and as a tumor deposit or perineural invasion in relation to the tumor location (distal, proximal and at the level of the tumor). Results. In the 245 tested specimens with colorectal cancer, a mean of 30 lymph nodes were assessed. There was no significant difference observed between neoadjuvantly treated or untreated rectal carcinomas. The main effect of AC was seen in lymph node size and metastatic status: In the 10 manually dissected samples, the number of lymph nodes which were <1  mm in diameter were less than 5% but increased to 25% after AC. In one case, initially only three 4–6 mm large lymph node metastases were found, but after AC in two further samples, three metasta-

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ses of <1 mm size were detected. In addition, tumor deposits as well as perineural tumor infiltrates were found. In neoadjuvantly treated rectal carcinoma, these findings could only be seen at the level of the tumor or proximal to it. Conclusions. Acetone compression is a time- and cost-efficient method which not only allows for standardised lymph node assessment but also provides an accurate picture of tumor deposits and perineural tumor infiltration. In up to 20% of the cases, these findings lead to a less favorable TNM classification.

SO-004 Wnt-pathway and CD44 in colorectal carcinogenesis: possible interactions? an immunohistochemical study from mouse to man F. Trapani1, U. Günthert1, L. Terracciano1, L. Tornillo1 University of Basel, Institute of Pathology, Basel, Switzerland

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Aims. Roughly 85% of sporadic colorectal carcinomas (CRCs) arise through mutations of the adenomatous polyposis coli gene (APC) and subsequent dysregulation of the Wnt-pathway. APC/Min- mouse are known to be prone to polyp formation, in most of cases adenomas, but not adenocarcinomas. The surface protein CD44 exists in different isoforms (10), which are generated through alternative splicing of the mRNA. CD44 has been shown to be involved in the development of different tumors, by interaction with several transduction cascades. In particular the variant isoforms CD44v6/v7 seem to have prognostic value in CRC. Our aim was to evaluate potential interactions between APC gene product, CD44 isoforms and the downstream signal transduction cascades in CRC Methods. APC/Min-CD44WT (group  1) and APC/Min-CD44v6/v7-/(group 2) mice were generated and microarray gene expression analysis of polyps vs. non-polyp region was performed. On the basis of the microarray expression analysis, immunohistochemistry was used to assess the expression of CTNNB1 (β-catenin), CDH1 (E-cadherin), CD44-pan, CD44v6, CD44v9/10 active CASP3 (caspase-3), PTK2 (FAK) and pSTAT3 in mice bowels. A tissue microarray (TMA) consisting of 1420, non-consecutive primary human colorectal cancers was subsequently immunohistochemically stained for CDH1, CD44v5, CD44-v9/10 and PTK2. Results. APC/Min-CD44v6/v7-/- mice had less polyps and increased survival in comparison to APC/Min-CD44WT ones. In general, immunohistochemical expression of CTNBB1, CDH1, CD44-pan and p-STAT3 was significantly different (0.0001
SO-005 Prevalence of mutations in signaling pathway components downstream of the epidermal growth-factor receptor (EGFR) in colorectal cancer J. Neumann1, L. Wehweck1, S. Maatz1, F. Mourched1, A. Hendrowarsito1, J. Engel2, T. Kirchner1, A. Jung1 1 Ludwig-Maximilians-Universität München, Department of Pathology, München, 2Ludwig-Maximilians-Universität München, Institut für medizinische Informationsverarbeitung, Biometrie und Epidemiologie, München Aims. In metastatic colorectal cancer (mCRC) KRAS-mutations are associated with clinical resistance to treatment with monoclonal antibodies targeting the epidermal growth factor receptor (EGFR). However, up to 50% of these patients do not respond to this therapy. To identify novel biomarkers, downstream effectors of the EGFR-pathway are currently validated according their predictive potential. Aim of this study was to determine the frequency and overlap of key-mutations in the EGFR-pathway. Furthermore, the concordance between primary tumor and distant metastasis was analyzed. Methods. Key mutations of KRAS, BRAF, AKT and PIK3CA were analyzed by pyrosequencing in a collection of 63 mCRC patients in primary tumor samples and corresponding metastases, respectively. Furthermore PTEN- and EGFR-Immunohistochemistry were applied. Results. Nine of the investigated 63 tumors (14.3%) had mutations in more than one gene of the EGFR-pathway. KRAS- and BRAF-mutations were detected in 50.8% and 7.9%, respectively, and were mutually exclusive. Mutations in the PIK3CA-gene (15.9%) showed huge overlap with KRASmutations (8 of 10 cases). Only one case with a mutation in the AKT-gene (1.6%) could be detected showing a simultaneous BRAF-mutation. Mutation analysis for KRAS, BRAF and AKT revealed a 100% concordance, whereas PIK3CA exhibited one discordant case showing a mutation in the primary tumor which could not be detected in the matched distant metastasis (Κ=0.9). Immunohistochemistry revealed in 49.2% high levels of EGFR-expression and in 42.9% loss of PTEN-expression, both showing huge overlap with KRAS-, BRAF-, AKT and exon 9 PIK3CA-mutations but not with exon 20 PIK3CA-mutations. Furthermore, high rates of discordant cases were found. Conclusions. We could demonstrate that KRAS- and PIK3CA-mutations as well as BRAF- and AKT-mutations can co-occur within a single tumor. However, the predictive value of these individual tumor profiles for anti-EGFR targeted therapies has to be validated in further clinical studies. Additionally, our data indicate that for molecular analysis of KRAS, BRAF and AKT either the primary tumor or the distant metastasis is suitable. Due to the lower concordance rates of PIK3CA-mutations the primary tumor site should be selected. Analysis of PTEN and EGFR protein expression are afflicted with a huge amount of discordant cases. Therefore, before these markers can be used in pathological routine diagnostics, standardized protocols are needed.

SO-006 Synergistic cytotoxicity of recombinant HMGB1 and pro-apoptotic drugs in colon carcinomas G. Gdynia1, C. Zhang1, M. Keith1, J. Kopitz2, P. Schirmacher2, W. Roth1 Institute of Pathology, University Hospital Heidelberg, and German Cancer Research Center, Heidelberg, 2Institute of Pathology, University Hospital Heidelberg, Heidelberg 1

electron microscopy, subcellular fractionation, liposome transfection, generation of stably Flag-/Myc-tagged-HMGB1 and stably Bcl-2 overexpressing cells, crystal violet cytotoxicity assay, oxygen consumption measurements. Results. Colon carcinoma cell lines were differentially sensitive to recombinant HMGB1. HMGB1 treatment resulted in the formation of giant mitochondria and a steady decline of ATP. Overexpressed HMGB1 specifically bound to cytochrome  c oxidase (COX IV) thus impairing mitochondrial respiration measured by a marked decrease of mitochondrial oxygen consumption. Colon carcinoma cells with depleted mitochondrial DNA (rho zero cells) were less susceptible to the cytotoxic effects of HMGB1. Combined treatment of colon cancer cells with subtoxic concentrations of HMGB1 and the death ligand TRAIL or the Bcl-2 inhibitor ABT-737 resulted in a strongly synergistic induction of cytotoxicity. The cell death was accompanied by an early activation of caspase-3 and a continuous decline of ATP. However, no cytochrome c release was measured in the co-treated cells, and the overexpression of the mitochondrial outer membrane associated anti-apoptotic Bcl-2 protein only moderately inhibited the HMGB1-mediated cytotoxicity. Conclusions. HMGB1 inhibits oxidative respiration of colon carcinoma cells thus depleting intracellular ATP levels. The administration of HMGB1 and pro-apoptotic compounds greatly increases their cytotoxic effects on colon carcinoma cells by activating both the necrotic and apoptotic cell death machinery.

SO-007 Opposite effects of tissue inhibitor of metalloproteinases- 1 (TIMP-1) overexpression and knockdown on colorectal liver metastases O.R. Bandapalli1, E. Paul1, P. Schirmacher1, K. Brand1 Institute of Pathology, Heidelberg

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Aims. Tissue inhibitors of metalloproteinases (TIMPs) and the corresponding metalloproteinases are integral parts of the protease network and have been shown to be involved in cancer development and metastasis. Paradoxically, for TIMP-1, tumor promoting as well as tumor inhibitory effects have been observed. Methods. To address this paradox, we utilized the BALB/c/CT26 mouse model that reliably leads to liver metastasis after splenic tumor cell injection and variegated the type of target cells for therapeutic intervention and the modalities of gene transfer. Since we have observed before that overexpression of TIMP-1 in liver host cells leads to efficient tumor growth inhibition in this model, we now examined whether targeting the tumor cells themselves will have a similar effect. Results. In concordance with the earlier results, TIMP-1 overexpression in tumor cells led to a dramatic reduction of tumor growth as well. To evaluate any influence of treatment modality, we further examined whether TIMP-1 knockdown in the same animal model would have the opposite effect on tumor growth than TIMP-1 overexpression. Indeed, TIMP-1 knockdown led to a marked increase in tumor burden. Conclusions. These data indicate that in the BALB/c/CT26 model, the modification of TIMP-1 has concordant effects irrespective of the type of target cell or the technique of modulation of TIMP-1 activity, and that TIMP-1 is unequivocally tumor inhibitory in this model.

Aims. Colon carcinoma cells are highly resistant to chemotherapeutic drugs. We recently described a new form of necrosis-like cell death in cancer cells induced by the HMGB1 protein. The aim of this study was to investigate whether the co-activation of cell death pathways by apoptosis inducers and HMGB1 can overcome the intrinsic resistance of colon carcinoma cells to pro-apoptotic therapeutics. Methods. ATP luciferase assay, LDH-linked lactate accumulation measurement, OXPHOS flux, FACS, western blot, immunoprecipitation, Der Pathologe Suppl 1 · 2012 

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Abstracts SO-008 KRAS mutational status is a prognostic biomarker in pancreatic ductal adenocarcinoma that is not influenced by p53 protein expression B .V . Sinn1, J .K . Striefler2, A . Lehmann1, M .A . Rudl1, M . Sinn2, A . Stenzinger3, M . Bahra4, W . Weichert3, H . Riess2, M . Dietel1, C . Denkert1 1 Charité Universitätsmedizin Berlin, Institut für Pathologie, Berlin, 2Charité Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Hämatologie, Onkologie und Tumorimmunologie, Berlin, 3Ruprecht-Karls-Universität Heidelberg, Institut für Pathologie, Heidelberg, 4Charité Universitätsmedizin Berlin, Klinik für Allgemein-, Viszeral- und Transplantationschirurgie, Berlin Aims. Mutations in the KRAS and p53 genes belong to the most frequently observed genetic alterations in pancreatic ductal adenocarcinoma (PDAC). Whereas p53 protein expression is of no prognostic value in most studies, the prognostic role of KRAS mutational status remains controversial. The aim of this study was to examine the frequency and prognostic impact of KRAS mutations in patients with PDAC (study group; n=153). In addition, we attempted to define molecular subgroups with distinct biological behaviour by combined analysis of KRAS sequencing data with p53 protein expression data. Methods. DNA was extracted from formalin-fixed and paraffin-embedded tissue cores using a fully automated extraction method. Codons 12 and 13 of the KRAS gene were sequenced using sanger sequencing technology (n=153). P53 immunostaining was performed on tissue microarrays from the same paraffin blocks. The impact of KRAS mutational status and nuclear p53 expression on patient outcome was evaluated. Results. KRAS mutations in codon 12 or 13 were found in 68% of cases. Nuclear positivity for p53 in at least 60% of tumor cells was observed in 47% of cases. We found no statistically significant association between KRAS mutational status and nuclear p53 positivity. KRAS mutational status, but not p53 expression, was an independent prognostic marker in the study group (p=0.011). Subgrouping of patients in four groups according to KRAS status and p53 expression failed to define subgroups with distinct biological behaviour and could not stratify patients beyond the impact of KRAS mutational status. Conclusions. In line with in vitro and in vivo data, we could demonstrate that the KRAS mutational status plays a crucial role in pancreatic cancer biology. KRAS may serve as a prognostic marker and potentially as a predictive marker for targeted therapies. P53 could not contribute to stratification of patients according to survival.

SO-009 Collagen type V affects the tumour-stroma interaction in pancreatic cancer S . Berchtold1, I . Esposito1 1 Technische Universität München, Institute of Pathology, München Aims. Pancreatic cancer (PDAC) is characterized by a dense stroma sustaining the cancer cells. The aim of this study is to understand the role that collagen V (Col V) plays in the interaction between stromal and epithelial cells during the progression of PDAC. Methods. The expression of Col V was analysed in human PDAC, precursor lesions and PDAC cells by immunohistochemistry and immunoblotting, respectively. To study the influence of Col V on PDAC cells, in vitro assays (adhesion, migration, invasion, chemoresistance) were performed. Col V-dependent signalling pathways were investigated by immunoblotting and immunofluorescence. The impact of Col V on angiogenesis was verified by tube formation assay in Col V knocked-down HUVEC cells. Results. A progressively increasing stromal expression of Col V could be shown during cancer progression. Moreover, Col V significantly affected adhesion, migration, invasion and promoted chemoresistance of PDAC cells. Tube formation was impaired in Col V-deficient cells; however, no

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significant correlation between Col V expression and neoangiogenesis was found. Conclusions. The malignant phenotype of pancreatic cancer cells is enhanced by stromal Col V, potentially through activation of the integrin signalling pathway.

Aktuelle Entwicklungen in der Forschung III – Translationale Forschung SO-010 The expression CDK4 and MDM2 in lipomas may point to a progression to GI liposarcomas M . Haab1, M . Buck1, L . Flossbach1, S . Brüderlein1, A . von Baer2, M . Schultheiss2, R . Mayer-Steinacker3, M . Wittau4, P . Möller1, T .F . Barth1 1 Ulm University, Institute of Pathology, Ulm, 2Ulm University, University Hospital, Department for Orthopaedic Trauma, Hand- and Reconstructive Surgery, Ulm, 3Ulm University, University Hospital, Internal Medicine III, Ulm, 4 Ulm University, University Hospital, Department of General, Visceral and Transplantation Surgery, Ulm Aims. Lipomatous tumors have the potential to progress from benign lesions to liposarcomas. Our goal was to specify changes during progression to distinguish progressing neoplasms from entirely benign lesions. Methods. We analyzed 31  lipomas of different regions including subcutaneous and deeply localized lesions, 42  liposarcomas GI and 8  hibernomas by morphology, immunohistochemistry with antibodies for MDM2, CDK4 and FISH with probes for the MDM2- and CDK4-region. Included were one lipoma with a recurrence, two ipomas that progressed to a GI liposarcoma after 8 years and 6 years respectively as well as one retroperitoneal lipomatous tumor with a lipoma and GI and GIII liposarcoma components. Furthermore, we included two own liposarcoma cell lines (LISA1 and LISA2) derived from dedifferentiated liposarcomas. Results. Of 31 lipomas 13 were CDK4+ and 8 were MDM2+ in various combinations. 19/19 showed no CDK4 aberration while two were amplified for MDM2. In GI liposarcomas 32/42 were CDK4+ and 31/42 were MDM2+. 9/13 GI sarcomas were amplified for CDK4 and 16/20 showed an amplification of MDM2. One/8 hibernoma each expressed MDM2 and CDK4 while no genetic aberrations of CDK4 or MDM2 genomic regions were detected. One lipoma with progression to GI liposarcoma showed a neoexpression of CDK4 and acquired an amplification of CDK4 and MDM2 in the GI sarcoma; in the other one an additional amplification of MDM2 was found. In one retroperitoneal lipomatous tumor we detected a neoexpression of CDK4/MDM2 in the sarcoma with an increase in copy numbers for CDK4 and MDM2. The cell lines LISA1 and LISA2 showed a heterogeneous expression of CDK4 and MDM2. Conclusions. The different patterns of CDK4 and MDM2 expression and gene amplifications in lipomas point to a progression to GI liposarcoma in morphologically unsuspicious tumors. Since hibernomas are generally negative for these markers they have different biology with no progression. LISA1 and LISA2 are in vitro models to functionally test the impact of CDK4 and MDM2 for the malignant phenotype. Immunohistochemistry and FISH-analysis for CDK4 and MDM2 may be crucial in lipomas for risk estimation.

SO-011 Methylation profiling and integrated genomic and transcriptional analysis reveal new tumor suppressor genes of human hepatocarcinogenesis O. Neumann1, M. Kesselmeier2, B. Radlwimmer3, P. Schemmer4, P. Lichter3, P. Schirmacher1, J. Lorenzo Bermejo2, T. Longerich1 1 University Hospital Heidelberg/Institute of Pathology, Heidelberg, 2Institute of Medical Biometry and Informatics, University Heidelberg, Heidelberg, 3Division of Molecular Genetics, German Cancer Research Centre, Heidelberg, 4Department of General, Visceral and Transplantation Surgery, University Hospital Heidelberg, Heidelberg Aims. We aimed at the identification of new tumor suppressor gene candidates of human hepatocarcinogenesis by vertical integration of genome-wide array-based CGH, methylation, and expression data from a cohort of well-characterized human hepatocellular carcinomas (HCC). Methods. Bisulfite converted DNA from 64 HCCs and 10 normal control livers was analyzed for the methylation status of about 14,000 genes using the Illumina Infinium 27k Methylation array. After determining the differentially methylated genes between HCC and normal liver, we integrated their genomic alterations as previously determined by array-CGH-data. The gene set that contained the genes with both hypermethylation and regional genomic losses was than correlated with gene expression data to select genes potentially silenced by promoter hypermethylation. Aberrant methylation of selected candidates was verified by pyrosequencing and methylation-dependent gene silencing was validated after treatment of suitable HCC cell lines with 5’-Azacytidine. Results. Methylation profiling revealed 2239 CpG-sites differentially methylated between normal control liver and HCCs. 540 CpG-sites of these were hypermethylated, whereas 1699 showed promoter hypomethylation. The hypermethylated group was enriched for genes known to be inactivated by the polycomb repressor complex 2 (PRC2), while the group of hypomethyated genes was enriched for imprinted genes showing loss of imprinting in the HCC samples. We identified 18 candidate genes matching both criteria hypermethylation and regional genomic loss. After integrating the expression data three candidates finally remained. Functional characterization of one of these promising candidates after re-expression in vitro was performed and the data will be presented during the meeting. Conclusions. Our data shows that vertical integration of methylation data with high resolution genomic and transcriptomic data is suitable for the identification of new tumors suppressor genes in human HCCs.

SO-012 AKT and N-Ras co-activation in the mouse liver promotes rapid development of hepatocellular carcinomas that are sensitive to Rapamycin treatment D.F. Calvisi1, C. Ho2, C. Wang2, S. Mattu1, G. Destefanis1, S. Delogu1, J. Armbruster1, X. Chen2, F. Dombrowski1, M. Evert1 1 University Medicine Greifswald, Institute for Pathology, Greifswald, 2 University of San Francisco, Liver Center, San Francisco, United States Aims. Activation of AKT and Ras pathways is often implicated in carcinogenesis. As yet unknown, the aim of this study is to unravel the mechanisms, underlying the oncogenic cooperation between these two cascades in relationship to hepatocellular carcinoma (HCC). Methods. Therefore, we generated a mouse model characterized by combined overexpression of activated forms of AKT and N-Ras protooncogenes in the liver via hydrodynamic transfection. Hepatocarcinogenesis and the anti-tumorigenic effect of Rapamycin treatment was investigated by morphological and molecular methods and these findings were verified in vitro, using HCC cell lines. Results. Co-expression of AKT and N-Ras resulted in a dramatic acceleration of liver tumor development when compared with mice overexpressing AKT alone, whereas N-Ras alone did not lead to tumor for-

mation. Accelerated hepatocarcinogenesis driven by AKT and N-Ras resulted from a strong activation of mammalian target of rapamycin complex  1 (mTORC1). Furthermore, elevated expression of FOXM1/ SKP2 and c-Myc also contributed to rapid tumor growth in AKT/Ras mice, yet via mTORC1-independent mechanisms. Of note, treatment of AKT/Ras mice with the mTORC1 inhibitor Rapamycin was able both to strongly constrain the growth of AKT/Ras preneoplastic lesions and to impede malignant transformation. However, liver tumors rapidly emerged in AKT/Ras mice following Rapamycin withdrawal. This was associated with induction of the MAPK/ERK cascade. The biological effects of co-activation of AKT and N-Ras can be recapitulated in vitro using HCC cell lines which supported the functional significance of mTORC1, FOXM1/SKP2 and c-Myc signaling cascades in mediating AKT- and NRas-induced liver tumor development. Conclusions. Thus, our data demonstrate the in vivo crosstalk between the AKT and Ras pathways in promoting liver tumor development, and the pivotal role of mTORC1-dependent and independent pathways in mediating AKT- and Ras-induced hepatocarcinogenesis.

SO-013 Contribution of keratin-18 in SH- and SH-induced HCC development A.K. Mehta1, K. Bettermann1, E. Lederer1, C. Diwoky2, A. Thüringer1, C. Stumptner1, H. Mueller1, T. Kolbe3, T.M. Magin4, C. Lackner1, H. Denk1, K. Zatloukal1, J. Haybaeck1 1 Medical University of Graz, Graz, Austria, 2Graz University of Technology, Austria, 3University of Veterinary Medicine Vienna, Biomodels Austria (Biat), Vienna, Austria, 4University of Leipzig, Translational Centre for Regenerative Medicine Leipzig, Leipzig Aims. Steatohepatitis (SH) is a liver disease morphologically characterized by steatosis, hepatocyte ballooning and occurrence of cytoplasmic protein aggregates termed Mallory-Denk bodies (MDBs). SH affects about 20% of alcoholics and up to 50% of obese type II diabetics. MDBs mainly consist of misfolded keratin (K) 8, 18 and in part p62 and ubiquitin. Keratin aggregates are known to be essential for MDB formation and thereby link keratins to SH which is a major precondition for the development of liver cirrhosis and hepatocellular carcinoma (HCC). In this study we aimed at elucidating the pathophysiological and molecular mechanisms of loss of K18 on hepatocarcinogenesis in mice and its functional implication in human NASH-induced liver cancer. Methods. 17 month-old krt18-deficient (krt18-/-) mice (129P2/Ola background) were investigated for the occurrence of SH- and SH-induced liver tumors by radiology, histology, immunohistochemistry, gene expression analysis and immunoblotting. Results. Aged krt18-/- mice developed the entire morphological spectrum of SH whereas aged wild-type mice displayed simple steatosis. Aminotransaminase levels were also elevated in aged krt18-/- mice. Interestingly, 91% of male and 46% of 17 months-old krt18-/- female mice developed liver tumors revealing morphological and genetic features of HCC. Moreover, 75% of male and 36% of female age-matched krt18+/mice developed HCC. Conclusions. Aged krt18-/- mice represent a new spontaneous SH- and SH-driven HCC model. Alterations of hepatocellular K18 therefore seem to determine the susceptibility towards SH and SH-induced HCC.

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Abstracts SO-014 Genome-wide mRNA expression analysis reveals massive transcriptional deregulation of cell proliferation and mitosis at multiple steps as a key factor for tumor progression in gastro­ intestinal stromal tumors (GISTs) F. Haller1, D.J. Zhang2, I.-M. Schaefer3, S. Cameron4, B.M. Ghadimi4, A. Agaimy5, S. Wiemann6, Ö. Sahin2 1 Albert Ludwigs University, Institute of Pathology, Freiburg, 2German Cancer Research Center, Heidelberg, 3Georg August University, Institute of Pathology, Göttingen, 4Georg August University, Göttingen, 5Friedrich Alexander University, Institute of Pathology, Erlangen, 6German Cancer Research Center Aims. Gastrointestinal stromal tumors (GISTs) carry mutations in the receptor tyrosine kinases KIT and PDGFRA, leading to ligand-independent autophosphorylation with constitutive activation of downstream intracellular signalling cascades and accelerated cell proliferation. Next to genotype, anatomical localisation and mitotic counts are two clinicopathological parameters with significant impact on clinical behavior in GISTs. The aim of the current study was to determine the effect of genotype, anatomical localisation and mitotic counts on global mRNA expression. Methods. Genome-wide mRNA expression analyses were performed using Sentrix HumanWG-6 arrays (Illumina, San Diego, CA) in a series of 20 GISTs with either KIT or PDGFRA mutation from different anatomical localisations, and with low and high mitotic counts. Results. Using two-dimensional principal component analysis, tumors were clearly separated according to genotype and anatomical localisation, with clustering of tumors from the stomach, duodenum, jejunum/ ileum and rectum, respectively. Moreover, tumors with high mitotic counts were separated from tumors with low mitotic counts. Group-wise comparison of gene expression levels revealed significant upregulation of 269 genes in GISTs with high mitotic counts, compared to 88 genes that were downregulated. Further functional enrichment analysis using this signature revealed a significant enrichment of genes allocated to 38 GO terms associated with cell proliferation and mitosis. Conclusions. Deregulation of several key players of cell cycle regulation at different steps of the cell cycle contributes to increased cell proliferation and tumor progression in GISTs, and determination of their expression may improve prognostication of clinical behavior.

SO-015 SRC signalling is crucial in the growth of synovial sarcoma cells S. Michels1, E. Sievers1, M. Trautmann1, D. Kindler1, S. Huss1, M. Renner2, R. Penzel2, O. Larsson3, A. Kawai4, S. Tanaka5, P. Schirmacher2, G. Mechtersheimer2, E. Wardelmann1, R. Büttner1, W. Hartmann1 1 University Hospital Cologne, Institute of Pathology, Köln, 2University Hospital Heidelberg, Institute of Pathology, Heidelberg, 3The Karolinska Institute, Department of Oncology & Pathology, Stockholm, Sweden, 4National Cancer Center Hospital, Division of Orthopaedic Surgery, Tokyo, Japan, 5 Hokkaido University Graduate School of Medicine, Laboratory of Molecular & Cellular Pathology, Sapporo, Japan Aims. Synovial sarcoma is a malignant soft tissue tumor, which affects mainly adolescents and young adults. It is molecularly characterized by a reciprocal balanced t(X;18) translocation, resulting in a chimeric transcriptional modifier. Several receptor tyrosine kinases including the IGF-IR and the EGFR have been shown to be expressed in synovial sarcomas, leading to an activation of common intracellular kinase signalling cascades. The SRC tyrosine kinase is an important interaction partner for different growth factor receptors and effectors of intracellular kinase signalling pathways and has been shown to be of particular importance in a variety of tumors. This study was performed to examine

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the functional relevance SRC in synovial sarcomas and to evaluate if it might represent a target for innovative therapeutic approaches. Methods. Immunohistochemical analyses of differentially phosphorylated SRC and the SRC regulators CSK and PTP1B were performed in a set of 30 synovial sarcomas. Functional aspects of SRC signals in synovial sarcomas and dependence of SRC activation on the SS18/SSX fusion proteins were subsequently analyzed in vitro. Results. Activated p-(Tyr416)-SRC was detected in the majority of synovial sarcomas; deregulation of CSK and PTP1B could be excluded to be the reason for the activation of the kinase. In a T-Rex293-based in vitro model, expression of the SS18/SSX fusion proteins was associated with increased p-(Tyr416)-SRC levels, at least partially due to an induction of the Insulin-like growth factor signalling pathway. Four human synovial sarcoma cell lines treated with the SRC inhibitor dasatinib displayed a significant and dose-dependent inhibition of cellular growth in MTT assays, which was accompanied by decreased phosphorylation of the SRC targets FAK, STAT3, IGF-IR and AKT. In flow cytometric analyses, the growth effects exerted by the inhibitor were shown to be due to a reduction of cellular proliferation combined with an increase of apoptosis. Concurrent exposure of synovial sarcoma cells to dasatinib and conventional chemotherapeutic agents resulted in positive interactions. Finally, synovial sarcoma cell migration and invasion was found to be dependent on signals transmitted by SRC. Conclusions. In summary, our data show that the SRC kinase might represent a promising therapeutic target in synovial sarcomas.

SO-016 Targeting endometrial stromal sarcoma: histone deacetylase and PI3K/Akt/mTOR signaling P. Quan1, E. Lederer1, I. halbwedl1, H. Denk1, K. Zatloukal1, J. Haybaeck1 1 Med. Uni. Graz/Department of Pathology, Graz, Austria Aims. Endometrial stromal sarcoma (ESS), derived from mesenchymal cells, is a rare gynecological malignancy with an unclear molecular pathogenesis and thus few therapeutic options. Previously, histone deacetylase (HDAC) 2 expression was shown to be upregulated in human ESS specimens. The HDAC inhibitor SAHA reduced in vitro growth of the ESS cell line ESS-1 through the inhibition of mTOR signaling. The PI3K/ Akt/mTOR signaling, central to translational regulation and vital to the growth and survival of cancer cells is an important target in cancer therapy. This study aims at investigating (1) if HDAC and the PI3K/Akt/ mTOR signaling are involved in ESS pathogenesis and (2) how altered HDAC levels regulate PI3K/Akt/mTOR signaling and its downstream translation regulators in ESS. Methods. The expression levels of HDAC1 and 2 in ESS were examined by using a tissue microarray. The mRNA and protein levels of HDAC1 and 2 were determined by Q-PCR and Western blotting in ESS cell lines (ESS-1 and MESSA) and the appropriate control endometrial stromal cell line HESC. The role of HDAC in regulating the PI3K signaling was checked in cells treated with SAHA. Results. 1.) HDAC1 and 2 are overexpressed in ESS tissues, compared to normal endometrium. 2.) Higher levels of HDAC1 and 2, increased cell growth, upregulated Akt and mTOR activation were detected in ESS cell lines, relative to HESC. 3.) SAHA inhibited cell growth of three cell lines. However, HESC is less sensitive to SAHA with a higher IC50 value than other cells. 4.) SAHA reduced phosphorylated 4EBP1 and BCL-2 protein levels in all cell lines. 5.) SAHA dose-dependently inhibited activation of Akt and p70S6k in ESS-1, but not in MESSA and HESC cells. Conclusions. HDACs and the PI3K signaling are involved in the ESS pathogenesis. Despite of different drug sensitivity and response rates among all cell lines, SAHA reduced cell growth via the PI3K/Akt/mTOR signaling and its downstream effectors 4EBP1 and p70S6K, indicating an integration of HDACs with the PI3K signaling and translation regulation. This connection offers a promise for a combination therapy, i.e. SAHA with various inhibitors for the PI3K signaling, which might be more effective than SAHA alone and possible for an individualized ESS therapy.

SO-017 High-resolution 3D visualization and semi-automated characterization of tumor angiogenesis J . Ehling1, B . Theek2, F . Gremse2, S . Baetke2, R . Knüchel3, F . Kiessling2, T . Lammers2 1 RWTH Aachen, Institute of Pathology & Institute of Biomedical Engineering, Aachen, 2RWTH Aachen, Institute of Biomedical Engineering, Aachen, 3 RWTH Aachen, Institute of Pathology, Aachen Aims. The visualization and quantification of functional tumor blood vessels is essential for assessing treatment responses to anti-angiogenic therapies. In experiments using tumor xenografts, anti-angiogenic effects are generally evaluated by immunohistochemistry (IHC). This method has several limitations: for example, the 3D architecture and the vessel functionality are not fully considered. To overcome these shortcomings, we established a protocol based on ex vivo high-resolution microcomputed tomography (µCT) for 3D visualization and characterization of tumor angiogenesis. Methods. Six different tumor models (A431, A549, Calu-6, DU145, MDAMB-231, MLS), differing in blood vessel density and maturation, were analysed. After tumors reached a diameter of ~6 mm, mice were intracardially perfused with the radiopaque contrast agent Microfil, which polymerises intravascularly. Subsequently, the tumor was harvested and scanned in a high-resolution µCT scanner (SkyScan, Belgium) with a maximal spatial resolution of 3 µM. Tumor microvessels were visualized, and vessel density, vessel size, number and order of branches, were analyzed using a 3D rendering software (MeVisLab). Histological validation was performed by CD31 and SMA staining. Results. Vascular casting and high-resolution µCT analysis of the vasculature in tumor models with large and highly mature vessels (CD31-positive and SMA-positive), such as A549 or MLS, enabled the detection of vessel branches up to the 7th order. Conversely, tumors primarily containing small and immature vessels (CD31-positive and SMA-negative), such as A431 or Calu-6, presented many randomly arranged small vessels, without proper hierarchy and architecture. The rising order of branches, the total number of branches and the distribution of branches correlated very well with the SMA levels. In addition, a highly significant correlation was observed between the vessel diameters measured by high-resolution µCT and by histology. Conclusions. An imaging protocol based on vascular casting and highresolution µCT has been developed for the 3D visualization of the micromorphology of tumor blood vessels. In addition, evidence is provided showing that high-resolution 3D µCT imaging of vascular casts allows for a semi-automated analysis of the micro-architecture of tumor vessels, thereby making it an exquisite and highly useful tool for supplementing IHC in translational focusing on tumor angiogenesis and antiangiogenic treatment responses.

SO-018 Eyetracking experiments identify “fast and frugal” heuristics during cancer grading D . Bombari1, B . Mora2, S . Schaefer3, F . Mast1, H .-A . Lehr4 1 University of Berne, Cognitive Psychology, Bern, Switzerland, 2CHUV, Lausanne, Institute of Pathology, Lausanne, Switzerland, 3Inselspital, Institute of Pathology, Bern, Switzerland, 4CHUV, Lausanne, Institute of Pathology, Lausanne, Switzerland Aims. In prior studies on prostate carcinomas we found that during nuclear grading, pathologists are heavily biased by the architectural growth patter of the carcinomas (Fandel et al., J Pathol. 2008). Methods. We asked 20 pathologists to assign nuclear grades to images of high power fields of 40 prostate carcinomas projected on a computer screen with an inbuilt eyetracking device. We wondered if pathologists fixate on different nuclei when the same circular high power fields (albeit turned and flipped to avoid recognition) were displayed before

background images of well-differentiated, tubule-rich or poorly differentiated, solid carcinomas. Using Photoshop-based image analysis, we analyzed nuclear size, chromasia, and roundness of those nuclei that the pathologists fixated, and compared these morphometric data to a random sample of nuclei contained within each high power field. Results. (i) The selection of fixated nuclei largely followed the confirmation bias induced by the tumor architecture. (ii) The selection of “matching” nuclei explained only about one tenth of the manipulation of nuclear grades induced by the tumor architecture, suggesting that it represents nothing but an unconscious effort of our minds to vindicate the gravitation of nuclear grades towards the tumor architecture. (iii) The majority of pathologists based their subjective nuclear grade on a single morphometric criterion (mostly nuclear size) and only few pathologists considered more than one nuclear criterion during nuclear grading. This observation has in the meantime been confirmed in a study on 44 pathologist asked to grade nuclei in breast carcinomas. Conclusions. (i) Counter the general expectation that pathologists rely in their diagnosis solely (or mostly) on what they see through the microscope, our experiments suggest that unconscious, poorly recognized biases influence not only the interpretation of the histological image but also the way we view the image (unwitting fixation on selected parts/aspects of the image). (ii) While we eloquently teach medical students that nuclei of aggressive tumors are enlarged, angulated, and hyper- as well as heterochromatic, we personally ignore most of these different morphometric criteria during our daily diagnostic routine and rather base nuclear grades on a single criterion (mostly size). (iii) It may appear judicious to recognize cognitive biases and heuristics as part of a consensuous error culture in diagnostic anatomical pathology and in translational research and to develop mechanisms to counteract/prevent ensuing blurs and/or outright diagnostic errors. This project was funded by a grant from the Fonds National Suisse (Proj .-N° 32000-120417) .

AG Gynäkopathologie und Mammapathologie I SO-019 p16/Ki-67 dual-stained cytology using ThinPrep® liquid-based cytology – sub-analyses of more than 9,000 PALMS participants H . Griesser1, F . Alameda2, C . Bergeron3, M . Labadie4, V . Maccalini5, M . Sideri6, R . Dachez7, R . Ridder8 1 Center for Pathology and Cytodiagnostics, Koeln, 2Hospital del Mar, Barcelona, Spain, 3Laboratoire Cerba, Cergy Pontoise, France, 4Laboratoire GRC, Limonest, France, 5Ospedale Atri, Unita Gestionale Screening Regionale, Atri, Italy, 6European Institute of Oncology, Milano, Italy, 7Institute Alfred Fournier, Paris, France, 8mtm laboratories, Heidelberg Aims. The PALMS trial (Primary ASC-US LSIL Marker Study) evaluated the diagnostic performance of the novel p16/Ki-67 dual-stained cytology testing in cervical cancer screening as well as in the triage of ASC-US or LSIL Pap cytology results. The outcomes were compared to Pap cytology and HPV testing in the screening setting, and to HPV testing in the triage of ASC-US or LSIL. For both Pap cytology and dual-stained cytology testing, liquid-based cytology and conventional cytology methods were included in the PALMS trial. Methods. We performed an analysis of the diagnostic performance of dual-stained cytology, Pap cytology and HPV testing limited to the subcohort of 9,231 women enrolled to the PALMS trial who had ThinPrep® (Hologic) liquid-based cytology testing for both Pap and dual-stain. Results. Test positivity rates for dual-stained cytology, Pap, and HPV testing over all ages were 5.7%, 5.6%, and 11.2%, respectively. Sensitivity of dual-stained cytology for CIN2+ (n=67 cases) was found at 95.6% (95%  CI 87.1–98.6%), significantly higher than Pap cytology (80.2%; Der Pathologe Suppl 1 · 2012  | 

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Abstracts 95%  CI 68.5–88.3%). Specificity levels were identical for both methods (95.0% for dual-stained cytology vs. 95.1% for Pap testing). In women aged 30 and older, sensitivity (specificity) of dual-stained cytology for CIN2+ was 91.2% (96.0%), compared to 77.9% (95.9%) for Pap testing and 96.1% (92.1%) for HPV testing. For the triage of abnormal Pap cytology results, dual-stained cytology showed identical (ASC-US triage: 100% for both tests) or similar (LSIL triage: 94.5 vs. 100%) sensitivity as HPV testing for the detection of underlying CIN2+, at significantly higher specificity rates. Conclusions. p16/Ki-67 dual-stained cytology performed on ThinPrep® liquid-based cytology may achieve a sensitivity level as high as 95% for underlying CIN2+ in primary screening of women of all ages, combined with a 95% specificity. Furthermore, dual-stained cytology was shown to be a highly efficient tool for the triage of abnormal Pap cytology results when performed as a reflex test out of ThinPrep liquid-based cytology specimens.

SO-020 Frequency of BRAF-p.V600E mutation in serous ovarian border­ line tumors and its peritoneal implants A.K. Höhn1, U. Siebolts1, J. Einenkel2, L.-C. Horn1 1 University of Leipzig, Institute of Pathology, Leipzig, 2University of Leipzig, Department of Obstetrics and Gynecology (Institute of Trier), Leipzig Aims. Genes of the RAF family, which mediate cellular responses to growth signals, encode kinases that are regulated by RAS and participate in the RAS/RAF/MEK/ERK/MAP-kinase pathway. Activating mutations in BRAF have been identified to play a major role in the pathogenesis of low-grade serous ovarian carcinomas (LG-OCA) via serous borderline tumors (s-BLT; Sieben et al. 2004, Mayr et al. 2006; Vang et al. 2009). But, limited information exists about a possible clonal relation comparing s-BLT and its peritoneal implants which we want to illuminate by BRAF p.V600E analysis. Methods. Thirteen cases of s-BLT with peritoneal implants (invasive and non-invasive) were identified from our files with subsequent macro- or microdissection followed by DNA extraction of the adequate tissue. To reveal the activating mutation of BRAF p.V600E we performed pyrosequencing of 48 samples with a sensitivity of at least 5% mutated alleles. Molecular analysis was performed from the ovarian tumor as well as within one to 6 peritoneal implants from different sites. Results. Five s-BLT (38.5%) showed BRAF-p.V600E mutation within the ovarian tumor. In three of those cases BRAF-p.V600E mutation was also identified within the peritoneal implants suggesting a clonal origin in terms of abdominal tumor spread. Conclusions. The frequency of BRAF-p.V600E mutation in s-BLT is concordant with the reported frequency within LG-OCA. In case of abdominal spread, peritoneal implants represent clonal origin of the primary tumor in about two thirds of the informative cases. Further studies, examining additional members of the RAS/RAF/MEK/ERK/MAP-kinase pathway and using laser-capture microdissection in cases of rare tumor epithelium by atrophy are required.

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SO-021 The relevance of steroid hormone receptors (estrogene alpha and beta, progesterone), luteinizing hormone and follicle-stimulation hormone receptor as well as aromatase activity in granulosa cell tumors (GCTs) of the ovary M.C. Jarrin Franco1, T. Kirchner1, J. Engel2, S. Lauf3, A. Mayerhofer4, D. Mayr1 1 University of Munich, Institute of Pathology, München, 2University of Munich, Munich Tumor Registry, München, 3University of Munich, Institute of Cell biology, München, 4University of Munich, Institute of Anatomy, München Aims. Granulosa cell tumors of the ovary (GCTs) are rare neoplasms from sex-cord stromal cells with a general trend toward late relapse and metastasis. In contrast to ovarian carcinomas, tumor stage is the only prognostic factor. The pathophysiology of ovarian tumors and relationship to hormonal control are not yet fully understood, but the ovary is the principal source of estrogens and one of their target organs. Some aromatase inhibitors are relatively well-tolerated oral drugs commonly used in breast cancer treatment. The aim of this study was to investigate the tumorigenesis of GCTs, regarding steroid hormone receptors, luteinizing hormone and follicle-stimulation hormone receptor and aromatase activity with correlation to clinical data and survival. Methods. 43 cases of GC-tumors of 36 patients from the archive of the Department of Pathology, LMU Munich were selected. Immunohistochemical assays (IHC) and RT-PCR were performed by standard methods. The IHC for ER alpha, ER beta and progesterone was evaluated by using the IRS-score. For aromatase activity, FSH and LH a 4-tired scoring system was developed. The results were correlated to each other and to clinical data (e. m. TNM-stage, Ki-67 proliferation index, progression) and survival. Results. Positive results for IHC: ER alpha in 79.1%, ER beta in 86%, PR in 97.7%, FSHR in 14%, LHR in 25.6% and aromatase in 51.2%. Positive results for RT-PCR: ER alpha in 88.4%, ER beta in 100%, PR in 86%, FSHR in 55.8%, LHR in 76.7% and aromatase in 74.4%. No statistical correlation between IHC and RT-PCR could be demonstrated. A significant correlation between T-stage and IHC for ER alpha (p=0.026), ER beta (p=0.01), progesterone (p=0.042) and Ki67 (p=0.046) was observed. Only for ER beta-IHC a high statistical significance (p=0.019) in correlation to progression could be demonstrated. No statistical significance between Tstage and RT-PCR results could be demonstrated in any case. Regarding the progression and survival, the only statistical significance could be demonstrated for RT-PCR of LHR (p=0.030). Conclusions. At the present time tumor stage and in some studies Ki-67 proliferation are the only established prognostic factors for GCTs. A long follow-up is the only way of validation the success of treatment. Regarding our results, ER beta and RT-PCR of LHR could be new prognostic signs and beyond that a basis for a new therapeutic strategy. Analyses of a larger number of cases, as well as studies of cell culture are already in progress.

SO-022 Specialized pathology review in patients with ovarian cancer: highly recommended to assure adequate treatment. Results from a prospective study S. Kommoss1, J. Pfisterer2, A. Reuss3, A. du Bois4, J. Diebold5, S. Hautmann6, D. Schmidt7, F. Kommoss7, for the AGO study group8 1 University of British Columbia, Department of Pathology, Vancouver, Canada, 2Klinikum Solingen, Dept of Gynecology, Solingen, 3Philipps-Universität Marburg, Koordinierungszentrum für klinische Studien, Marburg, 4Kliniken Essen Mitte (KEM), Dept Gynecology & Gyn.Oncology, 5Luzerner Kantonsspital, Institute of Pathology, Luzern, Switzerland, 6Institute of Pathology, Allgäu-Oberschwaben, Wangen i. A., 7Institute of Pathology, A2,2, Mannheim, 8AGO study group, Wiesbaden Aims. In view of retrospective findings on second opinion pathology in ovarian cancer it seems certain, that a considerable number of ovarian borderline tumors (BOTs) or metastatic non-ovarian primaries are being erroneously diagnosed as ovarian carcinomas. If BOTs are misdiagnosed as cancer, patients may not only suffer from non-beneficial morbidity at unnecessary high cost but may have to cope with an incorrect diagnosis of cancer for the rest of their lives. In cases of metastatic disease mistaken for an ovarian primary, more adequate therapeutic modalities may be withheld from some patients. Finally, clinical trials may be biased through disregarding of histological inclusion criteria. We hypothesized that 5% of all patients in clinical trials of ovarian carcinomas have lesions other than epithelial ovarian cancer. This is the first such study with a prospective approach. Methods. Patients who were enrolled into a chemotherapy trial of ovarian carcinoma were asked to consent to a translational subprotocol. Contributing pathologists were asked to submit all original slides as well as paraffin material. Specialized central pathology review of all cases was performed by two experienced gynecopathologists. In cases of clinically relevant diagnostic discrepancies, the contributing pathologist was contacted. If a given discrepancy could not be resolved, a panel of experts was available for clarification. Results. 454  patients with an outside diagnosis of ovarian epithelial cancer were recruited. In 6.8% (n=31), a major diagnostic discrepancy of potential clinical relevance was found. Most frequently (n=15), serous BOTs had been misdiagnosed as invasive cancer. Ovarian metastases constituted the second most frequent misdiagnosis (n=12). As minor discrepancies, a divergent histological typing of ovarian carcinomas was found in 28.2% (n=128). Conclusions. This study clearly shows that central pathology review by experienced gynecopathologists is highly recommendable if overtreatment with chemotherapy of patients with BOTs and inadequate treatment of patients with ovarian metastases is to be avoided in the future. Specialized pathology review should become standard procedure in study protocols prior to randomization. In order to further optimize the quality of care, a high throughput infrastructure for specialized pathology review will have to be established. The authors propose a internetbased ovarian cancer network, capable of providing specialized second opinion pathology within 10 working days.

SO-023 Development of a consortial database with pathological and clinical data for fresh frozen breast cancer specimen P. Bronsert1, E. Stickeler2, S. Schmid1, K. Aumann1, F. Haller3, C. Röcken4, N. Arnold5, C. Mundhenke5, F. Fend6, A. Stäbler6, T. Fehm7, U. Vogel6, M. Werner1, O. Opitz8 1 Albert-Ludwigs-University Freiburg, Institute of Pathology, Freiburg, 2 Albert-Ludwigs-University Freiburg, Department of Obstetrics and Gynecology, Freiburg, 3Friedrich-Alexander-University Erlangen-Nürnberg, Institute of Pathology, Erlangen, 4Christian-Albrechts-University, Institute of Pathology, Kiel, 5Christian-Albrechts-University, Department of Gynecology and Obstetrics, Kiel, 6Eberhard Karls University Tübingen, Institute of Pathology, Tübingen, 7Eberhard Karls University Tübingen, Department of Gynecology and Obstetrics, Tübingen, 8Albert-Ludwigs-University Freiburg, Tumour Center Ludwig Heilmeyer, Freiburg Aims. Over the past two decades biomedical research technology in tumor banking has enabled significant advances in the molecular characterization of cancers, especially in research projects. Essential for the functioning of a high quality tumor bank is a standardized implementation of clinical and pathological information of tumor tissue. Each specific specimen cohort reflects certain quality characteristics of a tumor bank database and has to be handled in a certain manner. By combining three independent breast cancer biobanks with two different disciplines per facility together, it has to be assured, that each attendant is working with synchronized and harmonized standard operating procedures (SOP) and datasets. Methods. At all three attended facilities rules of internal procedure were defined, a Shared Resources Advisory was established, SOPs were partially new elaborated, harmonized and synchronized. Minimal obligate and facultative breast-cancer-specific, datasets for pathological and clinical diagnoses were established. A periodically updated, secured database with an automated combination of all consortial data was developed. A web page for public presentation and research access was designed. To attest effective consortial operation, tissue micro arrays (TMA) of a well specified, facility research related, patient cohort were established, sectioned and send to each facility where immunohistochemical staining was performed. The results will be published in a consortial publication. Results. From 2001 to 2011, the consortial breast cancer tissue bank contains 3400 fresh frozen, prospectively collected and immunohistochemically classified breast cancer samples from all three facilities. Nearly half of the patients were diagnosed with a ductal carcinoma in situ. The median age was 61 years. The common pT category was pT1c (n=1063), the most frequently pN category was pN0 (1693) followed by pN2 (n=197), pN1 (194) and pN3 (82). 1113 patients were ER and/or PR and/or HER2/ neu positive. After written request, access for researchers to the consortial internet accessible breast cancer database can be granted. Conclusions. A well planed clinicopathological, IT linked infrastructure is the fundament of a consortial database and the basic principle for multicentric translational research.

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Abstracts SO-024 Evaluation of tumor proliferation and hormone receptor status in breast cancer. Comparison of quantitative real time PCR, image analysis of IHC, and visual scoring H.-P. Sinn1, M. Keller1, N. Waldburger2, A. Schneeweiss3, R. Wirtz4 University of Heidelberg, Institute for Pathology, Heidelberg, 2University of Heidelberg, Dept. of Pathology, Heidelberg, 3University of Heidelberg, National Center for Tumor Diseases, Heidelberg, 4St. Elisabeth Krankenhaus, Dept. of Pathology, Köln 1

Aims. The exact determination of hormone receptors, HER2 and proliferation is of outmost importance for clinical decision making in breast cancer. According to the 12th St Gallen Guidelines systemic therapy recommendations follow the subtypes classification originally defined by genetic array testing. These molecular subtypes can be approximated by clinicopathological parameters combining immunohistochemistry assessments of ER, PR, HER2 and Ki67. Methods. Matched fresh and fixed pretreatment biopsy samples were available from 90 patients participating in neoadjuvant clinical trial. RTqPCR data were available after extracting RNA using Qiagen kits. RNA was isolated from fixed tissue samples by using coated magnetic particles. Multiplex RT-qPCR was performed by TaqMan® based primer probe sets for ESR1, PGR, Ki67, as well as CALM2 as reference gene. Single Step RT-qPCR was performed by using Invitrogen reagents on a Stratagene MX3005p. RNA results were then reported as 40-CT values, which correlate proportionally to the mRNA expression level of the target gene. All cases were also used for quantitative immunohistology of nuclear antigens (ER, PR, Ki67) by automated image analysis on a virtual microscopy system (Aperio Technologies, Vista, CA, USA). Results. We observed a significant correlation of mRNA data from independent, matched fresh and fixed biopsy samples by using RT-qPCR (ER1 r=0,91; PR r=0,79, Ki67 r=0,72). When comparing automated image analysis results with conventional histological evaluation of IHC markers, there were only 3 cases misclassified for ER positivity or negativity, and 4 cases misclassified for PR positivity or negativity. Correlation of RT-qPCR data for ER and PR with quantitative immunohistology was highly significant (p<0.0001) with few cases classified as negative by image analysis but positive by RT-qPCR, but not the other way around. Correlation coefficients between image analysis and RT-qPCR on fresh frozen tissue, were 0.72 for ER, 0.73 for PR, and 0.51 for Ki67. Similar results were obtained for RG-qPCR of FFPE tissue. Conclusions. Determination of ER, PR, and Ki67 mRNA analysis of fresh and fixed tissue samples results in robust and comparable results when compared with quantitative immunohistochemistry. Both methods are more accurate than conventional visual analysis of IHC staining. Discrepancies in individual cases must be analyzed case by case to determine problems with either method.

SO-025 Breast cancer excision specimens evaluated by micro-computed tomography (micro-CT) with histopathological correlations E. Brachtel1, L.J. Fernandez1, J.M. Buckley1, O.P. Aftreth1, R. Tang1, M. Saksena1, Y. Yagi1, J.S. Michaelson1, F.C. Koerner1, B.L. Smith1 1 Massachusetts General Hospital and Harvard Medical School, Boston, United States Aims. Breast conserving surgery is standard of care in the treatment of breast cancer. Re-excision is required in 25–40% to provide negative margins on permanent histopathological evaluation. Intraoperative frozen sections are not done for breast specimens at many places. Improved methods to intraoperatively determine margin involvement by carcinoma would be desirable. In this study, we explore a novel method of imaging excision specimens by micro-CT for rapid visualization of the removed mass and its relationship to the margins.

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Methods. Fourteen breast excision specimens for breast cancer from 13 female patients who had given their informed consent were evaluated with a table top micro-computed tomography scanner (micro-CT), Skyscan 1173 (Skyscan, Belgium). Scanning took <15 minutes, followed by routine histopathological processing and reporting. Micro-CT images of the excision specimens were evaluated for mass size and relation to margins. Whole slide images of the histologic slides were obtained by Nanozoomer 2.0-HT (Hamamatsu LTD, Japan). Results. Average mass size was 1.9 cm by micro-CT (range 0.9–3.5 cm) and 1.3cm on pathology (range 0–2.2 cm). Margins were positive/<0.1 cm in 86% (n=12) by micro-CT and 64% by histology (n=9). Margins were free (0.2 cm) in 7% by micro-CT (n=1) and 14% (n=2) by histopathology. Additional shaved margins (not evaluated by micro-CT) were negative in 71% (n=10). Tumors consisted of 12 invasive ductal carcinomas (IDC), 1 invasive lobular carcinoma and 1 healing biopsy site with mass-like appearance on micro-CT but no residual carcinoma. 57% showed ductal carcinoma in-situ histologically (n=8). Conclusions. In this small pilot study, tumor size was slightly overestimated by micro-CT compared to pathology. Specimen margins were more often considered involved by tumor on micro-CT than confirmed histologically. Densely fibrous lesions, even if they do not contain tumor cells histologically, may be difficult to distinguish from tumor on micro-CT. Analysis of a larger cohort is necessary to determine predictive value of micro-CT assessment for breast excisions.

SO-026 Underestimation of DCIS and invasive breast cancer after ADH diagnosis on breast core and vacuum biopsies Z. Varga1, C. Rageth2, C. Braschler2, B. Papassotiropoulos2, R. Rubenov2, A. Noske1 1 University Hospital Zurich, Institute of Surgical Pathology, Zürich, Switzerland, 2Breast Center Seefeld Zurich, Zürich, Switzerland Aims. The diagnosis of atypical ductal hyperplasia (ADH) in breast biopsy is considered as an indication for surgical excision of the involved breast region. Underestimation of ADH on subsequent surgical specimens as in situ (DCIS) and invasive breast cancer varies from 10–40%. However, it is still a debated issue whether all ADH cases should undergo surgical excision or not. In this study we assessed ADH underestimation in a series of core and vacuum breast biopsies. Methods. We analyzed 107 consecutive breast biopsies with the histological diagnosis of atypical ductal hyperplasia (ADH) and correlated the histological and molecular findings with the frequency of higher grade malignant lesions in the consequent surgical specimens. The biopsies were taken in a 7-years period (2003 to 2010), 43 biopsies were core biopsies, 64 were vacuum biopsies. All cases were re-evaluated histologically using WHO (2003) criteria. Results. 77% of ADH cases on core biopsies were mass lesions on imaging, 90% of ADH cases on vacuum biopsies had suspicious calcification on mammography (p=0.0001). Invasive carcinomas in surgical specimens were detected in 27% of core and 10% of the vacuum biopsy cases (p=0.009). ADH and/or DCIS were detected almost equally after core and vacuum biopsies. Histological extension of the ADH lesion (number of involved ducts and extension in mm) did not correlate with the outcome of invasive carcinomas or DCIS in the surgical specimens. Molecular/immunohistochemical phenotype of ADH was identical in both groups. ADH lesions detected within flat epithelial atypia (FEA) in the biopsies, were associated with less invasive carcinomas or DCIS in the surgical specimens (p=0.009). Conclusions. The outcome of ADH lesions depends on the imaging findings and on the presence of other B3 lesions as FEA on the core/vacuum biopsies. These factors can be considered as additional decision tools if dealing with ADH on a core/vacuum biopsy. However further analyses are necessary to identify distinct molecular subgroups of ADH lesions.

SO-027 Ki-67 in mitotic score groups of the Nottingham Grading System for breast cancer C . Focke1, D . Gläser1, K . Finsterbusch1, T . Decker1 Dietrich-Bonhoeffer-Klinikum Neubrandenburg, Department of Pathology, Neubrandenburg

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Aims. The 2011 St. Gallen Consensus suggested the addition of Ki-67 for defining proliferation and thus the difference between luminal A and B clinicopathological subtypes. Whereas a Ki-67 cut-off of 14% is cited from literature it became obvious from the discussion that no standardized methodology or cut-off definition for Ki-67 is available so far. To estimate the capability of immunohistochemically quantified Ki-67 rates to discriminate the Nottingham Grading System (NGS) mitotic score groups and to determine respective cut-offs in breast cancer (BC). Methods. We retrospectively analyzed routinely H&E stained slides of 50 invasive BC (9 G1, 23 G2, and 18 G3). Immunohistochemistry for Ki-67 was done prospectively according to an in house protocol, evaluated in a national interlaboratory trial for quality assurance in immunohistochemistry. To rate Ki-67, 100 tumor cells within an area of the “hot spot “ were evaluated by counting all stained nuclei regardless of intensity. We used the mitotic activity index (MAI) per mm2 as gold standard for proliferation measurement. By assessing the MAI ranges within the mitotic score subgroups of the Nottingham Grading System (NGS) we determined respective MAI cut-offs. Using these MAI cut-offs we adjusted the observed Ki-67 rates. Results. Whereas the cut-offs of MAI for NGS mitotic scores 1, 2, and 3 were 0–32, 33–70, and 71–582, respectively, the resulting ranges of Ki-67 were 7–30%, 13–54%, and 33–98%. The median Ki-67 rates for NGS scores 1–3 came out with 21 (SD±7.5), 41 (SD±11), and 59 (±18.8), respectively. Conclusions. Whereas there is obviously a trend of higher Ki-67 rates in NGS score groups with higher MAI, our data indicate that 1) it was not possible to discriminate the NGS mitotic score groups by using Ki-67 rates, 2) the cited cut-off of <14% for “low proliferation” is not readily applicable in laboratory practice, and 3) in addition to technical QA, there is an urgent need for standardization of Ki-67 diagnostic assessment before using it for therapy decision making.

AG Gynäkopathologie und Mammapathologie II SO-029 St. Gallen 2011 intrinsic subtypes of breast cancer: concordance of core biopsies and related surgical specimens T . Decker1, C . Focke1 Dietrich Bonhoeffer-Klinikum, Department of Pathology, Neubrandenburg

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Aims. For systemic therapy decision the St Gallen consensus Conference 2011 recommends a simplified clinicopathological definition of intrinsic breast cancer subtypes (IBCST) rather than gene expression array criteria. There are many reasons to provide patients and doctors these information already preoperatively. To evaluate relevance of St. Gallen 2011 IBCST core biopsy (CB) results we estimated their concordance with postoperative IBCSTs. Methods. IBCSTs of CB and related surgical specimen (SSP) of 285 breast cancers (BC) (52 Grade 1, 122 Grade 2, 114 Grade 3) assessed using the prospectively estimated results for Estrogen-, Progesteron-, and Her2status. To differentiate between Luminal A and the Her2 negative subgroup of Luminal  B we retrospectively measured the mitotic activity index (MAI; WHO definition 2003) with a cut-off of ≥10 for high versus low as evaluated by Baak et al. 2008. Concordance between IBCST results of CB and SSP and PPV of IBCST of CB was calculated. Results. The 285 BCs were classified in SSP as follows: 60% (171) Luminal A, 25% (71) Luminal B [Her2− 18% (52), Her2+ 7% (19)], 3.5% (10) Her2

positive and 11.5% (33) triple-negative. Concordance of IBCSTs in CB vs. SP was 100% (171/171) for Luminal A, 29% (15/52) for Luminal B (Her2−), and 100% for Luminal B (Her2+) (19/19), Her2 positive (10/10), and Triplenegative subtypes (33/33). None of the Luminal A BCs was overestimated in CB as a more unfavourable subtype, whereas 52.1% (37/71) of all Luminal B or 71.2% (37/52) of the Luminal B (Her2−) BCs were underestimated as Luminal A in CB. For all other subtypes, the agreement was 100%, there was no overestimation as a more unfavourable IBCST in CB. The overall underestimation rate came out with 13%. Whereas the PPV in CB is 100% for Luminal B, Her2 positive, and Triple negative IBCSTs, it is only 82% for Luminal A (171/208). The reason is underestimation of proliferation in CB. Conclusions. Using the St. Gallen clinicopathological definition and the MAI as the most well reproducible tool for proliferation measurement to assess IBCST on CB both, the agreement with SSP results and the PPVs are generally high. This means that intrinsic subtyping on CB can be used for preoperative decision making in the vast majority of cases. However, for the Her2 negative subgroup of Luminal B subtype in CB the agreement with SSP is lower due to underestimation of proliferation in CB.

SO-030 Comparison of the EndoPredict test between core biopsies and corresponding surgery breast tumor blocks B .M . Müller1, J .C . Brase2, F . Haufe2, K .E . Weber2, C . Petry2, R . Kronenwett2, M . Dietel1, C . Denkert1 1 Charité – University Hospital Berlin, Institute of Pathology, Berlin, 2Sividon Diagnostics, Cologne Aims. The EndoPredict (EP) has been recently introduced as a novel RNA-based multigene classifier to predict the likelihood of distant recurrence in ER-positive, HER2-negative breast cancer patients treated with adjuvant endocrine therapy. This study was designed to compare the EP between formalin-fixed and paraffin embedded (FFPE) core biopsies and surgical specimen of breast cancer patients. Additionally, we investigated the influence of biopsy-induced tissue injuries on the quantification of the EP using the paired breast cancer samples. Methods. 80 FFPE tumor blocks composed of 40 paired samples (core biopsies and corresponding surgery specimen) from ER-positive, HER2negative patients were selected based on the presence of a biopsy channel. Total RNA was extracted from FFPE samples using a fully automated isolation method. Relative expression levels of 8 EP genes were assessed in triplicate by one-step reverse transcription qPCR and normalized to the expression level of 3 reference genes. The EP score was calculated for the biopsy and tumor section, respectively. Results. Mean Ct (cycle threshold) value of the 3  normalization genes was used as surrogate marker for RNA yield. With a median difference of 2 Ct units, RNA yield was considerably lower in core biopsies, but sufficient to measure the multigene assay in all recruited core biopsies and surgical tumor samples. The EP score, calculated from the gene expression values, was highly correlated between tumor sections and core biopsies (Pearson r=0.92) with a concordance of classification into low or high risk of metastasis of 95%. Conclusions. The EndoPredict score was highly correlated between core biopsies and corresponding surgical tissue specimen. Preoperative biopsy sampling has no considerable effect on risk categorization by the EP.

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Abstracts SO-031 Decentral gene expression analysis to predict outcome of ER+/ Her2− breast cancer – results of a proficiency testing program for the EndoPredict assay C. Denkert1, R. Kronenwett2, W. Schlake3, K. Bohmann2, R. Penzel4, K.E. Weber2, H. Höfler5, U. Lehmann6, P. Schirmacher4, K. Specht5, M. Rudas7, H. Kreipe6, P. Schraml8, G. Schlake3, Z. Bago-Horvath7, F. Tiecke3, Z. Varga8, H. Moch8, M. Schmidt9, J. Prinzler1, D. Kerjaschki7, B.V. Sinn1, B.M. Müller1, M. Filipits10, C. Petry2, M. Dietel1 1 Charité University Hospital, Institute of Pathology, Berlin, 2Sividon Dignostics GmbH, Köln, 3Institute of Pathology, Gelsenkirchen, 4University of Heidelberg, Institute of Pathology, Heidelberg, 5Technical University Munich, Institute of Pathology and Pathological Anatomy, München, 6Medizinische Hochschule Hannover, Institute of Pathology, Hannover, 7Medical University of Vienna, Institute of Pathology, Austria, 8University Hospital Zurich, Institute of Surgical Pathology, Zürich, Switzerland, 9University of Mainz, Department of Gynecology and Obstetrics, 10Medical University of Vienna, Institute of Cancer Research, Austria Aims. Gene expression profiles provide important information about the biology of breast tumors and can be used to develop predictive tests. However, the implementation of quantitative RNA-based testing in routine molecular pathology has not been accomplished, so far. The EndoPredict assay has recently been described as a quantitative RT-PCR-based multigene expression test to identify a subgroup of hormone-receptor positive tumors that have an excellent prognosis with endocrine therapy only. To transfer this test from bench to bedside it is essential to evaluate the testperformance in a multicenter setting in different molecular pathology laboratories. In this study, we have evaluated the EndoPredict assay in seven different molecular pathology laboratories in Germany, Austria and Switzerland. Methods. A set of ten formalin-fixed paraffin-embedded tumors from patients with breast cancer was tested in the different labs. The EndoPredict score (EP score) ranging from 0 to 15 was calculated and patients were classified into low or high risk for the occurrence of distant recurrence using a validated cut-off. The variance and accuracy of the EndoPredict assays were determined using predefined reference values. Results. Extraction of a sufficient amount of RNA and generation of a valid EP score was possible for all 70 study samples (100%). The EP scores measured by the individual participants showed an excellent correlation with the reference values, respectively, as reflected by Pearson correlation coefficients ranging from 0.987 to 0.999. The Pearson correlation coefficient of all values compared to the reference value was 0.994. All laboratories determined EP scores for all samples differing not more than 1.0 score units from the pre-defined references. All samples were assigned to the correct EP risk group, resulting in a sensitivity and specificity of 100%, a concordance of 100%, and a kappa of 1.0. Conclusions. Taken together, the EndoPredict test could be successfully implemented in all seven participating laboratories and is feasible for reliable decentralized assessment of prognosis in luminal breast cancer.

SO-032 Cyclin D1 gene amplification is rarely heterogeneous in breast cancer E. Burandt1, M. Grünert1, A. Lebeau1, M. Choschzick1, V. Müller2, C. Bokemeyer3, R. Simon1, G. Sauter1, F. Jänicke2, W. Wilczak1 1 University Medical Center Hamburg-Eppendorf, Department of Pathology, Hamburg, 2University Medical Center Hamburg-Eppendorf, Department of Gynecology, Hamburg, 3University Medical Center Hamburg-Eppendorf, Department of Internal Medicine II, Oncology Center, Hamburg Aims. Amplification of Cyclin D1 (CCND1) occurs in about 10–20% of breast cancers and has been suggested to predict resistance to anti-hormonal therapy. As the diagnostic accuracy of predictive biomarkers can be substantially limited by regional expression differences within tumors, heterogeneity of CCND1 amplification was assessed in this study. To assess heterogeneity, a novel tissue microarray based analysis platform was developed. Methods. To comprehensively asses the three-dimensional molecular composition of breast cancers, a “heterogeneity TMA” was constructed containing 8 different tissue cylinders from as many different cancer containing tumor blocks as possible (at least 4) from 147 primary breast cancers. Additional tissue samples were taken from 1–4 corresponding nodal metastases from 35 of these patients. CCND1 amplification was assessed by dual labeling fluorescence in situ hybridization (FISH). Results. The analysis revealed amplification in 28 of 133 (21.05%) patients with informative FISH results. CCND1 amplification was significantly associated with high tumor grade (p=0.042). The preference of ER positive tumors (p=0.061) was not statistically significant. No association was found between CCND1 amplification and tumor type (p=0.307), stage (p=0.540) and PR expression (p=0.871). A discordant Cyclin D1 amplification status was initially detected in 6 out of 28 (21.43%) amplified tumors by TMA analysis. Re-testing on large sections confirmed CCND1 heterogeneity in only 3 of them. Discordant results of the other cases were due to variable interpretation of the TMA cores within the borderline range (CCND1/centromer 11 ratios between 1.7 and 2.3). Overall CCND1 genetic heterogeneity was observed in 3 out of 133 informative tumors (2.3%). No discrepancies were detected between 22 primary tumors and their matched lymph node metastases. Conclusions. The high degree of homogeneity seen for CCND1 amplification suggests that this alteration represents an early event in breast cancer development in a small subset of breast cancers. Thus, CCND1 status determined in a core biopsy is highly representative of the entire tumor and appropriate for predicting treatment outcome if applicable.

SO-033 Prognostic relevance of AIB1 (NCoA3) amplification and over­ expression in breast cancer E. Burandt1, G. Jens1, F. Holst1, F. Jänicke2, V. Müller2, C. Bokemeyer3, W. Wilczak1, L. Terracciano4, R. Simon1, G. Sauter1, A. Lebeau1 1 University Medical Center Hamburg-Eppendorf, Department of Pathology, Hamburg, 2University Medical Center Hamburg-Eppendorf, Department of Gynecology, Hamburg, 3University Medical Center Hamburg-Eppendorf, Department of Internal Medicine II, Oncology Center, Hamburg, 4University of Basel, Department of Pathology, Basel, Switzerland Aims. AIB1 is an estrogen receptor co-activator, know to be amplified in a fraction of breast cancers. Aim of this study was to test the potential clinical significance of AIB1 expression levels and its relationship with ER alpha expression, tumor phenotype and prognosis. Methods. To analyze AIB1 expression and amplification immunohistochemistry and Fluorescence in situ hybridization (FISH) was performed on a pre-existing breast cancer tissue microarray (TMA) containing tumor samples of 2197 breast cancer patients. Results. AIB1 expression was found in 60% of our tumors including 29% weak, 7% moderate and 24% strong expressors. AIB1 expression was sig-

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nificantly associated with advanced tumor stage (p=0.003), high BRE grade (p<0.0001), poor prognosis (p=0.0018), and rapid tumor cell proliferation (Ki67 labeling index; p<0.0001). There was a strong inverse relationship with ER expression (p<0.0001). AIB1 amplification was found in 132 of 1253 interpretable cases (11%). AIB1 expression was significantly associated with presence of AIB1 gene amplification but there was also a substantial fraction of AIB1 amplified cases (28%) without detectable protein expression. This raises the possibility of AIB1 being silenced in a fraction of 20q13 amplified breast cancers, which are potentially driven by other 20q13 oncogenes. Conclusions. The strong inverse relationship between AIB1 and ER expression in breast cancer might suggest a compensatory upregulation of the ER co-activator AIB1 in cancers with low ER levels. Furthermore, our data support the arguments for other non-ER dependent functions of AIB1, especially including a significant impact on cell cycle regulation in breast epithelial cells, which may serve as an important rack-wheel in the complex escape-mechanisms of anti-hormonal treated breast cancers.

SO-034 FGFR1 amplification in breast cancers with unfavorable features K. Pfaltz1, S. Schneider2, U. Güth3, E. Kilic4, S. Eppenberger-Castori2, C. Tapia1 University Bern, Institute of Pathology, Bern, Switzerland, 2University Hospital Basel, Institute of Pathology, Basel, Switzerland, 3University Hospital Basel, Department of Gynecology and Obstetrics, Basel, Switzerland, 4University Charité Berlin, Institute of Pathology, Berlin 1

Aims. The fibroblast growth factor receptor 1 (FGFR1) gene is located at chromosome 8p12 encoding for a tyrosine kinase. FGFR1 is involved in cell proliferation, survival, migration, and differentiation. FGFR1 can be targeted by a small molecule (FGFR inhibitor) leading to significant tumor shrinkage. FGFR1 amplified tumors seem to be targetable/responsive to FGFR inhibitors. Therefore, we validated FGFR1 gene status in a large cohort of breast cancers to evaluate FGFR inhibitors as a possible therapeutic option in this disease. Methods. We hybridized tissue mirco-arrays (TMA) with 907 breast cancers using a commercially available fluorescent in-situ hybridization probe (FGFR1/CEN8; ZytoVision). Normal gene status was considered as a ratio (FGFR1/CEN8): 0.8–1.9, amplification was defined as ratio ≥2.0, polysomy was defined as >4 FGFR1 and CEN8 signals. Results. FGFR1 amplification was observed in 8.9% (n=81), a normal gene status was found in 80.7% (n=732), and a polysomy was detected in 10.2% (n=93) of all tumors. FGFR1 amplified breast cancers showed the following features: 72.8% (59/81) ductal, 45.5% (35/77) high grade (G3), 16% (12/75) HER2 of 2+/3+, 55.5% (35/63) positive lymph nodes, and 18% (13/72) recurrence. Comparing T-categories (T1, T2, T3) FGFR1 amplified breast cancers and non-amplified tumors showed the following T1: 25% vs. 34%, T2: 56% vs. 48%, T3: 9% vs. 6%. There was no significant difference in overall survival between patients with and without FGFR1 amplification. However, patients with FGFR1 amplified cancers and anti-hormonal therapy showed slightly better survival. Conclusions. FGFR1 amplification is prevalent (8.9%) in breast cancers, especially in tumors with unfavorable/aggressive features such as a high tumor grade (45.5%), large tumor diameter, and metastasis (55.5%). These patients are a clinically relevant group since they require aggressive adjuvant treatment. The detection of FGFR1 amplification could help in the identification of some patients already at higher risk which might benefit from a new therapeutic option with FGFR1 inhibitors.

SO-035 Influence of FGFR1, FGFR2 and FGFR3 gene status and protein expression on prognosis and therapy response in breast cancer – an assessment using FISH analysis and immunohistochemistry R. Erber1, D. Wachter1, P.A. Fasching2, A. Santiago3, R. Guzman3, A. Gasparyan3, I. Villalobos3, S. Davenport3, M. Gordon3, B. Meyer4, S. Hauke4, M.W. Beckmann5, M.F. Press3, A. Hartmann1 1 University Erlangen, Department of Pathology, Erlangen, 2University of California at Los Angeles, Department of Medicine, Division of Hematology and Oncology, David Geffen, Los Angeles, United States, 3University of Southern California, Norris Comprehensive Cancer Center, Department of Pathology, Los Angeles, United States, 4ZytoVision GmbH, Bremerhaven, 5Department of Gynecology and Obstetrics, Erlangen University Hospital, Department of Gynecology and Obstetrics, Erlangen University Hospital, Erlangen Aims. Deregulated FGFR signalling is frequently associated with human cancer. Amplification of FGFR1 is found in 9–15% of breast cancer and in 16–27% in the Luminal B subgroup, respectively, and is associated with ER-positive breast cancer and resistance to tamoxifen therapy. FGFR2 amplification occurs in 4–12% of breast cancer, especially in triple negative breast cancer known to have poor prognosis. FGFR2 single nucleotide pleomorphisms (SNPs) have been shown to be associated with higher risk of breast cancer. Since FGFR inhibitors could be a potential targeted therapy for breast cancer patients harbouring FGFR amplification and overexpression, the assessment of FGFR gene status and/or protein expression in certain subgroups of breast cancer patients might be important. In the ongoing project, the first aim is to assess amplification and overexpression rates of FGFR1/2/3 and analyze, whether there is any correlation with clinical and histopathological parameters, outcome and response to chemo and/or hormone therapy. Second, we want to investigate, if there is any association between FGFR2 amplification/overexpression and FGFR2 SNPs. Methods. Tumour tissue of a subgroup of 907 patients of the BBCC study was archived and tissue microarrays were created. Clinical and histopathological parameters, outcome data and the status for the most frequent FGFR2 gene polymorphisms are available. Gene status and overexpression is assessed using fluorescence-in-situ-hybridisation with ZytoLight gene/centromere probes for FGFR1/2/3 (ZytoVision) and immunohistochemistry analysis, respectively. Results. FISH analysis of FGFR1 shows an amplification rate of 6.6%. There is no amplification of FGFR3 found in the evaluated cases. Analysis of SNP arrays of 50 breast cancer cell lines shows no FGFR3 gene copy number higher than 4 and, therefore, supports the hypothesis that there is no FGFR3 amplification in breast cancer. The assessment of protein expression of FGFR1/2/3 and of FGFR2 gene status and the correlation to clinical and histopathological parameters are ongoing. Conclusions. FGFR1 amplification is present in relatively low frequency in unselected breast cancer. FGFR1 and FGFR2 might act as molecular markers in breast cancer that predict resistance to tamoxifen and poor prognosis and might indicate patients with possible benefit from targeted therapy using FGFR inhibitors. A trial to show the possible correlation between FGFR amplification/overexpression and response to FGFR inhibitors is planned.

SO-036 Tumour-suppressive functions of SFRP1 in vitro: decreased cell proliferation and increased adhesion in breast cancer cells of luminal and basal type L. Franken1, A. ten Haaf1, W. Winkens1, R. Knüchel1, E. Dahl1 University Hospital of the RWTH Aachen, Institute of Pathology, Aachen

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Aims. Breast cancer is characterized by aberrant WNT signalling. SFRP1, a putative tumour suppressor, is an important inhibitor of WNT signalling. A high frequency (>65%) of human breast tumours show hypermethylation and silencing of the SFRP1 promoter region. Nevertheless little Der Pathologe Suppl 1 · 2012 

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Abstracts is known about the functionality of SFRP1. In this project we have analyzed the functional consequences of a SFRP1 re-expression in human breast cancer cell lines. We also conducted a systematic expression analysis to determine possible links to other pathways after SFRP1 re-expression. Methods. Using standardized methods SFRP1 overexpressing clones of two human breast cancer cell lines, BT20 (basal-like) and SKBR3 (luminal-like), were generated and analyzed in cell-culture based assays. The ability of SFRP1 expressing BT20 clones to grow in nude mice will be also tested. Using DNA array expression profiling, we searched for genes activated or repressed after forced re-expression of SFRP1 in these two tumour cell lines (“SFRP1 target genes”). Validation of differential gene expression was performed by real-time PCR comparing stable SFRP1 and control clones. Results. SFRP1 re-expression in stable clones of two breast cancer cell lines (BT20 and SKBR3) was validated on mRNA and protein level. We found a correlation between SFRP1 expression and the growth behaviour of both breast cancer cell lines in functional assays. SFRP1 re-expression resulted in a significant reduced proliferation (p<0.001 and p=0.003) as well as in an increased ability for adhesion (p=0.03 and p<0.001). Chip expression profiling identified 127 “SFRP1 target genes”, i.e. genes that are controlled by SFRP1 expression and might explain the altered cellular behaviour observed in SFRP1-tranfectands. Conclusions. Our in vitro study indicates a functional involvement of SFRP1 in the proliferation and adhesion of human breast cancer cells, supporting the putative tumour-suppressive function of SFRP1 in this tumour entity. We are in the process of defining signalling pathways (besides WNT) affected after forced SFRP1 re-expression. This will increase our knowledge on the function of this important breast cancer related gene. Defining small molecule drugs that can mimic active SFRP1 signalling pathways may ultimately led to novel targeted therapies in breast cancer.

SO-037 Functional proteomic analysis reveals a possible role for RAD23B in breast cancer invasion A . Linge1, P . Maurya1, M . Henry1, M . Clynes1, P . Meleady1 Dublin City University, National Institute for Cellular Biotechnology, Dublin, Ireland

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Aims. The molecular mechanisms involved in tumour cell invasion and metastasis are complex multistep processes. Identification of protein targets, which play a role in breast cancer invasion may help in understanding the rapid progression of breast cancer and may ultimately lead to the development of new biomarkers for the disease or potentially novel drug targets. Methods. Two dimensional difference gel electrophoresis (2-D DIGE) and western blot analysis were performed in high and low invasive breast cancer cell lines in order to identify differentially expressed proteins that may be linked to the invasive phenotype in vitro. Functional effects on invasion, migration and adhesion were further examined using transient siRNA knockdown. Results. The UV excision repair protein RAD23 homologue B (RAD23B) was found to be among those differentially expressed proteins and was chosen for further functional follow-up. For the first time, we show that transient down-regulation of RAD23B using specific siRNA molecules leads to a significant increase in invasion of breast cancer cell lines in vitro whereas no significant effect on migration could be revealed. In addition, we demonstrate that down-regulation of RAD23B increases adhesion of breast cancer cells to fibronectin and collagen IV in vitro. Conclusions. Our study demonstrates that a proteomic approach using 2-D DIGE and mass spectrometry to identify proteins that may be related to the invasive phenotype can yield proteins, e.g. RAD23B, which have a functional effect on the invasive phenotype in vitro. However, further studies are required to gain a greater understanding of the impact of RAD23B and its potential role in breast cancer invasion and metastasis.

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AG Paidopathologie I SO-038 Change of diagnostic gold standard for primary ciliary dyskinesia H . Omran1 1 Kinderklinik der Universitätsklinik Münster, Münster Die primäre ciliäre Dyskinesie (PCD) ist eine klinisch und genetisch heterogene Gruppe hereditärer Erkrankungen, die durch eine Dysfunktion motiler Zilien charakterisiert sind. Die Kinder fallen durch RDS, Rhinitis und chronischen Infektionen der oberen und unteren Atemwege wie durch einen persistierenden feuchten Husten auf. In ca. der Hälfte der Fälle tritt ein Situs inversus auf. Betroffene Männer weisen häufig aufgrund einer Spermienschwanzfehlfunktion eine Infertilität auf. Die Diagnostik der PCD ist komplex. Dies erklärt, warum die Erkrankung häufig erst beim Auftreten irreversibler Bronchiektasien diagnostiziert wird. Kürzlich veröffentlichte die ERS Empfehlungen zur Diagnostik. Eine Diagnose ist durch Analyse von Zellen der Nasenschleimhaut möglich. Zur Diagnostik der PCD werden v.  a. drei Methoden eingesetzt: (i) Idealerweise erfolgt an direkt gewonnenen Atemwegszellen eine Analyse der Zilienfunktion mittels Hochfrequenzvideomikroskopie. Einige PCD-Varianten (z.  B. DNAH11) können ausschließlich anhand des Zilienschlagmusters diagnostiziert werden. (ii) Früher entsprach die Transmissionselektronenmikroskopie (TEM) dem Goldstandard der PCD-Diagnostik. Typische in der TEM nachweisbare Defekte umfassen das Fehlen äußerer Dyneinarme sowie Defekte der radialen Speichen mit tubulärer Disorganisation. Die Diagnose von Defekten der inneren Dyneinarme mittels Elektronenmikroskopie ist schwierig, da diese innerhalb eines Zilienquerschnitts auch bei gesunden Zilien nur vereinzelt nachweisbar sind. Aufgrund zahlreicher methodischer Limitationen erzeugt die TEM leider häufig falsch negative und falsch positive Befunde. (iii) Mittels der immunfluoreszenzmikroskopischen Diagnostik können Proteine unterschiedlicher Motorproteinkomplexe untersucht werden, und Defekte äußerer und innerer Dyneinarme wie auch Radialspeichendefekte nachgewiesen werden. Im Gegensatz zur TEM ist diese Methode für Fehlinterpretationen aufgrund sekundärer ziliärer Veränderungen weniger anfällig. Die IF erlaubt es auch die genetische Diagnostik gezielt durchzuführen. (iv) In Zusammenarbeit mit unserer Arbeitsgruppe konnten in den letzten Jahren zahlreiche genetische Defekte (DNAH5, DNAH11, DNAI1, DNAI2, TXNDC3, LRRC50, KTU, RSPH4A, RSPH9, OFD1, RPGR, CCDC39, und CCDC40) nachgewiesen werden. Zur weiteren Abklärung bieten wir allen Patienten im Rahmen einer Teilnahme an unserer Studie zur Erforschung der molekularen Ursachen der primären ciliären Dyskinesie eine genetische Diagnostik an.

SO-039 Prenatal incidence of GATA1 mutations in fetuses with trisomy 21 S . Höller1, M . Bihl1, A . Tzankov1, R . Chaffard1, T . Kuehne2, C . Potthoff2, E . Bruder1 1 University of Basel, Institute of Pathology, Basel, Switzerland, 2University of Basel, University Children’s Hospital, Basel, Switzerland Aims. Newborns with trisomy 21 are at risk to develop a transient myeloproliferative disorder. This transient myeloproliferative disorder may progress to acute megakaryoblastic leukemia (AML) in young children, and both TMD and AML are closely related to GATA1 exon 2 mutations. Methods. We screened 22 fetal autopsies (livers and lungs) or curettages with known trisomy 21 for GATA1 exon 2 mutations by gene sequencing at different time points in the fetogenesis. Moreover, fetal hematopoiesis was immunohistochemically analyzed. Results. GATA1 exon 2 mutations could be detected in 3 of 22 cases (14%): p.G37R, p.G40D and p.G31R. The earliest occurrence of this mutation was found in a curettage of the 13th week of gestation. The number of maturing megakaryocytes, granulocytes and erythrocytes in the fetal

liver did not correlate with the mutation status. However, the number of CD34, MPO, GlycophorinA and CD61 positive hematopoietic cells varied considerably from case to case and the number of mutated cases was rather small. No mutation could be found in the lung specimens of each autopsy. Conclusions. GATA1 exon 2 mutations are acquired mutations and may occur already in fetal hematopoiesis as early as 13th week of gestation. We could not demonstrate a phenotypic correlation with quantitative fetal liver hematopoiesis.

SO-040 Fetal acute myeloid leukemia in Down syndrome causing intrauterine fetal death H. Löser1, M. Engels1, S. Huss1, R. Büttner1, A. Müller2, J. Fries1 University of Cologne, Institute of Pathology, Köln, 2University of Bonn, Institute for Pathology

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Aims. Down syndrome (DS) is associated with a 10- to 20-fold higher risk for developing acute leukemia compared to non-DS children. About 10% of DS newborns are diagnosed with a “transient myeloproliferative disorder” (TMD) and up to 30% of these children sustain a progression to an acute myeloid leukemia (AML) within the following 3 years. Methods. We report of a fetal, intrauterine AML in trisomy 21. While no further fetal malformation was recognised in the pervious prenatal screening of the 42-years old woman the fetus died in utero in the 24th week of gestation without any detectable clinical cause. After informed consent was obtained by the mother an autopsy was performed at the Institute of Pathology, University of Cologne. Results. We saw a phenotypically female fetus with an almost gestational age-appropriate development without any apparent anomalies. The internal organs regular, the membranous part of the ventricular septum already closed. Histologically we found extensive tissue and vascular infiltrations with megakaryoblasts in all organs including thymus gland, heart, lungs, liver, pancreas, spleen, adrenal glands, kidneys, intestine, and organs of the lesser pelvis. Immunohistologically positive for CD61, this finding was consistent with an AML FAB-subtype M7. The placenta showed pronounced infiltrations of leukemic cells in the villous vessels, the larger villi and in the umbilical vein. The literature describes mutations of the exon 2 and 3 of the GATA1 gene, an erythroid transcription factor particularly in children with AML and DS. Conclusions. Until now, no data has been published about prenatal GATA1-mutations, although this is there most likely point in time of occurrence when AML develops in early childhood. In the present case, we are in the process of analysing whether GATA-1 mutations can be detected in the fetal tissue by sequencing. Considering that the heart was also affected by the massive intra- and extravascular dissemination of AML, it is very likely that this is the crucial reason for the intrauterine fetal demise. In accordance with published literature this is the first report of fetal AML in DS. It extends the spectrum of possible differential diagnosis of intrauterine fetal death in DS.

SO-041 On the correlation of coronal clefts and chromosomal aberrations E. Doberenz1, U. Gembruch2, R. Schumacher3, A.M. Müller4 1 University Bonn Medical Center, Institute of Pathology, Bonn, 2University Bonn Medical Center, Dept. of Prenatal Medicine and Obstetrics, Bonn, 3University Clinic Freiburg, Department of Pediatrics, Freiburg, 4University Bonn, Department of Pediatric Pathology, Bonn Aims. Coronal vertebral clefts, radiologically defined as vertical radiolucent bands in the middle of the vertebral body, are to our observation associated with chromosomal aberrations. Published studies concerning this are missing.

Methods. Hence 443 aborted fetuses were studied radiologically concerning the incidence of coronal clefts and their association with proven chromosomal aberrations. Furthermore they were studies histologically concerning remnants of the notochord which are still discussed as cause of coronal clefts. Results. In 44 cases coronal clefts were visualized radiologically. In 93.2% of these fetuses chromosomal aberrations were proven. On the other hand, of all fetuses with trisomy only 30% showed this diagnostic finding. Histologically coronal clefts displayed a missing ossification at the center of the vertebral body. Remnants of the notochord could be excluded. Conclusions. Summing up, coronal clefts represent a retarded ossification of vertebral bodies in fetal development, nearly solely found in fetus with chromosomal aberrations and malformations. On the other hand, the genetic diagnosis of chromosomal malformation, especially trisomy, does not automatically implicate coronal clefts.

SO-042 Molecular-genetic classification of glomerulocystic kidney disease J.K. Lennerz1, H. Liapis2 1 University Ulm, Institute of Pathology, Ulm, 2Washington University, Department of Pathology and Immunology, St. Louis, United States Aims. Glomerular cysts (GCs) are defined as Bowman space dilatation >2–3× normal that occupy >5% of glomeruli. GCs occur in pediatric and adult kidneys. GCs are associated with a plethora of genetic- and nongenetic diseases and present significant diagnostic challenges. We aim to develop a molecular-genetic classification scheme to facilitate diagnosis. Methods. We reviewed our biopsy and nephrectomy material and the medical literature of >100 years (20+230 cases). Additionally, we performed in silico experiments mapping gene-protein networks (IngenuitySystems; MetaCoreV4.5). Results. We identified 5 categories: Type I represents GCK in polycystic kidney disease (PKD). Type  II represents molecularly-recognized (UMOD/TCF2) and inherited subtypes of GCK (other than PKD). Type  III represents syndromic-GCK, associated with malformations/ syndromes. Type IV includes obstructive-GCK (±dysplasia). Type V encompasses sporadic-GCK, including ischemic- and drug-induced types, which lack an inheritance pattern, syndromic features or obstruction/ dysplasia. In this scheme, types I/II can be regarded primary-GCK whereas III–V represent secondary-GCK and emerge (not necessarily from constitutional mutations but) from a loss-of-function of ‘maintenance kidney genes’ involved in injury repair. Conclusions. The clinical relevance of the various associations forms the basis for this molecular-genetic classification scheme that provides a framework for a structured differential diagnosis, suggests screening for probable mutations, and opens new avenues in understanding common kidney injuries.

SO-043 RSV- and hMPV-infections in BALB/c mice M. Neumann1, J. Lüsebrink2, V. Ditt2, O. Schildgen2, A.M. Müller3 University Bonn Medical Center, Institute of Pathology, Bonn, 2University Bonn Medical Center, Institute of Virology, Bonn, 3University Bonn, Department of Pediatric Pathology, Bonn

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Aims. hRSV (human respiratory syncytial virus) and hMPV (human metapneumovirus) cause respiratory infections, leading to death in approximately 200,000 toddlers and old patients each year. Numerous aspects of the pathomechanisms and resulting pathomorphologic changes of infection are sparsely studied. Murine models differ concerning methodical parameters as well as inoculation methods resulting in a limited comparability of published studies. By using a mild inoculatiDer Pathologe Suppl 1 · 2012 

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Abstracts on method avoiding cough reflex, we studied, whether an infectiously sufficient viral load can be achieved, if it causes significant pulmonary pathomorphological findings and if those findings differ for each virus and can be correlated by age. Methods. 43 mice (group I: 4 weeks of age; group II: 16 months of age) were either mock infected, inoculated with hRSV, hMPV or half the quantity of both viruses. Five days after inoculation the lungs were analysed by RTq-PCR for containing viral loads and by light microscopy and immunohistochemistry concerning pathomorphological findings. Results. Only lungs of infected mice showed a significantly raised viral load. In young hMPV-infected mice pathomorphological findings (broadened septae, focal poor ventilation) were far more prominent than in older animals. RSV-infection and co-infection caused increased severity of pathomorphology in older animals, while only displaying focal poor ventilation in young mice. By immunohistochemistry, a more proximal hRSV-infestation of the bronchi was found for co-infections than for solitary hRSV-infections. Old infected animals displayed virus proteins within macrophages and an enhanced BALT-activation. Conclusions. The raised viral loads affirm the effectivity of the inoculation method under inhalative short time anaesthesia, suppressing cough reflex without putting a strain on the animals concerning side-effects of anesthesia, e.g. vomitus. In all, pathomorphological changes were mild. Nevertheless, viral- and age-specific differences were found which might be related to age-dependent immune responses. An explanation for the more proximal bronchial distribution of hRSV during co-infection could be a result of competing receptors for attachment (as they are in large parts identical with hMPV) or a mutual inhibition during the intracellular replication process.

SO-044 Genetic analysis of relapsed childhood germ cell tumors by CGH L. Wulf1, C. Vokuhl1, D. Schneider2, G. Calaminus3, I. Leuschner1 1 University of Kiel, Department of Pediatric Pathology, Kiel, 2Municipal Clinics Dortmund, Department of Pediatrics and Adolescent Medicin, 3University Hospital Münster, Department of Pediatric Oncology and Hematology Aims. Germ cell tumours are rare heterogeneous malignancies in childhood. Pure teratomas in childhood normally don’t show genetic changes in terms of aberrations and they are associated with a relapse-free prognosis. Higher grade of immaturity in teratomas increases probability of york sac tumour presence which concurrently decreases prognosis for relapse-free survival, especially if the tumour can not be completely excised. It is meaningful whether the rare cases of relapsing teratomas are genetic changes that are likely to predict recurrence of these tumours. This assumption disposed us to use chromosomal genomic hybridisation for primary tumours with relapse and comparing them to teratomas without relapse. Methods. Formalin-fixed, paraffin-embedded tissue blocks were retrieved from the files of the German Pediatric Tumor Registry. Sufficient DNA from 9 patients included in the Malignant Germ Cell Study Group (MAKEI) could be extracted, among them 9 primary tumors and 8 relapses. Tumor DNA was labeled with spectrum-green dUTPs, normal reference DNA with spectrum-red dUTPs. After co-hybridisation on normal male human metaphase spreads, CGH was analysed using ISIS CGH software (Metasystems). Results. All of the primaries were teratomas, beneath the relapses there were 4 teratomas and 4 tumors with at least a microfocus of YST. All but two of the primary teratomas had chromosomal aberrations detectable by CGH. The average number of chromosome arm aberrations per tumor was 1.9 (range: 0–5). Copy number changes were gain of chromosome 17, 7, 1q, 3 and 12p and loss of chromosome 13q, 5q and 5p. When comparing the two groups (primary tumor and relapse) most of the chromosomal imbalances detected in the primary tumor could also be found in the relapse. Furthermore some of the tumor relapses had additional changes (e.g. amplification of chromosome 8q).

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Conclusions. Pediatric germ cell tumors are a heterogeneous group with generally good relapse-free prognosis. Regardless most of the children are cured, some tumors relapse. In this study we wanted to search for differences between primary and relapsed tumors to find possible markers which could predict tumor relapse. In contrast to most teratomas which generally show a normal karyotype, the teratomas with relapse in our study showed chromosomal aberrations in 78% (7/9) of cases. Taken together the detection of chromosomal aberrations in teratoms could be a risk factor for tumor relapse. This assumption has to be evaluated on a bigger cohort of patients.

SO-045 Genetic and immunhistochemical analysis of embryonal rhabdomyosarcoma with good and poor prognosis T. Heilmann1, C. Vokuhl1, T. Dantonello2, E. Koscielniak2, I. Leuschner1 1 University of Kiel, Department of Pediatric Pathology, Kiel, 2Olgaspital, Klinikum Stuttgart, Pediatric 5 Aims. Embryonal rhabdomyosarcoma (ERMS) is the most common soft tissue sarcoma in children, typically affecting children younger than 5  years of age. Contrary to alveolar rhabdomyosarcoma, ERMS don’t show specific translocations or specific genetic changes. Prognosis depends on the tumor size, localization and the age of the patient. Even if the overall survival with approximately 70% is generally good, there are some cases of ERMS with unfavourable prognosis. In our study we used comparative genomic hybridization (CGH) and immunohistochemistry (IHC) to analyse the tumours with good prognosis comparing to tumors with unfavourable prognosis. Methods. Formalin-fixed, paraffin-embedded tissue blocks were retrieved from the files of the German Pediatric Tumor Registry. Sufficient DNA from 28 patients for the CGH included in the Cooperative Weichteilsarkom (Soft Tissue Sarcoma) Study Group (CWS) could be extracted. For the CGH tumour DNA was labelled with spectrum-green dUTPs, normal reference DNA with spectrum-red dUTPs. After co-hybridization on normal male human metaphase spreads, CGH was analysed using ISIS CGH software (Metasystems). Furthermore we analysed the expression of the cell-cycle-proteins p16ink4, pRb and cyclin D1 as well as p53 in 40 cases. Results. The most common chromosome arm copy number changes were loss of chromosome 4q (39%), 6q (32%) and 5q (29%). Frequent gains were on chromosome 20q (54%), 22q (50%), 8p (46%), 8q (43%) and 11q (39%). When comparing the two groups we found gain on chromosome 6p (2/14), 14q (4/14) and 18p (3/14) and loss on chromosome 3p (3/14) only in cases with unfavourable prognosis. Only in the group of good outcome we found gains on chromosome 1q (2/14), 2q (2/14) and 11p (2/14). The results from the IHC showed abnormal expression of p53 5/40, Cyclin D1 10/40, pRb 6/40 and p16 24/40. Comparing the two groups the 5 cases with abnormal p53 expression were only in the group with poor prognosis. Conclusions. Although embryonal rhabdomyosarcoma generally has a good prognosis there are cases with less favourable prognosis. In this study we were able to confirm the frequent genetic changes in embryonal rhabdomyosarcoma. Furthermore we found some genetic changes (3p, 6p, 14q and 18p) and abnormal expression of p53 only in the tumour group with less favourable prognosis. To find better tools for risk stratification in embryonal rhabdomyosarcomas, future studies should be concentrated on possible genes within the chromosomal regions we have found in our study.

AG Paidopathologie II SO-046 Overexpression of co stimulatory ICAM1 enhances killing of RMS cell lines by NKT and chimeric T cells K . Simon-Keller1, A .-L . Bohlender1, K . Mößinger1, S . Küffer1, P . Ströbel1, A . Marx1 1 University Medical Center Mannheim, Institute of Pathology, Mannheim Aims. Rhabdomyosarcomas (RMS) are the most common soft tissue sarcoma of childhood and adolescence. Recent efforts to enhance overall survival of patients with clinically advanced RMS have failed. Different types of immunotherapies have been suggested as new perspective. However, little is known about immune escape mechanisms in RMS. Killing of RMS cells with specific chimeric T (cTCs) and NKT cells was markedly attenuated in comparison to killing of CEA-expressing colon carcinoma cells by respective cTCs. Therefore, we wondered whether resistance to killing might be due to lack of co-stimulatory molecules and if so, whether it is possible to improve killing efficiency by overexpression of defective co-receptors. Methods. We compared four alveolar RMS cell lines and two embryonal RMS cell lines with 16 embryonal and 12 alveolar RMS biopsies. Expression status of different surface molecules was checked by FACS analysis, Western blot, and qRT-PCR to characterize RMS cell lines. Biopsies were analyzed by IHC, Western blot and qRT-PCR. Cell lines were transfected with CD54 by electroporation and checked by FACS for CD54 surface expression. Cell survival after co-cultivation of RMS and chimeric T cells was checked by MTT test and FACS analysis. Results. Studying expression levels of MHC class I, MHC class II, CD80, CD86, ICAM-1/CD54 in RMS cell lines and biopsies we found low to absent expression of crucial co-receptors, e.g. ICAM1 and CD86 both in vitro and in vivo. To functionally prove the influence of ICAM-1 expression on killing efficiency, we overexpressed ICAM1 in six RMS cell lines. On co-incubation with cTC and NKT cells, all RMS cell lines showed substantially increased susceptibility to cTC and NKT cell mediated killing after overexpression of ICAM1. Conclusions. The results imply that RMS cell lines lack expression of crucial co-receptors for cytotoxic T cell responses. Up-regulation of ICAM appears as promising strategy to enhance the cytotoxic effect of the immune system against RMS cells, e.g. following RMS-directed vaccination or adoptive transfer strategies.

SO-047 The role of homeobox genes in the development of nephroblastomas K . Koller1, M . Pichler2, K . Koch1, M . Zandl1, I . Leuschner1, G . Höfler1, B . Gürtl1 1 Medical University of Graz, Institute of Pathology, Graz, Austria, 2Medical University of Graz, Department of Internal Medicine, Graz, Austria Aims. Different homeobox genes and some of their binding partners feature already known tumorigenic properties, but their role in the pathogenesis of nephroblastomas has hardly been investigated. In our study we therefore focused on the expression of HOXA9 and its binding partners in different subtypes of nephroblastomas. Methods. The mRNA expression levels were investigated by quantitative REALtime PCR, protein expression levels by immunohistochemistry. Results. All nephroblastomas investigated so far had a significant upregulation of MEIS1 mRNA expression levels. In parallel in most of these cases a nuclear positivity with antibodies against MEIS1 was identified. The majority of nephroblastomas investigated so far also showed an overexpression of PBX2 mRNA, some of them additionally a distinct positive nuclear staining in the immunohistochemical investigation. The

mRNA expression levels of HOXA9 were significantly higher in most of the tumors investigated. Conclusions. Our results show that the upregulation of homoebox genes and their binding partners might play a role in the development of nephroblastomas.

SO-048 Gain of chromosome 8 and IGF1R in pleuropulmonary blastoma C . Vokuhl1, L . de Leon1, S . Kirsch2, E . Koscielniak2, I . Leuschner1 1 University of Kiel, Department of Pediatric Pathology, Kiel, 2Olgaspital, Klinikum Stuttgart, Pediatric 5 Aims. Pleuropulmonary blastoma (PPB) is a very rare, aggressive primary intrathoracic tumor of early childhood. This tumor is composed of a malignant mesenchymal component, namley a rhabdomyomatous, chondroid or fibrosarcomatous, and an epithelial component regarded as entrapped elements. Histopathologically, three different types are described: Type 1 PPB is composed exclusively of cysts lined by a benign epithelium. The cyst walls consist of small, malignant mesenchymal cells, type 2 is composed of cystic and solid areas and type 3 is an entirely solid tumor. The outcome is poor, with 5-years survival rates of 45-49% and 66%, respectively. Because of the rarity of this tumor type, the genetics are poorly understood. Methods. In this study we want to analyse the PPB cases of the Kiel Pediatric Tumor Registry. Sufficient DNA from 16 patients could be extracted. We analysed these cases by comparative genomic hybridisation and confirmed tumors with gain of chromosome 8 and of IGF1R by FISH. Results. The most frequently recurring change was gain of chromosome 8 and the short arm of chromosome 8, respectively (9/17). Six pleuropulmonary blastomas show gain of chromosome 20 (pq and p). Genomic amplification was observed in 5 cases, four related to 15q25qter, and one to 1p. We were able to perform Cen8 FISH in all but one of the cases with chromosome 8 gain. Two tumors show trisomy of chromosome 8, 4 tumors a polysomy (in average 4–5.8 signals per nucleus). The remaining one showed normal signal pattern. FISH could confirm three low-level amplifications and one high-level amplification of the IFG1R gene. Conclusions. Pleuropulmonary blastoma is a very rare pediatric tumor, therefore only case reports or small series of genetic studies were published. In our series of 18 PPBs we could show a gain of chromosome 8 in 51% (9/17). In addition we found five amplifications, four of which are in the 15q21qter region where the insulin-like growth factor type 1 (IGF1R) gene (15q26) lies. All of these were pleuropulmonary blastomas type III, indicating that it is a later event in tumor progression. In future we have to correlate these results with prognosis and these findings suggest the possibility of use of targeted agents in the therapy of a subset of these rare tumors.

SO-049 Expression of cancer testis (CT) antigens in fetal thymus A . Jungbluth1, D . Frosina1, M . Holz1, G . Spagnoli2, S . Gnjatic1, A .M . Müller3 Ludwig Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, United States, 2University Hospital Basel, Department of Biomedicine, Basel, Switzerland, 3University Bonn, Department of Pediatric Pathology, Bonn 1

Aims. CT antigens such as NY-ESO-1, MAGE, GAGE, and CT7 are named after their characteristic pattern of expression, since they are found in various types of cancer and in normal adult tissues are restricted to testicular germ cells. They are also present in fetal ovarian germ cells and occasionally in placenta. In cancer patients, some CT antigens are highly immunogenic and due to their limited expression in normal tissues, are used as vaccine targets for the immunotherapy of cancer. They also serve as markers of malignancy. Little is known about the biology of CT antigens and especially their role in the normal immune system. Der Pathologe Suppl 1 · 2012  | 

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Abstracts Consequently, here was analyzed the presence of CT antigens in a series of fetal thymic tissues. Methods. Archival thymus tissue from 40 cases (week 15–42) were available for analysis. Immunohistochemistry employing the following mAbs (to the following CT antigens) were used: MA454 (MAGE-A1), 57B (MAGE-A4), E978 (NY-ESO-1), #26 (GAGE), CT7-33 (CT7), and CT10#5 (CT10). Results. Expression of the tested CT antigens was highly variable. NYESO-1 remained completely negative. MAGE-A1 was solely found in single cells of three thymi. CT7 and CT10 were present in 10 and 11 thymi respectively, both showing solely focal staining. GAGE and MAGE-A4 were most abundantly expressed: GAGE was present in 22/40 and MAGE-A4 in 27/40 tissues; both antigens displaying larger groups of positive cells. For all tested antigens, immunopositive cells were restricted to the medulla and were exclusively epithelial cells. There was not predilection of any gestational age for any of the tested antigens. Conclusions. The present study shows that -irrespective of fetal development/gestational age- several CT antigens are consistently present in fetal thymus, albeit to a variable extent. Expression is restricted to thymus epithelial cells and ranges from a few cells to larger groups of cells; GAGE and MAGE-A4 are most abundantly present. Interestingly, there was no NY-ESO-1 expression in any of the tested thymi. These data complement serological data in cancer patients which show rare immune responses to those antigens, which were highly expressed in our series of thymus tissues. NY-ESO-1, however, is the most immunogenic antigen in cancer patients. The lack of NY-ESO-1 expression in fetal thymus could be the cause of lacking pre-existing immunotolerance to NY-ESO-1 rendering cancer patients more sensitive to the presence of NY-ESO-1 in cancer tissue and/or vaccine applications.

SO-050 Hepatobiliary rhabdomyosarcoma in a 2-year-old: a case report K. Wieczorek1, G. Fitze2, R. Knöfler3, G. Hahn4, G. Baretton1 1 University Hospital “Carl Gustav Carus”, TU Dresden, Department of Pathology, Dresden, 2University Hospital “Carl Gustav Carus”, TU Dresden, Department of Pediatric Surgery, Dresden, 3University Hospital “Carl Gustav Carus”, TU Dresden, Department of Pediatric Oncology, Dresden, 4University Hospital “Carl Gustav Carus”, TU Dresden, Department of Radiology, Dresden Aims. Although representing the most common sarcoma in the pediatric patient, rhabdomyosarcomas of the liver account for only 0.8% of all rhabdomyosarcomas, and 1% of all liver tumors. Unless they present in the characteristic setting of a botryoid mass in the biliary tree, they are difficult to diagnose, as they share many histomorphologic similarities with undifferentiated embryonal sarcomas of the liver. This case report summarizes clinical data and histomorphologic and immunohistochemical features of a hepatobiliary rhabdomyosarcoma in a 2-year-old boy. Methods. A 2-year-old boy presented to an external hospital with severe jaundice and hepatomegaly. Initially, an infectious process was suspected, and the boy was referred to the pediatrics department of the Dresden university hospital. MR imaging revealed a very large liver tumor and multiple metastases in the lung and bone. The gallbladder and the biliary tree were initially not visible due to the size and partially cystic appearance of the tumor. A biopsy of the liver was taken. Results. Liver biopsy showed a malignant mesenchymal neoplasia consisting of primitive round to spindled cells with multiple mitoses. A zonal architecture was noticed around small bile ducts. Very few (<1%) tumor cells showed myogenic differentiation with eosinophil cytoplasm. Immunohistochemistry confirmed the suspected diagnosis of a hepatobiliary rhabdomyosarcoma with expression of desmin and myf4. The tumor-free liver parenchyma showed severe cholestasic cirrhosis. After neoadjuvant chemotherapy, left-sided hemihepatectomy was performed, including resection of the left and common hepatic duct and the gallbladder.

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Conclusions. Distinction between hepatobiliary rhabdomyosarcoma and undifferentiated embryonal sarcoma is difficult based on histomorphology alone. As both prognosis and therapy differ between the two entities, a thorough histomorphologic and immunohistochemical analysis of pediatric malignant mesenchymal liver tumors is necessary. Myogenic markers such as myf4 and MyoD1 are a helpful and sometimes indispensable diagnostic tool for the correct diagnosis of rhabdomyosarcoma.

SO-051 Angiofibromyxoma of the umbilical cord A.M. Müller1, U. Gembruch2, M. Vogel3 1 University Bonn, Department of Pediatric Pathology, Bonn, 2University Bonn Medical Center, Dept. of Obstetrics and Prenatal Therapy, Bonn, 3 University of Leipzig, Institute of Pathology Aims. Tumours of the umbilical cord are extremely rare. Differential diagnosis includes hemangiomas, teratomas, abdominal wall defects and vascular lesions like hematoma and thrombosis. Hemangiomas are by far the most common umbilical cord tumours. Methods. In a 35-year-old women in the 25th week of gestation a tumour of the umbilical cord was ultrasonographically diagnosed. Ultrasound displayed a significantly broadened umbilical cord with hyperechogenic areas and increased whartons’ jelly and arteriovenous fistulas. Apart from a slight cardiomegaly fetal heart was regular. In week 38 a healthy girl was born by cesarean. Results. Histologically the placenta showed several chorangiomas. The umbilical cord displayed an angiofibromyxom with – taking into consideration ultrasound findings – intratumoral arteriovenous malformations. Conclusions. Angiofibromyxoma with intratumoral arteriovenous malformations is a very rare subtype of hemangiomatous lesion of the umbilical cord. Umbilical cord is formed between the sixth and eighth week of gestation by the approximation of the omphalomesenteric duct resp yolk sac and the allantoic duct within the body stalk. The two arteries and one vein derive from the allantoic vessels. Hence cord hemangiomas are recognized as arising from omphalomesenteric or allantoic vessels. As they often occur together with the more common chorangiomas – like in our case – an underlying congenital predisposition to vascular neoplasms has to be discussed.

SO-052 Pregnancy luteoma – an uncommon ovarian tumor: association with intrauterine growth retardation (IUGR)? C. Jayasinghe1, T. Ameziane2, C. Auerbach2, A. Müller3 Gummersbach Hospital, Institute of Pathology, Gummersbach, 2 St. Elisabeth Hospital Bonn, Department of Obstetrics and Gynecology, Bonn, 3University of Bonn, Institute of Pathology, Bonn

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Aims. Pregnancy luteomas are rare benign lesions of the ovary induced by hormonal alterations during pregnancy. They present as unilateral or bilateral tumorous masses and are usually incidental findings during imaging or caesarean section. Most of the patients are asymptomatic. However, virilization of the mother occurs in one third of cases and virilization of the female fetus is found with two thirds of virilized mothers. Pregnancy luteomas regress postpartum spontaniously, therefore conservative treatment is sufficient. Nevertheless pregnancy luteomas are challenging, particularly for clinicians, as they can mimic malignant ovarian tumors. Methods. We present the case of a 30-year-old primigravida with beta thalassemia major who underwent caesarean section at week 37 because of pathological CTG. A hypotrophic girl was delivered with normal Apgar score. Both mother and daughter showed no endocrine abnormalities. Intraoperative both ovaries were enlarged and multicystic. One ovary was resected for histopathologic examination.

Results. Histomorphology of the ovary displayed nodules of luteinized cells and multiple luteinized ovarian cysts within an edematous stroma, typical histological findings of a pregnancy luteoma. Placental examination revealed signs of insufficiency. Conclusions. Pregnancy luteoma is a rare ovarian lesion which can macroscopically be misinterpreted as malignancy. Awareness of this entity can avoid unnecessary adnexectomy in young patients as pregnancy luteomas regress spontaneously. Although hyperandrogenism can be associated with IURG it is hardly probable that in the present case fetal hypotrophy was due to pregnancy luteoma because the level of secreted androgens was not high enough to cause manifest hyperandrogenism. Instead IUGR in our case is more likely attributable to placental insufficiency and/or beta thalassemia major.

SO-053 Infantile digital fibromatosis (IDF) – A case report and review of the literature U. Titze1, R. Rödl2, G. Köhler1 University Hospital Münster, Gerhard-Domagk-Institute for Pathology, Münster, 2University Hospital Münster, Department of General and Tumor Orthopedics, Münster

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Aims. We report on a 7 months old male infant who received excisionbiopsy of a rapidly growing dermal tumor from the 2nd toe of the right side. Methods. Histological, immunohistological and ultrastructural examination revealed typical findings of an infantile digital fibromatosis (WHO: inclusion body fibromatosis). Results. Infantile digital fibromatosis (IDF) is a rare, distinctive benign fibroblastic/myofibroblastic tumor of infancy typically arising in the digits of the hands or feet. Characteristic morphological findings in the proliferating spindled cells are characteristic rounded eosinophilic paranuclear inclusions. Conclusions. Etiology of IDF remains uncertain. Despite of its benign behavior, local recurrence was seen in up to 60% of cases after surgical therapy. Local installations of corticosteroids do not reduce the size of these lesions. Current management of IDF recommends avoiding surgical intervention, as spontaneous involution is the rule.

SO-054 Male fetus with ectrodactyly ectodermal dysplasia clefting (EEC) syndrome F. Fronhoffs , S. Detering , S. Gerlach , C. Berg , M. Born , A.M. Müller 1 University Bonn Medical Center, Institute of Pathology, Bonn, 2University Bonn Medical Center, Institute of Pathology, Bonn, 3University Clinic of Cologne, Department of Prenatal Medicine and Ultrasound, Köln, 4University Bonn Medical Center, Institute of Radiology, Bonn, 5University Bonn, Department of Pediatric Pathology, Bonn 1

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Aims. Characteristics of the ectrodactyly ectodermal dysplasia clefting (EEC) syndrome, first described by Rüdiger et al in 1970, are ectrodactyly, dysplasia of skin, its adnexal structures and/or teeth as well as orofacial clefts. Its exact prevalence is unknown. Until now, about 300 cases have been reported in literature. In more than 90% of all cases, a missense mutation in the gene TP63 can be detected. Methods. 33-year-old mother, gravida 1, para 1. Proof of bilateral complete cleft of lip and palate and ectrodactyly of both feet and hands and tentative sonographic diagnosis of complex cloacal persistence and malformation. Confirmation of EEC-syndrome by genetic testing. Feticide in 24th+4 week of gestation. Results. Male fetus, appropriate for gestational age, with bilateral complete cleft of lip and palate accompanied by deformation of the nasal apex. Ectrodactyly of both feet and hands. Right hand with five metacarpals (I,

III–V regular, II shortened) and agenesis of phalanges II and III. At the left hand only a rudimentary anlage of digitus II but regularly formed digitus I and III–V. Right foot with five metacarpals but shortened metacarpal II. Left foot with five regularly shaped metacarpal bones, but only four phalanges (I and III–V), i.e. missing second toe. Time-adequate development of the nails. Histologically, in the skin biopsy only very few perspiratory and sebaceous glands. Very scarce scalp hair. Additionally, a megacystis with pseudodiverticulum and dilatated and sidled ureters. Conclusions. This fetus presented classic findings of the EEC syndrome. Because of the additional urogenital anomalies the diagnosis was expanded to ectrodactyly ectodermal dysplasia syndrome with urinary tract pathology (EECUT).

SO-055 Femur fibula ulna (FFU) complex A.M. Müller1, S. Detering2, U. Gembruch3 University Bonn, Department of Pediatric Pathology, Bonn, 2University Bonn Medical Center, Institute of Pathology, Bonn, 3University Bonn Medical Center, Dept. of Prenatal Medicine and Obstetrics, Bonn 1

Aims. Femur fibula ulna (FFU) complex is a sporadic, non-lethal malformation characterized by typical unilateral combination of defects of the femur and fibula and contralateral defect of the ulna. Methods. We present a fetus of 23 weeks of gestation of consanguine parents. Results. Macroscopically the fetus showed a short collar, hypertelorism, slightly down sloping palpebral fissures, short, flat nose, small lips and high arched palate and dysmorphic ear concha. In comparison to the right side, left sided upper and lower leg were significantly shortened, the foot appeared as clubfoot. Furthermore, in comparison to the left side, the right forearm was shortened, the right hand displaying four fingers and an aplasia of the thumb. Conclusions. Etiology of the sporadically occurring FFU complex is unknown. Up to now there are no signs a paternal age effect or an association with consanguinity. Neither could chromosomal studies reveal any abnormalities. Furthermore there is no evidence for an infectious or teratogenic cause. Children show normal mental development and normal life expectancy. As – dependent on the number of involved malformed limbs – the FFU complex is grouped in four groups (I–IV) this case can be assigned to type II.

SO-056 Fetal manifestation of the Peters’ plus syndrome associated with lenticular ectopia and occipital meningocele in one of the cases K. Schoner1, J. Kohlhase2, J. Steinhard3, R. Bald4, A. Schwan5, P. Wieacker6, H. Rehder1 1 Philipps University Marburg, Institute of Pathology, Marburg, 2Praxis of Human Genetics, Freiburg, 3Department of Obstetrics, Münster, 4Clinic of Gynecology, Leverkusen, 5Division of Human Genetics, Dortmund, 6Institute of Human Genetics, Münster Aims. Fetal pathology aims to recognize syndrome specific patterns of malformations and dysmorphic features for goal directed mutation analyses, genetic counselling of the parents and early prenatal molecular diagnoses in consecutive pregnancies. Here we report on four fetuses with Peters’ plus syndrome from two distinct families. Methods. We performed fetal autopsies after prenatal ultrasound diagnoses of malformations and termination of pregnancy and carried out molecular genetic investigations on fetal and parental DNA. Results. Four fetuses of 16 to 22 gestational weeks presented with multiple malformations and dysmorphic signs in addition to Peters’ anomaly of the eyes. The latter comprised central sclerocornea, absence of the posterior corneal stroma, and a variable degree of iris and lenticular attachments to the central posterior cornea in association with microphthalDer Pathologe Suppl 1 · 2012 

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Abstracts mia and lenticular ectopia. Additional features concerned hydrocephaly, a characteristic round face with cleft lip and palate, hypertelorism and prominent front, short stature, brachydactyly, and also cardiac, renal, genital and cerebral malformations including occipital meningocele. Peters’ plus syndrome was confirmed by sequence analysis of the B3GALTL gene revealing homozygosity for the common 660+1G>A donor splice site mutation in intron 8 in all four cases and heterozygosity for this mutation in the Caucasian, non-consanguineous parents. Conclusions. The four affected fetuses show a characteristic facial aspect that in association with the accompanying malformations should enable the diagnosis of a Peters’ plus syndrome. Peters’ anomaly of the eyes, representing an evolutive feature, is already evident at 18 weeks of gestation. However, manifestation of the disorder is variable. Occipital meningocele is a novel finding in Peters’ plus syndrome.

SO-057 Massive ovarian edema (MOE) V . Sailer1, S . Huss1, F . Fronhoffs1, E . Wardelmann1, A .M . Müller2 1 University Clinic of Bonn, Institute of Paidopathology and Institute of Pathology, Bonn, 2University Bonn, Department of Pediatric Pathology, Bonn Aims. Massive ovarian edema (MOE) is a very rare benign tumor-like condition found in young women resulting from accumulation of fluid mostly due to partial or intermittent torsion of the ovary or secondary to a pre-existing ovarian lesion. Methods. We report a case of a 13-year-old girl that presented with an ovarian mass measuring 16  cm in diameter. Ultrasound and CT-scan revealed a multilobulated cystic mass. CA-12-5 levels were increased. Concerns regarding underlying malignancy lead to unilateral salpingooophorectomy. Results. Pathological evaluation revealed a MOE and multiple thromboses of ovarian veins. Conclusions. Differentiation MOE from malignant tumor is crucial to prevent unnecessary surgery potentially resulting in hormonal dysfunction and infertility. Conservative treatment is possible and may be more appropriate in cases when histology on frozen section supports a benign lesion.

SO-058 Infantile myofibroma of the thyroid gland A . Agaimy1, D . Schmidt2, P . Klein3, R . Carbon4, W . Holter5 1 Friedrich-Alexander University of Erlangen, Institute of Pathology, Erlangen, 2 Friedrich-Alexander University of Erlangen, Department of Nuclear Medicine, Erlangen, 3Friedrich-Alexander University of Erlangen, Department of Surgery, Erlangen, 4Friedrich-Alexander University of Erlangen, Department of Surgery, Erlangen, 5Friedrich-Alexander University of Erlangen, University Children‘s Hospital, Erlangen, Erlangen Aims. Spindle cell lesions of the thyroid gland are rare and may thus be diagnostically challenging. They encompass a heterogeneous group of reactive mesenchymal lesions, and a variety of benign and malignant neoplasms of epithelial and mesenchymal origin. Methods. A 5-year-old girl presented with a rapidly growing firm nodular cervical mass localized to the right thyroid lobe associated with bilateral lymphadenopathy. Because of symptoms and concern about malignancy, an open surgical biopsy was performed followed by resection of the right lobe and biopsy of the cervical nodes. The patient is alive with no evidence of recurrence 18 months after surgery. Results. The specimen contained a 3.8  cm firm tan circumscribed nodular mass surrounded by a thin rim of thyroid tissue. Histological examination displayed a moderately cellular lesion composed of alternating fascicles of eosinophilic myoid spindled cells and primitive looking small rounded cells with hemangiopericytoma-like vascular pattern and a prominent myointimal proliferation at the periphery of the lesion. The

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myoid cells expressed strongly alpha-smooth muscle actin but were negative for desmin, h-caldesmon, epithelial membrane antigen, pankeratin, CK7, thyroglobulin, TTF-1, protein S100, TLE1, ALK-1, beta-catenin, CD31, CD34 and CD99. The lymph nodes showed reactive florid hyperplasia without evidence of tumor. Conclusions. To our knowledge, this case represents the first report of solitary myofibroma presenting as a thyroid mass. Awareness of this differential diagnosis is necessary to avoid misinterpretation as a sarcoma with the sequelae of unnecessary over-treatment.

AG Urologische Pathologie I SO-061 Mechanisms of VEGF-C and Neuropilin-2 induced therapy resistance in prostate cancer M . Muders1, S . Haberlau1, M . Stanton2, H . Zhang3, S . Dutta2, M . Krause4, K . Datta2, G .B . Baretton1 1 University Hospital Carl Gustav Carus at the University of Dresden, Institute of Pathology, Dresden, 2University of Nebraska Medical Center, Omaha, NE, United States, 3Mayo Clinic, Department of Urology, Rochester, MN, United States, 4University Hospital Carl Gustav Carus at the University of Dresden, Department of Radiation Oncology, Dresden Aims. Resistance to treatment is a major contributor to prostate cancer mortality in advanced stages. Therefore, in order to develop new treatment modalities and improve the efficacy of current ones, it is important to understand the molecular mechanisms that promote resistance to therapy in prostate cancer cells. Methods. To investigate the signaling pathways involved in induction of therapy resistance by VEGF-C and Neuropilin-2 we knocked down VEGF-C or Neuropilin 2 by RNA interference in standard prostate cancer cells lines and treated these cell lines with ionizing irradiation or Docetaxel. During or after treatment autophagic pathways were evaluated by studying autophagic flux. Results. VEGF-C and Neuropilin-2 are important molecules of radiation and chemotherapy resistance in prostate cancer. Furthermore, we have found that the VEGF-C/NRP-2 axis is involved in the activation of autophagy, which maintains cancer cell survival following treatment. Blocking autophagy also limits the ability of Neuropilin2 and VEGF-C to induce therapy resistance in prostate cancer cell lines. Conclusions. Together, these data suggest a link between the VEGF-C/ NRP-2 axis in prostate cancer cell survival in the presence of therapy-induced stress by activating autophagy. Effective targeting of this pathway may lead to the development of new cancer therapies.

SO-062 The ETS family of transcription factors and prostate cancer: The role of the family prototype ETS-1 Z . Shaikhibrahim1, N . Wernert1 University Hospital Bonn, Institute of Pathology, Bonn

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Aims. The ETS family of transcription factors plays important roles in both normal and neoplastic cells for various biological processes. In prostate cancer (PCa), recurrent gene fusions occurring between the androgen-regulated prostate-specific serine protease TMPRSS2 gene, and several ETS family members, most commonly ERG, are frequently reported. ETS-1, the prototype of the family is reported to be overexpressed in latent and clinically manifest PCas. The ETS-1 gene encodes three distinct proteins, ETS-1 p51 encoded by a full-length mRNA, ETS-1 p42 and ETS-1 p27 encoded by an alternatively spliced mRNA lacking exon VII and exons III–VI, respectively. Even though ETS-1 p42 and p27 have been investigated in functional terms, the presence and roles of ETS-1

p42 and p27 have not yet been investigated in PCa. Therefore, we aimed in this study at investigating the role of ETS-1 in PCa cell lines, and whether the ETS-1 splice variants p42 and p27 are expressed in PCa cell lines. Methods. We first examined the expression of all 27 ETS family members using quantitative RT-PCR in androgen-sensitive and insensitive PCa cell lines. As ETS-1 was found to be highly expressed in the androgeninsensitive PCa cell lines PC3 and DU-145, we investigated the effect of blocking ETS-1 in PC3 cells on genes involved in the metastatic cascade using comprehensive gene expression microarrays, and correlated these findings with PCa tissues. The expression of the ETS-1 splice variants p42 and p27 was assessed using an anti-ETS-1 antibody directed against the DNA-binding domain (DBD), and RT-PCR. Results. Assessment of ETS-1 blockade yielded many genes which are known to be implicated in PCa. Correlating these genes with findings from PCa tissues identified 16 genes that are up or down regulated in healthy compared to tumorous PCa glands. Bioinformatical analysis revealed that 13/16 of these genes have potential ETS-1 binding sites within their promoter regions, and 4 were reported to be regulated by members of the ETS family. Furthermore, our results show for the first time the novel identification of the ETS-1 splice variants p42 and p27 in both the androgen-dependent and the androgen-independent PCa cell lines. Conclusions. Future studies will address the roles of the ETS-1 splice variants in PCa cell lines and tissues. These findings provide in vitro and in vivo evidence for the importance of ETS-1 in development and progression of PCa

SO-063 Serum and prostate cancer tissue signatures of ERG rearrangement derived from quantitative analysis of the PTEN conditional knockout mouse proteome N.J. Rupp1, I. Cima2, R. Schiess3, P.J. Schüffler4, T. Fuchs5, N. Fankhauser2, M. Kälin6, S. Gillessen6, R. Aebersold3, W. Krek2, M.A. Rubin7, H. Moch1, P.J. Wild1 1 University Hospital Zurich, Institute of Surgical Pathology, Zürich, Switzerland, 2ETH Zurich, Institute of Cell Biology, Zürich, Switzerland, 3ETH Zurich, Institute of Molecular Systems Biology, Zürich, Switzerland, 4ETH Zurich, Department of Computer Science, Zürich, Switzerland, 5California Institute of Technology, Pasadena, CA, United States, 6Cantonal Hospital St. Gallen, Department of Oncology, St. Gallen, Switzerland, 7Weill Cornell Medical College, Department of Pathology and Laboratory Medicine, New York, NY, United States Aims. Applying a systems biology approach to assess serum and tissue signatures of ERG rearrangement in patients with prostate cancer with the goal of determining downstream molecular targets for gene fusion cancers. Methods. Prostate tissue from a conditional PTEN knockout mouse model of prostate cancer was investigated, using selective enrichment of N-glycopeptides and mass spectrometry-based label-free quantification. Mouse tissue signatures were validated in sera and tissue of mice (n=12) and humans (n=105) by selected reaction monitoring (SRM), ELISA, and immunohistochemistry. ERG rearrangement status was assessed using fluorescence in situ hybridization (FISH) on two independent tissue microarray based prostatectomy cohorts (n=41, n=348). A Random forest model was trained and validated to identify serum and tissue signatures of ERG rearrangement. Results. A comprehensive PTEN dependent protein catalogue representing over 700 glycoproteins was established. TMPRSS2-ERG gene fusions occurred in 41% (17/41) of analyzable prostate cancers of the training cohort. ERG dependent serum signatures could be found. Predicted serum signatures were systematically tested on two prostate cancer tissue microarrays (training and test cohort) using immunohistochemistry for 15 candidate proteins and members of the PI3K/AKT/mTOR pathway. Conclusions. This is the first study to demonstrate a proteomic signature of ERG rearrangement prostate cancer. Given the challenges of directly targeting ETS transcription factors, this study has potential clinical im-

plications providing important insights into future targetable downstream pathways.

SO-064 Landscape of chromosome number changes during prostate cancer progression J. Stomper1, M. Braun1, W. Vogel1, D. Böhm1, V. Scheble2, F. Fend3, S. Perner1 1 University Hospital of Bonn, Institute of Pathology, Bonn, 2University Hospital of Tübingen, Division of Oncology, Tübingen, 3University Hospital of Tübingen, Institute of Pathology, Tübingen Aims. Genetic instability resulting in both aneuploidy and polyploidy is discussed to be involved in prostate cancer (PCa) development and progression. However, a complete survey of numerical chromosomal changes in PCa is lacking so far. The aim of this study was to comprehensively characterize the ploidy level in PCa with regard to disease progression via fluorescence in situ hybridization (FISH). Since aneuploidy and aggressive disease are often associated with increased tumor cell proliferation, we also assessed the expression of two common mitosis markers within the same PCa cohort. Methods. We studied a cohort comprising 186 localized PCa, 75 PCa with 125 corresponding lymph node metastases, and 42 hormone-refractory distant metastases. Using dual-color FISH, we assessed the cohort for losses and gains of all 24 chromosomes. Conducting immunohistochemistry studies with the markers pHH3 and Ki67, we quantified the proliferation rate within the same cohort. Results. We observed a sharp and significant increase in aneuploidy with advancing tumor stage (p<0.01). Whereas 36.0% of localized tumors, 61.3% of lymph node metastasized primary PCa, and 80% of corresponding lymph node metastases displayed an aneuploid karyotype, almost all (97.6%) distant metastases were aneuploid. In general, gains (47.9%) of chromosomes were more common than losses (22.0%). Regarding characteristic copy number changes of single chromosomes, chromosomes X (26.6%), 21 (22.8%), Y (20.7%), 14 (19.2%), 16 (17.9%), 8 (17.7%), and 1 (17.4%) were most frequently altered. Additionally, we found that losses of chromosomes 20 (11.0%), 10 (4.1%), and 6 (4.0%) accounted for the most frequent monosomies. Interestingly, an increased degree of cell proliferation was significantly associated with the extent of aneuploidy and higher tumor stage (p<0.01). Conclusions. Disease progression from localized to hormone-refractory PCa is poorly understood on the molecular level. Here, we accumulate evidence that genomic instability leading to aneuploidy may be a crucial factor in carcinogenesis and the metastatic cascade. Our results further indicate a potential association of increased cell proliferation and numerical chromosomal changes. Together, we provide new insight into the incompletely elucidated chromosomal landscape of PCa. More directly, our study suggests an approach that may help to identify patients at risk of unfavourable clinical course.

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Abstracts SO-065 Mutation analysis of squamous bladder tumours N.T. Gaisa1, J. Korb1, N. Reimer1, S. Denzinger2, S. Koufou2, E. Eltze3, M. Toma4, S. Siegert5, A. Hartmann6, R. Stöhr6, R. Knüchel1 1 RWTH Aachen University, Institute of Pathology, Aachen, 2University Hospital Regensburg, Department of Urology, Regensburg, 3Institute of Pathology Saarbrücken-Rastpfuhl, Saarbrücken, 4University Hospital Dresden, Institute of Pathology, Dresden, 5LMU Munich, Institute of Pathology, München, 6University Hospital Erlangen, Institute of Pathology, Erlangen Aims. The identity and impact of genetic changes in non-Schistosoma associated squamous carcinoma of the bladder and urothelial carcinoma with squamous differentiation are still unknown. Therefore, in this study squamous tumours have been analyzed for frequent somatic mutations in urothelial cancer. Methods. Pure squamous carcinoma (n=34) and mixed urothelial cancers with additional squamous differentiation (n=42) as well as their precursor lesions have been screened for mutations in TP53, FGFR3 and PIK3CA. Sanger Sequencing was performed for TP53; FGFR3 and PIK3CA were analyzed by SnapShot-method. Results. 47% of pure squamous carcinoma (16/34) and 62% of urothelial cancer with squamous differentiation (26/42) showed TP53 mutations. The most frequent mutation was p.R175H (5 times, 3 in pure squamous carcinoma, 2 in mixed carcinoma). FGFR3 mutations (exclusively S249C) were detected in 9% (3/34) and 12% (5/42) respectively, PIK3CA mutations (E542K and E545K) in 18% (6/34) and 17% (7/42). Both FGFR3 and PIK3CA mutations were found in 2 patients (pure squamous carcinoma) only. TP53 and FGFR3/PIK3CA mutations were not mutually exclusive, and TP53 mutations associated with either FGFR3 or PIK3CA mutations were found in 9 cases (n=4 pure squamous carcinoma, n=5 mixed carcinoma). FGFR3 mutations were not related to any particular morphological phenotype. Conclusions. TP53 mutations occurred with slightly higher frequency in squamous parts of mixed carcinoma, but the overall incidence of TP53 mutations was similar to reports of pure urothelial carcinoma in the literature. TP53 mutations may play a critical role in the development of squamous bladder tumours, whereas FGFR3 and PIK3CA mutations seem to be less relevant.

SO-066 The impact of bladder cancer histology on overall survival of patients treated by cystectomy and adjuvant cisplatin-based chemotherapy B. Keck1, R. Stöhr2, S. Wach1, H. Taubert1, F. Kunath1, S. Bertz2, J. Lehmann3, M. Stöckle4, B. Wullich1, A. Hartmann2 1 University of Erlangen, Department of Urology, Erlangen, 2University Erlangen, Department of Pathology, Erlangen, 3Urology Practice Prüner Gang, Kiel, 4Saarland University, Department of Urology Aims. To evaluate the impact of conventional urothelial (UC), plasmacytoid (PUC) and micropappilary (MPC) histology on survival of bladder cancer patients treated by cystectomy and adjuvant cisplatin-based chemotherapy within the prospective and randomized trial AUO-AB05/95. Methods. Tumor samples of 221 patients with a majority treated within the randomized AUO-AB05/95 trial by radical cystectomy and adjuvant cisplatin-based chemotherapy were reviewed for identifying histologic subtypes of locally advanced bladder cancer. 191 UC, 20 PUC and 10 MPC of the bladder were identified. For the definition of PUC and MPC, at least 50% of the tumor had the specific histologic pattern. Kaplan Meier analysis and multivariate Cox’s proportional hazards regression analysis were performed to compare overall survival (OS) of UC, MPC and PUC. Of these 221 patients, 50 patients were treated with gemcitabine/cisplatin, 83 patients with M-VEC and 88 patients with cm. Results. Median OS of PUC was 29.9months and significantly worse compared to patients suffering from UC (62.8 months) or MPC (64.2 months;

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p=0.04). Multivariate Cox’s proportional hazards regression analysis adjusted to chemotherapy showed a hazard ratio of 1.9 (p=0.034) for UC (n=191) and 2.7 (p=0.083) for PUC (n=20) in contrast to patients suffering from MPC (n=10). Conclusions. Bladder cancer histology gives important information on the prognosis of patients suffering from locally advanced bladder cancer. As OS of UC, PUC and MPC differs in patients treated by radical cystectomy and adjuvant cisplatin-based chemotherapy further prospective studies comparing histologic variants of bladder cancer are necessary in order to tailor therapeutic strategies in the future.

SO-067 Prognostic role of androgen receptor in bladder cancer R. Wirtz1, S. Bertz2, B. Keck3, S. Claas2, L. Dyrskjøt4, T. Orntoft5, B. Wullich3, R. Hake6, S. Eidt6, A. Hartmann1 1 University Cancer Center Erlangen, Institute of Pathology, Erlangen, 2University Cancer Center Erlangen, Institute of Pathology, 3University Cancer Center Erlangen, Urology University Clinic, 4Aarhus University Hospital, Denmark, 5Aarhus University Hospital, Molecular Medicine, Denmark, 6Institute of Pathology at the St-Elisabeth-Hospital Cologne Aims. Hormone receptors are the prototype predictive marker in breast and prostate cancer. Hormone receptor positive cancers have a better prognosis, increased tropism to metastasize into the bones and respond to endocrine treatment options. The prognostic value of hormone receptor in urothelial carcinoma of the bladder (UCB) is less established. This may in part result from technical limitations of immunhistochemical detection methods. Interestingly, female gender has recently been identified as strong adverse factor in advanced UCB (May et al. 2011). By analyzing whole genome expression data from non-muscle invasive bladder cancer patients, we have evaluated the potential of top candidate genes (ESR1, PGR, AR, CYP19, HER2, RACGAP1) commonly used to stratify breast cancer patients to predict bladder cancer progression. In view of the gender specific effects, we have focused on the prognostic role of androgen receptor expression on tumor invasion, disease progression and survival. Methods. Affymetrix microarray data from 41 non-metastatic bladder cancer patients undergoing curative surgery were analyzed. Prognostic value of androgen receptor mRNA expression was analyzed by unsupervised Cluster analysis, partitioning tests, Mann Whitney tests and Kaplan Meier estimates of cancer specific survival. Results. Cluster analysis in the microarray date of the superficial UCB cohort identified a hormone receptor positive subtype and a proliferation dominated subtype of equal size. Androgen receptor expression was negatively associated with cancer specific death (r=−0.42; p=0.005), while proliferation correlated with increased risk of cancer specific death (r=0.46; p=0.003). In addition, low androgen receptor expression was associated with higher tumor stage (pTa vs pT1-4; p=0.017). In Kaplan Meier analysis, the cancer specific survival was significantly better in tumors exhibiting high androgen receptor levels (80% vs. 20%; p<0.0001). Conclusions. Resembling to some extent the situation in other cancer types, hormone receptors are prognostic factors in early stage bladder cancer. These results may also explain the recently described gender specific effects in bladder cancer. Moreover, these results raise the possibility, that UCB patients may be stratified according to their androgen receptor status in view of prognosis and putative endocrine therapy options.

SO-068 Recent advances in understanding the genetic and epigenetic networks of germ cell tumors D . Nettersheim1, B . Westernstroer2, S . Schlatt2, H . Schorle1 University of Bonn Medical School, Institute of Pathology, Bonn, 2 University of Münster, CERA, Münster

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Aims. In the past decades the incidence of germ cell tumors (GCT) has been rising constantly. It is believed, that GCT arise from an arrested progenitor to form an IGCNU (CIS). With puberty, CIS eventually progress into seminoma and nonseminoma. The genetics of this progression is poorly understood, so knowledge of genetic and epigenetic markers is mandatory. Further, the establishment of a murine model system for CIS/seminoma and embryonal carcinoma would further help in addressing functional questions relating to GCT initiation, progression and treatment. Methods. We used cell lines derived from seminoma (TCam-2) and embryonal carcinoma (2102EP) as well as paraffin sections from human fetal material and germ cell tumors to elucidate the specificity of the markers for primordial germ cells BLIMP1, PRMT5 and TFAP2C. Furthermore, we generated knock down cell lines as well as knockout-models for TCFAP2C in order to analyze the requirement of these factors. Finally, we transplanted various GTC cell lines to nude mice and analyzed tumor initiation and development. Results. In our hands, the markers BLIMP1 and TFAP2C are highly specific for CIS and seminoma. They are downregulated in embryonal carcinoma, forced loss of these factors leads to upregulation of somatic marker genes indicative for differentiation. A lack of Tfap2c in the murine model results in sterility. Transplantation of the TCam2 seminoma cell line revealed, that this cells grew as CIS/seminoma or embryonal carcinoma depending on the microenvironment. Conclusions. We demonstrate that TFAP2C and BLIMP1 might be suitable for the diagnostics of GCTs. We show that these proteins are required for the maintenance of the undifferentiated status of CIS and seminoma. Finally, the establishment of a murine model for GCT enables for in vivo studies relating to germ cell tumor biology.

SO-069 Progressive kidney lesions in mutant mouse models for uromodulin-associated kidney disease E . Kemter1, S . Sklenak1, P . Prueckl1, B . Rathkolb1, F . Habermann2, M . Hrabé de Angelis3, E . Wolf1, B . Aigner1, R . Wanke4 1 LMU Munich, Chair for Molecular Animal Breeding and Biotechnology, München, 2LMU Munich, Chair for Veterinary Anatomy, Histology, and Embryology, Department of Veterinary Sciences, München, 3Helmholtz Zentrum München, Institute of Experimental Genetics, Neuherberg, 4LMU Munich, Institute of Veterinary Pathology, Center for Clinical Veterinary Medicine, München Aims. Uromodulin-associated kidney disease (UAKD) is a heritable renal disease in humans caused by mutations in the uromodulin (UMOD) gene. Impaired maturation of mutant uromodulin with retention of uromodulin in the hyperplastic endoplamic reticulum is considered to be causative for thick ascending limb (TALH) cell dysfunction. Besides clinical symptoms like hyperuricemia, gout, and alteration of urine concentrating ability, histological kidney alterations like tubulointerstitial nephritis, cysts, and interstitial fibrosis are heterogeneously present in affected humans. Progression of disease leading to end-stage-kidney disease is a feature of UAKD, which pathophysiology is unknown. The objective of this study was to analyze the age-associated development of kidney alterations of two recently described mutant mouse lines carrying two different Umod mutations. Methods. Histopathology, immunohistochemistry, confocal laser scanning microscopy, quantitative stereology, and transmission electron microscopy.

Results. Both the kind of uromodulin mutation and the allelic status influence onset, severity, and progression of renal dysfunction, as demonstrated by a continuous age-related increase of plasma urea concentrations in mutant mice. Interstitial fibrosis and tubular atrophy (IFTA) as well as interstitial infiltrates of inflammatory cells were constantly present in kidneys of aged mutant mice of both lines. An association of different Umod mutations and allelic status with differences in onset, degree, and progression of these nephropathological alterations was detected. Further, glomerulocystic and tubulocystic changes were occasionally observed in kidneys of aged mutant mice. Conclusions. Both Umod mutant mouse lines represent valuable models for human UAKD and enable insights into the pathogenesis of this disease. Intracellular accumulation and release of mutated uromodulin might trigger an inflammatory response leading to interstitial fibrosis. Intracellular accumulation of the mutated misfolded protein in the hyperplastic ER might lead to TALH cell death leading to tubular atrophy. Supported by the German Research Foundation (KE1673/1-1) .

AG Urologische Pathologie II SO-071 Renal cell carcinoma – Implications for cooperation of pathologists and urologists T . Steiner1 1 HELIOS hospital Erfurt, Erfurt Aims. Over the last ten years various new options have been developed for the management of patients with renal cell carcinoma (RCC). Methods. Due to improved imaging techniques small renal masses (SRM) are detected more frequently. About 20% of renoparenchymal tumors with less then 3 cm in diameter have benign histology. Despite RCC histology a significant amount of the remaining tumors show only minimal growth over time. Therefore, in elderly patients or those with significant comorbidities active surveillance strategies should be considered. Renal mass biopsy enhanced by molecular profiling holds promise for assessing aggressive potential in this scenarium. Histologic subtypes and genetic aberrations of RCC predict different probability of development of local recurrence, lymph node and distant metastases and should be considered in follow up after local surgery for RCC. Results. The development of targeted therapy revolutionized the management of metastatic RCC. It was based on the detection of VHL mutations with consecutive expression of growth factors in clear cell RCC. For other histologic RCC subtypes VEGF targeted therapy seems to be less effective. But even in clear cell RCC primary and secondary resistance occurs. In the future predictive molecular markers have to be determined to give patients a chance of really individualized medicine. Conclusions. In conclusion, cooperation between urologists and pathologists is of increasing meaning for optimal management of RCC.

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Abstracts SO-072 Therapeutic and prognostic implications of an isolated Banff II acute cellular rejection without tubulointerstitial component in renal transplants V. Broecker1, M. Hirzallah2, C.L. Bockmeyer1, P.A. Agustian1, M.E. Dämmrich3, A. Schwarz4, J.U. Becker3 1 Hannover Medical School, Institute of Pathology, Hannover, 2Klinikum Region Hannover Krankenhaus, Oststadt-Heidehaus Medizinische Klinik I, Hannover, 3MHH, Institute of Pathology, Hannover, 4Hannover Medical School, Clinic for Nephrology and Hypertension, Hannover Aims. In order to avoid unnecessary therapy it is debated intensely, whether acute cellular rejection with Banff components v≥1, i0, t0 (v_only) has the same therapeutic and prognostic implications as acute cellular rejections with Banff components v≥1, i≥1, t≥1 (v_plus). We examined this question retrospectively in renal transplant biopsies from a single center. Methods. All 23 renal transplant biopsies (from 23 patients) from our biopsy archive with v_only were compared to 23 biopsies from 23 patients with v_plus. All patients, v_only and v_plus, received therapy. They were followed for 135 weeks ±106 (v_only) or 201 weeks ±151 (v_plus). Results. No significant difference was found between v_only and v_plus regarding donor sex, donor age, immunosuppressive regimen, recipient age, time between transplantation and biopsy, number of glomeruli in the biopsy, Banff components v, g, mm, ah, cg, cv, ci, ptc, ratio of of preglomerular vessels with endothelialits to sum of preglomerular vessels, C4d-positivity of preglomerular endothelium, of peritubular capillary endothelium (Banff C4d), of glomerular endothelium, eGFR at biopsy, kind of anti-rejection therapy, eGFR one week after initiation of anti-rejection therapy, eGFR slope per week between biopsy and end of followup. v_only had significantly more HLA-mismatches, a higher Banff ct component and less cortical tubular interstitial edema. Conclusions. The present data indicate marginal histological differences between v_only and v_plus (with the exception of the defining Banff components i and t). We could not find a difference regarding response to anti-rejection therapy, transplant function or prognosis between v_plus and v_only. The higher number of HLA-mismatches in v_only might suggest that this rare lesion could be associated with donor specific antibodies. We will examine this association further.

SO-073 Renal cell carcinoma and senescence: p400 as a novel prognostic marker S. Macher-Göppinger1, J. Lorenzo Bermejo2, M. Hohenfellner3, N. Wagener3, P. Schirmacher1, W. Roth1 1 University of Heidelberg, Institute of Pathology, Heidelberg, 2University of Heidelberg, Institute of Medical Biometry and Informatics, Heidelberg, 3University Heidelberg, Department of Urology, Heidelberg Aims. Mutations of the von Hippel-Lindau (VHL) tumor suppressor gene cause hereditary and sporadic renal cell carcinomas (RCCs). The best characterized function of VHL protein is suppression of the alpha subunit of hypoxia inducible factor (HIF). Additional VHL functions have been reported, including induction of senescence mediated by loss of chromatin remodelling factor p400. Induction of senescence either by oncogene activation or inactivation of tumor suppressors is considered as a critical feature of mammalian cells to suppress tumorigenesis. Here, we studied the expression of p400 in a large and well documented series of RCCs with long term follow-up information. Methods. Expression of p400 was examined by immunohistochemistry using a tissue micro array containing RCC tumor tissue samples and corresponding normal tissue samples from 932  patients. Regression models were used to investigate the possible relationship between p400 expression and Ki-67 proliferative index, clinical and pathologic parameters and disease-specific survival.

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Results. Loss of p400 expression was detected in 64% of all tumor specimens and was associated with advanced tumor stage, higher grade of malignancy and regional lymph node metastasis. Among well differentiated RCCs, high proliferation (Ki-67 index 10+) was found in 10% of carcinomas with a positive p400 expression, compared to 3% in p400 negative RCCs. Importantly, multivariate Cox regression indicated that patients with high-proliferative tumors, those with p400-positive RCCs have a 159% increased cancer-specific mortality risk compared to p400negative RCCs. Conclusions. Loss of p400 expression is common in RCCs and the proportion of carcinomas with loss of p400 increases alongside advancing tumor stage and decline of differentiation. In contrast, a subgroup of tumors defined by high proliferation and expression of p400 shows significantly shorter cancer-specific survival in multivariate analyses than the subgroup of tumors with high Ki-67 labeling index without co-expression of p400. Our data suggest that the highly proliferative, p400positive subgroup of RCC represent tumors that are characterized by a loss of the tumor-suppressive mechanism of senescence.

SO-074 Infiltration by tumor associated macrophages and CCR2/CCL7 expression correlates with brain metastases from clear cell renal cell carcinoma L.G. Wyler – von Ballmoos1, C. Urrejola2, P.H. Schraml1, H. Moch1 1 Institute of Surgical Pathology, University Hospital Zurich, Zürich, Switzerland, 2Institut of Pathology, University Hospital Basel, Basel, Switzerland Aims. Renal cancer patients with brain metastasis have a poor prognosis. The mechanisms, by which renal cancer metastasize to the brain is not entirely understood. The Goal of this study was to find out more about pathways of metastases from clear cell renal cell carcinoma (ccRCC) and whether the expression of CCL2, CCL7, CCR2 and CXCR4 by tumor cells and tumor associated macrophages (TAMs) correlates with brain metastasis and/or outcome. Methods. Among 40’021 routine autopsies, the reports from those 636 with metastatic renal cancer were rewied and the metastatic sites were analyzed. For imunohistochemical staining tissue microarrays were constructed from biopsy samples of 333 primary RCCs and 51 brain metastases from ccRCCs. Stainings for CCL2, CCL7, CCR2, CXCR4 and CD68 were analyzed and compared. Results. Hematogenous metastases were present in 39% of the autopsy cases, with most frequent involvement being lung (75%), liver and bone (40% each), and soft tissue (34%). Brain metastases were observed in 15%. The rate of brain metastases was higher in patients with lung metastases, but was also observed in patients without thoracic metastases and as isolated brain metastases. A strong expression of CXCR4 was seen in 31% of primary ccRCCs and 45% of brain metastases however a moderate expression was seen in over 80% in both. CCR2 and CCL7 showed a significantly higher expression in brain metastases of ccRCCs compared to primary ccRCCs (p<0.0001 each). 88.7% of all primary ccRCCs and 88.6% of the brain metastases showed a loose (20–40/HPF) to dense (>40) macrophage infiltrate. Within these CCR2 expressing TAMs correlated with higher Fuhrman grade (p=0.003) and were more frequent in brain metastases (p<0.0001). Conclusions. Together our data raise evidence that besides the cava-type of metastasis through lung passage there might be a backward venous spread to the brain. We suggest expression of CCR2 by tumor cells as well as by TAMs plays a pivotal role in brain metastases from ccRCC next to CXCR4. Also seems CCL7 to be involved in these processes, whether over an interaction with CCR2 or over other pathways.

SO-075 A novel, dual role of CCN3 in experimental glomerulonephritis: pro-angiogenic and anti-mesangioproliferative effects P . Boor1, C . van Roeyen1, E . Borkham-Kamphorst2, S . Rong1, U . Kunter1, I .V . Martin1, A . Kaitovic1, S . Fleckenstein1, B . Perbal3, R . Weiskirchen2, T . Ostendorf1, J . Floege1 1 RWTH Aachen University, Aachen, 2RWTH Aachen University, Inst . of Clinical Chemistry and Pathobiochemistry, Aachen, 3R&D L’Oréal, Clark, NJ, United States Aims. In contrast to factors promoting mesangial cell proliferation, little is known about their endogenous inhibitors. During experimental mesangioproliferative nephritis, glomerular CCN3 (also known as NOV or nephroblastoma overexpressed gene) expression is reduced prior to the proliferative phase and overexpressed in glomeruli and serum when mesangial cell proliferation subsides. Methods. To further elucidate its role in mesangioproliferative glomerulonephritis, CCN3 was systemically overexpressed by muscle electroporation in healthy or nephritic rats. This increased CCN3 serum concentrations more than 3-fold for up to 56 days. Results. At day 5 after disease induction, CCN3-transfected rats exhibited an increase in glomerular endothelial area and in glomerular mRNA levels of the pro-angiogenic factors VEGF and PDGF-C. In the mesangioproliferative phase (day 7), CCN3 overexpression decreased mesangial cell proliferation including expression of α-smooth muscle actin and matrix accumulation of fibronectin and type IV collagen. In progressive nephritis (day 56), overexpression of CCN3 resulted in decreased albuminuria, glomerulosclerosis and reduced cortical collagen type I accumulation. In healthy rat kidneys, overexpression of CCN3 induced no morphological changes but regulated glomerular gene transcripts (reduced transcription of PDGF-B, PDGF-D, PDGFR-β and fibronectin and increased PDGFR-β and PDGF-C mRNA). Conclusions. The above data identify a dual role of CCN3 in experimental glomerulonephritis with pro-angiogenic and anti-mesangioproliferative effects. Manipulation of CCN3 may represent a novel approach to help repair glomerular endothelial damage and mesangioproliferative changes.

Deutsch-Chinesisches Symposium Colorectal Carcinoma SG-129 ACSL5, a modifier of WNT activity C . Klaus1, U . Schneider1, R . Knuechel1, N . Gaßler1 1 RWTH Aachen University, Institute of Pathology, Aachen Aims. The mitochondrial localized Acyl-CoA synthetase 5 (ACSL5) converts free long-chain fatty acids into fatty acyl-CoA esters, and thereby plays a key role in lipid biosynthesis and fatty acid degradation. In particular, ACSL5 has been recently identified to be involved in apoptotic cell death of senescent enterocytes along the intestinal crypt-villus axis and to interact with mitochondrial proteins. The aim of this study was to investigate ACSL5-dependent effects on intestinal signalling pathways that coordinate proliferation and/or differentiation of enterocytes, particularly the Wnt pathway. Methods. Wnt signalling was analysed in an ACSL5 overexpressing cell culture model using luciferase assays, immunohistochemistry, qRT-PCR and Western blot. The findings were substantiated with expression studies in human colon carcinomas and in a Wnt-associated mouse model. Results. ACSL5 transgenic intestinal-derived cells displayed a strong susceptibility to pro-apoptotic stimuli. This phenomenon was accom-

panied by caspase-3 activation and significant down-regulation of Wnt signalling activity. In particular, Wnt pathway-associated mitochondrial localized molecules were identified as interaction partners of ACSL5 activity. Conclusions. Molecular mechanisms underlying ACSL5-dependent apoptosis susceptibility of enterocytes are probably bivalent including pro-apoptotic and anti-proliferative activities.

SG-130 Regulation of differential WNT activity in colorectal cancer D . Horst1, J . Chen2, T . Kirchner1, R . Shivdasani2 Ludwig-Maximilians-Universität München, Pathologisches Institut, München, 2Harvard Medical School, Dana-Farber Cancer Institute, Boston, United States

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Aims. Most colorectal cancers (CRCs) express the WNT-effector protein β-catenin in a heterogeneous pattern. Strong nuclear expression is often confined to a small fraction of tumor cells at the tumor’s leading edge. Recent data suggest a role for Mitogen Activated Protein Kinase (MAPK) signaling in nuclear accumulation of beta-catenin. We therefore investigated if MAPK activity regulates overtly differential WNT activity in CRC cell subpopulations. Methods. We used gene expression profiling, and immunohistochemistry to assess interdependence of MAPK and WNT pathway activity in CRC. Lentivirus- and drug-based pathway modification in CRC xenograft tumors of primary human colon cancers and colon cancer cell lines was used to study the effect of MAPK activation or repression on differential WNT activity. Results. CRC cells with high WNT activity showed coincident overexpression of MAPK target genes and high levels of phospho-ERK, indicating active MAPK signaling. Forced MAPK activation by lentiviral expression of constitutively active KRAS enhanced WNT pathway activity in vivo in CRC xenograft tumors, whereas drug based inhibition of EGFR signaling attenuated it. Conclusions. Although CRC is characterized by mutational activation of the WNT pathway, MAPK signaling influences intratumoral β-catenin heterogeneity, revealing a mechanism for external stimuli to modulate pathway activity. Because MAPK signaling does not merely coincide with nuclear β-catenin but also regulates it, this may account for the high frequency of KRAS mutations in CRC.

SG-132 IGFBP7 and WNT signaling pathway in tumor stroma interactions C . Rao1, J . Xu1, M . Liu1, H . Deng1 1 Department of Pathology, School of Medicine, Zhejiang, China Aims. To find out the mechanism of the up-regulation of IGFBP7and the biological changes in fibroblasts during the interactions with colorectal cancer cells. Fibroblasts (HELFs) were cultured in colorectal cancer cells conditioned media (SW620-CM), treated by TGF-β 1, TGF-β1 receptor antagonist (SB431542), TGF-β1 specific antibody (AF), Wnt signaling pathway agonist (LiCl) and inhibitor (DKK-1) respectively. Q-PCR, Western Blot, ELISA, Immunofluorescence microscopy and flow cytometry were used to detect the expression of related targeted genes and proteins of TGF-β and Wnt signaling pathways. HELFs cultured in SW620-CM were activated with abundant expression of α-SMA and showed strong proliferation and weak apoptosis and senescence. The expression of IGFBP7 of HELFs was up-regulated in timedependent and dose-dependent manners when cultured with SW620CM, while TGF-β signaling were activated as Smad2, P-Smad2 and TGF-βRΠ were up-regulated in HELFs. These effects could be strengthened by TGF-β1 and inhibited by SB431542 or AF. During the interactions, the downstream genes of Wnt signaling pathway such as c-myc, cyclinD1 Der Pathologe Suppl 1 · 2012  | 

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Abstracts were up-regulated and the proteins of Wnt signaling pathway such as β-catenin, Dvl3, Dvl2 and Naked2 were obviously up-regulated which suggested the canonical Wnt signaling pathway was also activated. TGF-β and Wnt signaling pathways were activated during colorectal cancer cells-fibroblasts interactions. Upregulating of IGFBP7 in HELFs was mainly through TGF-β1/ALK5/ Smad-2 signaling pathway. Wnt signaling pathway may also play an important role in this process.

SG-134 FMNL2 is regulated by MIR-137 and promotes actin assembly and cell invasion Y. Zeng1, Y. Qiao1, W. Zhao1, X. Zhu1, J. Wang2, X. Zhang2, X. Bian3, Y. Ding2, L. Liang2 1 Department of Pathology, School of Basic Medical Sciences, Guangzhou, China, 2Guangdong Province Key Laboratory of Molecular Tumor Pathology, Guangzhou, China, 3Institute of Pathology and Southern Cancer Center, Southwest hospital, Chongqing, China Aims. FMNL2 is a member of diaphanous-related formins which act as effectors of Rho family GTPases and control the actin-dependent processes such as cell motility or invasion. We previously validated FMNL2 as a positive regulator of cell motility and metastasis in colorectal carcinoma (CRC) but the mechanisms of FMNL2 in CRC remain unclear. The aim was to investigate the association of FMNL2 with Rho signaling pathway in the invasion of CRC. Methods. Rho family GTPase activity was tested by Rho pull-down assay. In vitro invasive ability of cells was detected by Boyden Chamber assay. And luciferase activities of MAL/SRF were detected using dualluciferase reporter assay. In addition, Immunofluorescent analyses were performed to examine F-actin stained by phalloidin and co-localization of FMNL2 and LARG or p115RhoGEF. Co-immunoprecipitation was used to determine the direct binding of FMNL2 and LARG. Finally, GST pull-down assay was used to detect the binding of LARG-CT with FMNL2 in the absence or presence of active RhoAV14. Results. In this study, we showed that FMNL2 activated Rho/ROCK pathway, and required ROCK to promote cell invasion. Moreover, FMNL2 promoted the formation of stress fiber and filopodia, and activated the SRF transcription factor in the Rho-dependent manner. We also demonstrated that FMNL2 was necessary for LPA-induced invasion, Rho/ ROCK activation, actin polymerization and SRF activation. FMNL2 is an essential component of LPA signal transduction toward Rho by directly interacting with LARG. Finally, we found FMNL2, LARG and RhoA constituted a positive feedback loop. LARG silencing inhibited Rho/ROCK pathway and cell invasion. Conclusions. Our findings provide evidence for the positive feedback between FMNL2 and RhoA, which promotes actin assembly and cell invasion of CRC.

SG-135 The p53 target gene desmocolin 3 acts as a novel tumor suppressor through inhibiting AKT signaling pathoway in human colon cancer T. Cui1, Y. Chen1, L. Yang1, T. Knösel1, K. Zöller1, O. Huber2, I. Petersen1 1 institute of pathology, Jena, 2Institute of Biochemistry II Aims. Desmocollin 3 (DSC3), a member of the cadherin superfamily and integral component of desmosomes, is involved in carcinogenesis. However, the role of DSC3 in human colorectal cancer (CRC) has not yet been established. Our aim of the study was to explore the role of DSC3 in human colorectal cancer. Methods. DSC3 expression in CRC cell lines was analyzed by RT-PCR and western blotting. Methylation status of DSC3 was examined by demethylation tests, methylation-specific PCR, and bisulfite sequencing (BS). The regulatory role of p53 was investigated by transfection.

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Results. DSC3 was downregulated in CRC cells at both mRNA and protein levels. DSC3 expression was restored in five out of seven cell lines after 5-aza-2’-deoxycytidine (DAC) treatment. A heterogeneous methylation pattern was detected by BS in promoter region and exon 1 of DSC3. Methylation of DSC3 genomic sequences was found in 41% (41 out of 99) of primary CRC, being associated with poor prognosis (p=0.002). Transfection of p53 alone or in combination of DAC increased the DSC3 expression. Similarly, treatment with p53 inducer adriamycin alone or in combination with DAC enhanced DSC3 expression. Conclusions. DNA methylation contributes to downregulation of DSC3 in CRC cell lines. Methylation status of DSC3 DNA is a prognostic marker for CRC. P53 appears to play an important role in regulating DSC3 expression.

SG-136 Connexin 43 reverses malignant phenotypes of glioma stem cells by modulating E-cadherin X.-W. Bian1, S.-c. Yu1, H.-l. Xiao1, X.-f. Jiang1, Q.-l. Wang1, Y. Li1, X.-j. Yang1, Y.-f. Ping1, J.-j. Duan1, X. Zhang1 1 Third Military Medical University, Institute of Pathology and Southwest Cancer Center, Chongqing, China Aims. The purpose of this study was to investigate the role of gap junction related proteins such as connexins for sustaining the malignant behavior of cancer stem cells (CSCs) in glioma. Methods. Tumorspheres from U87 cells and primary specimens were isolated and characterized as previously described. CD133-positive cells were sorted by flow sorting cytometry and termed as glioma stem cells (GSCs). Electron microscopy was used for observation of ultrastructure of GSCs. Laser confocal microscopy was used for fluorescence recovery after photobleaching (FRAP) on GSCs. Cells and tissues were immunocyto(histo)chemically stained by using stemness and differentiation markers. Real time RT-PCR and western blotting were used for detection of mRNA and protein levels of the stem cell markers. Cx43 or E-cadherin pulldown assays were performed with anti-Cx43 antibody or anti-E-cadherin antibody using the Co-immunoprecipitation. In addition, analyses for promoter methylation of GJA1 gene, overexpression and knock-down of Cx43, gene expression array and gene set enrichment analysis (GSEA) were performed. Proliferation assay Matrigel invasion assay were carried out in vitro and in vivo experiments were performed in nude mice. Results. We obtained tumorspheres formed by glioma stem cells (GSCs) and adherent GSCs, and then examined their GJIC. All GSCs showed reduced GJIC, and differentiated glioma cells had more gap junction-like structures than GSCs. GSCs expressed very low level of connexins, Cx43 in particular, which are key components of gap junction. We observed hypermethylation in the promoter of gap junction protein ***1 (GJA1), which encodes Cx43 in GSCs. Reconstitution of Cx43 in GSCs inhibited their capacity of self-renewal, invasiveness and tumorigenicity via influencing E-cadherin and its coding protein, which leads to changes of the expression of Wnt/***-catenin targeting genes. Conclusions. In this study, we provide evidence for the first time that dysfunction of GJIC is an important feature of GSCs. Reconstitution of Cx43, the key component to maintain the functional GJIC, does not alter functional status of GJIC in GSCs but ablates their self-renewal, invasiveness and tumorigenicity though the interaction with E-cadherin and its coding protein to downregulate the express of those Wnt/β-catenin targeting genes. Our results identify a potential role of Cx43 in maintaining the malignant phenotype of GSCs.

Pancreatic Adenocarcinoma SG-137 Serum MiR-192 and MiR-194 as tumor biomarker for pancreatic ductal adenocarcinoma J . Zhang1, C .-y . Zhao1, D .-h . Yu1, Y . Chen1, Q .-h . Liu1, M . Shi1, J .-t . Zhang1, G . Jin1, P . Cheng1, X .-g . Hu1, C .-r . Ni1, M .-h . Zhu1 1 Department of Pathology, Changhai Hospital, Shanghai, China Aims. To assess the validity of using serum miRNA signatures of PDAC as biomarkers for diagnosis. Methods. MiRNA microarray was used to detect the differences between PDAC samples and normal pancreatic tissues. MiR-192 and miR-194 were found in the tissues of human PDAC and in the explants in tumorbearing SCID mice by locked nucleic acid-based in situ hybridization (LNA-ISH). Serum levels of miR-192 and miR-194 in PDAC patients, duodenal adenocarcinoma patients and healthy controls, as well as six pancreatic cancer cell lines and their culture supernatants were determined by real-time PCR. Results. Eight miRNAs were found overexpressed and eight were lowly expressed in PDAC tissues compared with those in the normal pancreatic tissues. MiR-192 and miR-194 were found overexpressed in the tissues of human PDAC and in the explants in tumor-bearing SCID mice. The levels of miR-192 and miR-194 in the supernatants of six pancreatic cancer cell lines were positively correlated with their cellular expressions (r=0.849, p<0.05; r=0.806, p<0.05, respectively). The serum levels of miR-192 and miR-194 in 70 PDAC patients were significantly higher than those in 17 duodenal adenocarcinoma patients and 40 healthy controls (p<0.05 in both cases). Combined analysis of the three groups yielded a sensitivity of 84.0% and a specificity of 75.0% for the combined detection of miR-192 and miR-194 in the diagnosis of PDAC. Conclusion. Combined detection of serum miR-192 and miR-194 level may serve as a sensitive diagnostic biomarker for PDAC.

SG-138 Targeted degradation of KRas by engineered ubiquitin ligase suppresses pancreatic cancer cell growth in vitro and in vivo Y . Ma1, Y . Gu1, Q . Zhang1, Z . Lu1, W . Zhao1, J . Chen1 1 Department of Pathology, Peking Union Medical College Hospital, Beijing, China Aims. KRas oncogene mutation is one of the earliest genetic events seen in 30% human pancreatic intraepithelial neoplasia (PanIN) with the frequency rising to nearly 100% in advanced pancreatic ductal adenocarcinoma cancer (PDAC). KRas is an attractive therapeutic target of PDAC. “Target ubiquitination and degradation of oncoproteins via ubiquitin-proteasome pathway” may be an alternative therapeutic strategy. E3 ligase is thought to be the component of the ubiquitin conjugation system that is most directly responsible for substrate recognition. In the present study, an engineered ubiquitin E3 ligase (RC-U) was generated to target the KRas oncoprotein for ubiquitination and degradation. The effects of RC-U on pancreatic cancer cell growth in vitro and in vivo were investigated. Methods. The engineered ubiquitin E3 ligase (RC-U) was constructed as follows: the cDNA fragments encoding U-box and (RBD+CRD) Raf-1 were amplified by PCR amplification and then were inserted into pcDNA3.1 vector (pRC-U). The fusion cDNA was also subcloned into the pLenti6.3 to generate pLenti6.3-RC-U. Lentiviruses were packaged in HEK 293T cells. The KRas mRNA and protein expression after transfecting the pRC-U expression plasmid into human pancreatic cancer cells or HEK293T cells were detected by real-time reverse transcription polymerase chain reaction (real-time RT-PCR), Western blotting and immunofluorescence. After pRC-U transfection, with mg-132 or cyclo-

heximide treatments, the KRas protein expression was tested by Western blotting. The interaction between RC-U and KRas, whether KRas could be ubiquitinated by RC-U or not were both investigated by immunoprecipitation. The expression of HRas, NRas, phosphorylated extracellular signal-regulated protein kinases (pERK) and extracellular signal-regulated protein kinases (ERK) in pancreatic cancer cells was also examined by Western blotting after pRC-U transfection. The effects of RC-U on pancreatic cancer cell growth in vitro were detected by CCK-8 and soft agar colony formation assays after lentivirus infection. In vivo, with RC-U treatment, the volumes of the subcutaneous xenografts in nude mice were measured. Results. RC-U dramatically decreased the protein level of KRas compared to the controls. No significant change of KRas mRNA expression within different groups was observed. After pRC-U transfection, the stability of KRas was significantly increased in the presence of specific proteasome inhibitor mg-132. HEK293T cells were transfected by mutant KRas construct together with either pRC-U or empty vector and then incubated with cycloheximide to inhibit novel protein synthesis. The exogeneous mutant KRas oncoprotein in pRC-U transfected cells was degraded more quickly than that in controls. RC-U can bind with KRas. KRas can be ubiquitinated by RC-U. It was shown that RC-U also degradate Kras as well as HRas and NRas. The protein expression of pERK was also decreased, but there was no significant change in total ERK expression. When the pancreatic cancer cells infected with lentivirus expressing RC-U, the ability of the cell growth in vitro was decreased. In vivo, the reduced volumes of the subcutaneous xenografts in nude mice with RC-U treatment group versus the control group were observed. Conclusions. Our findings for the first time, implicate the chimeric ubiquitin ligase RC-U can decrease KRas protein expression. Destruction of KRas by RC-U through a ubiquitin-dependent, proteasome-mediated degradation pathway. RC-U degradates HRas and NRas simultaneously. RC-U inhibited pancreatic cancer cell growth in vitro and in vivo. This may represent an effective alternative strategy for the targeted therapy of KRas in pancreatic cancers.

SG-139 Neuropilin-2 in progression and therapy resistance of pancreatic cancer M . Muders1, M .J . Stanton2, S . Dutta2, P . Hönscheid1, D . Aust1, K . Datta2, G .B . Baretton1 1 Institute of Pathology, University Hospital “Carl-Gustav-Carus”, Dresden, 2 Department of Biochemistry, University of Nebraska Medical Center, Omaha, United States Aims. Exocrine pancreatic adenocarcinoma is a deadly disease with 5 year survival rates of 25 to 30 percent in patients without and 10 percent in patients with lymph node metastasis. Surgical resection is the only curative treatment option. Unfortunately only 15–20% of pancreatic cancer patients are eligible for curative surgical therapy due to advanced disease at presentation. Therefore, new treatment options are urgently needed. Methods. To investigate the role of Neuropilin-2 in therapy resistance of pancreatic cancer we knocked down Neuropilin-2 and its ligand VEGF-C by RNA interference in standard pancreatic cancer cells lines and treated these cell lines with Gemcitabine. After treatment the cell death was measured using a YO-PRO/PI apoptosis assay. The role of autophagy in the treatment resistance was tested by a standard autophagic flux assay. Results. Neuropilin-2 and its ligand VEGF-C are important molecules of chemotherapy resistance in pancreatic cancer. Furthermore, we have found that the VEGF-C/NRP-2 axis is involved in the activation of autophagy, which maintains cancer cell survival following treatment. Conclusions. Together, these data suggest a link between the VEGF-C/ NRP-2 axis in pancreatic cancer cell progression and therapy resistance. Effective targeting of this pathway may lead to the development of new cancer therapies. Der Pathologe Suppl 1 · 2012  | 

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Abstracts SG-140 Sialinomycin induces autophagic and apoptotic cell death in pancreatic carcinoma cell lines M . Vogt1, B . Verdoodt1, S .-T . Liffers1, A . Tannapfel1, A . Mirmohammadsadegh1 1 Ruhr-University of Bochum, Institute of Pathology, Bochum Aims. Salinomycin a polyether antibiotic, is produced by a strain of streptomyces albus and has anti-microbial and anti-coccodial activities. Recently, a number of studies described anti-tumor properties of salinomycin, in particular its effect on chemoresistant tumor initiating cells. In the present study, we investigated the impact of salinomycin mediated activation of MEK/ERK signalling pathway on autophagy and apoptotic cell death in pancreatic cancer cell lines. Methods. Two pancreatic cell lines MIAPaCa-2 and PaTu8902 were used to analyze the effect of salinomycin on cell viability, autophagy and apoptosis. The effect of salinomycin on autophgay was investigated by transmission electron microscopy (TEM) for detection of autophagic vesicles and processing of LC3B, microtubule-associated protein 1 light chain 3 isoform B (LC3B). Towards investigating the role of salinomycin on apoptotic cell death we used caspase3/7, FACS, Western blot analysis and immunofluorescence staining. Results. Salinomycin treatment inhibits cell viability and colony forming in MIA PaCa-2 and PaTu8902 in a time and concentration depending manner. In both cell lines salinomycin was able to induce autophagic cell death, detected by LC3 processing and formation of autophagic vacoules. Salinomycin was able to induce autophagic and apoptotic cell death in MIAPaCa-2 cell lines. In MIAPaCa-2 cells the salinomycin induced autophagic cell death was dependent on ERK1/2 pathway. In contrast, salinomycin induced autophagic cell death in PaTu8902 was independent of ERK1/2. Conclusions. Salinomycin, a novel anti-tumor drug is able to induce autophagic and apoptotic cell death depending on cellular background.

Mammary Cancer SG-141 Kindlin2 up-regulation promotes tumor progression W . Fang1, T . Zhao1, H .-q . Zhang2 Department of Pathology, Key Laboratory of Carcinogenesis and Translational Research, Beijing, China, 2Peking University Health Science Center, Beijing, China 1

Aims. Kindlin-2 has been confirmed as an essential element of bidirectional integrin signaling. In recent years, the relationship between Kindlin-2 expression and cancers has been a focus of interest. Our previous studies have shown that Kindlin-2 expression was up-regulated in several types of human cancers, and a strong correlation between Kindlin-2 expression and clinical outcome of breast cancer patients was found. However, the functional role of Kindlin-2 in breast cancer has not been studied. This study was designed to investigate the role of Kindlin-2 in the progression of human breast cancer cells. Methods. Firstly, Kindlin-2 expression at protein level was detected by Western blot in several breast cancer cell lines. Two luminal-like breast cancer cell lines, MCF-7 and T47D, expressed low level of Kindlin-2. Two basal-like breast cancer cell lines, MDA-MB-231 and HS578T, expressed moderate levels of the protein. Then, Kindlin-2 gene was overexpressed by transfected into MCF-7 cells. In comparison, short hairpin RNA (ShRNA)-mediated knockdown of Kindlin-2 was performed in HS578T cells. Vector controls were also done in the same cell lines. Ki67 Li, FCM cell cycle, anchorage-independent colony formation (in vitro tumorigenesis), and in vivo tumorigenesis in NOD/SCID mice were observed. Apoptotic cells were labeled by fluorescent annexin V assay and

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quantified by FACS. Array CGH analysis and spectral karyotyping were performed to detect the genomic instability of these cells. Results. The growth rate of Kindlin-2-transfected MCF-7 cells was much quicker than that of the controls. The proportion of G2-M phase cells, clone formation and tumorigenicity were significantly higher than these of the controls. The change of Kindlin-2-ShRNA transfected cells was just the reverse. Moreover, Up-regulation of Kindlin-2 can also reduce the rate of apoptosis induced by the chemotherapy drugs, and these cells showed much more genomic instability compared with the controls. Conclusions. These findings suggested that up-regulation of Kindlin-2 promotes the progression of human breast cancer cells by increasing their proliferation, drug resistance, genomic instability, and tumorigenesis.

SG-142 ECRG4 is frequently downregulated by CpG island hypermethylation in human breast cancer W . Zhang1 1 Zhejiang University, Institute of Pathology, Hangzhou, China Aims. Esophageal cancer related gene4 (ECRG4) is a recently reported candidate tumor suppressor gene frequently hypermethylated in several human tumor types, including esophageal squamous cell carcinoma, colorectal carcinoma, glioma and prostatic carcinoma. This study is to investigate if ECRG4 is transcriptionally silenced by promoter CpG island methylation in breast cancer. Methods. We analyzed several breast cell lines and 12 pairs of fresh samples from breast cancer patients for ECRG4 promoter CpG island methylation by COBRA and bisulfite sequencing. ECRG4 mRNA and protein expression analysis was carried out by semi-quantitative RT-PCR, realtime PCR, Western Blot and immunohistochemistry. Results. We found that all of three immortalized normal breast cell lines and three normal breast tissues expressed ECRG4 proteins, whereas ECRG4 expression were reduced or absent in most breast cancer cell lines and primary breast cancer samples. We also identified that ECRG4 promoter was frequently methylated in breast cancer samples. An inverse correlation between mRNA expression and methylation status of the ECRG4 promoter CpG island was observed in primary breast cancer samples. Conclusions. The expression of ECRG4 is frequently decreased due to promoter CpG island hypermethylation in breast cancer.

SG-143 Novel biomarkers in breast carcinoma: DKK3, ITIH5, SFRP-1 V . Kloten1, B . Becker1, M .G . Schrauder2, P .A . Fasching2, A . Hartmann3, J . Veeck1, R . Knüchel1, E . Dahl1 1 University Hospital of the RWTH Aachen, Institute of Pathology, Aachen, 2 University Hospital Erlangen, University Breast Center, Erlangen, 3 University Hospital Erlangen, Erlangen Aims. For early detection of breast cancer the development of robust blood-based biomarkers that accurately reflect the host tumor is mandatory and thus a growing field of research. The most common alterations in human cancers including breast cancer are changes in the status of DNA methylation, which are therefore quickly emerging as a new pool of potential biomarkers. Thus, we investigated the feasibility of detecting aberrant tumor suppressor gene methylation in cancer cell-derived free circulating DNA in the bloodstream of patients. Methods. Using qualitative MSP, we examined the methylation status of six biologically significant putative tumor suppressor genes, i.e. ITIH5, DKK3, WIF1, SFRP1, SFRP2 and SFRP5 in DNA extracted from serum. Clinical performance was determined in a large training study on 150 serum samples (120 breast cancers, 30 healthy controls). 20 benign di-

sease and 30 colon cancer serum samples were included for additional specificity testing. Results. Based on the training study we could evaluate the top candidate biomarkers with the best values for sensitivity and specificity. A marker panel of DKK3 and ITIH5 detected breast cancer with a sensitivity of 46% (55/120). Specificity of the panel was sufficient with 83%, 100% and 93% in colon cancer samples, benign and healthy control samples, respectively. Control samples revealed unacceptable high methylation rates of SFRP1 and SFRP5 in DNA extracted from colon cancer sera, whereas SFRP2 and WIF1 showed a considerable methylation frequency in sera from healthy controls. Conclusions. The current study suggests that cancer-specific methylation of ITIH5 and DKK3 in serum-derived tumor-borne DNA might be valuable biomarkers for the early detection of breast cancer. In the second phase of this project we are currently validating ITIH5 and DKK3 as reliable methylation biodiagnostic markers in an independent test set consisting of 160 breast cancer serum samples and 160 control samples.

SG-145 Slug and SOX9 in aggressive breast carcinoma V . Tischler1, A . Noske1, U . Zürrer-Härdi1, F . Ingold1, J .-P . Theurillat1, H . Moch1, Z . Varga1, W . Guo2 1 University Hospital Zurich, Institute of Surgical Pathology, Zürich, Switzerland, 2Albert Einstein College of Medicine, Ruth L . and David S . Gottesman Institute for Stem Cell Biology and Regenerative Medicine, New York, United States Aims. Co-expression of the transcription factors Slug and Sox9 was recently used to determine mammary stem cell state. Our intention was to study the expression of Slug and Sox9 in primary breast cancer and to correlate it with clinicopathological parameters including overall survival. Methods. Formalin-fixed paraffin embedded tumor tissue of 306  patients with primary breast cancer [pT1 132 (43.1%), pT2 134 (43.8%), pT3 21 (6.9%), pT4 19 (6.2%); pN0 92 (34.1%), pN1 136 (50.4%), pN2 22 (8.1%), pN3 20 (7.4%); G1 41 (13.4%), G2 144 (47.1%), G3 121 (39.5%)] were assembled on a tissue microarray (TMA). Mean follow-up was 40 months (range 4–324 months). Immunohistochemical Slug and Sox9 expression was semi-quantitatively evaluated. The median value was used as cut-off point for dichotomization into “low Slug”/”high Slug” and “low Sox9”/”high Sox9” expressing groups. Results. Slug and Sox9 expression was identified in the tumor cell nuclei. High Slug expression was found in 41% of the cases (126/306) and high Sox9 expression in 74% (226/306). High expression of both Slug and Sox9 was found in 92/306 (30.1%). Co-expression of Slug and Sox9 significantly correlated with higher tumor grade (p<0.001) and a shortened overall survival (p=0.001). Conclusions. Co-expression of Slug and Sox9 identifies a more aggressive phenotype in breast cancer which might be due to their role in promoting (cancer) stem cell properties.

Malignant Lymphoma SG-146 The molecular mechanism of Shp2 action in IL-6/gp130-inducing the re-differentiation of Burkitt lymphoma cell X . Jiang1, R . Zhou1, L . He1, C . He1, J . Wang1 Zhejiang University, School of Medicine, Hangzhou, China

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Aims. To investigate the potential role of Shp2 in the GC phenotype regulation of BL cell lines. Methods. We used either scrambled siRNA or Shp2 siRNA pools to transfect human BL cell lines (Daudi, Raji, Ramos). We also expressed a PTP-inactive Shp2 mutant (Shp2CS) that had the catalytic Cys-459 replaced with Ser in Raji cells to verify whether the Shp2 PTP activity is required in BL cells growth. And we analyzed the effects of Src inhibitor-1 (Sigma) and Mek inhibitors (U0126) in BL cells. Results. Shp2 had been effectively silenced by the Shp-2 siRNA. Shp2 knockdown led to a decrease in Erk1/2 phosphorylation. Concomitantly, we observed a decrease in the phosphorylation of the tyrosine residue Y416 in the activation loop of Src, whereas Stat3 and Akt phosphorylations were not affected. Similar to Shp2 knockdown in BL cell lines, the C/S protein in Raji cells inhibited ERK1/2 and Src phosphorylation. The abrogation of Shp2 in both cell lines significantly impaired cellular proliferation over time compared with cells grown to the control cell lines. Cell cycle analysis revealed that Shp2 knock down led to a significant increase in the percentage of BL cells in the G1 phase and a corresponding decrease in the S and G2-M phases, without changes in the apoptotic fraction as indicated by the sub-G0-G1 populations. Thus knockdown of Shp2 significantly inhibited BL tumor growth. We also found that shp2 inhibition led to a decrease in CD77 positivity in BL cell lines. Concomitantly, the protein levels of Bcl-6, E2A, AID and Pax-5 were substantially lower in Shp2-siRNA BL cells than in their Shp2-positive counterparts. Biochemical analysis also showed that Shp2 knockdown resulted in down-regulation of c-Myc in BL cells. And Src inhibitor-1 and U0126 caused decreases of c-Myc and GC factors as CD77, Bcl-6, AID, E2A and Pax-5 expression level in BL cells. These results indicate that Src and Erk1/2 activities are necessary for a constitutive GC phenotype in BL cells. Conclusions. In this study, we showed that Shp2 regulated BL cells proliferation in cell culture. Reduction of Shp2 expression led to a decrease of c-Myc and GC factors (Bcl-6, E2A, AID and Pax-5) in BL cell lines through Src and Erk1/2 pathway, which suggested Shp2 play a key role in the series of regulative factors of the GC phenotype in BL cells. Keywords: Shp2, Burkitt’s lymphoma, GC phenotype Grants: the NSF of Zhejiang Province (No.Y2090167) and Research Foundation in Zhejiang Scientific and Technical Bureau (No.2009C33039).

SG-147 Molecular expression profiles in malignant lymphomas D . Lenze1*, M .R . Schweiger2*, M . Piechotta3, K . Zimmermann4, A . Fischer2, S . Boerno2, C . Dieterich3, U . Leser4, M . Hummel1 1 Charité – Universitätsmedizin Berlin, Institute of Pathology, Berlin, 2Max Planck Institute for Molecular Genetics, Berlin, 3Max-Delbrueck-Center for Molecular Medicine, Institute for Medical Systems Biology, Berlin, 4Humboldt-University, Institute for Informatics, Berlin Aims. The molecular analyses of human cancers revealed that great heterogeneity on the molecular level exists even within the same tumor entity which might explain different therapeutic outcome to the same type of treatment. This holds also true for lymphoma. Therefore it is justified to assume that lymphomas carrying the same molecular features might benefit from the same targeted treatment modalities irrespective of their histological or immunophenotypical features. It was the goal of Der Pathologe Suppl 1 · 2012  | 

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Abstracts our study to identify overlapping molecular characteristics in a variety of histologically defined lymphoma entities. To achieve this goal, comprehensive expression profiles of non-coding and coding transcripts derived from a broad spectrum of lymphomas were established. Our data show that overlapping molecular features can be identified beyond the current lymphoma classification. Methods. RNA was extracted from frozen tissue blocks of distinct human B- and T-cell lymphoma entities as well as tonsils (n=116). The small (micro) RNA fraction was sequenced by next generation technology (SOLiD) and total RNA was subjected to Affymetrix Exon 1.0 ST array hybridisation. In addition immunohistochemical and clinical data was acquired. Bioinformatic analyses were then applied for subgroup detection. Results. Unsupervised clustering based on the mature micro RNA expression derived from deep sequencing demonstrated the presence of two distinct large clusters within the case collection. One cluster contained predominantly the so-called indolent lymphoma types, but also a considerable number of aggressive B-cell lymphoma cases. In the second cluster the majority of cases were aggressive B-cell lymphoma but interestingly intermingled with the T-cell lymphoma cases. Differentially expressed micro RNAs between the two clusters were determined and logistic regression identified a micro RNA classificator able to distinguish the two groups. The micro RNA expression data was combined with the coding RNA data in order to identify the involved pathways. Conclusions. Micro RNA expression profiles obtained by deep sequencing were able to identify two groups within a lymphoma collection that separated the cases beyond the histopathological classification system. Discriminative micro RNAs and associated pathways were identified. These findings give new insights into lymphoma biology and point to alternative treatment options. *These authors contributed equally to the study .

Poster Poster: Deutsch-Chinesisches Symposium SG-P-112 Mitochondrial mortalin (HSPA9) expression in enterocytes is regulated by ACSL5 N . Gaßler1, C . Klaus1, E . Kämmerer2, M . Adolf1, A . Reinartz1 1 RWTH Aachen University, Institute of Pathology, Aachen, 2RWTH Aachen University, Klinik für Kinder- und Jugendmedizin, Aachen Aims. Mitochondrial acyl-CoA synthetase 5 (ACSL5), a long-chain fatty acid activating enzyme, is preferentially found in small intestinal enterocytes. ACSL5 activities are associated with complex changes in the mitochondrial lipid metabolism and are important for epithelial differentiation and cell death. The aim of the present study was to identify and characterize ACSL5 related regulation of mitochondrial proteins. Methods. Mitochondria isolated from ACSL5 transfected CaCo2 cells and controls were homogenized and further separated with 2D-gel electrophoresis. Spots were analyzed using special proteomics software. Protein spots differentially expressed were further identified with MALDI-TOF. Additional techniques were used for target validation. Laser microdissected enterocytes from normal human small intestinal mucosa were investigated to substantiate the in vitro findings. Results. 14 mitochondrial protein species, probably regulated by ACSL5, were identified. ACSL5 dependent expression and synthesis of the candidate molecule mortalin (HSPA9) was confirmed using qRT-PCR, Western blotting, and siRNA ACSL5 knockdown. Using this approach, a positive correlation of ACSL5 and mortalin expression was verified. The positive correlation of ACSL5 and mortalin expression was additio-

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nally found in human normal small intestinal mucosa. Strong mortalin expression was seen in ACSL5-rich enterocytes isolated from intestinal villi, whereas mortalin was diminished in low ACSL5 expressing enterocytes isolated from intestinal crypts. Conclusions. In conclusion, ACSL5 activities are associated with changes in lipid metabolism as well as expression of mitochondrial proteins. ACSL5-dependent expression and synthesis of mitochondrial mortalin is probably a phenomenon of stress-response.

SG-P-113 ACSL5 as putative modifier of Wnt activity in intestinal cells C . Klaus1, U . Schneider1, R . Knuechel1, N . Gaßler1 1 RWTH Aachen University, Institute of Pathology, Aachen Aims. The mitochondrial localized Acyl-CoA synthetase 5 (ACSL5) converts free long-chain fatty acids into fatty acyl-CoA esters, and thereby plays a key role in lipid biosynthesis and fatty acid degradation. In particular, ACSL5 has been recently identified to be involved in apoptotic cell death of senescent enterocytes along the intestinal crypt-villus axis and to interact with mitochondrial proteins. The aim of this study was to investigate ACSL5-dependent effects on intestinal signalling pathways that coordinate proliferation and/or differentiation of enterocytes, particularly the Wnt pathway. Methods. Wnt signalling was analysed in an ACSL5 overexpressing cell culture model using luciferase assays, immunohistochemistry, qRT-PCR and Western blot. The findings were substantiated with expression studies in human colon carcinomas and in a Wnt-associated mouse model. Results. ACSL5 transgenic intestinal-derived cells displayed a strong susceptibility to pro-apoptotic stimuli. This phenomenon was accompanied by caspase-3 activation and significant down-regulation of Wnt signalling activity. In particular, Wnt pathway associated mitochondrial localized molecules were identified as interaction partners of ACSL5 activity. Conclusions. Molecular mechanisms underlying ACSL5-dependent apoptosis susceptibility of enterocytes are probably bivalent including pro-apoptotic and anti-proliferative activities.

SG-P-114 MAPK signaling regulates differential WNT activity in colorectal cancer D . Horst1, J . Chen2, T . Kirchner1, R . Shivdasani2 Ludwig-Maximilians-Universität München, Pathologisches Instiut, München, 2Harvard Medical School, Dana-Farber Cancer Institute, Boston, United States

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Aims. Most colorectal cancers (CRCs) express the WNT effector protein β-catenin in a heterogeneous pattern. Strong nuclear expression is often confined to a small fraction of tumor cells at the tumor’s leading edge. Recent data suggest a role for Mitogen Activated Protein Kinase (MAPK) signaling in nuclear accumulation of β-catenin. We therefore investigated if MAPK activity regulates overtly differential WNT activity in CRC cell subpopulations. Methods. We used gene expression profiling, and immunohistochemistry to assess interdependence of MAPK and WNT pathway activity in CRC. Lentivirus- and drug-based pathway modification in CRC xenograft tumors of primary human colon cancers and colon cancer cell lines was used to study the effect of MAPK activation or repression on differential WNT activity. Results. CRC cells with high WNT activity showed coincident overexpression of MAPK target genes and high levels of phospho-ERK, indicating active MAPK signaling. Forced MAPK activation by lentiviral expression of constitutively active KRAS enhanced WNT pathway activity in vivo in CRC xenograft tumors, whereas drug based inhibition of EGFR signaling attenuated it.

Conclusions. Although CRC is characterized by mutational activation of the WNT pathway, MAPK signaling influences intratumoral β-catenin heterogeneity, revealing a mechanism for external stimuli to modulate pathway activity. Because MAPK signaling does not merely coincide with nuclear β-catenin but also regulates it, this may account for the high frequency of KRAS mutations in CRC.

SG-P-115 Influence of an anti-TLR2-Intrabody on rheumatoid arthritis B. Küttner1, V. Seiffert1, S. Laggies1, G. Gross1, T. Böldicke1, A. Dellmann2, K. Donhuijsen2 1 Helmholtz-Centre, Infection Research, Braunschweig, 2academical hospital Braunschweig, department of pathology, Braunschweig Aims. Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease. Recent results showed that toll-like receptors (TLRs) particularly TLR2 and its signaling pathway is involved in the pathogenesis of rheumatic arthritis and might be a “key target” in prevention of RA. An intracellular antibody (intrabody) fused with an endoplasmatic reticulum retention sequence has been developed that inhibits the function of TLR2 by preventing the translocation of TLR2 from the ER to the cell surface. Methods. To test if the intrabody has the potential to inhibit inflammation during rheumatoid arthritis two mouse models were established: a collagen-induced arthritis model and a model based on the injection of inflammatory synovial fibroblasts into joints. Investigations by immunohistochemistry, confocal microscopy, flow cytometry and paw swelling showed the course of the disease. At first we analyzed the efficiency of the anti-TLR2 intrabody in the collagen arthritis mouse model. Results. A strong inflammatory response was seen in both rheumatoid mouse models in the absence of the intrabody. Anti-TLR2 intrabody inhibited significantly the paw swelling after adenoviral anti-TLR2 intrabody gene transfer into the joints. Joints with an inflammatory response showed migration of cells (HE staining) into the lamina synovialis and the fat-tissue, in fact macrophages (CD11b+) and fibroblasts (CD40+). Conclusions. Analysis of TLR2 surface representation and intracellular intrabody expression of cells prepared from joints of rheumatoid mice treated with intrabody-adenovirus and eGFP adenovirus (control) is currently under investigation. In the rheumatoid arthritis model using synovial cells with inflammation, we plan to analyze the influence of cellular anti-TLR2-intrabody expression on the manifestation of the disease.

SG-P-116 TGF-β and Wnt signaling pathways regulate the expression of IGFBP7 of fibroblasts in the tumor-stroma interactions C. Rao1, J. Xu1, M. Liu1, H. Deng1 1 Department of Pathology, School of Medicine, Zhejiang, China Aims. To find out the mechanism of the up-regulation of IGFBP7and the biological changes in fibroblasts during the interactions with colorectal cancer cells. Fibroblasts (HELFs) were cultured in colorectal cancer cells conditioned media (SW620-CM), treated by TGF-β 1, TGF-β1 receptor antagonist (SB431542), TGF-β1 specific antibody (AF), Wnt signaling pathway agonist (LiCl) and inhibitor (DKK-1) respectively. Q-PCR, Western Blot, ELISA, Immunofluorescence microscopy and flow cytometry were used to detect the expression of related targeted genes and proteins of TGF-β and Wnt signaling pathways. HELFs cultured in SW620-CM were activated with abundant expression of α-SMA and showed strong proliferation and weak apoptosis and senescence. The expression of IGFBP7 of HELFs was up-regulated in timedependent and dose-dependent manners when cultured with SW620CM, while TGF-β signaling were activated as Smad2, P-Smad2 and

TGF-βRΠ were up-regulated in HELFs. These effects could be strengthened by TGF-β1 and inhibited by SB431542 or AF. During the interactions, the downstream genes of Wnt signaling pathway such as c-myc, cyclinD1 were up-regulated and the proteins of Wnt signaling pathway such as β-catenin, Dvl3, Dvl2 and Naked2 were obviously up-regulated which suggested the canonical Wnt signaling pathway was also activated. TGF-β and Wnt signaling pathways were activated during colorectal cancer cells-fibroblasts interactions. Upregulating of IGFBP7 in HELFs was mainly through TGF-β1/ALK5/ Smad-2 signaling pathway. Wnt signaling pathway may also play an important role in this process.

SG-P-117 MicroRNA-137, a HMGA1 target, suppresses cell invasion and metastasis of colorectal carcinoma by directly targeting FMNL2 X. Li1, X. Zhang1, W. Zhao1, J. Wang1, X.W. Biang2, L. Liang3, Y. Ding3 Department of Pathology, School of Basic Medical Sciences, Guangzhou, China, 2Institute of Pathology and Southern Cancer Center, Southwest Hospital, Chongqing, China, 3Guangdong Province Key Laboratory of Molecular Tumor Pathology, Guangzhou, China 1

Aims. FMNL2, a member of diaphanous-related formins, has been strongly associated with tumor progression but the post-transcriptional regulatory mechanism of FMNL2 remains unknown. The aim of this study was to investigate whether increased FMNL2 expression is mediated by microRNAs in colorectal carcinoma (CRC). Methods. Real-time PCR or Western blot was performed to detect the expression level of miR-137 or FMNL2 in CRC cells and tissues. The in vitro and in vivo functional effect of miR-137 was examined further. A luciferase reporter assay was conducted to confirm the associations between miR-137 and FMNL2 3’UTR, HMGA1 and miR-137 promoter. CHIP was used to assess the direct binding of HMGA1 to miR-137 promoter. Results. We initially chose miR-137 and miR-142-3p as potential miRNAs targeting FMNL2 which were predicted by bioinformatics. Only miR-137 showed significant inverse correlation with FMNL2 protein level in CRC cell lines and tissues. We validated that FMNL2 was a target gene of miR137, and miR-137 could inhibit cell proliferation, invasion in vitro, hepatic or intestinal metastasis in vivo by targeting FMNL2. We further show that HMGA1 enhances miR-137 transcription by binding its promoter, and then negatively downregulates FMNL2 expression. Finally, miR-137 inhibited the activation of p-MAPK and p-Akt, followed by the suppression of MMP2, MMP9 and VEGF. Conclusions. Our findings demonstrate a novel mechanism of post-transcriptional regulation of FMNL2 expression and identified miR-137, induced by its upstream transcription factor HMGA1, can suppress its direct target FMNL2 and lead to tumor invasion and metastasis, at least in part through inhibiting PI3K/Akt and MAPK/ERK pathways.

SG-P-118 The positive feedback between FMNL2 and RhoA, promotes actin assembly and cell invasion of colorectal carcinoma Y. Zeng1, Y. Qiao1, W. Zhao1, X. Zhu1, J. Wang2, X. Zhang2, X. Bian3, Y. Ding2, L. Liang2 1 Department of Pathology, School of Basic Medical Sciences, Guangzhou, China, 2Guangdong Province Key Laboratory of Molecular Tumor Pathology, Guangzhou, China, 3Institute of Pathology and Southern Cancer Center, Southwest hospital, Chongqing, China Aims. FMNL2 is a member of diaphanous-related formins which act as effectors of Rho family GTPases and control the actin-dependent processes such as cell motility or invasion. We previously validated FMNL2 as a positive regulator of cell motility and metastasis in colorectal carcinoma (CRC) but the mechanisms of FMNL2 in CRC remain unclear.

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Abstracts The aim was to investigate the association of FMNL2 with Rho signaling pathway in the invasion of CRC. Methods. Rho family GTPase activity was tested by Rho pull-down assay. In vitro invasive ability of cells was detected by Boyden Chamber assay. And luciferase activities of MAL/SRF were detected using dualluciferase reporter assay. In addition, Immunofluorescent analyses were performed to examine F-actin stained by phalloidin and co-localization of FMNL2 and LARG or p115RhoGEF. Co-immunoprecipitation was used to determine the direct binding of FMNL2 and LARG. Finally, GST pull-down assay was used to detect the binding of LARG-CT with FMNL2 in the absence or presence of active RhoAV14. Results. In this study, we showed that FMNL2 activated Rho/ROCK pathway, and required ROCK to promote cell invasion. Moreover, FMNL2 promoted the formation of stress fiber and filopodia, and activated the SRF transcription factor in the Rho-dependent manner. We also demonstrated that FMNL2 was necessary for LPA-induced invasion, Rho/ ROCK activation, actin polymerization and SRF activation. FMNL2 is an essential component of LPA signal transduction toward Rho by directly interacting with LARG. Finally, we found FMNL2, LARG and RhoA constituted a positive feedback loop. LARG silencing inhibited Rho/ROCK pathway and cell invasion. Conclusions. Our findings provide evidence for the positive feedback between FMNL2 and RhoA, which promotes actin assembly and cell invasion of CRC.

SG-P-119 DSC3 expression is regulated by p53, and methylation of DSC3 DNA is a prognostic marker in human colorectal cancer T. Cui1, Y. Chen1, L. Yang1, T. Knösel1, K. Zöller1, O. Huber2, I. Petersen1 1 Institute of Pathology, Jena, 2Institute of Biochemistry II Aims. Desmocollin 3 (DSC3), a member of the cadherin superfamily and integral component of desmosomes, is involved in carcinogenesis. However, the role of DSC3 in human colorectal cancer (CRC) has not yet been established. Our aim of the study was to explore the role of DSC3 in human colorectal cancer. Methods. DSC3 expression in CRC cell lines was analyzed by RT-PCR and western blotting. Methylation status of DSC3 was examined by demethylation tests, methylation-specific PCR, and bisulfite sequencing (BS). The regulatory role of p53 was investigated by transfection. Results. DSC3 was downregulated in CRC cells at both mRNA and protein levels. DSC3 expression was restored in five out of seven cell lines after 5-aza-2’-deoxycytidine (DAC) treatment. A heterogeneous methylation pattern was detected by BS in promoter region and exon 1 of DSC3. Methylation of DSC3 genomic sequences was found in 41% (41 out of 99) of primary CRC, being associated with poor prognosis (p=0.002). Transfection of p53 alone or in combination of DAC increased the DSC3 expression. Similarly, treatment with p53 inducer adriamycin alone or in combination with DAC enhanced DSC3 expression. Conclusions. DNA methylation contributes to downregulation of DSC3 in CRC cell lines. Methylation status of DSC3 DNA is a prognostic marker for CRC. P53 appears to play an important role in regulating DSC3 expression.

SG-P-120 ECRG4 is frequently downregulated by promoter CpG island hypermethylation in human breast cancer W. Zhang1 1 Zhejiang University, Institute of Pathology, Hangzhou, China Aims. Esophageal cancer related gene4 (ECRG4) is a recently reported candidate tumor suppressor gene frequently hypermethylated in several human tumor types, including esophageal squamous cell carcinoma, colorectal carcinoma, glioma and prostatic carcinoma. This study is to

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investigate if ECRG4 is transcriptionally silenced by promoter CpG island methylation in breast cancer. Methods. We analyzed several breast cell lines and 12 pairs of fresh samples from breast cancer patients for ECRG4 promoter CpG island methylation by COBRA and bisulfite sequencing. ECRG4 mRNA and protein expression analysis was carried out by semi-quantitative RT-PCR, realtime PCR, Western Blot and immunohistochemistry. Results. We found that all of three immortalized normal breast cell lines and three normal breast tissues expressed ECRG4 proteins, whereas ECRG4 expression were reduced or absent in most breast cancer cell lines and primary breast cancer samples. We also identified that ECRG4 promoter was frequently methylated in breast cancer samples. An inverse correlation between mRNA expression and methylation status of the ECRG4 promoter CpG island was observed in primary breast cancer samples. Conclusions. The expression of ECRG4 is frequently decreased due to promoter CpG island hypermethylation in breast cancer.

SG-P-121 Clonality analysis of neuroendocrine cells in gastric adenocarcinoma L. Wang1, G. Yao1, Z. Zhao2, X. Wei1, R. Xu3 Institute of Pathology and Forensic Medicine, Zhejiang University, Hangzhou, China, 2Zhejiang Provincial People’s Hospital, Hangzhou, China, 3 Hangzhou First People’s Hospital, Hangzhou, China

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Aims. Gastric cancer remains one of the most common cancers and the leading causes of cancer death worldwide. Neuroendocrine differentiation (NED) is a common phenomenon in adenocarcinomas, but there have been only a few studies of NED in gastric adenocarcinoma. However, it remains unclear whether the glandular and endocrine cells expand from two distinct precursors, or arise from a single progenitor cell. Therefore, we studied the clonality of neuroendocrine (NE) cells in gastric adenocarcinoma. Methods. In this study, 120 cases of gastric adenocarcinoma and corresponding non-neoplastic gastric mucosal tissues were obtained, immunohistochemistry was carried out using the primary antibody against NE marker (chromogranin A). Frozen section immunohistochemistry samples were selected with a large quantity of NE cells. Then laser-capture microdissection (LCM) was performed to obtain NE cells from gastric adenocarcinoma, ready for DNA extraction and subsequent genetic analysis. DNA extraction from the captured cells and whole genome amplification (WGA) were performed using DNA Micro-kit and DNA Repli-g Midi kit to obtain a large quantity of DNA. Then we chose 26 microsatellite markers with genome-wide scope, and exon 7 and exon 8 of p53, designed primers, and performed PCR. Genome-wide microsatellite abnormalities (MSI and LOH) and p53 mutation were detected by PCR-SSCP silver staining and PCR sequencing to identify the clonality of NE cells. Statistical analyses were performed using SPSS for Windows version 15.0. Results. Thirty samples from a total of 120 that contained a large number of NE cells were chosen for LCM. Through LCM, about 500  NE cells were precisely captured from each sample. The total incidence rate of MSI was 27.4%, and LOH rate was 17.9%. The rates for adenocarcinoma and NE cells were similar. There was no significant relationship between the incidence rate of MSI or LOH and clinicopathological characteristics. According to the coincidence of microsatellite changes, cases 2, 3, 5, 6, 11, 12, 18, 24, 27 and 30 had highest the concordance for the two types of cells. The other samples had similar microsatellite changes, except for cases 7 and 10. Most p53 mutations were detected in exons 7 and 8. Concordant mutations were observed in samples 4, 14, 21 and 27, and there were different mutations in the two types of cells in case 7. In case 17, mutation was seen only in adenocarcinoma cells. p53 mutation occurred six times in adenocarcinoma cells (20.0%) and five times in NE cells (16.7%). Clinicopathological data showed that mutations were present in poorly

differentiated and TNM stage III or IV tumours. p53 mutation was closely related with the degree of differentiation, TNM stage, vessel invasion and lymph node metastasis. By combining the results with microsatellite changes and p53 mutation, NE and adenocarcinoma cells showed the same MSI, LOH or p53 mutation in most cases (27/30). In the other three cases, different MSI, LOH or p53 mutation occurred. Conclusion. we suggest that, in 27 of 30 cases, NE and adenocarcinoma cells were generated from the same stem cells. The multipotent stem cells differentiate to NE and adenocarcinoma cells, initiated by hormonal change, microenvironmental change, and genomic instability. NE cells act as parenchyma of carcinoma, and excrete hormones to promote carcinoma. The remaining three cases might have had different ancestral cells, and this needs further study.

SG-P-122 IGFBP7 high expression in the colorectal cancer cells is related to Wnt signaling pathway inhibition during the interactions between colorectal cancer cells and fibroblasts J. Xu1, H. Deng*1, C. Rao1, S. Lin1 Zhejiang University, School of Medicine, Hangzhou, China

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Aims. To study the Wnt signaling pathway in interactions between the colorectal cancer cells and fibroblasts and its impact on the expression of IGFBP7 (Insulin-like growth factor binding protein 7) in the colorectal cancer cells. Methods. Colorectal cancer cells SW480 or SW620 were cultured in HELF cells or activated HELF cells conditional media, treated by Wnt signaling pathway agonist (LiCl) or Wnt signaling pathway inhibitor (DKK-1). RT-PCR, Real-time PCR, Western blot and immunofluorescence microscopy were used to detect the related protein and target genes of Wnt signaling pathway and the expression of IGFBP7. Results. IGFBP7 expression was increased with times in the colorectal cancer cells treated by the activated HELF conditional media. And activation of Wnt signaling pathway reduced IGFBP7 expression, inhibition of Wnt signaling pathway induced IGFBP7 expression. Conclusions. The interactions between tumor cells and fibroblasts could inhibit Wnt signaling pathway in the tumor cells, and induce the expression of IGFBP7.

SG-P-123 TGF-beta and Wnt signaling pathways regulate the expression of IGFBP7 of fibroblasts in the tumor-stroma interactions C. Rao , J. Xu , M. Liu , H. Deng* Zhejiang University, School of Medicine, Hangzhou, China 1

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Aims. To find out the mechanism of the up-regulation of IGFBP7and the biological changes in fibroblasts during the interactions with colorectal cancer cells. Methods. Fibroblasts (HELFs) were cultured in colorectal cancer cells conditioned media (SW620-CM), treated by TGF-beta1, TGF-beta1 receptor antagonist (SB431542), TGF-beta1 specific antibody (AF), Wnt signaling pathway agonist (LiCl) and inhibitor (DKK-1) respectively. QPCR, Western Blot, ELISA, Immunofluorescence microscopy and flow cytometry were used to detect the expression of related targeted genes and proteins of TGF-beta and Wnt signaling pathways. Results. HELFs cultured in SW620-CM were activated with abundant expression of alfa-SMA and showed strong proliferation and weak apoptosis and senescence. The expression of IGFBP7 of HELFs was up-regulated in time-dependent and dose-dependent manners when cultured with SW620-CM, while TGF-beta signaling were activated as Smad2, P-Smad2 and TGF-beta R2 were up-regulated in HELFs. These effects could be strengthened by TGF-beta1 and inhibited by SB431542 or AF. During the interactions, the downstream genes of Wnt signaling pathway such as c-myc, cyclinD1 were up-regulated and the proteins of

Wnt signaling pathway such as beta-catenin, Dvl3, Dvl2 and Naked2 were obviously up-regulated which suggested the canonical Wnt signaling pathway was also activated. Conclusions. TGF-beta and Wnt signaling pathways were activated during colorectal cancer cells-fibroblasts interactions. Upregulating of IGFBP7 in HELFs was mainly through TGF-beta1/ALK5/ Smad-2 signaling pathway. Wnt signaling pathway may also play an important role in this process.

SG-P-124 Serum MiR-192 and MiR-194 as tumor biomarker for pancreatic ductal adenocarcinoma J. Zhang1, C.-y. Zhao1, D.-h. Yu1, Y. Chen1, Q.-h. Liu1, M. Shi1, J.-t. Zhang1, G. Jin1, P. Cheng1, X.-g. Hu1, C.-r. Ni1, M.-h. Zhu1 1 Department of Pathology, Changhai Hospital, Shanghai, China Aims. To assess the validity of using serum miRNA signatures of PDAC as biomarkers for diagnosis. Methods. MiRNA microarray was used to detect the differences between PDAC samples and normal pancreatic tissues. MiR-192 and miR-194 were found in the tissues of human PDAC and in the explants in tumorbearing SCID mice by locked nucleic acid-based in situ hybridization (LNA-ISH). Serum levels of miR-192 and miR-194 in PDAC patients, duodenal adenocarcinoma patients and healthy controls, as well as six pancreatic cancer cell lines and their culture supernatants were determined by real-time PCR. Results. Eight miRNAs were found overexpressed and eight were lowly expressed in PDAC tissues compared with those in the normal pancreatic tissues. MiR-192 and miR-194 were found overexpressed in the tissues of human PDAC and in the explants in tumor-bearing SCID mice. The levels of miR-192 and miR-194 in the supernatants of six pancreatic cancer cell lines were positively correlated with their cellular expressions (r=0.849, p<0.05; r=0.806, p<0.05, respectively). The serum levels of miR-192 and miR-194 in 70 PDAC patients were significantly higher than those in 17 duodenal adenocarcinoma patients and 40 healthy controls (p<0.05 in both cases). Combined analysis of the three groups yielded a sensitivity of 84.0% and a specificity of 75.0% for the combined detection of miR-192 and miR-194 in the diagnosis of PDAC. Conclusion. Combined detection of serum miR-192 and miR-194 level may serve as a sensitive diagnostic biomarker for PDAC.

SG-P-125 Targeted Degradation of KRas by Engineered Ubiquitin Ligase Suppresses Pancreatic Cancer Cell Growth In vitro and In vivo Y. Ma1, Y. Gu1, Q. Zhang1, Z. Lu1, W. Zhao1, J. Chen1 Department of Pathology, Peking Union Medical College Hospital, Beijing, China

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Aims. KRas oncogene mutation is one of the earliest genetic events seen in 30% human pancreatic intraepithelial neoplasia (PanIN) with the frequency rising to nearly 100% in advanced pancreatic ductal adenocarcinoma cancer (PDAC). KRas is an attractive therapeutic target of PDAC. “Target ubiquitination and degradation of oncoproteins via ubiquitin-proteasome pathway” may be an alternative therapeutic strategy. E3 ligase is thought to be the component of the ubiquitin conjugation system that is most directly responsible for substrate recognition. In the present study, an engineered ubiquitin E3 ligase (RC-U) was generated to target the KRas oncoprotein for ubiquitination and degradation. The effects of RC-U on pancreatic cancer cell growth in vitro and in vivo were investigated. Methods. The engineered ubiquitin E3 ligase (RC-U) was constructed as follows: the cDNA fragments encoding U-box and (RBD+CRD) Raf-1 were amplified by PCR amplification and then were inserted into pcDNA3.1 vector (pRC-U). The fusion cDNA was also subcloned into Der Pathologe Suppl 1 · 2012 

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Abstracts the pLenti6.3 to generate pLenti6.3-RC-U. Lentiviruses were packaged in HEK 293T cells. The KRas mRNA and protein expression after transfecting the pRC-U expression plasmid into human pancreatic cancer cells or HEK293T cells were detected by real-time reverse transcription polymerase chain reaction (real-time RT-PCR), Western blotting and immunofluorescence. After pRC-U transfection, with mg-132 or cycloheximide treatments, the KRas protein expression was tested by Western blotting. The interaction between RC-U and KRas, whether KRas could be ubiquitinated by RC-U or not were both investigated by immunoprecipitation. The expression of HRas, NRas, phosphorylated extracellular signal-regulated protein kinases (pERK) and extracellular signal-regulated protein kinases (ERK) in pancreatic cancer cells was also examined by Western blotting after pRC-U transfection. The effects of RC-U on pancreatic cancer cell growth in vitro were detected by CCK-8 and soft agar colony formation assays after lentivirus infection. In vivo, with RC-U treatment, the volumes of the subcutaneous xenografts in nude mice were measured. Results. RC-U dramatically decreased the protein level of KRas compared to the controls. No significant change of KRas mRNA expression within different groups was observed. After pRC-U transfection, the stability of KRas was significantly increased in the presence of specific proteasome inhibitor mg-132. HEK293T cells were transfected by mutant KRas construct together with either pRC-U or empty vector and then incubated with cycloheximide to inhibit novel protein synthesis. The exogeneous mutant KRas oncoprotein in pRC-U transfected cells was degraded more quickly than that in controls. RC-U can bind with KRas. KRas can be ubiquitinated by RC-U. It was shown that RC-U also degradate Kras as well as HRas and NRas. The protein expression of pERK was also decreased, but there was no significant change in total ERK expression. When the pancreatic cancer cells infected with lentivirus expressing RC-U, the ability of the cell growth in vitro was decreased. In vivo, the reduced volumes of the subcutaneous xenografts in nude mice with RC-U treatment group versus the control group were observed. Conclusions. Our findings for the first time, implicate the chimeric ubiquitin ligase RC-U can decrease KRas protein expression. Destruction of KRas by RC-U through a ubiquitin-dependent, proteasome-mediated degradation pathway. RC-U degradates HRas and NRas simultaneously. RC-U inhibited pancreatic cancer cell growth in vitro and in vivo. This may represent an effective alternative strategy for the targeted therapy of KRas in pancreatic cancers.

SG-P-126 Neuropilin-2 in progression and therapy resistance of pancreatic cancer M. Muders1, M.J. Stanton2, S. Dutta2, P. Hönscheid1, D. Aust1, K. Datta2, G.B. Baretton1 1 Institute of Pathology, University Hospital “Carl-Gustav-Carus”, Dresden, 2 Department of Biochemistry, University of Nebraska Medical Center, Omaha, United States Aims. Exocrine pancreatic adenocarcinoma is a deadly disease with 5-year survival rates of 25 to 30 percent in patients without and 10 percent in patients with lymph node metastasis. Surgical resection is the only curative treatment option. Unfortunately only 15 to 20 percent of pancreatic cancer patients are eligible for curative surgical therapy due to advanced disease at presentation. Therefore, new treatment options are urgently needed. Methods. To investigate the role of Neuropilin-2 in therapy resistance of pancreatic cancer we knocked down Neuropilin-2 and its ligand VEGF-C by RNA interference in standard pancreatic cancer cells lines and treated these cell lines with Gemcitabine. After treatment the cell death was measured using a YO-PRO/PI apoptosis assay. The role of autophagy in the treatment resistance was tested by a standard autophagic flux assay.

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Results. Neuropilin-2 and its ligand VEGF-C are important molecules of chemotherapy resistance in pancreatic cancer. Furthermore, we have found that the VEGF-C/NRP-2 axis is involved in the activation of autophagy, which maintains cancer cell survival following treatment. Conclusions. Together, these data suggest a link between the VEGF-C/ NRP-2 axis in pancreatic cancer cell progression and therapy resistance. Effective targeting of this pathway may lead to the development of new cancer therapies.

SG-P-127 Salinomycin induces autophagic and apoptotic cell death in pancreatic carcinoma cell lines M. Vogt1, B. Verdoodt1, S.-T. Liffers1, A. Tannapfel1, A. Mirmohammadsadegh1 1 Ruhr-University of Bochum, Institute of Pathology, Bochum Aims. Salinomycin a polyether antibiotic, is produced by a strain of streptomyces albus and has anti-microbial and anti-coccodial activities. Recently, a number of studies described anti-tumor properties of salinomycin, in particular its effect on chemoresistant tumor initiating cells. In the present study, we investigated the impact of salinomycin mediated activation of MEK/ERK signalling pathway on autophagy and apoptotic cell death in pancreatic cancer cell lines Methods. Two pancreatic cell lines MIAPaCa-2 and PaTu8902 were used to analyze the effect of salinomycin on cell viability, autophagy and apoptosis. The effect of salinomycin on autophgay was investigated by transmission electron microscopy (TEM) for detection of autophagic vesicles and processing of LC3B, microtubule-associated protein 1 light chain 3 isoform B (LC3B). Towards investigating the role of salinomycin on apoptotic cell death we used caspase3/7, FACS, Western blot analysis and immunofluorescence staining. Results. Salinomycin treatment inhibits cell viability and colony forming in MIA PaCa-2 and PaTu8902 in a time and concentration depending manner. In both cell lines salinomycin was able to induce autophagic cell death, detected by LC3 processing and formation of autophagic vacoules. Salinomycin was able to induce autophagic and apoptotic cell death in MIAPaCa-2 cell lines. In MIAPaCa-2 cells the salinomycin induced autophagic cell death was dependent on ERK1/2 pathway. In contrast, salinomycin induced autophagic cell death in PaTu8902 was independent of ERK1/2. Conclusions. Salinomycin, a novel anti-tumor drug is able to induce autophagic and apoptotic cell death depending on cellular background.

SG-P-128 Up-regulation of Kindlin-2 promotes progression of human ­breast cancer cells by increasing their proliferation, drug resistance, genomic instability, and tumorigenesis W. Fang1, T. Zhao1, H.-q. Zhang2 Department of Pathology, Key Laboratory of Carcinogenesis and Translational Research, Beijing, China, 2Peking University Health Science Center, Beijing, China 1

Aims. Kindlin-2 has been confirmed as an essential element of bidirectional integrin signaling. In recent years, the relationship between Kindlin-2 expression and cancers has been a focus of interest. Our previous studies have shown that Kindlin-2 expression was up-regulated in several types of human cancers, and a strong correlation between Kindlin-2 expression and clinical outcome of breast cancer patients was found. However, the functional role of Kindlin-2 in breast cancer has not been studied. This study was designed to investigate the role of Kindlin-2 in the progression of human breast cancer cells. Methods. Firstly, Kindlin-2 expression at protein level was detected by Western blot in several breast cancer cell lines. Two luminal-like breast cancer cell lines, MCF-7 and T47D, expressed low level of Kindlin-2. Two basal-like breast cancer cell lines, MDA-MB-231 and HS578T, ex-

pressed moderate levels of the protein. Then, Kindlin-2 gene was overexpressed by transfected into MCF-7 cells. In comparison, short hairpin RNA (ShRNA)-mediated knockdown of Kindlin-2 was performed in HS578T cells. Vector controls were also done in the same cell lines. Ki67 Li, FCM cell cycle, anchorage-independent colony formation (in vitro tumorigenesis), and in vivo tumorigenesis in NOD/SCID mice were observed. Apoptotic cells were labeled by fluorescent annexin V assay and quantified by FACS. Array CGH analysis and spectral karyotyping were performed to detect the genomic instability of these cells. Results. The growth rate of Kindlin-2-transfected MCF-7 cells was much quicker than that of the controls. The proportion of G2-M phase cells, clone formation and tumorigenicity were significantly higher than these of the controls. The change of Kindlin-2-ShRNA transfected cells was just the reverse. Moreover, Up-regulation of Kindlin-2 can also reduce the rate of apoptosis induced by the chemotherapy drugs, and these cells showed much more genomic instability compared with the controls. Conclusions. These findings suggested that up-regulation of Kindlin-2 promotes the progression of human breast cancer cells by increasing their proliferation, drug resistance, genomic instability, and tumorigenesis.

SG-P-129 DKK3 and ITIH5 gene methylation as novel biomarkers for bloodbased breast cancer screening: Improving early detection of breast cancer V. Kloten1, B. Becker1, M.G. Schrauder2, P.A. Fasching2, A. Hartmann3, J. Veeck1, R. Knüchel1, E. Dahl1 1 University Hospital of the RWTH Aachen, Institute of Pathology, Aachen, 2 University Hospital Erlangen, University Breast Center, Erlangen, 3University Hospital Erlangen, Erlangen Aims. For early detection of breast cancer the development of robust blood-based biomarkers that accurately reflect the host tumor is mandatory and thus a growing field of research. The most common alterations in human cancers including breast cancer are changes in the status of DNA methylation, which are therefore quickly emerging as a new pool of potential biomarkers. Thus, we investigated the feasibility of detecting aberrant tumor suppressor gene methylation in cancer cell-derived free circulating DNA in the bloodstream of patients. Methods. Using qualitative MSP, we examined the methylation status of six biologically significant putative tumor suppressor genes, i.e. ITIH5, DKK3, WIF1, SFRP1, SFRP2 and SFRP5 in DNA extracted from serum. Clinical performance was determined in a large training study on 150 serum samples (120 breast cancers, 30 healthy controls). 20 benign disease and 30 colon cancer serum samples were included for additional specificity testing. Results. Based on the training study we could evaluate the top candidate biomarkers with the best values for sensitivity and specificity. A marker panel of DKK3 and ITIH5 detected breast cancer with a sensitivity of 46% (55/120). Specificity of the panel was sufficient with 83%, 100% and 93% in colon cancer samples, benign and healthy control samples, respectively. Control samples revealed unacceptable high methylation rates of SFRP1 and SFRP5 in DNA extracted from colon cancer sera, whereas SFRP2 and WIF1 showed a considerable methylation frequency in sera from healthy controls. Conclusions. The current study suggests that cancer-specific methylation of ITIH5 and DKK3 in serum-derived tumor-borne DNA might be valuable biomarkers for the early detection of breast cancer. In the second phase of this project we are currently validating ITIH5 and DKK3 as reliable methylation biodiagnostic markers in an independent test set consisting of 160 breast cancer serum samples and 160 control samples.

SG-P-130 Forced expression of ITIH5 in a luminal-type breast cancer model confers growth suppressive properties S. Huth1, R. Knüchel1, E. Dahl1 1 University Hospital of the RWTH Aachen, Institute of Pathology, Aachen Aims. Inter-α-trypsin inhibitors are protease inhibitors which consist of one light chain (bikunin) and different heavy chains (ITIHs). We recently characterized ITIH5 as a novel member of the ITIH gene family and showed that its loss in human breast cancer is associated with parameters of tumour invasion and distant metastasis. Thus, we aimed to analyze the biological function of ITIH5 in an in vitro model of breast cancer, the luminal-type breast cancer cell line T47-D. Methods. T47-D clones expressing ITIH5 and corresponding mock (empty vector) clones were generated using standard methods and subsequently validated on DNA, mRNA and protein level. Functional analyses of ITIH5 clones were performed by standardized assays including XTT, colony formation, cell-matrix-adhesion and Apo-ONE® Homogeneous Caspase-3/7 Assay. Results. Forced ITIH5 expression in T47-D transfectants was successfully achieved validating integration and expression of ITIH5 on the DNA, mRNA and protein level, respectively. Using functional assays we observed significantly reduced proliferation (p=0.01) as well as significantly increased cell-matrix-adhesion (p<0.001) and apoptosis (p=0.01) in ITIH5 clones compared to mock clones. Conclusions. Our study indicates a functional involvement of ITIH5 in cell proliferation, cell-matrix-adhesion and apoptosis of luminal-type human breast cancer cells. These processes are important in tumour development and metastatic spread. The in vitro functional analysis of ITIH5 expressing tumor cells present further evidence that ITIH5 is a novel tumour suppressor gene in human breast cancer.

SG-P-131 Co-expression of Slug and Sox9 identifies a more aggressive phenotype in primary breast cancer V. Tischler1, A. Noske1, U. Zürrer-Härdi1, F. Ingold1, J.-P. Theurillat1, H. Moch1, Z. Varga1, W. Guo2 1 University Hospital Zurich, Institute of Surgical Pathology, Zürich, Switzerland, 2Albert Einstein College of Medicine, Ruth L. and David S. Gottesman Institute for Stem Cell Biology and Regenerative Medicine, New York, United States Aims. Co-expression of the transcription factors Slug and Sox9 was recently used to determine mammary stem cell state. Our intention was to study the expression of Slug and Sox9 in primary breast cancer and to correlate it with clinicopathological parameters including overall survival. Methods. Formalin-fixed paraffin embedded tumor tissue of 306  patients with primary breast cancer [pT1 132 (43.1%), pT2 134 (43.8%), pT3 21 (6.9%), pT4 19 (6.2%); pN0 92 (34.1%), pN1 136 (50.4%), pN2 22 (8.1%), pN3 20 (7.4%); G1 41 (13.4%), G2 144 (47.1%), G3 121 (39.5%)] were assembled on a tissue microarray (TMA). Mean follow-up was 40 months (range 4–324 months). Immunohistochemical Slug and Sox9 expression was semi-quantitatively evaluated. The median value was used as cut-off point for dichotomization into “low Slug”/”high Slug” and “low Sox9”/”high Sox9” expressing groups. Results. Slug and Sox9 expression was identified in the tumor cell nuclei. High Slug expression was found in 41% of the cases (126/306) and high Sox9 expression in 74% (226/306). High expression of both Slug and Sox9 was found in 92/306 (30.1%). Co-expression of Slug and Sox9 significantly correlated with higher tumor grade (p<0.001) and a shortened overall survival (p=0.001). Conclusions. Co-expression of Slug and Sox9 identifies a more aggressive phenotype in breast cancer which might be due to their role in promoting (cancer) stem cell properties. Der Pathologe Suppl 1 · 2012 

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Abstracts SG-P-132 SiRNA inhibition of Shp-2 reduces the GC phenotype of Burkitt’s lymphoma cell lines X . Jiang1, R . Zhou1, L . He1, C . He1, J . Wang1 Zhejiang University, School of Medicine, Hangzhou, China

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Aims. To investigate the potential role of Shp2 in the GC phenotype regulation of BL cell lines. Methods. We used either scrambled siRNA or Shp2 siRNA pools to transfect human BL cell lines (Daudi, Raji, Ramos). We also expressed a PTP-inactive Shp2 mutant (Shp2CS) that had the catalytic Cys-459 replaced with Ser in Raji cells to verify whether the Shp2 PTP activity is required in BL cells growth. And we analyzed the effects of Src inhibitor-1 (Sigma) and Mek inhibitors (U0126) in BL cells. Results. Shp2 had been effectively silenced by the Shp-2 siRNA. Shp2 knockdown led to a decrease in Erk1/2 phosphorylation. Concomitantly, we observed a decrease in the phosphorylation of the tyrosine residue Y416 in the activation loop of Src, whereas Stat3 and Akt phosphorylations were not affected. Similar to Shp2 knockdown in BL cell lines, the C/S protein in Raji cells inhibited ERK1/2 and Src phosphorylation. The abrogation of Shp2 in both cell lines significantly impaired cellular proliferation over time compared with cells grown to the control cell lines. Cell cycle analysis revealed that Shp2 knock down led to a significant increase in the percentage of BL cells in the G1 phase and a corresponding decrease in the S and G2-M phases, without changes in the apoptotic fraction as indicated by the sub-G0-G1 populations. Thus knockdown of Shp2 significantly inhibited BL tumor growth. We also found that shp2 inhibition led to a decrease in CD77 positivity in BL cell lines. Concomitantly, the protein levels of Bcl-6, E2A, AID and Pax-5 were substantially lower in Shp2-siRNA BL cells than in their Shp2-positive counterparts. Biochemical analysis also showed that Shp2 knockdown resulted in down-regulation of c-Myc in BL cells. And Src inhibitor-1 and U0126 caused decreases of c-Myc and GC factors as CD77, Bcl-6, AID, E2A and Pax-5 expression level in BL cells. These results indicate that Src and Erk1/2 activities are necessary for a constitutive GC phenotype in BL cells. Conclusions. In this study, we showed that Shp2 regulated BL cells proliferation in cell culture. Reduction of Shp2 expression led to a decrease of c-Myc and GC factors (Bcl-6, E2A, AID and Pax-5) in BL cell lines through Src and Erk1/2 pathway, which suggested Shp2 play a key role in the series of regulative factors of the GC phenotype in BL cells. Keywords. Shp2, Burkitt’s lymphoma, GC phenotype Grants. the NSF of Zhejiang Province (No.Y2090167) and Research Foundation in Zhejiang Scientific and Technical Bureau (No.2009C33039).

SG-P-133 Identification of lymphoma subtype independent micro RNA expression profiles D . Lenze1*, M .R . Schweiger2*, M . Piechotta3, K . Zimmermann4, A . Fischer2, S . Boerno2, C . Dieterich3, U . Leser4, M . Hummel1 1 Charité – Universitätsmedizin Berlin, Institute of Pathology, Berlin, 2Max Planck Institute for Molecular Genetics, Berlin, 3Max-Delbrueck-Center for Molecular Medicine, Institute for Medical Systems Biology, Berlin, 4Humboldt-University, Institute for Informatics, Berlin Aims. The molecular analyses of human cancers revealed that great heterogeneity on the molecular level exists even within the same tumor entity which might explain different therapeutic outcome to the same type of treatment. This holds also true for lymphoma. Therefore it is justified to assume that lymphomas carrying the same molecular features might benefit from the same targeted treatment modalities irrespective of their histological or immunophenotypical features. It was the goal of our study to identify overlapping molecular characteristics in a variety of histologically defined lymphoma entities. To achieve this goal, comprehensive expression profiles of non-coding and coding transcripts de-

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rived from a broad spectrum of lymphomas were established. Our data show that overlapping molecular features can be identified beyond the current lymphoma classification. Methods. RNA was extracted from frozen tissue blocks of distinct human B- and T-cell lymphoma entities as well as tonsils (n=116). The small (micro) RNA fraction was sequenced by next generation technology (SOLiD) and total RNA was subjected to Affymetrix Exon 1.0 ST array hybridisation. In addition immunohistochemical and clinical data was acquired. Bioinformatic analyses were then applied for subgroup detection. Results. Unsupervised clustering based on the mature micro RNA expression derived from deep sequencing demonstrated the presence of two distinct large clusters within the case collection. One cluster contained predominantly the so-called indolent lymphoma types, but also a considerable number of aggressive B-cell lymphoma cases. In the second cluster the majority of cases were aggressive B-cell lymphoma but interestingly intermingled with the T-cell lymphoma cases. Differentially expressed micro RNAs between the two clusters were determined and logistic regression identified a micro RNA classificator able to distinguish the two groups. The micro RNA expression data was combined with the coding RNA data in order to identify the involved pathways. Conclusions. Micro RNA expression profiles obtained by deep sequencing were able to identify two groups within a lymphoma collection that separated the cases beyond the histopathological classification system. Discriminative micro RNAs and associated pathways were identified. These findings give new insights into lymphoma biology and point to alternative treatment options. *These authors contributed equally to the study .

Poster: GI-Trakt: Ösophagus, Magen FR-P-036 Acanthosis nigricans: a paraneoplastic condition of the esophagus D . Karimi1, J . Siveke2, M . Ringelhahn2, M . Bettstetter3, M . Sarbia1 Pathology München-Nord, 2Department of Gastroenterology Technical University München, 3Institute of Molecular Pathology Südbayern

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Aims. Presentation of endoskopic and histologic findings of esophageal acanthosis nigricans. Methods. The case of a 69-year-old man with dysphagia and weight loss will be presented. Results. Endoscopic examination revealed an adenocarcinoma of the esophagogastric junction as well as multiple small polyps in the middle and the lower thirds of the esophagus. Histological examination showed papilloma-like proliferations without atypia, which were diagnosed as acanthosis nigricans of the esophagus. After completion of the staging investigation regarding the cardiac carcinoma, combination chemotherapy was started because of the presence of liver metastases. Subsequently, partial regression of the carcinoma as well as of the esophageal lesions was noted. Conclusions. Acanthosis nigricans is a rare paraneoplastic disease of the esophagus. As an indicator lesion, its detection should prompt a search for a malignant tumor in the gastrointestinal tract.

FR-P-037 MALDI imaging reveals COX7A2, TAGLN2 and S100-A10 as novel prognostic markers in Barrett’s Cancer M. Elsner1, S. Rauser1, S. Maier1, C. Schöne1, B. Balluff1, S. Meding1, G. Jung1, M. Nipp1, H. Sarioglu1, G. Maccarone2, U. Jütting1, A. Feuchtinger1, R. Langer3, M. Feith3, B. Küster4, M. Ueffing1, H. Zitzelsberger1, H. Höfler3, A. Walch1 1 Helmholtz-Zentrum München, Neuherberg, 2Max Planck Institute of Psychiatry, München, 3Technische Universität München, Neuherberg, 4Technische Universität München, Chair of Proteomics and Bioanalytics, München Aims. In order to characterize proteomic changes found in Barrett’s cancer and its premalignant stages, the proteomic profiles of histologically proposed precursor and invasive carcinoma lesions were analyzed by MALDI imaging mass spectrometry (MALDI imaging). Methods. For a primary proteomic screening, a discovery cohort of 38 fresh frozen Barrett’s Cancer samples was used, with the goal to find intestinal metaplasia and Barrett’s cancer specific proteins that might be used as markers for cancer development as well as for lymph node metastasis and disease outcome. Based on this cohort we studied 33 areas of Barrett’s Cancer and 11 areas of intestinal metaplasia. Protein identification was done by mass spectrometry. To validate the identified proteins, immunohistochemistry was performed on an independent validation set of 102 Barrett’s Cancer cases and the results were correlated to clinical data. Results. We detected 60 m/z species that are significantly differentially expressed in intestinal metaplasia and invasive carcinoma (p<0.05). Two of them were identified as COX7A2 and S100-A10. Furthermore, among 22 m/z species that are significantly differentially expressed in Barrett’s Cancer cases with and without lymph node metastasis, one was identified as TAGLN2. Similarly in the validation set, we found a correlation of the expression levels of COX7A2 (p=0.008) and TAGLN2 (p=0.022) with a poor prognosis while S100-A10 could be confirmed by multivariate analysis as so far unknown independent prognostic factor in Barrett’s Cancer (p=0.003). Conclusions. Our results underscore the high potential of MALDI imaging for revealing new biologically significant molecular details from cancer tissues which might have potential for clinical application. Insitu proteome analysis by MALDI imaging uncovered TAGLN2 and COX7A2 as novel prognostic and S100-A10 as independent prognostic marker for Barrett’s Cancer.

FR-P-038 Apoptosis as a useful grading-tool in lymphocytic oesophagitis L. Veits1, T. Schulz1, M. Vieth1 1 Klinikum Bayreuth, Institute of Pathology, Bayreuth Aims. Lymphocytic oesophagitis is a poorly understood disease of unknown origin of the esophageal mucosa, said to be characterized by an infiltration of lymphocytes within the squamous epithelium. An increased number of intraepithelial lymphocytes can also be found in gastrooesophageal reflux disease and Crohn’s disease. Furthermore lymphocytic inflammation can be seen in oesophageal motility disorders, e.g. achalasia, and moniliasis. In contrast to other lymphocyte-rich inflammatory lesions of squamous epithelium (e.g. allergic contact dermatitis) lymphocytic oesophagitis shows only few and non-specific histological changes. This study was performed to investigate, if there are more histological findings in lymphocytic oesophagitis. Methods. We collected 28 biopsies of 21 patients with lymphocytic oesophagitis, which were diagnosed between 2010 and 2011. The group consisted of 14 men and 7 women, the mean age was 56,8 years (23 – 79) and 55,9 years (11 – 79), respectively. Intraepithelial lymphocytes were counted per high power fields and classified as mild and severe (according to Purdy et al.).

Results. Beside the hitherto described histological changes, apoptotic keratinocytes could be detected in 12 out of 18 cases, which were graded as severe, (66,6%), whereas in contrast apoptosis was present in only in 4 out of 10 cases, which showed a mild lymphocytic infiltrate (40%). Classified by intraepithelial lymphocytes per high power field (IEL/HPF) in severe (>50 IEL/HPF) and mild (<50 IEL/HPF) inflammation, apoptotic cells were seen in 12 out of 16 severe cases (75%) and 4 out of 12 mild cases (33%). In all cases the apoptotic bodies were found in all epithelial layers. Conclusions. Apoptosis has not been described in lymphocytic oesophagitis so far. Our data show that apoptosis is useful in grading of lymphocytic oesophagitis. Furthermore the presence of apoptosis in all epithelial layers distinguishes it from oesophageal lichen planus, where apoptosis is strictly limited to the basal epithelial layers. Further studies should focus on the question, whether lymphocytic infiltration and apoptotic keratocytes are a hint to an autoimmunologic mechanism or not.

FR-P-039 Subsquamous Extension of Barrett Epithelium Is a Regular Event A.H. Marx1, Y. Luchs1, M.A. El-Masry1, M. Anders1, G. Schachschal1, D. von Reteln1, U. Denzer1, U. Gauger1, M. Mirlacher2, J.R. Izbicki3, G. Sauter1, T. Rösch1 1 University Medical Center Hamburg-Eppendorf, Institute of Pathology, Hamburg, 2University Medical Center Hamburg-Eppendorf, Pathology, Hamburg, 3University Medical Center Hamburg-Eppendorf, Department of Surgery, Hamburg Aims. Endoscopic resection and/or ablation of Barrett esophagus (BE) is directed by the endoscopic visual impression of pinkish Barrett versus whitish esophageal mucosa; microscopically however, BE may extend underneath squamous epithelium. Aim of the present study is to evaluate subsquamous BE extension in a large cohort of patients with endoscopically resected neoplastic BE. Methods. Resection specimens of 97 patients treated by endoscopic mucosal resection (EMR) for suspected or confirmed neoplastic BE (83 men, 14 women, mean age 65 years) were retrospective reanalyzed by two experienced GI histopathologists. Orientation of resected specimens pinned on cork was done prior to histopathological workup with squamous epithelium oriented proximally. Subsquamous extension was measured in all specimens containing squamous epithelium. Endoscopic and other histopathologic data were also analyzed. Results. 442 specimens of the 97 study cases were analyzed and subsquamous BE was found in 42.5% of specimens or 97.9% of patients. Mean length of subsquamous BE extension was 3.5 mm (0.0–9.6 mm) with 27.8% of cases measuring 5 mm or more. In 56 cases (57.7%), subsquamous BE consisted entirely or partially of neoplasia (42 cancers, 12 HGIN, 2 LGIN) with a mean subsquamous tumor extension of 1.2 mm (range 0.0–5.2 mm). Conclusions. Subsquamous extension of BE including all forms of neoplasia is a regular event. A minimal safety margin of 1 cm has to be kept with Barrett ablation techniques in order to avoid BE and/or tumor recurrence.

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Abstracts FR-P-040 MicroRNA expression profiling for prediction of resistance to neoadjuvant radiochemotherapy in squamous cell carcinoma of the oesophagus J. Slotta-Huspenina1, E. Drecoll1, M. Feith2, C. Wagner3, H. Höfler1,4, K. Becker1, R. Langer1 1 Technical University of Munich, Institute of Pathology, München, 2Technical University of Munich, Department of Surgery, 3IMGM Laboratories GmbH, Martiensried, 4Institute of Pathology, Helmholtz-Zentrum München, Oberschleissheim Aims. MicroRNA (miRNA) expression has been shown to play an important role in biology of malignant tumours, including sensitivity to chemotherapy and radiation. Neoadjuvant radiochemotherapy (RCTX) followed by surgery is a standard treatment strategy for locally advanced oesophageal squamous cell carcinoma (ESCC). However, a subset of patients does not respond to RCTX. In the present study we evaluated whether miRNA profiles can predict resistance to RCTX in ESCC. Methods. 31  patients with locally advanced ESCC (cT3-4, cN1-3, M0-1) underwent preoperative radiochemotherapy with cisplatin, 5-fluorouracil and 30-45 Gy, followed by resection of the oesophagus. Tumour response was evaluated by histopathological tumour regression. MiRNA profiling was done in pre-therapeutic formalin-fixed and paraffin embedded (FFPE) biopsies using the Agilent Human Microarray platform (Release 16.0), encompassing 1205 human miRNAs. Differential expression was identified in responders (n=15) and non-responders (n=16) by applying appropriate biostatistics to the data and validated by real-time quantitative PCR (qRT-PCR). Results. Even if the miRNA expression profiles of pre-therapeutic ESCC biopsies within and between non-responders (n=16) and responders (n=15) were highly similar (average correlation coefficients r=0.96, 0.94 and 0.95), ten differentially expressed miRNAs could be identified by microarray analysis in non-responders and responders of ESCC (p<0.025). In particular, non-responders showed an upregulation of six miRNAs (miR-1323, miR-3678-3p, miR-194*, miR-3152, miR-665, miR-3659) from 22 to at least 2-fold and a downregulation of four miRNAs (miR-126*, miR-484, miR-330-3p, miR-3653) from 10 to at least 2-fold. Conclusions. This indicates that miRNAs are involved in therapy response and suggests that miRNA profiles could be used to predict response to RCTX with the aim of better adapting treatment to tumour biological specificities and also to identify new potential therapeutic strategies. Validation of the discriminating miRNA profiles by qRT-PCR in the 31 samples of the training collective and an independent validation collective is currently in progress.

FR-P-041 Analysis of EGFR, HER2 and HER3 expression, colocalization and dimerization in esophageal carcinomas and cell lines* C.D. Fichter1, S. Timme1, A. Schoepflin1, L. Bogatyreva2, D. Hauschke2, L. Tang3, D. Klimstra3, H. Geddert4, G. Faller4, O. Opitz5, M. Werner1, S. Lassmann1 1 University Medical Center Freiburg, Institute of Pathology, Freiburg, 2University Medical Center Freiburg, Institute of Medical Biometry and Medical Informatics, Freiburg, 3Memorial Sloan Kettering Cancer Center, Dept. Pathology, New York, United States, 4St. Vincentius-Kliniken, Institute of Pathology, Karlsruhe, 5University Medical Center Freiburg, Tumorzentrum Ludwig Heilmeyer – CCCF, Freiburg Aims. The receptor tyrosine kinases EGFR, HER2 and HER3 are in the focus of targeted therapy for epithelial tumors. Differential receptor interaction may affect cellular biology and responses to EGFR or HER2 targeted therapy. However, little is known about co-expression and dimerization of EGFR, HER2 and HER3 in the two main histotypes of esophageal cancer – esophageal squamous cell carcinoma (ESCC) and

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Barrett’s adenocarcinoma (BAC). Therefore, we here examined EGFR, HER2 and HER3 protein expression and dimerization in esophageal cancer tissues and cell lines. Methods. Serial sections of 110 esophageal carcinomas were examined for EGFR, HER2 and HER3 expression by immunohistochemistry. Thereof, selected BACs (n=9) were used for double immunofluorescence (dIF; colocalization) and in situ proximity ligation assay (PLA; dimerization) of EGFR/HER2 or HER2/HER3. In normal esophageal epithelium (EPC-hTERT), ESCC (OE21) and BAC (OE33) cell lines expression, colocalization and dimerization of EGFR/HER2 and HER2/HER3 was similarly examined. Results. Normal esophageal epithelium had marginal EGFR, HER2 or HER3 expression. In ESCCs and BACs, EGFR appeared more frequent in ESCCs (11/49, 22.4%) than BACs (6/61, 9.9%; p=0.088). However, HER2 and HER3 protein expression were significantly more frequent in BACs than in ESCCs (both p<0.0001). This was accompanied by HER2/HER3 colocalization and dimerization in single BACs. Similar, in vitro EPChTERT cells had marginal EGFR, HER2 and HER3 expression. In contrast, OE21 cells predominantly expressed EGFR, whereas OE33 cells expressed EGFR, HER2 and HER3. Correspondingly, EGFR/HER2 and HER2/HER3 heterodimers were more frequent in OE33 than in OE21 cells and were negligible in EPC-hTERT cells. Conclusions. Our data show that some BACs with preferential overexpression of HER2 show colocalization and dimerization of HER2/HER3. In ESCCs and BACs overexpressing EGFR or HER2 (with or without HER3 expression), it is most likely that EGFR or HER2 form homodimers. Inhibition of EGFR (ESCCs) and/or HER2 (BACs) may represent a therapeutic option. However, further detailed investigation of EGFR, HER2 and/or HER3 signaling events is required, since distinct dimerization may affect signaling events and therapeutic efficiency in ESCCs and BACs. *Study supported by Deutsche Forschungsgemeinschaft DFG CRC850 project C5, and in part Mushett Family Foundation, Chester, NJ, US.

FR-P-042 MALDI Imaging identifies mitochondrial proteins as markers for the prediction of response to neoadjuvant chemotherapy in Barrett’s cancer M. Elsner1, S. Rauser1, M. Aichler1, N. Ludyga1, S. Maier1, C. Schöne1, B. Balluff1, S. Meding1, H. Sarioglu1, A. Feuchtinger1, R. Langer2, M. Feith2, B. Küster3, M. Ueffing1, H. Höfler2, A. Walch1 1 Helmholtz-Zentrum München, Neuherberg, 2Technische Universität München, Neuherberg, 3Technische Universität München, Chair of Proteomics and Bioanalytics, München Aims. In advanced stages of esophageal adenocarcinoma (Barrett’s cancer), neoadjuvant chemotherapy is the most common treatment strategy, but it improves survival only in a subgroup of patients. So far, there are no reliable biomarkers available that predict therapy response before treatment. To identify predictive proteomic markers, MALDI Imaging was performed. Methods. To work out a proteomic signature of response to neoadjuvant chemotherapy, pretherapeutic Barrett’s cancer biopsies (n=23) were analyzed by MALDI Imaging and proteomic profiles were correlated with response data of the patients. Candidate species discriminating between responders and non-responders were identified by LC-MS/MS. Predictive impact of the proteins was validated by immunohistochemistry on an independent set of pretherapeutic, endoscopic biopsies (n=46). Results. MALDI Imaging revealed 22 m/z species correlating with therapy response in Barrett’s Cancer (p<0.05). Hierarchical clustering showed that MALDI Imaging profiles can be used to accurately define responders to neoadjuvant therapy from non-responders. By LC-MS/ MS, six proteins were identified from which four are mitochondrial proteins (COX7A2, COX6B1, COX6C, Complex I-MLRQ). These proteins show a reduced expression in responders and haven’t been recognized

in the context of therapy response before. Immunohistochemistry of the mitochondrial protein COX7A2 in the validation set confirmed the MALDI Imaging results and revealed the predictive impact of COX7A2 (p=0.0015). By electron microscopy we found, that the loss of these proteins is strongly associated with a severe mitochondrial damage. Conclusions. MALDI Imaging is able to detect novel proteomic patterns distinguishing responders from non-responders. These protein patterns provide new insights in mechanisms of response. For the first time we could show that mitochondrial dysfunction is a predisposition for response to neoadjuvant chemotherapy in Barrett’s cancer.

FR-P-043 Heterogeneity of TP53 mutations in gastric adenocarcinomas as well as their corresponding lymph node metastases D. Mones1, G. Cadeddu1, K.-L. Schäfer1, H.E. Gabbert1, S.E. Baldus1 University of Düsseldorf, Institute of Pathology, Düsseldorf

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Aims. The frequency of TP53 mutations in gastric adenocarcinomas as well as the prognostic and predictive value of this biomarker is still controversially discussed. In order to elucidate if genetic mosaicism may explain at least in part the conflicting results obtained in previous studies, we investigated the intratumoral heterogeneity of TP53 mutations analysing tissue specimens from tumor center, invasion front and lymphonodal metastases. Methods. We studied a series of 75 gastric adenocarcinomas comprising 25 diffuse type, 24 intestinal type and 26 mixed type carcinomas according to Laurén. DNA of the paraffin-embedded tissue samples from tumor center and invasion front (n=75) as well as corresponding lymph node metastases (n=47) was obtained by microscopically controlled manual microdissection. DNA was purified and subjected to PCR amplification of TP53 exon 5–8 followed by direct sequencing. Results. TP53 mutations were observed in 34 of the 75 primary tumors (45%). Intratumoral heterogeneity of TP53 mutations was found in 13 primary tumors (17%): Two samples showed the mutation only in the invasion front, ten cases only in the tumor center and one case showed different mutations in invasion front and tumor center. There was no significant difference between the mutation rates in diffuse type (40%) compared to intestinal type adenocarcinomas (50%), while mixed type carcinomas showed a mutation in 46% of the cases. In addition, there was no difference between pT1/pT2 tumors (46%) and pT3/pT4 tumors (45%). Mutations in the lymph node metastases were found in 19 of 47 specimens (40%). Genetic heterogeneity between primary tumor and lymph node metastases was observed in 12 of the 47 cases (26%) comprising six cases showing the TP53 mutation only in the primary tumor, but not in the lymph node metastasis as well as four cases exhibiting a mutation in the lymph node metastasis, but not in the primary tumor. In two cases different mutations in primary tumor and corresponding lymph node metastasis were observed. Conclusions. Intratumoral heterogeneity of TP53 mutations is present in 17% of gastric adenocarcinomas and may therefore contribute to the controversial results regarding the prognostic value of the TP53 status. In addition, in 26% of the cases heterogeneity between TP53 mutations in primary tumors and lymph node metastases was observed. This should be considered in future studies on the predictive and prognostic value of TP53 mutation analysis in gastric adenocarcinomas.

FR-P-044 Gastric Polyvinylpyrrolidon (PVP) Athrozytosis in chronic hemodialysed patients S.A. Thies1, B. Kaduk2, R. Ott3, S. Turi4, M. Sarbia5 University of Zurich, Institute of Surgical Pathology, Zürich, Switzerland, 2 Kaduk Medical Service (KMS) GmbH, Baar, Switzerland, 3Gastroenterologie Bogenhausen, Gemeinschaftspraxis Dr. Schatke, Munich, 4Medizentrum Erlangen, Gemeinschaftspraxis Internist/Gastroenterologie, Erlangen, 5 Pathologie München Nord, Munich

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Aims. The originally as plasma expander developed and applied high molecular Polyvinylpyrrolidone (PVP) – a polymer of the monomer N-Vinylpyrrolidon cannot be eliminated by diuresis nor metabolised by liver. These PVP polymers were phagocytosed by macrophages and permanently arrested. According to the type and topos of application of PVP – intradermal, subcutaneous, intracavitary, intraperitoneal, interpleural or intravenous – the phenotype of this storage disease differs and is predictive. The only non-predictive manifestation and severity of the accumulation and conditio sine qua non is due to the entrance of the high molecular PVP polymers into the blood circulation. This induces a high risk situation for the patients. For that reason high molecular PVP is not yet used as plasma expander. But nevertheless there is an ubiquitary (generalised) PVP-thesaurismosis, e.g. using PVP-coating fibre filters in human hemodialysis. Methods. We examined gastric biopsies of two patients with chronic hemodialysis in history without defined clinical question. The main histological topic was aggregates of isolated or grouped large macrophages with a certain but non-characteristic staining pattern (indirect indicators for PVP; i.e. advice): – H&E: intracytoplasmic blue-gray globules – Sirius red: brightly red deposits on a pale yellow background – Argentic impregnation: markedly coloured dark black deposits – PAS: negative – v. Kossa: negative – Immunocytochemically: CD68+++ Results. The definitive morphological typing of the macrophages as high molecular PVP-containing macrophages could only approved by the investigation of the ultrastructure in transmission electron microscopy (direct indicator for PVP; i.e. evidence): intracytoplasmic globules of different size and periodic lamellate internal structure. Conclusions. The results of this study are essential for the manufacture of dialyse membranes by advancement the adhesion of PVP and for the patients to prohibit the generalized PVP-thesaurismosis. Also the findings are helpful for the clinicians, notably for nephrologists in the surveillance of chronic hemodialysed patients, for gastroenterologists and for pathologists to receive an algorithm of the diagnostic relevant panel.

FR-P-045 Interaction of cathepsin X with galectin-2 in H. pylori dependent gastric carcinogenesis A. Teller1, D. Kuester1, A. Roessner1, S. Krueger1 Otto-v.-Guericke University, Department of Pathology, Magdeburg

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Aims. Our previous studies have shown an association between Helico­ bacter pylori infection, the strong up-regulation of cathepsin X (CTSX), and the development of gastric cancer. The physiological function of CTSX still needs to be clarified. In a yeast two-hybrid screen we could identify galectin-2 as a novel interaction partner of cathepsin X. Now we suppose an interactive role of CTSX and galectin-2 in modulation of the immune system in response to H. pylori-infected gastric epithelial cells. Methods. Background for further experiments was the screening of the yeast two-hybrid system, where we could identify galectin-2 as a novel interaction partner of CTSX. Using Western blots and immunohistochemistry we want to analyze galectin-2 levels in biopsy specimens of H. pylori-infected and non-infected patients as well as in gastric cancer Der Pathologe Suppl 1 · 2012 

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Abstracts samples which showed increased CTSX levels in disease progression. Functional consequences of the interaction of CTSX and galectin-2 were tested on siRNA treated primary human epithelial and immune cells in confrontation and migration experiments with and without infection with H. pylori. Results. Interaction of CTSX and galectin-2 was clearly characterized by immunoprecipitation, double immunofluorescence and pull-down assays. Immunohistochemistry and western blots on tissue samples indicated an inversely expression of CTSX and galectin-2. H. pylori-negative samples showed low expression of CTSX but high expression of galectin-2, whereas galectin-2 expression decreased and CTSX increased with progression of disease. Macrophages with high expression of CTSX rapidly migrate into epithelial cell monolayers and down regulate whereby their galectin-2 levels. Conclusions. The interaction of CTSX and galectin-2 seems to be a major regulatory element to regulate the activity of the immune system against H. pylori colonization and cancer development. As both enzymes known to be effecting T-cell function and spreading our further experiments will focus on possible mechanisms to induce an efficient anti-tumoral immune response by influencing CTSX/galectin-2 signalling.

FR-P-046 The impact of HER2 amplification in the dysplasia-carcinoma-­ sequence in the stomach T. Vlajnic1, S. Eppenberger1, S. Schneider1, L. Terracciano1, L. Tornillo1, G. Cathomas2 1 Institute of Pathology, University Hospital Basel, Switzerland, 2Cantonal Institute of Pathology, Liestal, Switzerland Aims. HER2 amplification and overexpression was demonstrated in gastric carcinoma soon after its discovery in breast carcinoma. There is growing evidence that HER2 has an important role in tumorigenesis in gastric cancer with a reported prevalence of amplification/overexpression in 7–34%. However, the role of HER2 in the progression of dysplasia to gastric carcinoma has not yet been investigated. The aim of this study was to determine the HER2 status in precursor lesions of gastric carcinoma, i.e. in gastric dysplasia and early cancer. Methods. A tissue microarray consisting of gastric carcinomas (n=370) was evaluated immunohistochemically and by FISH and SISH analysis for the HER2 status. Whole tissue sections with gastric cancer (n=206) were then re-evaluated for gastric dysplasia and in case of presence of dysplasia next to carcinoma an immunohistochemical analysis was performed. Additionally, immunohistochemistry and SISH analysis was performed on gastric biopsies with dysplasia (n=62) without coexisting carcinoma. Results. HER2 amplification was found in 7.9% of gastric carcinomas. 50 cases showed gastric dysplasia next to carcinoma and the HER2 status in the dysplasia was the same as in the respective invasive carcinoma. However, the prevalence of HER2 amplification in the cases of dysplasia alone was only 3.2%. Conclusions. Interestingly, our data indicate that HER2 amplification/ overexpression may be an early event and may induce a rapid progression from dysplasia to invasive carcinoma in the stomach. Further studies are needed to elucidate the potential role for the anti-HER2 targeted therapy in patients with gastric dysplasia.

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FR-P-047 Epstein-Barr virus (EBV) in the development of gastric cancer M. Cathomas1, V. Genitsch1, L.M. Terracciano2, L. Tornillo2, A. Lugli2, A.H. Marx3, G. Sauter3, F. Carneiro4, F. Hofstädter5, N. Willi1, G. Cathomas1 1 Institute of Pathology, Liestal, Switzerland, 2Institute of Pathology, University Basel, Switzerland, 3Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, 4Institute of Molecular Pathology of the University of Porto (IPATIMUP), Portugal, Portugal, 5Institute of Pathology, University Regensburg, Regensburg Aims. Epstein-Barr virus (EBV) infections are associated with a number of tumours, including lymphoproliferative diseases and nasopharyngeal carcinomas. In addition, a subset of gastric tumours has been associated with EBV, ranging from 1.3% to 20.2% of all gastric cancers. The role of EBV in the development and progression of gastric cancer, however, remains to be elucidated. Aim of the study was the assessment of the prevalence of EBV associated gastric cancers and the presence of the virus in the development of individual tumours. Methods. Based on tissue micro arrays (TMA), the presence of EBV was evaluated in gastric cancers of 5 institutions within Europe (Liestal and Basel, Switzerland; Hamburg and Regensburg; Porto, Portugal) by using a commercial in situ hybridization assay for EBER. In a second step, nontumorous and tumorous tissue including dysplasia, intramucosal and invasive carcinomas as well as metastasis were analyzed for the presence of EBV. Results. A total of 610 (91.6%) of 666 tumours on TMAs were available for analysis. Overall, 4.9% of gastric cancers were positive for EBER, ranging form 2.2% to 9.1% within the different institutions. No age difference was observed within EBV positive and negative patients [68.2 vs. 66.2 (n=23/515)], but EBV positive tumours showed a male predominance [87.0% vs. 60.6%; p<0.02 (n=23/515)]. No difference was observed in the tumour stage of the two groups. Tissue of 22 EBV positive tumours was available for further studies. All invasive cancers showed diffuse EBER positivity and in 21 lymph nodes derived from 10 patients, only 2 nodes revealed a complete or focal loss of EBER. In contrast, 2 cancers with remote foci of adenomatous low grade dysplasia and in 11 tumours with epithelial atypia consistent with low grad dysplasia adjacent to invasive cancer, no or only focal EBER positivity was observed. Conclusions. EBV positive cancer is a small subgroup of gastric tumours with no obvious difference in tumour stage but with a male predominance. In invasive cancer, EBV is diffusely expressed in tumour cells and only occasionally gets lost in metastasis. In low grade dysplasia, however, EBV is often missing, indicating that EBV infection may be associated with progression from low grade dysplasia to invasive cancer.

FR-P-048 Effects of cathepsin X knockout on galectin-2 expression in a mouse model of gastric carcinogenesis M. Baldensperger1, D. Kuester1, A. Roessner1, S. Krueger1 Otto-von-Guericke-University Magdeburg, Department of Pathology, Magdeburg

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Aims. Using a yeast-two-hybrid approach we found out that cathepsin X, which plays a crucial role in migration processes of immune cells, interacts with galectin-2. Interestingly, galectins have also been described as novel regulators in inflammation. Recent studies showed that galectin-2-expression is decreased in colitis but there is nothing known about galectin-2 expression in gastritis and gastric carcinogenesis. Methods. To find out if there is a variation of galectin-2 expression in different stages of gastritis we performed immunohistochemical stainings on wild-type and cathepsin-X knockout mice with 24, 36 and 50 weeks post infection with H. pylori compared to uninfected controls. Quantitative real-time PCR was performed on frozen tissue samples to detect mRNA-levels of galectin-2. Western blot analysis was used to determine galectin-2 levels in corresponding protein lysates. In vitro experiments

on primary cells of wild-type and cathepsin X knockout epithelial cells with infection by H. pylori were performed to confirm the descriptive data on tissue samples. Results. Galectin-2 is constitutively expressed in gastric mucosa of wildtype and cathepsin-X knockout mice. Galectin-2 expression is decreased in gastric inflammation and spasmolytic polypeptide-expressing metaplasia (SPEM). H. pylori infection leads to lower mRNA- and protein-levels of galectin-2 in wild-type and cathepsin-X knockout mice. However, levels of galectin-2 were significantly higher in cathepsin-X knockout mice compared to wild-type mice independent of H.  pylori infection. Infection of primary cells revealed comparable results. Conclusions. Because galectin-2 expression is decreased in gastric inflammation and cathepsin-X is increased we propose an inverse regulation of these molecules in gastric inflammatory disease. Since there is an intense and smooth expression of galectin-2 in healthy gastric mucosa, treatment of patients with galectin-2 could help to prevent epithelial damage and thus decelerate gastric carcinogenesis.

FR-P-049 Krukenberg tumor with partial yolk sac differentiation or collision tumor? A case report A. Weber1, A. Schmieder1, K.-H. Berghaeuser1, T. Friedrich1 Institut of Pathology, Saalfeld

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Aims. A 28-year-old woman suffered from a diffuse infiltrating gastric carcinoma. After chemotherapy with ECF, subtotal gastrectomy with Roux-Y reconstruction was performed before. Methods. In microscopic examination of the tumor diffuse carcinoma without any other differentiation was diagnosed. Tumor regression was estimated to 40% at time of surgery staging was ypT2b ypN0 cm1 (PER). Despite of adjuvant chemotherapy six months later malignant ascites and peritoneal dissemination of the tumor with the same differentiation occurred. Two years later two metastases were removed from the peritoneum with the same histological appearance. At that time both enlarged ovaries were removed. Results. Histologically, in both ovaries metastases of the diffuse carcinoma were found. In addition in the left side was a definite area with large clear cells according to yolk sac differentiation. Immunhistologically this area with cells reacted positively with AFP and CK 7. Conclusions. Adenocarcinomas of stomach with yolk sac differentiation are extremely rare. It remains to be clarified if in this case there is one of this rare adenocarcinomas or a collision tumor combining metastases of signet ring cell carcinoma of the stomach with a primary yolk sac tumor of the ovary.

FR-P-050 FoxO6 expression in gastric cancer J. Haag1, B. Ingold-Heppner2, H.-M. Behrens1, T. Aigner3, C. Röcken1 1 Christian-Albrechts-University Kiel, Institute of Pathology, Kiel, 2Charité Campus Mitte, Institute of Pathology, Berlin, 3Klinikum Coburg, Institute of Pathology Aims. The expression of the transcription factor FoxO6 in gastric cancer and the correlation of FoxO6 expression levels to clinicopathological tumor characteristics were evaluated. Methods. Study population: 485 cases with cancer of the stomach or oesophagogastric junction were characterized for type of surgery, age at diagnosis, gender, tumor localization and tumor size, tumor type, tumor grade, depth of invasion, number of lymph nodes resected, and number of lymph nodes containing metastases. Tumors were classified according to the Laurén classification and the mucin phenotype. pTNM-stage of all study patients was determined according to the 7th edition of the UICC guidelines and our recent proposal (“Kiel-stage”). Mismatch DNA repair capacity and microsatellite instability was analyzed by immuno-

histochemistry and a pentaplex PCR assay. Analysis of FoxO6 expression: polyclonal rabbit antisera were generated against human FoxO6. A FoxO6 specific peptide (NH2-)CAQDAYGPRARAGTP(-CONH2) was synthesized, coupled to a carrier molecule and injected into two rabbit hosts. Antisera were obtained at day 84 of immunization and purified by peptide affinity chromatography using a FoxO6 specific peptide affinity column (Innovagen, Lund, Sweden). Tissue micro arrays (TMA) were prepared to study the expression of FoxO6 in the study population. FoxO6 immunostaining of the TMAs was evaluated by applying an immunoreactivity scoring system [0 (no immunostaining), 1 (weak), 2 (moderate), and 3 (strong)]. Statistics: Statistical analyses were done with SPSS 18.0. With respect to continuous variables, cases were divided into two groups by splitting at the median value. Significance of correlation between clinicopathological parameters and FoxO6 expression was tested using Fisher’s exact test. For parameters with ordinal scale (T, N, stage) we applied Kendall’s tau test instead. Generally, p-values less than 0.05 were considered statistically significant. Results. FoxO6 staining was found exclusively in the cytoplasm, no nuclear staining was detected. Expression levels were heterogeneous when different cases were compared. Statistically significant correlation to the FoxO6 expression levels were found for the Lauren phenotype, the Tcategory, the mucin phenotype and the microsatellite instability status. Conclusions. The correlation of FoxO6 expression levels with major clinicopathological tumor characteristics warrants further analysis of the biological role of FoxO6 in gastric cancer.

FR-P-051 Differential expression of LGR4, LGR6, GPR34, GPR160 and GPR171 in gastric carcinoma J.S. Steffen1, E. Simon1, K. Balschun1, C. Böger1, C. Röcken1 Christian-Albrechts-University, Institute of Pathology, Kiel

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Aims. Gastric cancer (GC) is one of the most common causes of cancerrelated deaths worldwide. Due to its poor prognosis, the identification of novel therapeutic targets is of high priority. G-protein-coupled receptors (GPCRs) are prime candidates for novel cancer prevention and treatment-strategies, since they play an important role in regulating physiological and pathophysiological processes. These receptors form the largest family of human membrane-bound proteins and represent a target for more than 40% of all drugs. We aimed to elucidate the differential expression and putative tumor biological significance of LGR4, LGR6, GPR34, GPR160 and GPR171, in GC. Methods. Based on our previous microarray analysis we identified five candidate genes in human gastric cancer samples. Real time RT-PCR was carried out to validate their expression in malignant and non-malignant tissue on an independent collective comprising 32 GC patients with and without lymph node metastases. Selected protein targets LGR4 and LGR6 were further validated on paraffin-embedded sections of 10 intestinal and 10 diffuse type gastric carcinomas and their corresponding non-malignant tissue using immunohistochemistry. Additionally, the putative tumour biological significance of LGR4 and LGR6 was studied using tissue micro arrays obtained from a cohort of 488 GCs. Results. GPR34, GPR160 and GPR171 did not show any differential expression in real-time RT-PCR analyses. LGR4 and LGR6 were markedly up-regulated on transcriptional (real-time RT-PCR) and translational (immunohistochemistry) level in GC. Furthermore, in tissue micro array analysis LGR6 expression was significantly associated with local tumor growth (T-category) and correlated with patient survival. LGR4 expression was significantly correlated with the lymph node metastases (N-category). Conclusions. LGR6 has recently been identified as stem-cell marker in the skin whereas LGR4 is of known tumor biological relevance in colon carcinoma and other malignancies. Our systematic analysis indicates that these two genes also play a role in gastric cancer biology. Future stu-

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Abstracts dies will have to demonstrate, whether these are also putative diagnostic, prognostic and/or therapeutic targets for GC.

FR-P-052 Differential expression and abnormal cytoplasmic distribution of E-cadherin and the desmosomal cadherin desmoglein 2 in gastric carcinomas: Prognostic relevance of desmoglein 2-plaques E.C. Scharbatke1, A. Gamboa-Dominguez2, F. Fend3, A. Brunner4, A. Schmidt1, R. Moll1 1 Philipps University of Marburg, Institute of Pathology, Marburg, 2Instituto Nacional de Sciencias Medicas y Nutricion Salvador Zubiran, Institute of Pathology, Mexico City, Mexico, 3University Hospital Tübingen, Institute of Pathology, Tübingen, 4University of Würzburg, Lehrstuhl für Volkswirtschaftslehre, insbesondere Wirtschaftsordnung und Sozialpolitik, Würzburg Aims. In gastric carcinomas, expression of E-cadherin has been shown to be important in pathogenetic and prognostic terms. We compared expression and subcellular distribution of E-cadherin and of another cellcell adhesion molecule of the cadherin family, the desmosomal cadherin desmoglein 2 (Dsg2), in a series of gastric adenocarcinomas of Mexican patients. Methods. Specimens of 63  cases of sporadic gastric adenocarcinomas (26 of intestinal type, 37 of diffuse type; three cases showing CHD1 mutations) were analyzed immunohistochemically for E-cadherin and Dsg2, using monoclonal antibodies AEC (clone 36; BD Transduction Laboratories) and G129 (Progen, Heidelberg), respectively, on paraffin sections after heat-induced antigen retrieval. Expression and subcellular distribution (membrane staining, intracytoplasmic plaques ≤5 µm and >5 µM) of these proteins were statistically correlated with clinicopathological parameters and patient survival. Results. 53 cases (84%) were positive for E-cadherin [19 cases (30%) with plaques] and 62 cases (98%) were positive for Dsg2 [54 cases (84%) with plaques]. Large intracytoplasmic plaques (>5 µM) were found for E-cadherin in 5 cases (8%) and for Dsg2 in 9 cases (14%; three of intestinal and six of diffuse type). The presence of E-cadherin or Dsg2 plaques of any size and of large E-cadherin plaques did not show prognostic relevance. However, the 9 patients with large Dsg2 plaques exhibited significantly shorter survival (p=0.020) in multivariate regression analysis. This correlation was independent from other parameters such as tumor stage. Conclusions. Both cadherins studied are expressed differentially in gastric carcinomas. Abnormal subcellular distribution of Dsg2 in the form of large intracytoplasmatic plaques appears to be an independent predictor of poor prognosis in gastric carcinomas. Prospective studies on larger cohorts are required to confirm these results.

FR-P-053 Unexpected role of caspases in the survival of human colon epithelial cells upon oxidative stress A. Just1, K. Reissig1, R. Hartig2, P. Steinberg3, T. Guenther1, A. Roessner1, A. Poehlmann1 1 Otto-von-Guericke University Magdeburg, Department of Pathology, Magdeburg, 2Otto-von-Guericke University Magdeburg, Department of Molecular and Clinical Immunology, Magdeburg, 3Institute of Food Toxicology and Analytical Chemistry, Hannover Aims. Current evidence suggests an increasing role for inflammation-associated oxidative stress in colorectal cancer initiation. We found that human colon epithelial cells (HCEC) survive oxidative stress through cell cycle arrest. Moreover, caspases were found not to be involved in apoptosis but in mediating the survival of HCEC. Thus, we aimed to unravel their unexpected role. Methods. We simulated acute inflammation by treating HCEC with a pathophysiologic concentration of H2O2. We performed inhibition stu-

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dies using a pan-caspase inhibitor. The cells were further analyzed by immunoblotting and FACS. Results. H2O2 induces DNA damage in HCEC. As a consequence, HCEC underwent apoptosis or cell cycle arrest to repair DNA damage. In addition, a proportion of cells may progress through the cell cycle irrespective of DNA damage (cellular survival). Surprisingly, caspase 3, 8 and 9 expression was found to be up-regulated, but did not precede cells into apoptosis. To unravel the role of caspases in cell survival, we performed inhibition studies using a pan-caspase inhibitor. Immunoblotting showed (i) p21 and (ii) y-H2AX up-regulation following caspase inhibition. Cell cycle analysis revealed the accumulation of cells in the G1 phase of the cell cycle as the first response and increased apoptosis following caspase inhibition as the second response. Because of these findings, we suggest caspase-mediated inactivation of the tumor suppressor p21 and the DNA damage sensor y-H2AX. Among others, this led us to suggest that caspases rather play a role in survival than in apoptosis. Conclusions. HCEC provide a model to analyze the molecular relationship between inflammation-associated oxidative stress and colorectal cancer initiation by mimicking the in vivo conditions in vitro. We suppose that caspases promote cell survival upon acute colonic inflammation through inactivation of p21 and y-H2AX. We presume (i) increased proliferation due to p21 down-regulation and (ii) accumulation of DNA damage because of y-H2AX down-regulation. Consequently, caspases seem to mediate the progression of cells through the cell cycle irrespective of DNA damage, and this might cause the cell to turn on a one way street to malignant transformation.

FR-P-054 Uncommon coincidence of B-cell chronic lymphatic leukemia with rare transversal-colon intussusception on older age flanked by a simultaneous carcinoma of the right and left flexure – successful management (only slight complication) with curative intent M. Petersen1, R. Kube2, S. Dalicho1, D. Wolleschak3, H. Lippert1, A. Roessner4, F. Meyer1 1 University Hospital, Dept. of Surgery, Magdeburg, 2Municipal Hospital, Dept. of Surgery, Cottbus, 3University Hospital, Dept. Hematology & Oncology, Magdeburg, 4University Hospital, Institute of Pathology, Magdeburg Aims. By means of an extraordinary case report, the rare hematological case with a chronic lymphatic leucemia (CLL) and an associated malignoma of the GI tract with the patient-related clinical finding and treatment characteristics incl. “outcome” out of the daily clinical practice with currently intermediary, interdisciplinary therapeutic success is described. Methods. Patient and diagnostic finding: approximately 4  weeks after puncture of the bone marrow resulting in the diagnosis B-cell leukemia (stage  IIa according to the classification by BINET and RAI; initially: Leucocytosis, hypochromic microcytic anemia), the patient (accompanying diseases: Coronary heart disease, former acute myocardial infarction, arterial hypertension, erosive gastritis) underwent surgical intervention because of a simultaneously diagnosed double carcinoma of the colon. Endoscopy revealed carcinoma (confirmed by histopathological investigation of a specimen) of the descending colon and a suspicious lesion of the ascending colon. CT scan excluded distal metastases and described an intussusception of the transversal colon, infiltrationg tumor growth through the whole colonic wall (1) and thickening of the wall of the right colonic flexure (2). Results. Treatment and course: intraoperatively, simultaneous double colonic carcinoma of the right flexure and aborally of the left flexure (no detection of an intussusception) was found prompting to a subtotal colectomy with a 2-row ileosigmoideostomy to reconstruct intestinal tract (1: pT2G2; 2: pT3N1[1/71]M0L0V0G2). There was only a summative anemia through the postoperative course and following need of blood transfusion. According to the oncological overall concept, an adjuvant

chemotherapy was recommended and continuing the follow-up observation of the B-cell CLL with no current need for a specific treatment. Conclusions. Despite a challenging combination of simultaneous neoplasias of different and/or the same entit(ies)y, in addition with complicating factors (2 primary tumor manifestations of the same entity with the need of an extended standard surgery due to tumor sites, subileus, perioperatively persisting, initially diagnosed hemoblastosis), the treatment was successful with curative intention and preserved mid- to longterm curative potential by abdominal surgery with a low complication rate. This confirms the indicated primary surgical care of the GI cancers, also because of the coincidence of a colonic carcinoma at 2 sites. The rare transversal-colon intussusception in this age, flanked by a carcinoma of the right and left flexure, is the first report of this type in the literature.

FR-P-055 Seltenes Langerhans-Zell-Sarkom der Milz mit ungewöhnlicher klinischer Manifestation J . Arend1, D . Küster2, H . Lippert1, F . Meyer1 1 University Hospital, Dept . of Surgery, Magdeburg, 2University Hospital, Institute of Pathology, Magdeburg Aims. Abklärung eines pathohistolischen Zufallsbefundes nach Splenektomie im Rahmen einer Operation bei Leberparenchymblutung (primär V. a. akute Cholangitis bei Choledochocystolithiasis). Methods. Zunächst therapeutische ERCP mit Stentimplantation im D.  hepatocholedochus nach Papillotomie unter antibiotische Abschirmung. In der Folge-CT multiple Leberherde, die zur histologischen Abklärung sonographiegestützt bioptiert wurden. Komplikation: intra- und perihepat. Hämatom, welches bei symptomatischer Größenzunahme und drohendem sept. KH-Bild (Ursache nur teils durch Staphylokokkenkolonisation eines i.v.-Portsystems zur Chemotherapie bei Mamma-Ca erklärt) eine notfallmäßige expl. Laparotomie zur sept. Fokussanierung erforderte: i) Intraabdominal kein sept. Fokus; ii) Entlastung des Leberkapselhämatoms und lokale Thermokoagulation/ Hämostyptikaapplikation; iii) intraop. multiple, unklare, teils massiv blutende fokale Milzläsionen (vulnerable Parenchymoberfläche), die zur Blutungsbeherrschung und pathohistologische Abklärung die Splenektomie erforderten; i.v.) Explantation des infizierten i.v.-Ports. Results. Bei Staphylokokkensepsis (zusätzl. Bakteriämie durch Streptokokkus hominis) und multiplen chologenen Leberabszessen zunächst kalkulierte und später resistenzgerechte Antibiotika (keine klein. Befundbesserung). Im Leberbiopsiepräparat multiple kleine Abszesse mit schaumzellig-histozytärer, resorptiver Entzündungskomponente. Das Splenektomiepräparat war diffus mit zytologisch malignen Zellen bei aufgehobener Milzarchitektur durchsetzt mit einer Zellproliferationsfraktion (Ki-67-Ag) von ca. 50% (pos. Immunhistologie für S-100, CD1a und teils CD68; Lysozym, CD3, Cd20, CD30, Alk 1, Myeloperoxidase und Chlorazetatesterase hingegen negativ). Trotz techn. Operationserfolgs tolerierte die Patientin die Intervention nur mäßig, durch zunehmende AZ-Verschlechterung keine weiterführend ggf. diagnosespezifisch indizierte Therapie möglich. Trotz max. int.-therapeut. Maßnahmen erlag die Patientin 13 Tage nach stationärer Aufnahme dem foudroyanten Verlauf im MOV. Conclusions. Die zur Aufnahme nicht bekannte, seltene Erkrankung eines Langerhans-Zell-Sarkoms trug wesentlich zum fulminanten Cholangitis-Verlauf bei. Zusätzliche Komplikationen wie Portinfektion und Blutung nach Leberpunktion verschlechterten die Prognose weiter. Offen bleibt, ob klinischer Verdacht oder Sarkomfrühdiagnose den KHVerlauf suffizient beeinflusst hätte. Die Seltenheit der Erkrankung und damit fehlende Erfahrungen in Diagnostik und Therapie erschweren eine individuelle Therapie massiv.

Poster: GI-Trakt: GIST, Dünndarm, Kolorektum FR-P-056 Value of epithelioid histomorphology and PDGFRA immunostaining patterns for prediction of PDGFRA mutated genotype in GISTs A . Agaimy1, C . Otto2, H . Geddert3, I .-M . Schaefer4, A . Braun2, R . SchneiderStock1, F . Haller2 1 Friedrich Alexander University, Institute of Pathology, Erlangen, 2Albert Ludwigs University, Institute of Pathology, Freiburg, 3St . Vincentius Hospital, Institute of Pathology, Karlsruhe, 4Georg August University, Institute of Pathology, Göttingen Aims. A majority of gastrointestinal stromal tumors (GISTs) carry mutations in the receptor tyrosine kinases KIT or PDGFRA. In a recent study, we demonstrated upregulated expression of KIT in KIT mutated GISTs, in contrast to upregulated PDGFRA expression in PDGFRA mutated GISTs, on mRNA (qRT-PCR) and protein (Western BloT) level. However, most routinely processed GISTs are formalin-fixed and paraffin-embedded; thus, these methods are not applicable in daily pathology routine. Reliable determination of PDGFRA expression by immunohistochemistry might help to identify GISTs with PDGFRA mutation, without the necessity for complete genotyping of KIT and PDGFRA by mutational analysis. The aim of the current study was to evaluate the predictive value of a combination of histomorphology and PDGFRA immunohistochemistry in comparison to mutational analyses. Methods. In order to conduct a tissue microarray, 109 surgically resected GISTs with known mutation status of KIT (74%) and PDGFRA (16%) were used. The histomorphological phenotype (spindled, epithelioid, mixed growth pattern) was determined on H&E sections without knowledge of the genotype. The staining intensity (negative, 1–25%, 26–50%, >50%) and the staining pattern (paranuclear, cytoplasmic, membranous) of PDGFRA were determined without knowledge of the genotype. Results. PDGFRA-mutated GISTs were significantly more often of epithelioid phenotype and had a significantly higher expression of PDGFRA protein, compared to KIT-mutated GISTs. The paranuclear stainig pattern was almost exclusively observed in PDGFRA mutated GISTs. A combination of histomorphology, staining intensity and staining pattern of PDGFRA was a reliable predictor for PDGFRA genotype. Conclusions. A combination of histomorphology and PDGFRA immunostaining is a reliable predictor of PDGFRA genotype. The use of this PDGFRA genotype predictor may help to reduce costs and shorten processing time of GIST genotyping by excluding KIT mutational analysis in PDGFRA-overexpressing GISTs. This might be even more important in less developed countries with restricted health budgets.

FR-P-057 Expression of CD34 in GIST is site-dependent and genotype- associated A . Braun1, C . Otto1, H . Geddert2, D .J . Zhang3, A . Agaimy4, Ö . Sahin3, F . Haller1 Albert Ludwigs University, Institute of Pathology, Freiburg, 2St . Vincentius Hospital, Institute of Pathology, Karlsruhe, 3German Cancer Research Center, Heidelberg, 4Friedrich Alexander University, Institute of Pathology, Erlangen 1

Aims. Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. In addition to immunopositivity for KIT (CD117), 60–70% of cases are positive for CD34. CD34 is a cell surface glycoprotein, which is expressed especially in early hematopoietic stem and progenitor cells. Its function is still not yet sufficiently clarified. Our aim was to gain a better understanding of the regulation of CD34 expression in GISTs. Methods. From the archives of our institutes, 109 surgically resected GISTs were compiled. Mutation analyses of KIT (exon 9, 11, 13 and 17), Der Pathologe Suppl 1 · 2012  | 

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Abstracts PDGFRA (exons 10, 12, 14 and 18), and BRAF (exon 15) were performed. The expression of CD34 was assessed semi-quantitatively using immunohistochemical staining on tissue microarrays. Methylation analyses were performed using pyrosequencing of two CpG loci in the promoter binding region of the CD34 gene (CD34_P339_R and CD34_P780_R). Furthermore, GIST cell lines 882 and 48B were treated with different concentrations of the demethylating agent 5-azacitidine (5-azaC), and the methylation status as well as the mRNA and protein expression of CD34 were determined. Results. KIT-mutated tumors of the stomach and rectum were at both CpG loci of the CD34 gene significantly less methylated compared to KIT-mutated small intestinal GISTs (p<0.001 and p<0.003), or compared to gastric GISTs with a PDGFRA mutation (p<0.001 and p<0.003). Notably, the CD34 expression was inversely correlated with the degree of CD34 methylation: KIT-mutant GISTs of the stomach and rectum had a significantly higher CD34 expression than KIT-mutant GISTs of the small intestine (p<0.001 and p=0.005), or PDGFRA-mutant GISTs of the stomach (p=0.002 and p=0.017). Treatment of cells with 5-azaC resulted in a concentration-dependent demethylation of CD34, with reciprocally increasing mRNA and protein expression at the same time. Conclusions. The expression of CD34 in GIST is epigenetically regulated and correlates with both genotype and anatomic localization of the tumor.

FR-P-058 Epigenetic regulation of CD133/PROM1 expression in GIST H. Geddert1, A. Braun2, C. Otto2, D.J. Zhang3, A. Agaimy4, Ö. Sahin3, F. Haller2 1 St. Vincentius Hospital, Institute of Pathology, Karlsruhe, 2Albert Ludwigs University, Institute of Pathology, Freiburg, 3German Cancer Research Center, Heidelberg, 4Friedrich Alexander University, Institute of Pathology, Erlangen Aims. Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Recently, high expression of CD133/PROM1 in gastric GISTs has been reported to be of prognostic impact for malignant behavior in GIST. CD133/PROM1 is a stem cell marker, and upregulation of CD133/PROM1 might be associated with a less differentiated phenotype in GIST tumor cells. Our aim was to gain a better understanding of the regulation of CD133/PROM1 expression in GISTs. Methods. From the archives of our institutes, 109 surgically resected GISTs were compiled. The expression of CD133/PROM1 was assessed semi-quantitatively using immunohistochemical staining on tissue microarrays. Methylation analyses were performed using pyrosequencing of a CpG loci in the promoter binding region of the CD133/PROM1 gene (CD133_P44_R). Furthermore, GIST cell lines 882 and 48B were treated with different concentrations of the demethylating agent 5-azacitidine (5-azaC), and the methylation status as well as the mRNA expression of CD133/PROM1 were determined. Results. KIT-mutated GISTs of the stomach had a significantly lower methylation status of the CD133/PROM1 gene, as well as a significantly higher expression of the CD133/PROM1 protein compared to KIT-mutated GISTs of the small intestine (p<0.01). Moreover, gastric GISTs with a PDGFRA mutation had a significantly higher methylation status, as well as a significantly lower CD133/PROM1 expression compared to gastric GISTs with a KIT mutation (p<0.01). Functional analyses in GIST cell lines revealed epigenetic regulation of CD133/PROM1 expression, with increasing mRNA expression after concentration-dependent demethylation of CD133/PROM1. Conclusions. The expression of CD133/PROM1 in GIST is epigenetically regulated and correlates with both genotype and anatomic localization of the tumor.

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FR-P-059 Comparison of interobserver mitotic counting reproducibility by light-microscopy on H&E stained sections vs. assistance by immunohistochemical staining of phospho-Histone H3 in GISTs: a multicenter experience A. Agaimy1, A. Gaumann2, C. Otto3, I.-M. Schaefer4, H.-U. Schildhaus5, C. Sick6, E. Wardelmann5, F. Haller3 1 Friedrich Alexander University, Institute of Pathology, Erlangen, 2Group Practice of Pathology, Kaufbeuren, 3Albert Ludwigs University, Institute of Pathology, Freiburg, 4Georg August University, Institute of Pathology, Göttingen, 5University Medical Center, Institute of Pathology, Köln, 6Novartis Pharma GmbH, Nürnberg Aims. Risk classification of gastrointestinal stromal tumors (GISTs) is based on tumor size, anatomical localization, and mitotic counts. While the first two parameters are easily determined, correct identification and counting of mitotic figures may be less reproducible, and also time consuming in daily pathological routine. The aim of the current study was to evaluate the reproducibility of mitotic counting in GISTs in a multicenter study, and to identify possible factors leading to incorrect counting. Moreover, counting on H&E stained routine sections was compared to counting on sections with immunohistochemical staining against Histone H3, which is specifically phosphorylated during mitosis. Methods. From the sample collections of four different GIST centers, 124 surgically resected primary GISTs with complete clinicopathological data and long-term follow-up were assessed. Fresh serial sections were cut from the paraffin blocks and used for H&E staining as well as for immunostaining against phospho-Histone H3 (pHH3). Mitotic figures were counted by seven pathologists without knowledge of clinicopathological data in conventional 50 high-power fields, and also in 10 mm2. Furthermore, histomorphology, grade of inflammation, as well as quality of staining were classified by each pathologist. Results. There was a good overall agreement on the mitotic counts in all cases. The variation in mitotic counts between the different pathologists was significantly correlated to the absolute number of mitotic figures in a single tumor, as well as to the quality of the slide, and the occurrence of possible artifacts. Yet, this had mostly no impact on the determination of risk classification in individual cases. As a prominent factor with influence on results of mitotic counting, the diverse microscopes could be identified, which led to unlike sizes of high-power fields. In individual tumors, staining against pHH3 was helpful in differentiation of true mitotic figures vs. artifacts (e.g. apoptotic figures, inflammatory cells). Conclusions. Correct counting of mitotic figures in GISTs is an important part of the pathology report, and has strong impact on therapy, e.g. adjuvant treatment. Reproducibility of mitotic counting correlates to quality of tissue proceeding, and can be improved by pHH3 immunohistochemical staining in individual (difficult-to-count) cases. Standardization of the field size to be counted, e.g. 10 mm2, is also an important factor for improved counting of mitotic figures in GISTs.

FR-P-060 ETV1 is expressed in GISTs and other mesenchymal tumors of the gastrointestinal tract M. Bihl1, F. Trapani1, P. Hirschmann1, L. Insabato2, S. Dirnhofer1, L. Terracciano1, L. Tornillo1 1 University of Basel, Institute of Pathology, Basel, Switzerland, 2University “Federico II”, Department of Biomorphological and Functional Sciences, Neapel, Italy Aims. Gastrointestinal stromal tumors (GISTs) are the most frequent mesenchymal tumors of the gastrointestinal tract, originating from the interstitial cells of Cajal (ICC) and characterised in most of cases by constitutive activating mutations in KIT or PDGFRA genes. ETV1, a transcription factor whose role has been demonstrated in several tumors, is expressed in a subset of ICCs probably giving rise to GISTs. Our

aims are twofold: 1)  to verify the immunohistochemical expression of ETV1 in a large series of mesenchymal tumors of the gastrointestinal tract; 2) to verify its possible relationship with clinicopathologic parameters (risk classification, survival, mutational status). Methods. 424 GISTs (381 primary and 43 metastases), 30 leiomyomas, 8 leiomyosarcomas and 1 schwannoma in TMA format. Mean age was 66±1.4 ys. For the risk classification was used the schema of Miettinen in 5 groups (probably benign,18%, very-low risk, 17%, low-risk, 14%, moderate risk, 12% high-risk, 10%) adding overtly malignant tumors, 20% and metastases, 9%. Immunohistochemical staining for ETV1 (AbCam®, dilution 1:100) was performed. Immunoreactivity was scored by evaluating the number of positive nuclei over the total number of tumor cells. For survival analysis the series was dycotomised, using 0 as cut-off. Results. ETV1 was expressed in the majority of primary GISTs (76% of cases) and of metastases (86%), but also in leiomyosarcomas (50%) and in leiomyomas (38%). The maximum score was observed in metastases (mean 68%) and in primary GISTs (mean 48%). Leiomyosarcomas (mean 37%) and leiomyomas (mean 14%) had a lower score. The differences were statistically significant (0.0001
FR-P-061 Rhabdoid differentiation in gastrointestinal stromal tumors (GISTs) of the stomach I.-M. Schaefer1, S. Cameron2, L. Füzesi1 1 University Medical Center Göttingen, Department of Pathology and Legal Medicine, Göttingen, 2University Medical Center Göttingen, Göttingen Aims. Partial rhabdoid differentiation in epithelioid gastric GISTs is a rare finding and little is known about the clinicopathologic and molecular genetic characteristics of this subtype of GISTs. Methods. We examined 3  cases of primary gastric GISTs with epithelioid differentiation and focal rhabdoid changes. Mitotic count, immunohistochemical staining, mutation analyses of KIT (exon 9, 11, 13, 17), and PDGFRA (exon 12, 14, 18), and comparative genomic hybridization (CGH) were performed, separately in epithelioid and rhabdoid areas. The risk of progression was estimated according to the updated Miettinen criteria. Results. All patients were male, aged 63, 56, and 56 years, respectively. The tumor size was 9, 5.5, and 3.3 cm, with 9, 4, and 2 mitoses/50 high power fields, respectively. The tumors were assessed as high, low, and very low risk. All tumors immunohistochemically expressed CD117, and Ki67 (in 15%, 15% and 5%), strongly PDGFRA, variable DOG-1 and CD34, but were negative for SMA, desmin, and S100. Two tumors (1 and 3) harbored a PDGFRA exon 18 mutation (c.2525A>T; p.D842V), whereas the second tumor showed a PDGFRA exon 14 mutation (c.1977C>G; p.N659K). CGH revealed -14q in all cases, and a tendency towards a higher frequency of chromosomal aberrations in the rhabdoid areas as compared with the epithelioid components. Conclusions. Partial rhabdoid differentiation in GIST has to be kept in mind in the differential diagnosis of mesenchymal tumors. A strong expression of PDGFRA, PDGFRA mutations, and chromosomal imbalances typical of gastric GISTs, in particular -14q are characteristic findings of the examined gastric GISTs with rhabdoid features. Additionally, CGH analysis revealed a tendency towards a higher degree of chromosomal instability in the areas of rhabdoid differentiation.

FR-P-062 Evolution of mutational pattern in metastasizing gastrointestinal stromal tumours K. Schierle1, U. Siebolts1, A. Markwarth1, C. Wickenhauser1 University of Leipzig, Institute of Pathology, Leipzig

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Aims. Gastrointestinal stromal tumour (GIST) is the most mesenchymal tumour of the intestinal tract. Although immunohistochemistry allows a precise diagnosis in most cases it may be difficult to exclude other sarcomas in some cases. As imatinib mesylate prolongs recurrence-free survival (RFS) in localised high risk GISTs it is extremely important to accurately confirm each diagnosis. Also, a precondition for effective long term treatment is the stability of the mutational status. Therefore, we evaluated 40 autopsies and routine examinations of the last 10 years for GIST tumours, rechecked the diagnosis and analysed the evolution of mutational patterns in primary tumours and metastasis. Methods. Sanger sequencing of primary tumours and metastasis for KIT and PDGFR mutations. Results. (1) Mutational analysis was helpful in identifying GISTs in those cases, where histomorphology and immunohistochemistry provided unusual constellations. (2) Mutational analysis was helpful in identifying secondary tumours responsible for metastases. (3) Synchronous GISTs may bear congeneric as well as different mutations. (4) All cases of relapsing GISTs retained their specific mutations. (5) In metastasizing GISTs the specific mutation was retained in most but not all of the metastases. Additional mutations in other exons of the same gene or in other genes appeared in other cases. (6) Identity in mutational pattern does not imply identity in histomorphology and vice versa. Conclusions. Mutational analyses are urgently necessary to validate the diagnosis of GIST and should also be performed in the course of the disease as they clearly predict outcome under imatinib mesylate treatment.

FR-P-063 Chromosomal aberrations in PDGFRA-mutated gastrointestinal stromal tumors (GISTs) I.-M. Schaefer1, C. Delfs1, B. Gunawan1, A. Agaimy2, S. Cameron3, M. Ghadimi3, L. Füzesi1, F. Haller2 1 University Medical Center Göttingen, Department of Pathology and Legal Medicine, Göttingen, 2University Medical Center Erlangen, Institute for Pathology, Erlangen, 3University Medical Center Göttingen, Göttingen Aims. Approximately 7% of gastrointestinal stromal tumors (GISTs) harbor mutations of the PDGFRA gene. The aim of this study was to investigate the clinicopathologic characteristics, types of mutations and chromosomal imbalances of PDGFRA-mutated GISTs, and identify prognostic implications. Methods. 53 patients with primary GISTs harbouring a mutation of the PDGFRA receptor tyrosine kinase were selected for this study. Assessment of maximal tumor size, localization, histologic growth pattern and mitotic count per 50 high power fields (HPFs) was performed and the malignant potential estimated according to the updated Miettinen criteria. Comparative genomic hybridization (CGH) was performed in all cases. Results. The mean tumor size was 7.1 cm, and 47 tumors were located in the stomach, 3 in the intestine, and 3 in the mesentery/omentum. 18 tumors showed epithelioid, 19 spindled, and 16 mixed-type differentiation. The mean mitotic count was 5.7 mitoses/50  HPFs, and the risk of progression very low in 12, low in 30, intermediate in 7, and high in 4 cases. PDGFRA mutations included 44 point mutations, with D842V (c.2525A>T) being the most frequent, as well as 8 deletions, and 5 deletion-insertions. CGH revealed -14q, -1p, and -22q as most the common aberrations (39, 15, and 10 cases, respectively). The mean number of clonal net changes was 2.68 (0.6 gains, 2.1 losses), and 10 tumors revealed no chromosomal imbalances.

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Abstracts Conclusions. PDGFRA-mutated GISTs are rather large tumors of gastric site with a very low/low risk of progression. D842V is the most common mutation. Overall, GISTs with PDGFRA mutations exhibit a low degree of chromosomal instability, compared to KIT-mutated GISTs. Although PDGFRA-mutated GISTs principally display the same chromosomal aberrations as KIT-mutated GISTs, the observed low degree of chromosomal instability might contribute to their generally low malignant phenotype.

FR-P-064 Rare inflammatory pseudotumor with primary (intrapulmonal, mediastinal, subpleural) manifestation and subsequent occurrence within mesenteric fatty tissue of the small intestine – secondary tumor lesion or metastasis? M. Petersen1, H. Lippert1, K. Schütte2, A. Roessner3, F. Meyer1 University Hospital, Dept. of Surgery, Magdeburg, 2University Hospital, Dept. of Gastroenterology, Magdeburg, 3University Hospital, Institute of Pathology, Magdeburg 1

Aims. The rarely occurring inflammatory pseudotumor (IPT) is considered a relevant differential diagnosis in regard to frequency, identifiability and adequate management in diagnostic and therapy of intestinal tumor lesions. Methods. By means of an extraordinary case report, this tumor finding is characterized with occurrence, finding the correct diagnosis, therapeutic measures and outcome. Results. In a 39-year-old man, a multifocal recurrent tumor growth within the thorax (intrapulmonary, mediastinal, subpleural lesions – predominating within the right upper segment) and a singular abdominal tumor lesion were diagnosed 12 years after a former successfully resected pulmonary first manifestation of an IPT. An in-toto resection of the middle jejunal segment was achieved because of a manifest inflammatory tumor conglomerate consisting of mesenteric fatty tissue and attached jejunal loops including a jejunojejunostomy with an uneventful postoperative course. A surgical re-intervention for the pulmonary/thoracic tumor lesions was not favored by the thoracic surgeons because of a superior vena-cava-neighbored tumor site with mediastinal infiltration. Initially, the patient did not agree to an externally recommended immunosuppressive treatment with cyclophosphamide and steroids because of a wish for a baby despite repeatingly expressed demands. Finally, the patient underwent a 5-month systematic steroid therapy and subsequently, a periodic repeat of such therapeutic cycles but, however, with no CT follow-up as recommended. A slight tumor progression was diagnosed. Histopathological investigation revealed myofibroblastic cell proliferation and inflammatory infiltrates; in addition, the immunohistochemical test marker profile (CD117-, Alk1-, chromogranine-negative; vimentinand partially SM-actin-positive) led to the diagnosis IPT. Conclusions. After appropriate literature search, the presented patient is one of the only few cases with an IPT of the described unusual tumor site within the jejunal mesenteric tissue, a rare multifocal recurrent tumor growth after former surgical resection through a long-term course of 12 years.

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FR-P-065 Tissue damage through MRI histopatology of small intestine in swine model due to high frequency radiation of different intensity and duration S. Griff1, T. Mairinger1, G. Stoltenburg-Didinger2 1 HELIOS Klinikum Emil v. Behring, Berlin, 2Institute for Cellbiology and Neurobiology Charité Berlin Aims. Magnetic resonance imaging (MRI) is an important diagnostic tool in daily clinical praxis. Regarding the physical aspects, the majority of radiofrequency (RF) power transmitted for imaging is transformed into heat within the patient’s body. The thresholds of exposure (specific absorption rate, SAR) have been defined in the early 1980s without investigation of changes of internal organs. The changes of organs due to the impact of high frequency radiation have not been investigated since. Based on these facts we evaluated the morphological effects in small intestine in swine in correlation to SAR and core temperature. Methods. By autopsy, small intestine tissue from 14 pigs was examined by histology and immunohistochemistry. The animals have been exposed to long-time whole-body SAR. There were four groups of animals with different SARs and duration of exposure. The control group (group 0) was not exposed. The highest exposure was beyond the SAR thresholds. Results. All exposed animals irrespective of dose and duration of exposure showed marked histological changes. These were: – Loss and/or necrosis of surface epithelium – Hyperaemia – Focal fibrin thrombi – Retraction of villi – Lymphocytic and eosinophilic infiltration of lamina propria Conclusions. The results of the present study question the so far valid SAR thresholds. The casuistic reports of complications were considered as handling error or patient’s erratic behaviour. Such cases have to be re-evaluated and newly discussed, as there are no histological studies of internal organs in these situations.

FR-P-066 Her2/neu in gastric cancer: comparison of tissue micro arrays with corresponding whole tissue sections C. Böger1, V. Warneke1, H.-M. Behrens1, C. Röcken1 1 Christian-Albrechts-University, Kiel, Institute of Pathology, Kiel Aims. Gastric cancer (GC) is one of the most common causes of cancerrelated deaths worldwide. The introduction of trastuzumab for Her2/ neu-positive gastric cancer is a recent advancement, with Her2/neu currently being the only predictive biomarker for gastric cancer. Her2/neu status is currently assessed by biopsy or resection material, but it is not validated which influence tissue sampling has on the assessment of the Her2/neu status. Methods. 485 patients (299 men, 186 women; median age 68 years) with GC had undergone either total or partial gastrectomy for adenocarcinomas of the stomach or oesophago-gastric junction. Formalin-fixed and paraffin-embedded tissue samples were used to generate tissue micro arrays (TMA). Briefly, five representative regions of the paraffin “donor” blocks were chosen. Tissue cylinders of 1.5 mm diameter were punched from these areas and arrayed into a new “recipient” paraffin block. Afterwards, 4 µM sections of the resulting tumor TMA-block were cut for further analysis. The Her2/neu-status was assessed using the monoclonal anti-Her2/neu-antibody (clone 4B5) and HER2-SISH double labelling in situ hybridization-system, according to the gastric cancer scoring system by Rüschoff et al. (Virch Arch. 2010 Sep; 457(3):299–307) separately for resection specimens (whole tissue sections) and for biopsies (TMAs) in order to test which influence tissue sampling has on the Her2/neu-status. Statistical analyses: For measuring the inter-rater agreement of Her2/neu scores between two observers, Cohen’s kappa coefficient was calculated.

Results. Her2/neu status in TMAs and corresponding whole tissue sections were investigated from the same GCs by two surgical pathologists. 37 (8.2%) and 38 (8.5%) GCs were classified as Her2/neu-positive by the two observers using whole tissue sections. Using the gastric cancer scoring system for biopsies, Her2/neu was positive in 26 (5.9%) and 27 (6.1%) GCs. The interobserver agreement was high (98.5% for whole tissue sections and 98.0% for TMAs; p<0.001). The kappa value was lower when whole tissue sections were compared with the corresponding TMAs (76.4% and 78.6%, respectively). TMAs carried the risk of a false negative result. Conclusions. We recommend that the Her2/neu status should be validated in resection specimens when tissue biopsies yielded a negative Her2/ neu-status. Further studies are needed to assess the appropriate number of tumor-carrying biopsies in order to reduce the number of false negative biopsy specimens.

FR-P-067 RECK expression: an independent prognostic marker of survival in colorectal cancer M. von Winterfeld1, A. Stenzinger2, A. Rabien3, A. Warth2, C. Kamphues4, M. Dietel1, W. Weichert2, F. Klauschen1, D. Wittschieber5 1 University Hospital Charité Berlin, Institute of Pathology, Berlin, 2University Hospital Heidelberg, Institute of Pathology, Heidelberg, 3University Hospital Charité Berlin, Department of Urology, Berlin, 4University Hospital Charité Berlin, Department of General, Visceral and Transplantation Surgery, Berlin, 5 University Hospital Münster, Institute of Legal Medicine, Münster Aims. Patient prognosis in colorectal cancer is determined as in most solid cancers by the extent of local invasion and the presence of lymph node and distant metastases. The invasive potential of a tumor depends on the ability to degrade extracellular matrix proteins, for example by matrix metalloproteinases (MMPs). An important inhibitor of MMPs is RECK (reversion-inducing cysteine-rich protein with Kazal motifs), a membrane-anchored glycoprotein. This study investigated the prognostic relevance of RECK expression in colorectal cancer. Methods. A total of 283 tissue samples from patients with colorectal carcinomas were enclosed in this study. Tissue microarray sections were stained immunhistochemically. To examine staining intensity immunoreactivity score (IRS) was used. Results. Analysis of immunohistochemical tissue microarray data showed that RECK protein levels did not seem to correlate with clinicopathological parameters (Spearmans rank correlation coefficients between −0.14 and −0.18) and that decreased RECK expression was significantly correlated with poor prognosis and a mean survival of 70  months in RECK negative (146 cases) vs. 97 months in RECK positive patients (137 cases; log rank test, p=0.002). Conclusions. The data show that decreased RECK expression is significantly prognostic of reduced survival in colorectal cancer. Moreover, this study demonstrates the independence of this association of standard clinicopathological parameters.

FR-P-068 Intussusception of the caecum caused by lymphoid hyperplasia in association with adenovirus infection in a 5-year-old boy E. Gradhand1, N.A. Wong1, T. Rogers2, P. Ramani1 1 University Hospitals Bristol, Histology Department, Bristol, United Kingdom, 2University Hospitals Bristol, Paediatric Surgery, Bristol, United Kingdom Aims. The most commonly described causes of childhood intestinal intussusception are Meckel’s diverticula, polyps and cystic fibrosis. Lymphoid hyperplasia containing adenovirus and causing intussusception has rarely been reported and in all these cases the lymphoid hyperplasia was restricted to the small bowel or appendix. We report the first known

case of intussusception secondary to caecal lymphoid hyperplasia containing adenovirus. Methods. The ileocaecal resection specimen of a 5-year-old boy presenting with intussusception was examined macroscopically and microscopically. Immunohistochemistry was performed after paranuclear inclusion bodies were identified in colonocytes. Results. Macroscopic examination showed a sessile polyp (70  mm in maximum diameter) arising from the mucosa of the caecum, enlarged mesenteric lymph nodes but a normal appendix and terminal ileum. Histologically, the sessile polyp corresponded to a focus of lymphoid hyperplasia. Occasional colonocytes overlying the caecal lymphoid tissue contained eosinophilic paranuclear inclusion bodies. Immunohistochemistry for adenovirus was therefore performed on the following tissues: normal caecum; the focus of lymphoid hyperplasia; the appendix; normal ileum; and two enlarged mesenteric lymph nodes. The presence of adenovirus was confirmed in occasional colonocytes overlying the caecal lymphoid tissue and within the appendix, but not in the immunostained sections of normal caecum, normal ileum and enlarged mesenteric lymph nodes. Conclusions. This is the first known case of an intussusception caused by mucosal lymphoid hyperplasia which contained adenovirus and was restricted to the caecum. It is not clear whether the adenovirus infection caused the lymphoid hyperplasia and subsequently the intussusception or whether the abnormal presence of lymphoid tissue within the caecum predisposed to adenoviral infection as a secondary phenomenon.

FR-P-069 IMP3 expression in polypoid lesions in ulcerative colitis – a diagnostic marker? T.T. Rau1, J. Koelle1, M. Vieth2, C. Geppert1, K. Knobloch1, R. Atreya3, R. Schneider-Stock1, A. Hartmann1, M.-O. Riener4 1 University Hospital Erlangen, Institute of Pathology, Erlangen, 2Institute of Pathology, Bayreuth, 3University Hospital Erlangen, Department of Medicine 1 , Erlangen, 4Clinic for Pathology, Frankfurt a. M. Aims. IMP3 has shown to be a biomarker of diagnostic value in the differentiation between low and high grade dysplasia. This accounts especially for lesions of the bile duct and ductal pancreatic lesions. In ulcerative colitis ALM or DALM lesions bear sometimes similar diagnostic challenges in distinguishing low from high grade intraepithelial neoplasia. For this reason we planned to test the application of IMP3 in this setting. Methods. 182 consecutive polypoid lesions from 138 patients with ulcerative colitis were analysed within a dedicated GI pathology institute (2). The series contained sporadic and CU associated lesions and consisted of 42 hyperplastic polyps, 5 sessile serrated adenomas, 87 classical adenomas, 5 traditional serrated adenomas, 38 CU associated IEN – generally with a clinical appearance as DALM lesions – and 4 colorectal cancers. All lesions were analysed by two independent GI pathologists. In summary 47 lesions had no dysplasia, low grade IEN was visible in 87 cases and high grade and carcinoma was found in 10 lesions. IMP3 immunohistochemistry was performed following routine protocols. Percentage of positive cells and the staining intensity was evaluated. For statistics student’s-t-test and calculations of sensitivity and specificity were performed. Results. We could show that IMP3 amount and intensity can significantly distinguish between low grade and high grade lesions (p=0.001). However this distinction is more accurate in sporadic lesions (p=0.001) than in CU associated lesions (p=0.07). In the latter it could be regarded as a trend. This is also reflected by the values of sensitivity (0.75) and specificity (0.86) in sporadic lesions in comparison to the lower sensitivity (0.50) and specificity (0.72) in CU associated lesions. Conclusions. IMP3 as a possible diagnostic marker in the setting of ulcerative colitis achieves moderate values in its sensitivity and specificity. However it might contribute to the distinction between low and high grade lesions. In regard to the diagnostic needs it does not reach the neDer Pathologe Suppl 1 · 2012 

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Abstracts cessary accuracy in the differentiation of CU associated IEN and could therefore only be used with care in this setting.

FR-P-070 Inter-observer variability in the diagnosis of colorectal polyps under special consideration of serrated lesions – a European study T.T. Rau1, A. Agaimy1, A. Gehoff2, C. Geppert1, K. Jung3, K. Knobloch1, C. Langner4, A. Lugli5, I. Nagtegaal6, J. Rüschoff2, X. Saegert7, M. Sarbia8, M. Vieth9, A. Hartmann1 1 University Hospital Erlangen, Institute of Pathology, Erlangen, 2Institute of Pathology Nordhessen, Kassel, 3Department of medical statistics, Göttingen, 4Medical University Graz, Institute of Pathology, Graz, Austria, 5University Bern, Institute of Pathology, Bern, Switzerland, 6UDC St. Radboud, Institute of Pathology, Nijmegen, Netherlands, 7Catholic University Leuven, Institute of Pathology, Leuwen, Belgium, 8Institute for Pathology and Cytology, München, 9Institute of Pathology, Bayreuth Aims. A huge number of different criteria in the diagnosis of serrated lesions of the colon are known from the literature. In 2010, a German consensus conference redefined and simplified criteria for the diagnosis of SSA, hyperplastic polyp, traditional serrated adenoma and mixed polyps. As a scientific amendment we aimed to reflect their appliance in daily practice and to prove their ability to achieve inter-observer concordance. Methods. Consecutive colorectal polyps of one month (n=1926) were analysed within a dedicated GI pathology institute (9). All consecutively diagnosed serrated adenomas such as TSA, SSAs and mixed polyps were included. To complete the diagnostic spectrum of colorectal lesions a consecutive number of hyperplastic polyps and classical adenomas were added and completed with a few probes of normal colonic mucosa to reach a study set of 200 lesions. Virtual microscopy was enabled with a Zeiss Mirax Scanner. Hard drives were sent to 10  pathologists with GI experience in 5 European countries in three rounds. The first round blanked, the second providing clinical data, the third round after a conference meeting agreeing upon the recently proposed German consensus guidelines (Virchow’s Archive, 2010 Sep;457(3):291–7). Results. Distribution of gender, age and localization highlighted predominance for certain lesions, but no exclusiveness. Kappa-statistics revealed a basically moderate average agreement (κ=0.56) in the first round. Providing clinical data a slight increase could be achieved (κ=0.63), which was nearly equal to the values under the use of German consensus criteria (κ=0.61). Single criteria of SSA and TSA diagnosis showed a divergence in inter-observer reliability (from κ=0.06 to κ=0.82). Conclusions. Using the simplified German consensus guidelines for serrated lesions the inter-observer certainty of diagnosing serrated lesions is maintained. Using the degree of concordance the single criteria of SSA and TSA diagnosis could be assembled in a hierarchical algorithm. Training in the diagnosis of TSA and mixed polyp seems to be most promising to increase quality in GI pathology.

FR-P-071 Role of VEGF-B in the progression of colorectal cancer C. Jayasinghe1, N. Simiantonaki1, C.J. Kirkpatrick2 Gummersbach Hospital, Institute of Pathology, Gummersbach, 2 University Medical Center, Institute of Pathology, Mainz

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Aims. The relevance of VEGF-B, a VEGF (vascular endothelial growth factor) family member, in the progression of colorectal cancer is unclear. Methods. Therefore, using immunohistochemistry we have investigated the expression of this VEGFR (VEGF receptor) -1 ligand in 91 non-metastatic (n=38), lymphogenously metastatic (n=26) and haematogenously metastatic (n=27) colorectal carcinomas.

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Results. Independent of the metastatic status,VEGF-B was expressed in endothelial as well as epithelial cells in colorectal carcinomas. In 89% of the cases with or without distant metastasis a vascular expression was found. In contrast, only 62% of the nodal-positive tumors had a VEGF-B positive vasculature. In relation to the non-metastatic (p=0.01) and haematogenously metastatic (p=0.027) cases this difference was statistically significant. Concerning the topological staining distribution, in 2/3 of the cases a homogenous pattern was seen without differences in immunostaining between the intratumoral vessels and the vascular fraction throughout the invasive tumor margin. Using the general endothelial marker, CD31, and the lymphatic endothelium-specific marker, D2-40, all VEGF-B positive vessels were of blood but not of lymphatic vascular origin. Interestingly, distal of the tumor a vascular VEGF-B expression was not demonstrable. Positive endothelial VEGF-B expression was not correlated with the metastatic status. In 46% of the investigated cases an epithelial VEGF-B expression was also seen, in 30%, 46% and 67% of the tumors without, with lymph node and with distant metastasis, respectively. In this context positive VEGF-B immunoreactivity was correlated with haematogenous metastasis (p=0.006). The epithelial VEGF-B immunostaining positivity was independent of the grade of the tumor cell dissociation and localization of tumor necrosis. Conclusions. These morphological observations provide evidence for a probable pathobiological significance of VEGF-B in the tumor progression of colorectal cancer, especially in the case of haematogenous metastasis.

FR-P-072 Is there a rationale to record lymphatic invasion in node-positive colorectal cancer? N. Schneider1, J. Betge1, M.J. Pollheimer1, R.A. Lindtner1, P. Kornprat2, P. Rehak3, C. Langner1 1 Medical University of Graz, Institute of Pathology, Graz, Austria, 2Medical University of Graz, Department of Surgery, Graz, Austria, 3Medical University of Graz, Research Unit for Biomedical Engineering & Computing, Graz, Austria Aims. The invasion of tumour cells into lymphatic channels represents a crucial step in the metastatic process. The detection of lymphatic invasion in histological slides has been associated with decreased survival and higher recurrence rates. Our study aimed to evaluate prognostic significance of lymphatic invasion in colorectal cancer when lymph node metastasis is already present. Methods. 168 patients with node-positive colorectal cancer (colon, n=98; rectum, n=70) were retrospectively evaluated. Lymphatic invasion was assessed on H&E stained slides. Presence of lymphatic invasion was correlated with different pathological variables applying the chi square test. Progression-free and cancer-specific survivals were assessed using the Kaplan-Meier method. Results. Lymphatic invasion was detected in 95 (57%) cases. There was a significant association of lymphatic invasion with the number of examined tissue blocks: 1–3 blocks (n=50): 46% presence of L1, 4–5 blocks (n=68): 53% presence of L1, and 6 or more blocks (n=50): 72% presence of L1 (p=0.02). Furthermore, L1 was associated with poor tumour differentiation (p=0.009) and the number of involved lymph nodes (p<0.001). Patients with tumours showing lymphatic invasion had decreased progression-free survival (p=0.025) and cancer-specific survival (p=0.082). When stratified by location, lymphatic invasion was significantly associated with decreased progression-free (p=0.01) and cancer-specific survival (p=0.02) in colon primaries, yet not in rectum primaries. Conclusions. The detection of lymph vessel invasion was significantly associated with tumour differentiation and the extent of lymph node metastasis. Moreover, presence of lymph vessel invasion proved to be a relevant prognostic variable in node-positive colon cancer, while no

prognostic impact was found in rectal cancer patients. The association of lymphatic invasion with the number of examined tissue blocks is an important finding that should be considered establishing practice guidelines for pathologic work-up of cancer specimens.

FR-P-074 Loss of enteroendocrine cells in autoimmune polyendocrine candidiasis ectodermal dystrophy (APECED) syndrome with gastrointestinal dysfunction

FR-P-073 Inflammatory bowel diseases (IBD) are associated with decreased colonic expression of PEPT1 in humans and mice

T .F .E . Barth1, G . Lahr2, J . von Schnurbein2, S . Buderus3, A . Findeisen4, C . Schröder4, C . Schütz2, A . Schulz2, K .-M . Debatin2, M . Wabitsch2, C . Posovszky2 1 ulm university, pathology, Ulm, 2ulm university, University Medical Center Ulm, Ulm, 3St . Marien hospital, Department of Pediatrics and Adolescent Medicine, Bonn, 4University of Greifswald, Department of Pediatrics and Adolescent Medicine, Greifswald

T . Wuensch1, S . Schulz2, N . Schaltenberg1, N . Lill1, D . Haller1, H . Daniel1 Technische Universität München, ZIEL – Research Center for Nutrition and Food Science, Freising-Weihenstephan, 2Charité – Universitätsmedizin Berlin, Pathology, Berlin 1

Aims. The intestinal peptide transporter PEPT1 is found in the brush border membrane of enterocytes in the small intestine where it contributes to amino acid absorption from luminal protein digestion. Beside that, PEPT1 is proposed to be aberrantly expressed during colonic inflammation. Because of its capability to transport bacteria-derived chemotacticacting agents such as fMLP (formyl-methionyl-leucine-phenylalanine), MDP (muramyl dipeptide) or L-Ala-D-Glu-meso-DAP, PEPT1 might act as an amplifier of inflammation. However, a systematic analysis of PEPT1 expression along the healthy colon has never been described. Here we provide information on PEPT1 mRNA and protein expression along the healthy colon of different species and during intestinal inflammation. Methods. PEPT1 mRNA and protein expression levels were determined by qRT-PCR, immunofluorescence and Western blotting, respectively, in intestinal samples from healthy mice, rats and humans. Furthermore, germ-free mice and three mouse models, resembling Crohn’s-like ileitis (TNFdeltaARE/WT) and colitis (IL-10-/-, IL- 10XTLR2-/-) as well as intestinal tissue samples from IBD patients were analyzed by immunohistochemistry. PEPT1 function in colon was determined by transport studies using [14C]-Glycyl-sarcosine (Gly-Sar). Moreover, the susceptibility of PEPT1-deficient (Pept1-/-) mice to dextran sodium sulfate (DSS)-induced colitis was investigated. Results. Colonic PEPT1 expression showed a distinct pattern of distribution with marked differences between proximal and distal segments. No PEPT1 expression was detectable in the proximal colon but prominent expression was found from mid colon to rectum of mice rats and humans. Functional Gly-Sar uptake rates were highest in jejunum and distal colon (19.76 nmol/20 min/mg protein vs. 1.71–0.42 nmol/20 min/ mg protein) and significantly lower (p<0.05) in proximal colon (0.30– 0.22  nmol/20  min/mg protein). Intestinal inflammation in the mouse models and human IBD samples revealed a markedly decreased expression of PEPT1 or even its absence. Moreover, in Pept1-/- mice susceptibility to DSS-induced colitis was unaltered as displayed by changes in body weight, disease activity index or tissue damage. Conclusions. PEPT1 is expressed in healthy colon in a segment-specific pattern and is down-regulated during intestinal inflammation and Pept1-/- mice show no altered response to DSS-induced colitis. Thus, PEPT1 does not alter the progress of colonic inflammation.

Aims. Enteroendocrine (EE) cells are necessary for the regulation of gastrointestinal function. The lack of intestinal enteroendocrine cells in enteroendocrine cell dysgenesis (ECD) causes severe malabsorptive diarrhea. Autoimmune-polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is often accompanied by gastrointestinal (GI) symptoms. We hypothesized that an autoimmune attack against the cells of the gastrointestinal (GI)-associated diffuse endocrine system may be a specific feature of GI dysfunction in APECED disorders. Methods. Biopsies were obtained during routine diagnostic endoscopy from 35 pediatric patients with gastrointestinal symptoms as well as from 5 healthy controls; biopsies were immunostained for chromogranin A and serotonin. Four patients were classified as APECED syndrome on molecular and clinical grounds. Results. Immunohistological analysis of biopsies along the GI tract (stomach, duodenum, colon) immunostained with chromogranin  A and serotonin revealed a widespread reduction or complete loss of EE cells in all four patients with APECED syndrome suffering from severe diarrhea, vomiting, malabsorption, or constipation. In contrast, EE cells were present in pediatric patients with similar gastrointestinal symptoms caused by inflammatory bowel disease, celiac disease, lymphocytic colitis, and autoimmune disorders without endocrinopathy or graft versus host disease of the gut. Conclusions. The reduction of EE cells is a specific and important early event in the pathogenesis of APECED with GI dysfunction. We propose a diagnostic algorithm integrating clinics, genetics and immunohistology (accepted JCEM, September 2011).

Poster: GI-Trakt: Kolorektum FR-P-075 Nuclear expression of thymidylate synthase predicts pathological response of rectal cancers to neoadjuvant radio/chemotherapy H . Geddert1, J . von Brocke2, F . Makowiec3, M . Henke4, A . Dimmler1, G . Faller1, U . Hopt3, M . Werner2, S . Lassmann2 1 Institute of Pathology, St . Vincentius-Kliniken, Karlsruhe, 2Institute of Pathology, University Medical Center Freiburg, 3Dept . of Surgery, University Medical Center Freiburg, 4Dept . of Radiotherapeutics, University Medical Center Freiburg Aims. The aim of this study was to evaluate the predictive value of thymidiylate synthase (TS), Survivin and Aurora-A for the tumor response to neoadjuvant radio/chemotherapy (nRCTx; 5×1Gy and 3×1.8Gy with 5-Fluorouracil/leucovorin/mitomycin C) in patients with rectal cancer. Methods. Pre-treatment biopsies (pre-Bx) of 25 rectal cancer patients treated by nRCTx were subjected to TS, Survivin and Aurora-A immunohistochemistry (IHC). Analysis was done by a semi-quantitive IHC score including intensity and percentage of positive stained tumor cells. TS, Survivin and Aurora-A IHC scores of pre-Bx were correlated to pathological tumour regression defined in matched resection specimens (pCR≤10% viable tumour cells; responders/n=18; non-responders/n=8.

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Abstracts Statistical analyses included Spearman and Mann-Whitney Rank Sum Tests (significance at p<0.05). Results. There were no significant differences in TS, Survivin or Aurora-A protein expression in the (pre-)bx between the two separate cohorts of patients. In pre-Bx of patients receiving nRCTx, neither Aurora-A (p=0.429) nor nuclear (p=0.259) or cytoplasmic (p=0.433) Survivin protein expression were significantly associated with the pathological tumor response to nRCTX. Also, cytoplasmic TS protein expression was not of predictive value for response to nRCTx (p=0.590). However, low nuclear TS protein expression in pre-Bx was significantly associated with tumor response to nRCTx (p=0.011). Conclusions. These data show that the response of rectal cancers to neoadjuvant treatment by a 5-Fluorouracil-containing chemotherapy may be predicted by immunohistochemical analysis of the target enzyme thymidylate synthase. In contrast, other proteins associated with radiotherapy (Survivin) or general cell proliferation (Aurora-A) have no predictive value in this setting.

FR-P-076 Colon carcinoma – classification into right- and left-sided cancer or according to colonic subsite? F. Benedix1, I. Gastinger2, F. Meyer1, H. Lippert1, R. Kube2 1 University hospital Magdeburg, Magdeburg, 2Carl-Thiem-Klinikum Cottbus Aims. Zahlreiche Studien der letzten beiden Jahrzehnte haben bemerkenswerte Unterschiede zwischen Karzinomen des rechten und linken Kolon herausgearbeitet. Dabei wurde die Hypothese entwickelt, dass es sich hierbei um verschiedene Tumorentitäten handeln könnte. Wenig Erfahrung besteht jedoch unverändert bezüglich des Einflusses der einzelnen anatomischen Kolonsegmente auf tumorbiologische Eigenschaften des Kolonkarzinoms. Methods. Im Rahmen der multizentrischen Qualitätssicherungsstudie (Kolon/Rektumkarzinom) wurden innerhalb eines 5-Jahres-Zeitraums 29.568  Patienten analysiert. Entsprechend der Karzinomlokalisation wurden 7  Gruppen in Analogie zu den anatomischen Segmenten gebildet und diese dann hinsichtlich demographischer und histologischer Parameter untersucht. Results. Die Analyse des Differenzierungsgrades und des histologischen Subtyps zeigte eine lineare Korrelation mit zunehmendem Abstand von der Ileocoecalklappe. Somit unterstützen diese Ergebnisse die Klassifikation in rechts- bzw. linksseitige Tumoren. Im Gegensatz dazu fanden sich deutliche Unterschiede bei der Analyse des Tumorstadiums mit dem höchsten Anteil lokal fortgeschrittener und lymphknoten-positiver Tumoren bei Karzinomen des Coecums (52.3%) und der linken Flexur (51.0%). Kolonkarzinome mit Ursprung im rechtsseitig gelegenen Colon ascendens (46.5%) sowie linksseitig gelegenen Colon descendens (44.7%) hatten hingegen signifikant weniger Tumoren mit prognostisch ungünstigeren Stadien, was eine simple Klassifikation in rechte und linke Kolonkarzinome in Frage stellt. Conclusions. Die Analyse von Alter, Differenzierungsgrad und histologischem Subtyp unterstützt die Klassifikation in rechts- bzw. linksseitige Kolonkarzinome. Im Gegensatz dazu fanden sich bei der Auswertung von UICC Stadium, T- und N-Status, Metastasierungsverhalten und Lymphangioinvasion erhebliche Unterschiede zwischen den einzelnen anatomischen Kolonsegmenten, unabhängig von der Tumorlokalisation rechts bzw. links. Die Ergebnisse der vorliegenden Untersuchung zeigen damit, dass eine simple Einteilung in rechts- bzw. linksseitige Kolonkarzinome die Heterogenität der Tumorentität Kolonkarzinom nicht in vollem Maße erfasst und damit in Frage gestellt werden muss.

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FR-P-077 Proliferation associated protein expression of Ki-67 and Repp 86 in colorectal cancer: high Ki-67 index is prognostic factor for clinical outcome C. Schrader1, F. Gieseler2, J.H. Bräsen3, B. Sipos4, C. Röcken3, W. Klapper3, H. Kalthoff5, W. v. Schönfels5, R. Lucius6, C. Pflüger1, S. Hinz5, H. Held7, T. Raff1, G. Klöppel8, J. Claasen1, S. Nazzal1, R. Worth1, C. Schafmayer5 1 University Hospital of Kiel, 2nd Department of Medicine, Kiel, 2UKSH, Campus Lübeck, I. Department of Medicine, 3UKSH, Campus Kiel, Department of Pathology, 4University of Tübingen, Department of Pathology, 5UKSH, Campus Kiel, Department of Surgery, 6UKSH, Campus Kiel, Department of Anatomy, 7Hospital Neumünster, Department of Medicine, Kiel, 8University Munich, Department of Pathology Aims. Proliferation indices are important prognostic factors in for the clinical outcome of patients with cancer. The clinical relevance of Ki67 Expression in colorectal cancer is discussed controversial in the literature. We investigated whether the expression of Ki-67 and repp86 (restrictedly expressed proliferation-associated protein 86 kDa), a new proliferation specific marker expressed in cell cycle phases G2, S, and M, but not in G1, correlates to with the clinical course in patients with colorectal cancer. Methods. Biopsy specimens from 359 untreated patients (184 men, 175 women) with colorectal cancer were investigated immunohistochemically with the monoclonal antibody against Ki-67 (Ki-S5) and repp86 (Ki-S2). Results. Ki-67 expression had a range from 9.4% to 94.2% with a median of 57% and a mean of 57%. Patients with >50% Ki-67 expression had a median overall survival time that was undefined, compared to with 43 months for patients with <50% positive cells. Kaplan-Meier analysis revealed a significant difference in the overall survival time between patients with very high (>50%) and low (<50%) Ki-67 expression (p<0.0001) in the tumor cells. Repp 86 expression had a range of 7.6% to 61% with a median of 29% and a mean of 29%. Kaplan-Meier analysis showed no significant difference in the overall survival time between patients with high or low Repp86 expression. Conclusions. Based on these findings, high expression of Ki-67 is a positive prognostic factor in patients with colorectal cancer.

FR-P-078 Testing KRAS and BRAF in colorectal carcinoma: a cost-effectiveness analysis P. Blank1, H. Moch2, T. Szucs3, M. Schwenkglenks3 1 University of Zurich, Institute of Social- and Preventive Medicine, Zürich, Switzerland, 2University Hospital Zurich, Institute of Surgical Pathology, Department of Pathology, Zürich, Switzerland, 3University of Basel, Institute of Pharmaceutical Medicine, ECPM, Basel, Switzerland Aims. Monoclonal antibodies against the epidermal growth factor receptor (EGFR) have led to considerable clinical benefits for metastatic colorectal cancer (mCRC) patients but also increased treatment costs. KRAS and BRAF gene mutations have a highly predictive value in identifying patients who are likely to benefit from anti-EGFR drugs such as cetuximab. Health economic data on testing for these gene mutations in cancer patients are scarce. The present study assessed the cost-effectiveness of predictive testing for KRAS and/or BRAF mutations, prior to cetuximab treatment of chemorefractory mCRC patients. Methods. We constructed a life-long Markov simulation model to estimate costs (EUR) and clinical effectiveness (quality adjusted life years, QALYs) of various testing strategies: 1) KRAS testing, 2) KRAS testing with subsequent BRAF testing of KRAS wild-types (KRAS/BRAF), 3) cetuximab treatment without testing. Reference strategy was no cetuximab treatment. In wild-type patients, cetuximab treatment was initiated. All patients received best supportive care. Published pivotal trials

provided information on survival times and utilities. Costs were assessed from the Swiss health care system perspective. Results. Remaining life-time costs ranged from EUR 3’983 (no cetuximab) to EUR 38’662 (no testing). Gains of 0.491 QALYs compared to the reference strategy were achieved by KRAS/BRAF testing. Compared to KRAS/BRAF, the KRAS testing strategy yielded an additional gain of 0.002 QALYs. Testing with KRAS/BRAF was the most cost-effective approach (incremental cost-effectiveness ratio; EUR 62’653/QALY) compared to the reference strategy. In Switzerland, KRAS and BRAF testing could lead to annual savings of EUR 591’170 and a loss of 1.89 QALYs compared with KRAS alone (1,003 new mCRC patients annually). Compared with no cetuximab, the usage of KRAS and BRAF testing would require an annual net investment of about EUR 30.9 million to acquire a gain of 493 QALYs. Conclusions. While new predictive test are introduced in pathology, the health economic implications of these tests remain mainly unknown. This study has shown that testing for KRAS and BRAF mutations in mCRC patients prior to cetuximab treatment would be favourable in a Swiss health care context. This model could also be used for comparable decision problems arising with other predictive tests, which are of highest relevance for oncologists, pathologists, and health policy makers. References

1. Blank PR, Moch H et al (2011). Clin Cancer Res

FR-P-079 HER-2/neu expression in locally advanced rectal cancer (cUICC II/ III) and its correlation with long-term prognosis L.-C. Conradi1, H. Stycen1, T. Sprenger1, J. Gaedcke1, K. Homayounfar1, H.A. Wolff1, H. Becker1, B.M. Ghadimi1, J. Rüschoff2, P. Middel1, T. Beissbarth1, T. Liersch1 1 University Medical Center Göttingen, Göttingen, 2Pathologie Nordhessen, Kassel Aims. With the implementation of multimodal treatment strategies including neoadjuvant radiochemotherapy (RCT), the prognosis of locally advanced rectal cancer has been improved over the last two decades. Most actual clinical trials aim to further develop therapy by intensification of agents administered. We examined Her2/neu in rectal cancer patients to evaluate its expression as a potential drug target as well as its capacity as predictive and prognostic biomarker. Methods. Two-hundred-sixty-four patients (192 male, 72 female; median age 64 years) with locally advanced rectal cancer (cUICC-II/III) were included in this study. Preoperative RCT (50.4 Gy and concomitant either 5-FU or 5-FU and oxaliplatin) was administered in 217 patients followed by surgical resection with total mesorectal excision (TME). Another 75 patients received postoperative RCT. All patients were treated within phase-II/III trials of the German Rectal Cancer Study Group or analogous to these standardized protocols. Her2-/neu expression from pretreatment biopsies and corresponding resection specimens were assessed by immunohistochemical staining and SISH-analysis Results. A positive Her-2/neu expression was found in 12.4% of all pretreatment tumor biopsy samples and in 29.3% of the resection specimens. The correlation of the pre-treatment Her-2/neu status did not show a significant correlation with the grade of tumor regression. However, patients with a higher Her-2/neu protein expression as measured in the resection specimen showed a significant survival benefit (p=0.045) and a prolonged disease free survival (p=0.05) compared with patients having low intratumoral Her-2/neu expression during the follow-up (median 41 months). The 5-year survival rate was 96.5% (Her-2/neu positive) versus 79.9% (Her-2/neu negative). Conclusions. Her-2/neu might be a promising drug target in a significant proportion of rectal cancer patients and should be further assessed within prospective clinical trials. Furthermore the Her-2/neu status seems to be a valuable prognostic marker within the multimodal treatment of advanced rectal cancer.

FR-P-080 Functional characterization of necroptosis in colorectal cancer M. Metzig1, D. Fuchs2, S. Macher-Goeppinger1, P. Schirmacher3, W. Roth1 Institute of Pathology, University of Heidelberg; German Cancer Research Center (DKFZ), Heidelberg, Heidelberg, 2German Cancer Research Center (DKFZ), Heidelberg, 3Institute of Pathology, University of Heidelberg, Heidelberg 1

Aims. Necroptosis is a recently discovered, RIP1-dependent form of programmed necrosis. Recent evidence indicates that this type of cell death plays an important role in both physiologic and pathologic cellular processes. While different stimuli, e.g. members of the tumor necrosis factor (TNF) family, have been identified as inducers of necroptosis, only little is known about the underlying signaling cascades and effector molecules of necroptosis. The aim of our project is to further clarify the mechanism of programmed necrosis and to analyze its biological significance in colorectal carcinoma. Methods. Cytotoxicity and apoptosis assay, FACS analysis, immunohistochemistry, RNA isolation, quantitative real-time PCR, cloning of expression vectors, co-immunoprecipitations. Results. We screened multiple cytotoxic stimuli for the induction of necroptosis in various colorectal carcinoma cell lines. We found a differential increase in sensitivity to cell death induced by specific drugs when co-treated with a pancaspase-inhibitor. Since a RIP1-specific inhibitor (necrostatin 1) diminished this effect we concluded that cells were dying due to necroptosis. Drug-induced necroptosis was further dependent on TNF receptor 1 (TNFR1) signaling. Overexpression of RIP3 has been shown to reconstitute the ability to undergo necroptosis in response to different stimuli in so far insensitive colon carcinoma cell lines. To assess the biological significance of RIP3 in colon cancer we analyzed protein and mRNA expression levels in human colorectal carcinoma samples. RIP3 expression was significantly reduced in colon carcinoma specimens compared to normal tissue. Conclusions. Chemotherapeutic drugs induce necroptosis in colon carcinoma cells in a TNFR1 and RIP3 dependent manner. RIP3 expression is reduced in colon carcinoma suggesting a physiologic function of necroptosis in tissue homeostasis. The possibility to re-sensitize carcinoma cells to necroptosis-inducing stimuli points towards novel therapeutic options in cancer therapy.

FR-P-081 High HSP70 and HSP27 expression levels are independent adverse prognostic factors in primary resected colon cancer K. Bauer1, U. Nitsche2, J. Slotta-Huspenina1, E. Drecoll1, C. Hann von Weyhern1,3, R. Rosenberg2, H. Höfler1,4, R. Langer1 1 Technische Universität München, Institute of Pathology, München, 2Technische Universität München, Klinikum rechts der Isar, München, 3Städtisches Klinikum München, Klinikum Harlaching, München, 4Institute of Pathology, Helmholtz-Zentrum München, Oberschleissheim Aims. The expression of Heat Shock Proteins (HSPs) is increased in various cancers and has been shown to correlate with biological tumor behaviour. We aimed to assess the impact of HSP70, HSP60 and HSP27 expression patterns with pathological features of colon carcinomas and patients survival in a large collection of colon cancer. Methods. HSP expression was determined by immunohistochemistry on a tissue microarray containing cancer tissue of 355 patients with primary resected colon carcinomas. Expression patterns were correlated with pathologic features (UICC pTNM category, tumor grading) and survival. Expression of HSP27, HSP60 and HSP70 was assessed in cancer tissue ranging from negative to high. Results. Expression of HSP27, HSP60 and HSP70 can be detected in colon carcinomas. High HSP70 expression was associated with worse overall survival (p<0.001) and was an independent prognostic factor (p=0.004) in multivariate analysis including the pathological parameters menDer Pathologe Suppl 1 · 2012 

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Abstracts tioned above. For patients without lymph node or distant metastases (UICC stages I/II) and with complete tumor excision, HSP70 expression was the only independent prognostic factor for survival (p=0.001) and superior to UICC pT category. In left sided UICC stage I/II carcinomas, high HSP27 expression also had adverse prognostic impact and was an independent prognostic factor (p=0.016) besides HSP70 (p=0.002). There was no correlation between HSP27, HSP60 and HSP70 expression among each other and with UICC pT category, presence of lymph node, distant metastases or tumor grading. Conclusions. High HSP70 and HSP27 expression is associated with worse clinical outcome. Determination of tumoral HSP70 and HSP27 may be used as additional biomarker for risk stratification in colon cancer patients especially in UICC stage I/II patients.

FR-P-082 p.G13D mutations in the KRAS oncogene are associated with sensitivity of colorectal carcinoma cells to anti-EGFR antibody treatment I. Messner1, S.E. Baldus1, H.E. Gabbert1, K.-L. Schäfer1 1 Heinrich-Heine-University Düsseldorf, Inst. of Pathology, Düsseldorf Aims. Targeted therapies with the anti-EGFR antibodies panitumumab (Pmab) or cetuximab (Cmab) have shown significant benefit for patients suffering from metastatic colorectal carcinoma (mCRC). According to the approval by the American Food and Drug Administration and the European Medicines Agency, application of these antibodies is restricted to tumors without mutation in the KRAS oncogene. However, a recent metaanalysis of 579 CRC patients treated with cmab indicated that patients with p.G13D-mutated tumors (exchange of glycine at codon 13 to aspartic acid) showed a longer overall survival compared to patients with tumors mutated in KRAS codon 12. In order to study the functional impact of the subtype of KRAS mutation on the efficiency of EGFR-targeted therapies in CRC, we have determined the KRAS mutation status of colorectal carcinoma cell lines and correlated these data to the in vitro sensitivity of these cells to cmab and Pmab. Methods. Using a set of 15 colorectal cancer cell lines, the KRAS mutation status was evaluated by Sanger sequencing. Mutations in the potential predictive biomarkers BRAF and PIK3CA as well as protein expression of EGFR and PTEN were also determined. In vitro sensitivity of tumor cells to anti-EGFR antibodies was measured by standard MTT assays. Results. Seven of 15 cell lines showed a KRAS mutation including four cell lines exhibiting the p.G13D mutation. If these cell lines were treated using panitumumab or cetuximab, a significant growth inhibition was observed, while cell lines showing a KRAS mutation at codon 12 or 61 were not sensitive to anti-EGFR treatment. Only three of eight cell lines showing KRAS wild type status were sensitive to Pmab and cmab. Noteworthy, all of the five resistant KRAS wild type cell lines were either characterized by BRAF mutation or by absence of EGFR or PTEN protein expression, respectively. No correlation between PIK3CA mutation status and sensitivity to anti-EGFR treatment could be demonstrated. Conclusions. Our in vitro data from colon carcinoma cell lines indicate that tumor cells showing the KRAS p.G13D mutation may respond to EGFR-targeted therapy. Therefore, subtype analysis of KRAS mutation should be included in prospective clinical trials analyzing the responsiveness of CRC to cmab or Pmab. In KRAS wild type tumor cells, additional aberrations like BRAF mutation and loss of EGFR or PTEN expression may lead to resistance to EGFR-targeted therapy and should be considered as additional negative predictive biomarkers.

FR-P-083 UBCH10 overexpression in human colorectal cancers S. Piscuoglio1, P. Pallante2, L. Tornillo3, A. Fusco2, L. Terracciano3 1 University of Basel, Department of Biomedicine, basel, Switzerland, 2University of Naples, Istituto di Endocrinologia ed Oncologia Sperimentale “G. Salvatore” (IEOS-CNR) c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, naples, Italy, 3University of Basel, Institute of Pathology, Basel, Switzerland Aims. Colorectal cancer is the result of the accumulation of different genetic modifications including critical genes involved in the control of cell proliferation. In a large number of carcinomas with worst prognosis, lesions are not diagnosed until the disease is at an advanced stage. To diagnose cancer at an early stage and to establish more effective therapies featuring of key molecules in carcinogenesis is a critical step. UBCH10 (also known as E2C or UBE2C) is a cell cycle-related protein involved in mitosis completion. UbcH10 participates in proper metaphase to anaphase transition, and abrogation of UbcH10 results in the premature separation of sister chromatids. Thus, UbcH10 is essential for cell cycle progression, and his expression is cell-cycle dependent and related to proliferation. The aim of this study is to investigate the association of UbcH10 gene expression with clinicopathological features and tumor progression of colorectal cancer. Methods. We investigated the expression of the UBCH10 genes in a tissue microarray platform consisting of normal and neoplastic colorectal cancers (CRC) tissues by immunohistochemistry. UBCH10 was detectable in 889 patients. Immunoreactivity was scored semi-quantitatively by evaluating the number of positive tumor cells over the total number of tumor cells. Scores were assigned using 5% intervals and ranged from 0% to 100%. Median protein expression levels were used as cut-off scores to define protein marker positivity and the findings were associated with clinical-pathological parameters. Results. Our results demonstrated that UBCH10 is overexpressed in CRCs tissues compared to normal colon (p<0.001), and more specifically the UBCH10 expression is significant higher in tumor location: left- vs. right-sided (p=0.005). Moreover we found a significant relationship between overexpression of UBCH10 with T stage (p<0.001), N stage (p=0.022), tumor border configuration: pushing vs. infiltrating (p=0.035) and also UBCH10 overexpression was significantly correlated with shorter patient survival. Conclusions. Our study suggests that the overexpression of UbcH10 gene plays a critical role in the carcinogenesis and tumor progression of colorectal cancer. Also, our findings indicate that in colorectal carcinomas low expression of UBCH10 may be linked to a more aggressive tumor phenotype.

FR-P-084 Topoisomerase II alpha expression in colorectal cancer: a clinicopathological investigation of 430 tumor specimens C. Schrader1, F. Gieseler2, J.H. Bräsen3, B. Sipos4, C. Röcken3, W. Klapper3, H. Kalthoff5, W. v. Schönfels5, R. Lucius6, C. Pflüger1, S. Hinz5, H. Held7, T. Raff1, G. Klöppel8, J. Claasen1, S. Nazzal1, C. Schafmayer5 1 University Hospital of Kiel, 2nd Department of Medicine, Kiel, 2UKSH, Campus Lübeck, I. Department of Medicine, 3UKSH, Campus Kiel, Department of Pathology, 4University of Tübingen, Department of Pathology, 5UKSH, Campus Kiel, Department of Surgery, 6UKSH, Campus Kiel, Department of Anatomy, 7Hospital Neumünster, Department of Medicine, Kiel, 8University Munich, Department of Pathology Aims. Topoisomerase IIα is an enzyme that is needed whenever uncoiling of DNA is necessary during the cell cycle. The enzyme is a marker of cell proliferation. We analyzed the expression of topo IIα in relation to the clinical outcome in patients with colorectal cancer.

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Methods. Biopsy specimens from 430 untreated patients were investigated immunohistochemically with monoclonal antibodies against topo IIα (Ki-S4). Results. Patients with low (<50%) topo IIα expression had a median overall survival time of 66.1 months, compared to 130.5 months for patients with high (>50%) topo IIα expression. The Kaplan-Meier analysis showed a significant difference in the overall survival time related to the percentage of topo IIα (p=0.0012) positive tumor cells. Conclusions. Patients with colorectal cancer and high expression level of topo IIα have a superior clinical outcome compared to patients with low expression levels.

FR-P-085 Stem cell marker cancer testis antigen (CT 45) expression in colorectal cancer: an immunhistochemical study of 704 patients C. Schrader , F. Gieseler , J.H. Bräsen , B. Sipos , C. Röcken , W. Klapper , H. Kalthoff5, W. v. Schönfels5, R. Lucius6, C. Pflüger1, S. Hinz5, H. Held7, T. Raff1, G. Klöppel8, J. Claasen1, S. Nazzal1, C. Dreyer1, C. Schafmayer5 1 University Hospital of Kiel, 2nd Department of Medicine, Kiel, 2UKSH, Campus Lübeck, I. Department of Medicine, 3UKSH, Campus Kiel, Department of Pathology, 4University of Tübingen, Department of Pathology, 5UKSH, Campus Kiel, Department of Surgery, 6UKSH, Campus Kiel, Department of Anatomy, 7Hospital Neumünster, Department of Medicine, Kiel, 8University Munich, Department of Pathology 1

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Aims. A subset of colorectal cancer derived from stem cells. Cancer testis antigen (CT45) is expressed in immature gonocytes, spermatogonia and also in germ cell tumors. There are only limited data about CT 45 expression in colorectal cancer and no data if the expression in tumor cells has an influence on the clinical course of the disease. Methods. We investigated immunohistochemically embedded tissue from 704 patients with a monoclonal antibody against CT 45 (Ki-A10). The percentage of CT45 expressing cells was quantified according to the following classification: negative, 0–25%, 25–50%, 50–75% and more than 75% positive tumor cells. Results. The majority of cases were negative (n=633). 71 tumor specimens were CT45 positive. The Kaplan Meier analysis revealed no significant difference (p=0.11) in the clinical outcome of CT 45 positive and negative cases Conclusions. Cancer testis antigen (CT 45) is expressed in a subset of colorectal cancer. The CT 45 expression is no prognostic factor for the clinical outcome of the disease, but it might have clinical implication for therapy decision of stem cell derived cancer.

FR-P-086 SOX9 promotes cell migration and anchorage-independent growth of colorectal cancer cell lines C. Hui1, X. Enping1 Zhejiang University, School of Medicine, Hangzhou, China

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Aims. SOX9 is a high-mobility group (HMG) domain transcription factor that plays a critical role in multiple aspects of development and differentiation. There is a close connection between normal development and oncogenesis. Recent studies have revealed that SOX9 is implicated in several types of cancer progression, but SOX9 does not have consistent function roles in different cancers. Importantly, the function and role of SOX9 in colorectal cancer (CRC) exactly remains unclear. In our previous study we identified that SOX9 overexpression in CRC as an independent predictor of an unfavorable outcome for CRC in Chinese population. This study is to show the role of SOX9 in CRC cell lines. Methods. Western blotting, RT-PCR and Q-PCR were performed to detect the expression of SOX9 in CRC cell lines. Stably SOX9-overexpression RKO cell was constructed. Subsequently, Cell Counting Kit-8

assay and Soft agar colony formation assay, Annexin v-PE/7-AAD Double Stain Apoptosis Detection assay, xCELLigence system, cell migration and 5-Fu cell toxicity test were performed to detect the roles of SOX9 in vitro. Results. After performing gain-of-function studies, it was clarified that the SOX9-overexpression RKO cell had no significant effects on cell proliferation, cell apoptosis and 5-Fu cell toxicity; In migration assay, it is revealed that SOX9 could promote cell migration by transwell chamber and xCELLigence system. Also it was assessed that the SOX9-overexpression RKO cell had impact on anchorage-independent growth by using soft agar colony formation assay. Conclusions. The expression of SOX9 can promote cell migration and anchorage-independent growth, but it has no effect on cell proliferation.

FR-P-087 RegIV promotes migration and invasion of CRC cell lines Z. Jie1, W. Jingyu1, L. Maode1 Zhejiang University, School of Medicine, Hangzhou, China

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Aims. The regenerating gene IV (RegIV) plays an important role in colorectal tumorigenesis. However, its biological functions have not been well elucidated. Methods. RT-PCR and western blotting analysis were performed to detect the expression of RegIV in colorectal carcinoma (CRC) cell lines HT29, RKO and LOVO, then the stably RegIV overexpressed and Reg4 shRNA-silenced cell lines were constructed in the RegIV negative-expressed and positive-expressed cell lines respectively. Subsequently, Cell counting Kit-8 (cck-8) assay, EdU assay and xCELLigence system assay were performed to detect cell proliferation. Transwell chamber and matrigel assays were used to detect cell migration and invasion. Results. RT-PCR and western blotting showed that RKO and HT29 cell lines do not expressed RegIV, while LOVO cell lines expressed RegIV. After performing gain-of-function and loss-of-function studies, it has been shown that both RegIV overexpressed and RegIV shRNA-silenced cell lines have no significant effects on cell proliferation. In contrast to control, migration and invasion abilities are significantly increased on RegIV overexpressed cell line and are significantly decreased on RegIV shRNA- silenced cell lines. Conclusions. The expression of RegIV can promote CRC cell lines to migrate and invade, but it has no effect on proliferation.

FR-P-088 Minichromosome maintenance protein 6 (MCM 6) expression in colorectal cancer: a proliferation marker and a prognostic factor for clinical outcome C. Schrader1, F. Gieseler2, J.H. Bräsen3, B. Sipos4, C. Röcken3, W. Klapper3, H. Kalthoff5, W. v. Schönfels5, R. Lucius6, C. Pflüger1, S. Hinz5, H. Held7, T. Raff1, G. Klöppel8, J. Claasen1, S. Nazzal1, C. Schafmayer5 1 University Hospital of Kiel, 2nd Department of Medicine, Kiel, 2UKSH, Campus Lübeck, I. Department of Medicine, 3UKSH, Campus Kiel, Department of Pathology, 4University of Tübingen, Department of Pathology, 5UKSH, Campus Kiel, Department of Surgery, 6UKSH, Campus Kiel, Department of Anatomy, 7Hospital Neumünster, Department of Medicine, Kiel, 8University Munich, Department of Pathology Aims. MCM6 is one of six proteins of the MCM family which are involved in the initiation of DNA replication and is a marker of proliferating cells. Proliferation markers and their expression levels as prognostic factors are discussed controversial in colorectal cancer. Methods. We investigated paraffin embedded tissue from 570  patients with colorectal cancer with stage 1 to 4  immunohistochemically with a monoclonal antibody against MCM6. 500  tumor cells were counted and the percentage of positive cells was calculated. Overall survival time Der Pathologe Suppl 1 · 2012 

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Abstracts was calculated from the date of diagnosis until death or loss to follow-up evaluation. Univariate survival analysis was computed by means of the Kaplan-Meier method and significance levels were assessed by means of the log-rank test. Results. The percentage of MCM6 expressing tumor cells ranged from 27.6% to 97.0%, with a mean of 82.8%. A high MCM6 expression level of more than 80% positive cells was associated with a significantly longer overall survival time (130.5 months) compared to patients with a low MCM6 expression level of less than 80% (58.7 months, p=0.0016). Conclusions. Immunohistochemical detection of MCM6 seems to be a promising marker for predicting the outcome in patients with colorectal cancer.

FR-P-089 Lymphangiogenesis in regional lymph nodes is an independent prognostic marker in rectal cancer patients after neoadjuvant treatment M. Muders1, C. Jakob1, D. Aust1, G. Folprecht2, K. Datta3, G.B. Baretton1 University Hospital Carl Gustav Carus at the University of Dresden, Institute of Pathology, Dresden, 2University Hospital Carl Gustav Carus at the University of Dresden, Department of Internal Medicine, Dresden, 3University of Nebraska Medical Center, Omaha, NE, United States 1

Aims. One of the major prognostic factors in rectal cancer is lymph node metastasis. The formation of lymph node metastases is dependent on the existence of a premetastatic niche. An important factor preceding metastasis are lymph vessels which are located in the lymph node. Accordingly, the occurrence of intranodal lymphangiogenesis is thought to indicate distant metastasis and worse prognosis. In this study we evaluate the significance of lymph node lymphangiogenesis in a cohort of rectal cancer patients who are treated with neoadjuvant radiochemotherapy. Methods. We studied formalin fixed, paraffin embedded adenocarcinomas and regional lymph nodes of 203 rectal cancer patients who were treated with neoadjuvant radiochemotherapy and consecutive curative surgery with cancer free surgical margins (R0). Regional lymph nodes were detected by immunohistochemistry for podoplanin (D2-40). These results were then correlated with disease free survival and stage of disease using standard statistical methods. Results. The presence of lymphatic vessels in regional lymph nodes significantly affects the disease-free survival in univariate and multivariate analyses. In contrast, there was no correlation between peritumoral or intratumoral lymph vessel density and prognosis. Conclusions. Our study demonstrates the importance of lymphangiogenesis in regional lymph nodes after neoadjuvant radiochemotherapy and consecutive surgery as an independent prognostic marker. Staining for intranodal lymphangiogenesis and methods of intravital imaging of lymphangiogenesis and lymphatic flow may be a useful strategy to predict long-term outcome in rectal cancer patients. Furthermore, addition of VEGF-blocking agents to standardized neoadjuvant treatment schemes might be indicated in advanced rectal cancer.

FR-P-090 FGFR1 amplification in primary and lymph node metastatic colorectal cancer A. Göke1, F. Goeke1, D. Boehm1, R. Menon1, W. Weichert2, R. Buettner3, S. Perner1 1 Universitätsklinikum Bonn/Institut für Pathologie, Institute of Prostate Cancer Research, 2University Hospital of Heidelberg/Institute of Pathology, 3 University Hospital of cologne/Institute of Pathology Aims. Previous studies showed that up to 20% of squamous cell lung cancers display an amplification of the fibroblast growth factor receptor 1 (FGFR1) gene and that these cases are potentially sensitive to treatment

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with a specific inhibitor. By exploring the largest publicly available database of somatic copy number changes in tumors (www.broadinstitute/tumorscape.org), we found evidence that also a subset of colorectal cancers (CRC) might be characterized by FGFR1 amplification. Consequently, we evaluated the frequency of FGFR1 amplifications in both primary and lymph node metastatic CRC by applying in-situ detection. Moreover, our objective was to determine whether FGFR1 can be used as a potential therapeutic drug target in primary and progressed CRC. Methods. We assessed 450 paraffin embedded primary CRC samples embedded in a tissue microarray format. Of these, 94 cases had corresponding lymph node metastases. A target probe located on the FGFR1 locus spanning 8p11.23 to 8p11.22 and a centromeric reference probe were used to determine the FGFR1 amplification status using fluorescence in situ hybridization (FISH). Furthermore, CRC cell lines are currently being tested for their FGFR1 amplification status. FGFR1 amplified cell lines will then be treated with a FGFR specific inhibitor. Results. 4.2% (19/450) of primary CRC displayed FGFR1 amplification. Of primary CRCs with corresponding lymph node metastasis 4.2% (4/94) harbored FGFR1 amplification in both, and 2.1% (2/94) cases showed FGFR1 amplification in only the primary tumor, but not in the metastasis. Conclusions. FGFR1 amplification is a recurrent event found in CRC. Furthermore, we suggest that FGFR1 amplification is a clonal event in tumor progression since the FGFR1 amplification status transferred to the corresponding lymph nodes in most of our cases. Our study may indicate novel therapeutic possibilities for patients suffering from primary and metastatic CRC.

FR-P-091 Neoadjuvant treated liver metastasis of colorectal cancer: Is the histopathological regression grade a useful predictor for survival? P. Bronsert1, S. Ruhm2, H. Neef3, S. Timme1, M. Werner1, G. Illerhaus2, F. Makowiec3 1 Albert-Ludwigs-University Freiburg, Institute of Pathology, Freiburg, 2 Albert-Ludwigs-University Freiburg, Department of Haemato-Oncology, Freiburg, 3Albert-Ludwigs-University Freiburg, Department of General and Visceral Surgery, Freiburg Aims. Surgical treatment of resectable liver metastasis of colorectal cancer (CRC-LM) after neoadjuvant therapy (NACT) is a common method worldwide. For several tumour entities (e.g. esophageal, gastric and breast cancer) histopathological tumor regression grade (TRG) is well known as a significant predictive marker. In terms of CRC-LM less is known about the predictive potential of TRG in relation to overall survival (OS). In this study we determined the TRG of neoadjuvant treated CRC-LM and correlated those data with OS and other clinicopathological parameters. Methods. The collective contains 89  patients, 68 (76%) of which were treated with an Oxaliplatin or Irinotecan containing NACT and 21 (27%) of which were treated with a “targeted therapy” using antibodies. Median NACT period time was 6  month (1–24  months) and the average number of liver metastasis per patient was 2 (1–11 metastasis). Complete hepatical resection (R0) was observed in 80 patients (89%), a complete overall resection (R0, cm0) in 69 patients (77%). From formalin fixed paraffin embedded tumor tissue of the resection specimen haematoxylin and eosin staining was performed. We determined the TRG according to a previously published score from Rubbia-Brandt et al. – a quantitative scoring system ranging from score 1 (complete regression) to score 5 (no cytopathic effect). After scoring, the regression was correlated to clinicopathological parameters including patient survival. Results. Most patients (62/89; 69%) showed a poor TRG (score 4/5) and 24/89 (27%) showed a moderate response (score 2/3). A mere 3/89 patients (4%) had a complete regression (score 1). The 5-year survival rate of all 89 patients was 41%. OS of patients with complete regression was 100%;

the OS rate was 53% for patients with a moderate and 33% for patients with a poor regression (p=0.08). Other factors such as gender, total tumour count and size, nodal status, localisation of the primaries, as well as duration and mode of NACT did not correlate in uni- or multivariate analysis with OS. Multivariate analysis showed an associated correlation between overall-resection (p<0.02) and TRG (p=0.06). Conclusions. As shown previously in a small number of publications, our results, generated out of a much lager study cohort, underline that a complete histological response after NACT is rare. Besides quantifying TRG proves a clear prognostic trend and should be integrated into clinicopathological diagnostics.

FR-P-092 G-beta proteins in colorectal carcinomas: Mechanisms of cell death resistance and novel therapeutic targets D . Fuchs1, M . Metzig1, S . Macher-Göppinger1, G . Gdynia1, P . Schirmacher2, W . Roth1 1 Institute of Pathology, University Hospital Heidelberg, and German Cancer Research Center, Heidelberg, 2Institute of Pathology, University Hospital Heidelberg, Heidelberg Aims. Resistance to apoptotic cell death is a major contributing factor for the development of cancer as well as for therapy resistance. G proteins are heterotrimeric proteins consisting of alpha, beta and gamma subunits. They mediate signals of G protein-coupled receptors and are involved in important biological processes such as proliferation, apoptosis, and migration. However, the functional relevance of beta subunits of G proteins in cancer is largely unknown. The aim of the project was to elucidate the contribution of G beta proteins to cell death resistance in colorectal carcinomas. Methods. Cell culture, Western blotting, quantitative RT-PCR, apoptosis assays (Annexin-V-FACS and PI), siRNA transfection, cytotoxicity assays, ectopic transgene expression, Luciferase assays, cell cycle analysis (flow cytometry), immunohistochemistry, co-immunoprecipitations. Results. Here, we show that G protein beta 5 induces resistance to cell death induced by chemotherapeutic drugs (e.g. oxaliplatin) and death ligands (e.g. TRAIL) in colon carcinoma cells. This is associated with a reduced cell surface expression of death receptors as well as with an increased expression of the anti-apoptotic protein XIAP. Moreover, G protein beta 5 activates NF-kappa-B signaling, as demonstrated by luciferase assays and immunoblotting of I-kappa-B-alpha. Finally, pharmaceutical inhibition of beta G proteins results in a strong sensitization to cell death in colon carcinoma cells. Conclusions. Our results describe a novel mechanism of apoptosis resistance which might provide strong survival advantages for colon cancer cells overexpressing G protein beta  5. At the same time, these results identify G protein beta 5 as a novel therapeutic target in colon cancer. Currently, we are evaluating G protein inhibitors as a new experimental treatment strategy for colorectal carcinomas.

FR-P-093 The tissue compatibility of barrier materials in peritoneal adhesion prevention is not predicted by CD 68-positive macrophages C . Brochhausen1, V .H . Schmitt1, A . Mamilos1, C .N .E . Planck2, B . Krämer2, T .K . Rajab3, H . Hierlemann4, H . Planck4, C .J . Kirkpatrick1 1 University Medical Centre, Institute of Pathology, Mainz, 2Eberhard Karls University, Clinic of Gynecology, Tübingen, 3Harvard Medical School, Brigham and Women’s Hospital, Boston, United States, 4Institute of Textile Technology and Process Engineering, Denkendorf Aims. Macrophages (M*) are involved in inflammation and foreign body response. Postoperative peritoneal adhesions are a clinical burden and the optimal approach to prevent adhesions has not yet been found. The-

refore, the biocompatibility of barrier materials has to be assessed. To evaluate whether the total population of CD68-positive M* correlates with the degree of fibrosis or inflammation we compared five approaches which are clinically used as adhesion barriers with an untreated control group. Methods. Via standardised method Wistar rats underwent peritoneal damage and were treated with either Adept® (n=14), Intercoat® (n=33), Spraygel® (n=8), Seprafilm® (n=29), SupraSeal® (n=14) or remained untreated (control, n=14). 14 days postoperatively, the treated areas were explanted and underwent standardised methods for histological and immunohistological assessment of fibrosis, inflammation (haematoxylin & eosin, chloracetate esterase) and CD68-positive M* (CD68 staining). The evaluation was undertaken by two independent investigators. Results. Between the population size of CD68-positive M* and the extent of fibrosis or inflammation no correlation was found. Intercoat® and SupraSeal® revealed no fibrosis next to a moderate infiltration of M*. Within Seprafilm®, moderate inflammation correlated with a likewise moderate infiltration of macrophages. In contrast, the control and Adept® groups revealed minimal numbers of M* next to minimal fibrosis. In the SupraSeal® group, both the inflammatory reaction and the account of M* were moderate. Minimal inflammation concomitant with moderate M* infiltration was seen within Intercoat® and Seprafilm®, whereas the control and the Adept® revealed minimal inflammation next to minimal infiltration with M*. Altogether, no correlation could be established between the number of CD68-positive M* and the subsequent tissue response. Conclusions. In adhesion prevention, the tissue compatibility with respect to inflammation and fibrosis can not be predicted by CD68-positive M* infiltration. However, CD68-poistive M* can be divided into three subpopulations according to their functions: immune response, wound healing including the generation of fibrosis and the regulation of both. Hence, as the number of CD68-positive M* gives no information about the biocompatibility of adhesion barriers, future studies should investigate whether these subpopulations can predict the tissue response to adhesion barriers and reveal their role in peritoneal adhesion formation.

Poster: GI-Trakt: Hepatobiliäres System und Pankreas FR-P-094 Amplification of fibroblast growth factor receptor 1 (FGFR1) in pancreatic ductal adenocarcinoma N .C . Lehnen1, A . von Mässenhausen1, T . Glowka2, H . Zhou1, S . Perner1, I . Gütgemann1 1 University of Bonn, Institute of Pathology, Bonn, 2University of Bonn, Department of General, Visceral, Thoracic and Vascular Surgery, Bonn Aims. Pancreatic ductal adenocarcinomas are rarely curable with an extremely poor survival confined to cases with disease localized to the pancreas. Tyrosine kinase receptor inhibitors (TKIs) are currently evaluated in several carcinomas including pancreatic adenocarcinoma. Therefore, expression and/or amplification of tyrosine kinase associated receptors, such as members of the fibroblast growth factor receptor (FGFR) family, even in a small subset of patients, becomes increasingly important. Our aim was to identify cases of pancreatic ductal adenocarcinoma with amplification and possible overexpression of FGFR1 in order to improve patient selection prior to treatment with TKIs. Methods. We performed fluorescence in situ analysis (FISH) of the FGFR1 region (8p12) and corresponding centromeric probe on 128 formalin fixed tissues of pancreatic ductal adenocarcinomas (primary resection specimens) using tissue microarrays. Protein expression was assessed by immunohistochemistry and the results as well as clinicopathologic parameters were correlated with the above. Currently, FISH analysis and protein expression studies are performed in several pancreatic cell lines. Der Pathologe Suppl 1 · 2012  | 

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Abstracts Results. Amplification of the FGFR1 region was rare, but could be found in three of 128 cases of pancreatic ductal adenocarcinoma (3/128; 2.3%) in our collective. Slightly more protein expression of FGFR1 was observed with commercially available FGFR1 antibodies by standard immunohistochemistry. So far, none of the pancreatic carcinoma cell lines showed FGFR1 amplification. Conclusions. FGFR1 amplification can be identified in a small subset of pancreatic adenocarcinomas. FGFR1 is a tyrosine kinase receptor protein which is able to promote tumor progression by altering angiogenesis, tumor cell proliferation, migration and cell survival. In the future, it will be necessary to characterize the biological role of FGFR1 amplification in pancreatic adenocarcinoma. Further studies are needed to determine, whether the patients with FGFR1 amplification and/or overexpression need to be pre-selected prior to TKI treatment.

FR-P-095 Paraduodenal pancreatitis: mini-series with regard to vessel obliteration T. Hansen1, F. Vitali2, R. Kießlich3, S. Heinrich4, P. Mildenberger5, A. Kumar5, L. Frulloni2, C.J. Kirkpatrick1 1 University of Mainz, Institute of Pathology, Mainz, 2University of Verona, Department of Medicine, Verona, Italy, 3University of Mainz, Department of Internal Medicine, Mainz, 4University of Mainz, Department of General and Abdominal Surgery, Mainz, 5University of Mainz, Department of Radiology, Mainz Aims. Paraduodenal pancreatitis comprises a form of chronic pancreatitis involving the duodenal wall close to the minor papilla within the surrounding parenchymal tissue and common bile duct (also called groove area). This disorder most commonly affects male patients in the 5th decade with a history of alcohol and/or nicotine abuse. Concerning the pathogenesis, it is believed that alcohol or smoking leads to a resistance of the pancreatic juice flow and ischemia of the paraduodenal tissue, giving relapsing episodes of pancreatitis. We report on a series of patients with paraduodenal pancreatitis with emphasis on vascular changes. Methods. We describe ten patients (all male, mean age 45.4 yrs). Most of them presented with abdominal pain (n=8), and weight loss was additionally found in eight patients as well. All patients were smokers; history of alcohol abuse could be confirmed in seven cases. In all patients, a pancreatico-duodenectomy was performed. For histological evaluation, tissue specimens were routinely processed. Results. The following characteristic histological phenomena of paraduodenal pancreatitis were observed: Brunner’s gland hyperplasia occurred in all cases, while cystic changes of the duodenal wall and adenomyomatosis of the duodenal wall were found in 9/10 patients. Variable numbers of a mixed inflammatory infiltrate were present in all patients analyzed. In 50%, we found foreign body giant cell reaction in the neighbourhood of some pseudocysts. However, most interestingly obliteration of segmental arteries was present in 6/10 cases. Conclusions. This histological study confirms the common morphological changes in paraduodenal pancreatitis. Interestingly, we found vessel obliteration in several cases, which has not been described for this subtype of chronic pancreatitis so far. It remains to be investigated whether this specific finding might reflect a particular subgroup of paraduodenal pancreatitis.

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FR-P-096 Diagnostic value of immunohistochemical IMP3 expression in core needle biopsies of pancreatic ductal adenocarcinoma D.L. Wachter1, A. Schlabrakowski2, J. Hoegel3, G. Kristiansen4, A. Hartmann1, M.-O. Riener1 1 University Hospital Erlangen-Nuremberg, Institute of Pathology, Erlangen, 2 Nuremberg Clinic Center, Insitute of Pathology, 3Institute of Human Genetics, University Hospital Ulm, 4University Hospital Zurich, Zürich, Switzerland Aims. The oncofetal protein, insulin-like growth factor-II messenger ribonucleic acid-binding protein 3 (IMP3), has been analyzed in many different tumors. Various studies have found that IMP3 is a marker for malignancy and is correlated with increased tumor aggressiveness and reduced overall survival. The diagnosis of pancreatic ductal adenocarcinoma (PDAC) in core needle biopsies can be challenging, and immunohistochemical markers are needed. Methods. We studied IMP3 expression in 177 core needle biopsies of the pancreas, including 112 PDACs, 55 cases with chronic sclerosing pancreatitis, and 10 biopsies with tumor-free pancreatic tissue without inflammation. An additional 18 biopsies of PDAC metastases (16 liver biopsies and 2 lymph node biopsies) were analyzed. To study IMP3 expression in large tissue sections, 45 pancreatic resection specimens (26 with PDAC and 19 with chronic sclerosing pancreatitis) were investigated. Results. In contrast to normal or inflamed pancreatic tissue, which was negative in 47 of 65 (72.3%) cases and weakly positive in 15 of 65 (23.1%) cases, strong IMP3 expression was found in 99 of 112 (88.4%) PDACs. Therefore, sensitivity and specificity of IMP3 expression in the differential diagnosis of PDAC and chronic sclerosing pancreatitis using core needle biopsies were found to be 88.4% and 94.6%, respectively. These results were confirmed in the pancreas resection specimens. Furthermore, strong IMP3 expression was found in 17 of 18 (94.4%) of the PDAC metastases that were analyzed. Conclusions. Our study shows that IMP3 is an easy to use and potentially new immunohistochemical marker for the diagnosis of PDAC in core needle biopsies.

FR-P-097 Molecular analysis of putative therapeutic targets in pancreatic acinar cell carcinomas F. Bergmann1, H. Bläker2, A. Harjung1, P. Mayer1, B. Sipos3, G. Klöppel4, P. Schirmacher1 1 University of Heidelberg/ Institute of Pathology, Heidelberg, 2Charité Berlin, Institute of Pathology, 3University of Tübingen, Institute of Pathology, 4Technical University Munich, Institute of Pathology Aims. Pancreatic acinar cell carcinomas represent aggressive tumors who frequently display metastases at the time of diagnosis. Because they are rare, established therapeutic concepts other than surgery practically do not exist. The aim of the present study was to evaluate established and innovative therapeutic markers in these to date poorly understood tumors. Methods. Putative therapeutic targets were investigated by means of direct sequencing, immunohistochemistry, and/or Western blot analyses in a series of 60 pancreatic acinar cell carcinomas. Results. 4% of the tumors displayed k-ras mutations. While 40% of the acinar cell carcinomas showed immunohistochemical expression of EGFR, no EGFR mutations were detected. Heat shock protein 90, heat shock protein 70, L1CAM, and Her2/neu were expressed in 100%, 90%, 65%, and 0% of the tumors, each. mgMT deficiency was found in 28% of the tumors. Conclusions. EGFR, heat shock proteins 90 and 70, L1CAM, and mgMT represent promising targets and predictive markers for the therapy of inoperable or progressive pancreatic acinar cell carcinomas.

FR-P-098 Immunohistological staining with the monoclonal antibody Em2G11 is highly specific and sensitive for Echinococcus multilocularis larvae in human tissue T.S. Herrmann1, D. Tappe2, L. Stark1, B. Grüner3, K. Buttenschön4, D. HenneBruns5, P. Kern6, P. Möller1, P. Kern3, P. Deplazes7, T.F.E. Barth1 1 University of Ulm, Institute of Pathology, Ulm, 2University of Würzburg, 3 University Hospital and Medical Center Ulm, Division of Infectious Diseases, Ulm, 4University of Alberta, Department of Surgery, Edmonton, Canada, 5 University Hospital of Ulm, Department of General, Visceral, and Transplantation Surgery, Ulm, 6University of Ulm, Institute of Epidemiology and Medical Biometry, Ulm, 7University of Zurich, Institute of Parasitology, Zürich, Switzerland Aims. Alveolar echinococcosis (AE) and cystic echinococcosis (CE) are two parasitic diseases in humans caused by the metacestode stages of Echinococcus multilocularis and Echinococcus granulosus, respectively. Differential diagnosis is fundamental for the choice of specific therapy strategies and prognosis. Methods. We have analyzed 96 archived formalin-fixed, paraffin-embedded tissue samples, including 5 cutting needle biopsies and 3 fine needle aspirates from patients with AE or CE with the monoclonal antibody MAbG11, specific for the antigen Em2G11 in the laminated layer of the metacestode of E. multilocularis. Results. We show that, in human tissue, staining with MAbG11 is highly specific for the laminated layer and the calcareous corpuscles of the E. multilocularis metacestode while no staining was observed in the metacestode stage of E. granulosus. Furthermore, the antibody marks small particles of E. multilocularis (Spems) of less than 1 µM in the necrotic tissue surrounding the main lesion. Spems are also detected in the liver sinusoids and lymphatic tissue outside the main lesion, most probably due to shedding from the laminated layer. In 6 of 96 samples, the conventional histological diagnosis based on haematoxylin and eosin and PAS stainings alone had to be adjusted when revised by immunohistology with MAbG11. The specific staining was proved on bioptic material fixed for more than 60 years in formalin. Conclusions. We conclude that the MAbG11-antibody is a highly specific tool in the diagnostic algorithm of AE also on long-time archived formalin-fixed or paraffin-embedded tissue. The staining modality with small particles outside the main lesion of E. multilocularis sheds new light on the interaction of the parasite with the human host and suggests a systemic effect.

FR-P-099 Chronic HEV hepatitis after liver transplantation: a clinicopathological follow up over 3 years with implications for the differential diagnosis of chronic hepatitis J. Friemel1, F. Böhm1, M. Bawohl1, E. Marques-Maggio1, J. Seebach2, P. Dutkowski3, B. Müllhaupt4, U. Protzer2, A. Weber1 1 University of Zurich, Pathology, Zürich, Switzerland, 2Technical University (TU) Munich, Institute of Virology, München, 3University of Zurich, Visceraland Transplant Surgery, Zürich, Switzerland, 4University of Zurich, Hepatology and Gastroenterology, Zürich, Switzerland

ORF2 gene region. Liver enzymes and viral infections were serologically monitored. Results. The patient developed elevated liver enzymes eight months after LTX. Liver biopsy by that time showed spotty eosinophilic necrosis, but no inflammation. After 8  month, HEV-RNA was detected serologically and in the liver biopsy. Follow-up biopsies revealed a chronic hepatitis with mild portal and lobular inflammation, ductular reaction and portal fibrosis. No other viruses (hepatitis viruses A, B, C and D; EBV, ADV, cmV) were detectable. Whereas retrospective testing of the explanted liver and day 1 biopsy were negative, follow-up biopsies were HEV-positive for now more than 3 years. Conclusions. The case presented illustrates that HEV infection is an upcoming cause of chronic hepatitis in particular in immunosuppressed patients. The histologic pattern is similar to the chronic hepatitis C infection. Histologic diagnosis is challenging in cases of unusual histology, e.g. with little inflammation. Molecular testing is helpful in such cases.

FR-P-100 eIF3a as a putative gall bladder cancer target A.K. Mehta1, R. Spilka2, C. Lackner1, H. Müller1, S. Lax3, P. Obrist2, J. Haybaeck1 1 Institute of Pathology, Medical University of Graz, Austria, 2Pathologylab Dr. Obrist & Dr. Brunhuber OG, Zams, Austria, 3Department of Pathology, General Hospital Graz West, Graz, Austria Aims. Gallbladder carcinoma (GBC) is the most common type of biliary tree carcinomas. GBC is an aggressive and often lethal cancer which is usually diagnosed at advanced stage with a 5-year survival rate <10%. eIF3a is the largest subunit of eukaryotic initiation complex eIF3a, which regulates a subset of mRNA involved in regulation of cell cycle. eIF3a is up-regulated in many cancer types and suppression of eIF3a expression leads to regression of tumour cells in vitro. We investigated the expression of eIF3a in GBC with the intention to improve currently used prognostic markers of GBC and in order to elucidate its possible implication as therapeutic target. Methods. eIF3a expression was determined by semiquantitative immunohistochemistry on paraffin embedded tissue from 74  GBC patients and compared with the patient survival times. Expression of eIF3a mRNA and protein levels were analysed in three GBC cell lines and one extrahepatic bile duct carcinoma cell line using immunocytochemistry, Western blotting and quantitative real-time PCR (qPCR). Results. Higher intensity scores of isolated focal areas in patient samples of GBC showed a 16% worse 5 year survival rate when compared to GBC with lower intensity and homogenously stained areas. The difference was significant. Expression levels of eIF3a mRNA was in all cell lines approximately 2-fold higher than in the normal gallbladder tissue, which was confirmed on protein level. Conclusions. Overexpression of eIF3a in GBC seems to be an indicator of a more aggressive type of GBC with lower 5-year survival rates. Gallbladder and extrahepatic bile duct cancer cell lines provide a model for studying the mechanistic role of eIF3a in tumour progression of GBC. Currently potential eIF3a dependent effects upon treatment with various substances are studied.

Aims. Hepatitis E virus (HEV) infection usually manifests as an acute and self-limiting hepatitis. Recently, cases of chronic HEV infection following (solid) organ transplantation have been reported. Here, we present the case of a 57-year-old man who de novo developed a chronic HEV infection following liver transplantation (LTX). Methods. The formalin-fixed and paraffin-embedded (FFPE) tissues of the explant liver and six liver biopsies (taken 1 day to >3 years after LTX) were routinely processed for histology. In addition, immunostains (CMV and ADV) and in situ hybridisation (EBV) were performed as well as RT-PCR testing on FFPE material for HEV, targeting the HEV Der Pathologe Suppl 1 · 2012 

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Abstracts FR-P-101 Metachronic coincidence of a neuroendocrine carcinoma of the gall bladder after former endometrioid ovarian adenocarcinoma M. Petersen1, T. Kalinski2, H. Lippert1, J. Bischoff3, U.R. Bohr4, F. Meyer1 1 University Hospital, Dept. of Surgery, Magdeburg, 2University Hospital, Institute of Pathology, Magdeburg, 3University Hospital, Dept. of Gynecology & Obstetrics, Magdeburg, 4University Hospital, Dept. of Gastroenterology, Hepatology & Infectious Diseases, Magdeburg Aims. Along the demographic development, there is an increasing agedependent multimorbidity. In particular, tumor diseases and second malignancies will have an impact on the future profile of medical care. Simultaneous or subsequent (within a short-term interval) occurrence of two tumor manifestations is always a clinical challenge, especially with regard to finding the correct diagnosis clarifying malignancy of the same or different entity. Methods. By means of an extraordinary and exemplary case report, the challenge is demonstrated to precisely diagnose a tumor lesion based on available results of investigations and to differentiate between recurrent tumor growth and a metastasized tumor manifestation of a second (different) tumor lesion. Results. Medical history and clinical finding: In a 73-year-old woman, histopathological investigation of a specimen after cholecystectomy revealed a slightly differentiated adenocarcinoma including a lymph node metastasis. To exclude a metastasis of a known endometrioid ovarian adenocarcinoma, comparing immunhistological investigations resulted in: i) CK7-/CA125-negative, CK20-/chromogranin-A-/synaptophysinpositive (counting for gall bladder and lymph node metastasis); ii) CK7-/ CA125-positive, CK20-/Chromogranin-A-/synaptophysin-negative (counting for ovar). Therefore, tumor lesion of the gall bladder and the lymph node was rather associated with a neuroendocrine carcinoma of the gall bladder than with a metastasis of an ovarian carcinoma. Based on the diagnosis, re-laparotomy was performed including resection of the cystic stump and atypical resection of hepatic segment V (“liver bed” of the gall bladder) as well as lymph node dissection of the hepatoduodenal ligament. Clinical course: After 14  months, a further metastasis of a neuroendocrine carcinoma within an additional lymph node was diagnosed. There had been no recurrent tumor growth of the left-sided endometrioid ovarian adenocarcinoma since 8 years. Conclusions. Due to the increased incidence of tumor diseases and second neoplasias, histopathological and immunohistochemical analyses are very important in the diagnostic process. According to the available literature, the presented case is the first description of a metachronous coincidence of an endometrioid ovarian adenocarcinoma and a neuroendocrine carcinoma of the gall bladder.

FR-P-102 No crush artifacts: statistical analysis of the histological changes in the donor bile ducts with special regard to ischemic-type biliary lesion (ITBL) T. Hansen1, D. Hollemann1, M. Heise2, M. Hoppe-Lotichius2, C.J. Kirkpatrick1, G. Otto2 1 University of Mainz, Institute of Pathology, Mainz, 2University of Mainz, Department of Transplantation/Hepatobiliopancreatic Surgery, Mainz Aims. Ischemic-type biliary lesion (ITBL) is a feared complication after liver transplantation as it often leads to graft loss. Recently, we presented the histological changes of the donor bile ducts taken during liver transplantation. We now performed a scoring system and statistically analysed the results. With special reference to ITBL, significant findings are now demonstrated. Methods. The histomorphology of the donor bile ducts was studied 84  patients (recipients: 68  male, 16  female, median age 58  yrs; donors: 40 male, 44 female; median age 52.5 yrs). For the characteristic histological findings, a histological scoring system was established. The single

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phenomena and the sum score were then statistically reviewed with regard to the development of ITBL. χ2-test was used to compare the different score values; p<0.05 was considered significant. Results. The recently described common findings could be confirmed in this larger cohort: most commonly, we found mucosal loss and intramural bleeding in the bile ducts. Furthermore, we observed some distinct vessel alterations such as subendothelial edema in about 77%, and arteriolonecrosis in 44% of the cases. In addition, there was vessel thrombosis in 25% of the cases studied. Slight inflammation was a common finding as well. Fourteen patients of this cohort (16.7%) developed ITBL. In the donor bile ducts of ITBL patients, bile duct necrosis, and arteriolonecrosis were significantly more frequently found than in the non-ITBLgroup (p values 0.001 and 0.007 respectively). Furthermore, the overall sum score for all histological subphenomena was significantly higher in the ITBL-group as compared to the non-ITBL group (p=0.007). Conclusions. These results confirm our previous report on common histological findings in the donor bile ducts. Most interestingly, some of the histological phenomena as well as the histological sum score are significantly associated with a latter development of ITBL. Further investigation should elucidate whether histological examination of the donor bile ducts might help to estimate the risk of ITBL in transplanted patients.

FR-P-103 Deleted in malignant brain tumors 1 (DMBT1) expression in biliary tract cancer and correlation with clinical data B. Goeppert1, L. Frauenschuh1, M. Zucknick2, A. Stenzinger1, A. Warth1, C. End3, J. Mollenhauer4, M. Hafezi5, A. Mehrabi5, G. Mechtersheimer6, P. Schirmacher1, W. Weichert1, M. Renner6 1 Ruprecht-Karls-University, Institute of Pathology, Heidelberg, 2Department of Biostatistics, German Cancer Research Center (DKFZ), Heidelberg, 33Division of Molecular Analysis, German Cancer Research Center (DKFZ), Heidelberg, 4Medical Biotechnology Center, University of Southern Denmark, Odense, Denmark, 5Department of General Visceral and Transplantation Surgery, University Hospital Heidelberg, 6Institute of Pathology, University Hospital Heidelberg Aims. The secreted scavenger receptor cysteine-rich (SRCR) protein DMBT1 is differentially expressed in various cancer types and exerts functions in innate pathogen defence and the regulation of inflammation. In biliary tract cancers (BTCs) several risk factors, including cholangiolithiasis, liver fluke infection, and anatomical abnormalities, all associated with inflammation of the biliary tract, have been described. Therefore, the aim of the study was to analyze the expression of deleted in malignant brain tumors 1 (DMBT1) in BTCs and to correlate the expression with clinicopathological data including patient survival. Methods. Tissue microarrays containing 205 samples of BTCs [79 intrahepatic (ICCs), 83 extrahepatic cholangiocarcinomas (ECCs), and 43 gallbladder adenocarcinomas], premalignant lesions (biliary intraepithelial neoplasia, BilIN), and histologically normal biliary epithelium were stained immunohistochemically for DMBT1 and the level of expression was quantified semi-quantitatively using the Remmele-score. Results. Compared to non-malignant intrahepatic large and small bile ducts, DMBT1 expression was significantly increased in BilINs (p=0.006) and significantly decreased in BTCs (p=0.04). Existence of DMBT1 expression in non-malignant biliary epithelial cells in BTC-patients was associated with improved survival (log-rank test, p=0.027). DMBT1 expression was positively correlated with high MHC class I expression in tumor cells (p>0.001). Correlation of DMBT1 expression with age identified significant higher expression in older patients (p>0.001). Conclusions. Our data suggest that DMBT1 seems to play a diverse role in chronic inflammation of bile ducts and in the development of BTC dependent on spatiotemporal expression. DMBT1 expression might be of importance in the progression of BTC and ultimately have an influence on the outcome of BTC-patients.

FR-P-104 Histopathological evaluation of the iron content in liver biopsies J. Kelterborn1, K. Zöller1, J. Biet1, Y. Chen1, A. Herrmann2, M. Kiehntopf3, A. Stallmach4, I. Petersen1 1 Friedrich-Schiller-University of Jena, Institute of Pathology, Jena, 2FriedrichSchiller-University of Jena, Institute of Internal Medicine, Jena, 3FriedrichSchiller-University of Jena, Institute of Clinical Chemistry, Jena, 4FriedrichSchiller-University of Jena, Institute for Internal Medicine, Jena Aims. Hereditary hemochromatosis is the most common autosomal recessive genetic disease in Northern Europe. Iron overload leads to serious damage of various organ systems. Early diagnosis is desirable due to the possibility of prevention of severe organ dysfunction. However, the majority of patients are diagnosed in adulthood in the context of an uncertain liver pathology. Liver biopsy is an essential tool of analysis. In the present work two new iron scores were tested for their validity in the diagnosis of hemochromatosis. Methods. At the Institute for Pathology of the University Hospital of Jena all liver samples of a 2-year period were retrieved from the pathology files and reviewed. In total, 2326 cases were assessed of which 60 were selected for an in-depth study on iron content. The mean age of the patients was 59 years, 72% originated from male patients. In all 60 samples a mutation analysis of the two most common gene loci in hemochromatosis were performed, i.e. HFE282 and HFE63. Moreover, using the clinical diagnosis documentation system, laboratory values of liver diseases and iron storage diseases were analyzed. The assessment of the iron content of the liver biopsies were done by the Prussian blue stain applying two scores with comprised a published 21-tier and a newly developed 5-tier grading system. Results. It was shown that both iron scores were highly correlated with each other (p<0.001). In addition, both tests indicated a significant correlation for the HFE282 mutation status, but not for the compound HFE282/HFE63 allele status. The histological diagnosis of hemochromatosis was clearly associated with the HFE282 mutation. Both scoring systems resulted in a higher specificity and sensitivity for establishing the diagnosis. Conclusions. To assess the iron content of liver samples both iron scoring systems are suitable. The clinical relevance of the compound mutation could not be confirmed by our study and remains controversial. Clinically, an iron score with a grading scale from zero to four is more practicable. Based on the already established 5-tier grading systems for assessing inflammation and fibrosis we would also recommend our 5-tier system for analyzing the iron content in liver biopsies.

FR-P-105 Detection and typing of Echinococcus species in formalin fixed tissue by PCR U. Krüger1, M. Baumann1, P. Graber2, P.E. Tarr3, C.A. Maurer4, G. Cathomas1 1 Cantonal Institute of Pathology, Liestal, Switzerland, 2Basel University Medical Clinic Liestal, Unit of Infectious Diseases, Switzerland, 3Infectious Diseases Service, Kantonsspital Bruderholz, Switzerland, 4Department of Surgery, Hospital of Liestal, Switzerland Aims. Echinococcosis in human is caused by the two species E. granulosus (cysticus) and E. multilocularis (alveolaris). In tissue samples, the diagnosis is based on the detection of protoscoleces and the characteristic parasite membrane. Subtyping of the two species, which are associated with a different clinical course, is not possible by histology. The aim of the study was to evaluate the use of PCR in detecting and typing of Echinococcus in formalin fixed, paraffin embedded (FFPE) tissue. Methods. FFPE tissue of biopsies and surgical specimen were retrieved from the files of the Cantonal Institute for Pathology, Liestal. DNA was extracted and the quality of DNA recovered confirmed by amplification of a cellular control gene. Nested PCR was performed by species specific

primers of the mitochondrial and 12S rRNA gene. Clinical and radiographic data were retrieved from the clinical files as available. Results. From 1993–2011, a total of 25 cases (5 biopsies and 20 resection specimen) were available for testing. A positive PCR result was obtain in 24 (96%) of 25 samples, including 17 (68%) samples in which no protoscoleces were detected by histology. In respect to species-specific primers, 5 (20%) samples were positive by primers for E. granulosus and 9 (36%) were positive for primers for E. multilocularis. In 10 (40%) samples a positive PCR result was obtained by both primers. Detailed sequence analysis of these double positive cases assigned additional 2 samples to E. granulosus and 2 to E. multilocularis, respectively. In addition 3 further samples, which were seronegative, could be verified and classified by PCR. Conclusions. For the diagnosis of Echinococcus infection in FFPE tissue samples, PCR is a sensitive method independent of the presence of protoscolices. Using specific primers and DNA sequencing, species detection can be obtained in up to 75% of positive samples, rendering PCR a helpful tool in the diagnosis of echinococcal diseases.

FR-P-106 Delta like ligand 4 expression in the endothelium of hepatocellular carcinoma is associated with poor tumor differentiation C. Mogler1, H. Weng2, S. Singer1, T. Longerich1, P. Schirmacher1 1 Institute of Pathology Heidelberg, Heidelberg, 2Molecular Alcohol Research in Gastroenterology, Department of Medicine II, University Hospital Mannheim Aims. Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Most tumors are stroma-poor but highly vascularised. Overexpression of the tip cell marker and Notch ligand delta like ligand 4 (DLL4) has been described in many tumor entities but not yet investigated in human HCC, thus to date no clear data exist about the influence of (sprouting) angiogenesis on HCC progression or prognosis. Methods. Tissue microarrays (TMA) including 172 HCC, 9 Dysplastic nodules (DN) and 12 normal liver samples (NL) were investigated for DLL4 and vascular endothelial growth factor receptor 2 (VEGFR2) expression. In addition, microvessel density (MVD) was calculated by CD34 Immunohistochemistry. Findings were correlated with clinical data (tumor stage and grading). Results. DLL4 expression was neither present in NL nor DN but in 22 of 172 HCC (12.72%), distributed in 2 different expression patterns: a “single cell positive” pattern (SC), where only few (<10) endothelial cells appeared to be positive and a more diffuse (D) expression pattern with multiple positive endothelial cells. A diffuse expression pattern could be associated with a poor tumor differentiation (p<0.05), while SC pattern showed lower MVD compared to DLL4 negative HCC. SC pattern could also be linked to advanced tumor stages compared to diffuse expression pattern (p<0.05). In comparison, VEGFR2 staining showed an opposite staining profile: in DLL4 negative HCC VEGFR2 was found in almost half of the cases (48.76%), while DLL4 positive HCC however co-expressed VEGFR2 in only one fifth to one third of samples. Conclusions. Compared to pre-existing DLL4 expression data from other types of carcinomas (colon, breast), DLL4 expression in HCC is less frequently detected. Its molecular counterpart, VEGFR2 shows an opposite staining profile indicating that the negative crosstalk between DLL4 and VEGFR2 is also active in HCC angiogenesis. Both DLL4 expression patterns might play different roles in angiogenic processes. Further investigations in vitro an in vivo are needed to better understand the role of DLL4 in HCC progression.

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Abstracts FR-P-107 Comprehensive histological characterization of murine hepatocellular tumors – towards a classification of murine and human hepatocellular carcinomas L. Frick1, F. Böhm2, Y. Boege1, J. Friemel2, M. Heikenwaelder3, A. Weber2 1 University Zurich, Department of Pathology, Zürich, Switzerland, 2University Zurich, Institute of Surgical Pathology, Zürich, Switzerland, 3Munich, Institute for Virology and Helmholtz Zentrum, München Aims. The aim of our study is to characterise and compare different mouse models of hepatocellular carcinoma (HCC) which may reflect different ways of hepatocarcinogenesis in humans (including viral and toxic) in order to test their applicability to human HCC. Methods. We have implemented high-throughput, high-quality scanning technology to make digital copies of all our histological slides. A viewing software is used to display sequential sections side-by-side on a computer screen, and to measure tumours and annotate them for later reference. This enables us to determine the morphological and immunohistochemical characteristics of each individual lesion efficiently and comprehensively, even if there are many tumours and dysplastic lesions per slide, and thus discover correlations that may otherwise have been missed. Results. We have found clear differences among the mouse models, not only in the underlying liver pathology, but also in the spectrum of liver tumours that arises. In addition, certain regularities and correlations of tumour morphology and immunophenotype suggest a possible classification of liver tumours in mice. Using tissue microarrays of human HCC, we have discovered that the classification of murine tumours has cross-species applicability to human tumours. Conclusions. The results of our morphological and immunophenotypic studies, together with the data from molecular analyses, provide the basis for a classification of liver tumours that applies to both mice and humans. We also intend to show which mouse models recapitulate which aspects of hepatocarcinogenesis in humans, and may thus provide suitable models for testing therapeutic interventions.

FR-P-108 LGR5 is differentially expressed in hepato-gastrointestinal tumors E. Simon1, D. Petke1, C. Böger1, V. Warneke1, H.-M. Behrens1, C. Röcken1 Christian-Albrechts-University, Institute of Pathology, Kiel

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Aims. Carcinomas of the hepato-gastrointestinal tract are still leading cause of cancer related deaths worldwide. The orphan G-protein-coupled receptor (GPCR) and Wnt-target protein LGR5 was recently identified as a stem cell marker for cells with intestinal differentiation. In this study we generated a polyclonal anti-LGR5 antibody to investigate protein expression in various hepato-gastrointestinal carcinomas and its correlation with clinicopathological patient characteristics. Methods. Differential expression of LGR5 was studied on transcriptional (real time-polymerase chain reaction) and translational level (immunohistochemistry) in carcinomas and corresponding normal mucosal specimens comprising seven different primary tumor sites, i.e. oesophagus, pancreas, stomach, liver, colon and rectum. The putative clinicopathological relevance of LGR5 expression in terms of patient survival and the histoanatomical distribution of the protein was studied in 100 patients with gastric carcinoma. Results. We succeeded to establish and characterize a highly specific antibody that recognizes the C-terminal tail of LGR5. LGR5 was differentially expressed on transcriptional and translational level in adenocarcinomas of the oesophagus, pancreas, stomach, colon, rectum, hepatocellular and cholangiocellular carcinoma of the liver compared with the adjacent non-neoplastic tissue. However, in intestinal type gastric cancer the localization and number of LGR5+ cells changed during tumorigenesis.

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Conclusions. Our results substantiate the significance of LGR5 on the biology of hepatogastrointestinal carcinomas and provide evidence for its function as potential stem cell marker and candidate therapeutic target in the stomach. The strikingly changed histoanatomical distribution of LGR5+ cells during tumorigenesis could be an important observation in understanding tumor formation and progression. With our anti-LGR5 antibody we now have a useful tool for detection and further analyzes of LGR5 biological function.

FR-P-109 Gene expression analysis of the Mcl-1delHEP mouse model of hepatocarcinogenesis reveals overlapping expression profiles with human hepatocellular carcinoma F. Böhm1, Y. Böge2, R. Maire2, J. Friemel1, M. Heikenwälder3, A. Weber1 1 University Hospital Zurich, Institute of Surgical Pathology, Zürich, Switzerland, 2University Hospital Zurich, Institute of Neuropathology, Zürich, Switzerland, 3Institute of Virology, Helmholtz Center, Munich Aims. Apoptosis is regulated by a counterbalancing network of proapoptotic and pro-survival proteins. Mcl-1 is a crucial pro-survival factor, preventing cells to undergo apoptosis. Many chronic liver diseases are characterized by a constant loss of hepatocytes. Recently, we have shown that a mouse model with hepatocyte-specific deletion of Mcl-1 (Mcl-1delhep) reveals increased apoptosis, regeneration, and development of hepatocellular carcinomas (HCC). To better characterize apoptosis-driving tumorigenesis, we analyse gene expression patterns in the Mcl-1delhep model, and compare these to gene expression patterns in human liver tissues including HCC. Methods. RNA from livers of Mcl-1delhep mice, several other genetic mouse models and human HCCs of various etiologies were isolated, analysed and compared by quantitative real-time PCR. Results. RNA microarray of livers at 2 months of age uncovered significantly up- and downregulated genes in Mcl-1delhep mice compared to wild-type mice. Expression of Top 5 genes was also found to be significantly upregulated in 12 months old Mcl-1delhep mice (non-tumor and tumor tissue) and HCCs of various genetic mouse models for hepatocarcinogenesis as well as in human liver RNA from HCCs with different etiology. Conclusions. The Mcl-1delhep mouse model shows that increased apoptosis might serve as a main trigger of tumorigenesis, and thus recapitulates the human pathogenesis of chronic liver diseases such as liver tissue destruction and subsequent cancer formation. Overlapping gene expression patterns in the Mcl-1delhep mouse model, human tissues of chronic liver diseases and HCC confirms that the Mcl-1delhep mouse model is a valuable tool for studying human hepatocarcinogenesis, and thus also might be suitable for the identification of new molecular targets and interventional studies.

FR-P-110 Clear cell foci of altered hepatocytes in human liver show an overexpression of the AKT/mTOR and Ras/Raf1 pathways as well as the lipogenic phenotype (similar to hepatocarcinogenesis in the rat and to human hepatocellular carcinoma) S. Ribback1, D.F. Calvisi1, C.-D. Heidecke2, M. Birth3, F. Dombrowski1 1 Universitätsmedizin Greifswald, Institut für Pathologie, Greifswald, 2Universitätsmedizin Greifswald, Klinik und Poliklinik für Chirurgie, Greifswald, 3 Hanse-Klinikum Stralsund, Klinik für Allgemein-, Viszeral-, Thorax- und Gefäßchirurgie, Stralsund Aims. AKT/mTOR and Ras/Raf1 pathways as well as the lipogenic phenotype have been shown to be activated in a model of hepatocarcinogenesis in the rat induced by hyperinsulinemia and in human hepatocellular carcinomas. In the rat model the activation of these pathways starts

within the earliest morphologic detectable alterations, i.e. clear cell foci (CCF) of altered hepatocytes. CCF are described in humans indeed, but it is unclear whether these proto-oncogenic pathways are already activated within these foci. Methods. 241 liver resections were examined by using electron microscopy, histology, enzyme- and immunohistochemistry. Results. CCF are present within 35% of the extrafocal tissues of non cirrhotic livers. Electron microscopy illustrates massive glycogen storage within CCF, largely due to reduced activity of the glycogenolytic enzyme glucose-6-phosphatase. Hepatocytes of the CCF overexpress the insulin receptor and the glucose transporter proteins GLUT1 and 4. Insulin induced protooncogenic pathways AKT/mTOR (AKT, chREBP, activated mTOR, inactivated AMPKalpha, activated RPS6, inactivated 4EBP1) and Ras/Raf1 (IRS1, Ras, Raf1, MEK-1, ERK1/2, MKP-3, RALA), enzymes of glycolysis (Glucokinase, PFK, Pyruvate kinase), de novo lipogenesis (ACLY, ACAC, FASN, USP2, AKR1B10, SCD1), beta-oxidation (ACADM) and cholesterol synthesis (HMGCoAR, SQS) are upregulated in the CCF compared to normal liver tissue. Due to activation of these proto-oncogenic factors the proliferation activity in the CCF is 2-fold higher than in extrafocal tissue (Ki67-Index 0.75% vs. 0.36%; p=0.002). Conclusions. CCF reveal a phenotype which is known caused by hyperinsulinism in experimental hepatocarcinogenesis models as well as in human hepatocellular carcinomas. These metabolic changes and activation of proto-oncogenic pathways have not been observed in human CCF so far. Our results indicate that CCF represent precursor lesions of hepatocellular carcinoma also in humans.

FR-P-111 Adjuvant fluorescent in situ hybridization in equivocal biliary and pancreatic duct cytology M . Schramm1, A . Thieme1, N . Pomjanski1, H . Neuhaus2, A . Böcking1, S . Biesterfeld1 1 Heinrich Heine University, Institute of Pathology, Düsseldorf, 2 Evangelical Hospital, Düsseldorf Aims. The information whether a stricture or compression of the biliary and pancreatic ducts is of benign or malignant nature is important for therapy planning. Brushing of the suspect lesion in the context of an endoscopic retrograde cholangiopancreaticography is an established diagnostic procedure with moderate sensitivity and high specificity of conventional cytological diagnosis. Equivocal cytology due to artefacts or insufficient sampling is frequent. The detection of aneusomy by multicolour fluorescent in situ hybridization (FISH) is strongly associated with malignancy. The concern of our study was to determine if the addition of FISH subsequent to the cytological investigation could substantially decrease the number of equivocal cytology diagnoses. Methods. A cohort of 235 biliary/pancreatic duct brushings sent to the Department of Cytopathology during January 2005 and December 2007 was enrolled in this study to determine the diagnostic accuracy of cytology. In a second series of 143 brushings, consisting of all positive specimens (positive control) and all equivocal specimens in the above mentioned period and negative specimens in the period from January to December 2007 (negative control), aneusomy was analysed with the UroVysion™ FISH multiprobe. A histological and/or clinical follow up according to a reference standard, defined in advance had been available for 219 brushings. Results. In the whole cohort, cytology achieved a sensitivity and specificity of 75.3% and 93.1%, respectively. The reduced specificity, compared with the literature was attributable to the use of cytology as a screening test; all equivocal specimens were assigned as positive for statistics. FISH identified 29 out of 38 (76.3%) evaluable cytologically equivocal specimens as true positive and 9 out of 9 (100%) as true negative, compared to the follow up. All cytological negative (January to December 2007) and 28 out of 29 positive specimens were confirmed by FISH. Aneusomy was not observed in benign lesions, according to follow up data.

Conclusions. Adjuvant analysis of aneusomy with multicolour FISH in biliary/pancreatic duct brushings reduces equivocal cytology rate to a great extent and is 100% specific.

Poster: Herz- und Gefäßpathologie FR-P-112 The impact of genetic inactivation of the LIM-ony-protein FHL2 on cardiac remodelling D . Goltz1, E . Ramadori1, S . Huss2, M . Besmens3, R . Meyer4, R . Büttner2 University of Bonn, Dept . of Pathology, Bonn, 2University of Cologne, Dept . of Pathology, Köln, 3University of Bonn, Physiology II, Bonn, 4University of Bonn, Physiology II, Bonn

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Aims. LIM-domain containing proteins play a decisive role during ontogenesis and cellular differentiation. The LIM-only protein Fhl2 associates with cytoplasmatic scaffolding proteins and acts intranuclearly as a co-activator and co-repressor of transcription factors. Fhl2 is specifically expressed within the cardiovascular system during ontogenesis and high expression levels continue throughout lifetime. This suggests that Fhl2 is also of importance during cardiovascular remodelling. Fhl2 deficiency is known to induce an exacerbated cardiac hypertrophy upon chronic beta- adrenergic stimulation. This study investigates cardiac remodelling in the Fhl2 deficient organism caused by an increase in cardiac afterload. Methods. Experimental transverse aortic constriction (TAC) was induced in control animals and Fhl2-/- mice. After 14 days, morphometric and hemodynamic evaluations using a Millar catheter were performed. Vascular contraction and relaxation was examined using a Mulvany myograph and renal renin-mRNA-expression was analysed by quantitative real time PCR. Cardiac fibrosis was addressed by histologic techniques. Results. TAC induced a rise in pre-stenotic diastolic arterial blood pressure and a significantly more severe increase in pre-stenotic systolic arterial blood pressure in the Fhl2-/- animal compared to the control animal. The transstenotic pressure gradient, however, was identical in the Fhl2/- animal and the control animal. In vitro vascular contraction measurements proved that aortic rings of Fhl2 deficient mice featured combined contractile dysfunction and disturbed relaxation that was independent of receptor mediation. As a consequence, renal renin-mRNA-expression was significantly suppressed in Fhl2-/- mice. Cardiac hypertrophy, however, was less distinct in Fhl2 deficient animals than in wild type animals as was cardiac fibrosis. Conclusions. Fhl2 deficiency encompasses cardiac protection against chronic increases in cardiac afterload, in this context due to a suppression of the renin angiotensin axis.

FR-P-113 The impact of Crp2 deficiency on cardiovascular adaptation and remodelling D . Goltz1, E . Ramadori1, S . Huss2, R . Meyer3, R . Büttner2 University of Bonn, Dept . of Pathology, Bonn, 2University of Cologne, Dept . of Pathology, Köln, 3University of Bonn, Physiology II, Bonn

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Aims. Lim domain containing proteins among them Crp2 play a central role in organogenesis and cellular differentiation. Crp2 is specifically expressed in cardiovascular tissue. Its up-regulation in cardiomyocytes coincides with vascular smooth muscle differentiation in vitro. We investigated the vascular phenotype of Crp2 deficient mice in vitro and addressed the issue of cardiac remodelling under chronic elevation of left ventricular afterload in vivo.

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Abstracts Methods. Phenylephrine and potassium induced vascular contraction and endothelially mediated relaxation was investigated in Crp2 deficient mice and control animals by using the Mulvany Myograph. Transverse aortic stenosis (TAC) was induced in Crp2-/- mice and wild type animals. After 14 days, cardiac function was evaluated by cardiac catheterisation using a Millar catheter. Cardiac hypertrophy and fibrosis was addressed my morphometric analysis. Results. Aortic rings of Crp2 deficient animals displayed an isolated contractile disorder. Vascular contraction was enhanced both under stimulation with phenylephrine and under increased extracellular potassium concentrations. Endothelially mediated relaxation was unaffected by Crp2 deficiency. 14 days after TAC Crp2-/- animals developed exacerbated cardiac hypertrophy and moderate myocardial fibrosis compared to the control group. We observed a significantly more severe elevation of blood pressure levels in Crp2-/- animals compared to the control group. At the same time the pressure gradient across the stenosis was unchanged in wild type and Crp2-/- animals. Functional analysis of cardiac contraction revealed an acute diastolic dysfunction and reduced contractile reserve in Crp2 deficient mice. Conclusions. Crp2 deficiency leads to an aggravated vascular contraction upon both receptor mediated and receptor independent stimuli in vitro. In vivo, TAC induces an exacerbated cardiac hypertrophy in Crp2-/- animals. Crp2 plays a central role in cardiac and vascular homoeostasis, its loss leads to disturbed cardiac remodelling.

FR-P-114 Functional dichotomy of myofibroblasts in chronic cardiac diseases M. Franz1, A. Renner2, M. Ditze3, K. Grün4, D. Neri5, H. Kosmehl6, H.R. Figulla1, I. Petersen3, A. Berndt3 1 University Hospital Jena, Department of Internal Medicine I, Jena, 2 Ruhr- University of Bochum, Heart Center North Rhine-Westphalia,, Bad Oeynhausen, 3University Hospital Jena, Institute of Pathology, Jena, 4University Hospital Jena, Department of Cardiothoracic Surgery, Jena, 5Swiss Federal Institute of Technology, Institute of Pharmaceutical Sciences, Zürich, Switzerland, 6HELIOS-Klinikum Erfurt, Institute of Pathology, Erfurt Aims. Chronic rejection following heart transplantation is accompanied by allograft vasculopathy (CAV) and fibrosis (CIF) and represents a valuable model for the cardiac remodelling during ischemia. Processes are accompanied by extracellular matrix remodelling and the recruitment of myofibroblasts (MyoFb). ED-A+ fibronectin was recently suggested as a co-regulator of MyoFb development. The functional role of MyoFb in the context of hypoxic damage is not fully understood up to now. Aim of the study was to analyse the relation of MyoFb to the grade of chronic rejection and to ED-A+ fibronectin deposition in a rat heart transplantation model. Furthermore, the influence of the MyoFb secretom on cardiac myocytes under hypoxic conditions was investigated in vitro. Methods. A model of chronic rejection after rat heart transplantation (rHTX) was used. Allografts, recipient and control hearts were subjected to histological assessment of rejection grade and to immunofluorescence co-localization analysis of ED-A+ Fn and alpha-SMA. For in vitro investigations, HL-1 cells were used as a model for cardiomyocytes and the hTERT-BJ1 fibroblasts cell line was as a model for fibroblasts. alpha-SMA positive myofibroblasts were generated by stimulation with transforming growth factor-beta 1 (TGF-beta 1. Hypoxia in HL-1 cells was simulated by cultivation in presence of deoxyglucose. HL-1 cells were incubated with unconditioned medium and Fb or MyoFb supernatant. LDH as well as Troponin-I levels were measured in the supernatant by ELISA. Results. In the rHTX model the grade of chronic rejection showed a clear association to the amount of alpha-SMA positive cells. Extensive codepositions of ED-A+ Fn and alpha-SMA occurred in CAV and also in fibrosis. TGF-beta 1 treated hTERT-BJ1 fibroblasts showed a substantial increase in alpha-SMA and were therefore designated as MyoFbs. Pre-

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culturing of HL-1 in MyoFb-conditioned medium resulted in a protection against deoxyclucose caused cell damage. Conclusions. The process of chronic rejection accompanied by tissue hypoxia entails MyoFb activation and associated ED-A+ fibronectin deposition speaking well for a perpetuating process. alpha-SMA is suggested as a valuable marker to detect and quantify tissue remodelling in CAV and fibrosis. A protective effect of MyoFb on myocytes under hypoxic stress could be evidenced in vitro. With respect to this result, MyoFb based diagnostic and therapeutic approaches for ischaemic heart failure should consider this dichotomy.

FR-P-115 Tumours of the heart, great vessels and mediastinum: a retrospective study H. Al-Mohamed1, R. Hetzer1, K. Wassilew1 1 Deutsches Herzzentrum Berlin Aims. Primary cardiac and pericardial tumours are rare, with a prevalence ranging from 0.001–0.3%. A retrospective study was conducted with the focus on epidemiology and pathological features of cardial, pericardial and mediastinal tumours. Additionally, the results were compared to those of already existing similar studies. Methods. In a review of all pathological records of our institution from 2000 to 2010 268  cardial, pericardial and mediastinal tumours were identified. Results. The majority of cases (n=209, 78%) were benign neoplasms, myxomas being the most frequent histological type (n=105, 50.24%). Among the primary malignant tumours (n=59, 22.01%), sarcomas (n=18), carcinomas (n=10) and tumours of the haematopoietic system (n=9) were the most frequent. Conclusions. This study gives an overview of primary and secondary neoplasms in the heart, great vessels and mediastinum, with their preferential localizations and their epidemiological distributions. As found by other studies, myxoma is the most common tumour.

FR-P-116 Pitfalls treating tumors close to carotid artery bifurcation – a case report H.P. Kuhne1, M. Hegenscheid1, E. Fietze2, A. Lieber1 Hospital of the Armed Forces Germany, Berlin, 2Clinic for Pathology Berlin, Berlin

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Aims. A 55-year-old patient presented with neuralgia pain in area of right face for further diagnostic and therapy. He reported about an operation in the region of lateral neck in his youth with no further information. There was no tumor palpable, no local pain. Beside this patient is deaf since birth and suffers of a gynecomastia with hyperprolactinemia treated with Cabergolin at the moment. An MRI of brain and skull was without pathological findings. Methods. An MRI in the region of right bifurcation of carotid artery showed a tumor with diameter 1.8×2.7×3.6 cm suspected to be a so called glomus tumor. In a color duplex sonography there was seen a normal flow, also a normal MRI of brain and supraaortic vessels was done. During operation we could identify the tumor beginning at the vagus nerve. We did a complete resection in toto macroscopical, an instantaneous section showed no signs of malignancy. Results. Histological result showed a ganglioneuroma with regressive changes. Tumorspread was into a cleft transection. Most cells showed in an immunohistology coloration strong expression of S-100 proteine and very few strongly regressive changed gangliocytes, furthermore with grained expression of Synaptophysin and marginal CD-56. After inconspicuous healing process patient was presented in a interdisciplinary tumor board where no further need of surgery was decided.

Conclusions. In primary imaging a glomus tumor was number one diagnosis as most frequent entity of tumors in region of bifurcation of carotid artery. During operation we found a close relationship to vagus nerve which was confirmed in histology. For differential diagnosis there has to be considered a hemangioma, myoepithelioma, peripheral paragangliomas (f. e. M. von Recklinghausen), schwannoma, clotted aneurysms of carotid artery interne/externe or tumors out of the members with multiple endocrine neoplasias. Because most of these tumors are found accidentally by blood pressure disorders, dizziness attacks or syncopes a fast imaging is being done which detects the majority of these tumors. Most authors in literature recommend a surgical resection to salve clinical symptoms and to exclude a malignant tumor. It should be made clear, that a apparent clear imaging not always can be confirmed in a histological examination so a definite diagnosis should always be forced.

FR-P-117 Antibody-mediated rejection is associated with microvasculopathy after heart transplantation N.E. Hiemann1, E. Wellnhofer1, S. Kretschmer1, C. Christan1, H. Lehmkuhl1, C. Knosalla1, R. Hetzer1, R. Meyer1 1 Deutsches Herzzentrum Berlin, Berlin Aims. Antibody-mediated rejection (AMR) and microvasculopathy are associated with poor survival after heart transplantation (HTx). Following the new guidelines of the ISHLT we tested the effect of AMR on the development of microvasculopathy (MVP) in biopsy. Methods. We prospectively studied 134 pts (117 men, mean age 50  yrs) who underwent endomyocardial biopsy at 4 weeks (n=134), 1 yr (n=107) and 3 yrs (n=61) after HTx. Acute cellular rejection (ACR; ISHLT), MVP (ratio of luminal radius to diameter of vessel wall) and endothelial swelling were evaluated in H&E stainings. AMR was assessed by immunohistochemistry (CD31-positive capillaries to CD68, IgG, IgA, IgM, C1q and C3c; all x200). Results. At 4 weeks, 1 yr and 3 yrs, MVP affected 36%, 48% and 43% of pts, and AMR was present in 37%, 8% and 10% of pts, respectively. Pts with AMR more frequently presented with MVP at 4 weeks (47% vs. 22%; p=0.010), 1 yr (74% vs. 46%; p=0.006) and 3 yrs after HTx (81% vs. 45%; p=0.013). AMR was significantly correlated to ACR, e.g. at 4 weeks 43% (p<0.001) and at 1  yr 50% (p=0.006) of pts presented with concurrent ACR. However, only pts with MVP at 3  yrs post-transplant presented more often preceding ACR of any grade at 4 weeks after HTx (29% vs. 5%; p=0.025). Otherwise ACR was not correlated to MVP. Endothelial swelling was significantly correlated to concurrent MVP at 4 weeks (74% vs. 33%; p<0.001) and 1 yr (61% vs. 32%; p=0.001) and also to future development of MVP at 3 yrs post-transplant (62% vs. 32%; p=0.009; 52% vs. 32%; p=0.066). Only at 1 yr post-transplant was endothelial swelling correlated to AMR (100% vs. 45%; p=0.002). Conclusions. AMR is associated with microvasculopathy after HTx in the early, mid- and long-term follow-up. Microvasculopathy in biopsy should trigger screening for AMR and additional biopsy follow-up to identify high risk patients.

FR-P-118 Correlation between endomyocardial IgA deposition and antibody-mediated rejection in cardiac transplant recipients N.E. Hiemann1, D. Kemper1, K. Wassilew1, R. Hetzer1 Deutsches Herzzentrum Berlin, Berlin

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Aims. The International Society for Heart and Lung Transplantation (ISHLT) recently proposed new guidelines to diagnose antibody-mediated rejection (AMR). We studied AMR using our established criteria and those recommended by the ISHLT to determine markers that might improve the reliability of pathologic diagnosis.

Methods. We studied prospectively all endomyocardial biopsies harvested since 01/2011 (n=205) for acute cellular rejection (ISHLT), endothelial swelling and microvascular deposition of C4d, C3d and IgA/M/G in paraffin sections (immunohistochemistry). AMR was tested in the following models: pathologic AMR including endothelial swelling and C4d+C3d+Ig/A/M/G+ or C4d+C3d+ or C4d+ and immunopathologic AMR without endothelial swelling but with C4d+C3d+IgA/M/G+ or C4d+C3d+ or C4d+. Left ventricular systolic function (LVEF) was studied by echocardiography and presence of HLA antibodies (abs) was assessed during each biopsy harvest (Luminex®). Results. Pathologic AMR as detected by C4d+C3d+Ig/A/M/G+ was positive in 3%, as detected by C4d+C3d+ in 3% and as detected by C4d+ alone in 14% of biopsies. Immunopathologic AMR as defined by C4d+C3d+Ig/ A/M/G+ was present in 2%, by C4d+C3d+ in 2% and by C4d+ in 6% of biopsies. IgA alone affected 70%, IgM 96% and IgG 93% of biopsies. Only IgA correlated significantly to C3d (94% vs. 69%; p=0.026) and C4d depositions (88% vs. 67%; p=0.005). This was more pronounced in cases with endothelial swelling than in cases without. There was no correlation between pathologic or immunohistochemical findings and LVEF except for IgA, which was frequently detected in patients with preserved LVEF (72% vs. 53%; p=0.08). Immunopathology positivity for C4d+C3d+Ig/ A/M/G+ or C3d+C4d+ correlated to the presence of HLA class I abs (8% vs. 2%; p=0.05) while C4d+ alone was more frequent in patients with HLA class II abs (19% vs. 5%; p=0.065). Patients with donor-specific HLA class II abs were less likely to have IgA deposition in biopsy (29% vs. 74%; p=0.020). Conclusions. C3d and C4d deposition is associated with antibody-mediated alloimmune response. However, IgA correlates to C3d and C4d deposition and might indicate mucosal alloimmune response mimicking AMR.

FR-P-119 Impact of cardiotropic viruses on alloimmune response and microvasculopathy in cardiac transplant recipients N.E. Hiemann1, E. Wellnhofer1, K. Klingel2, R. Kandolf2, C. Proch1, C. Knosalla1, H. Lehmkuhl1, R. Hetzer1, R. Meyer1 1 Deutsches Herzzentrum Berlin, Berlin, 2University Hospital Tübingen, Institute for Pathology and Neuropathology, Tübingen Aims. Retrospective studies describe cardiotropic viruses as being a risk factor for acute cellular rejection and the development of epicardial vasculopathy after heart transplantation (HTx). Longitudinal analyses that consider the impact of viruses on alloimmune response and microvasculopathy in endomyocardial biopsy have not yet been performed. Methods. Prospective analysis was done in 199 biopsies obtained from 78 consecutive HTx recipients at 4 weeks, 1 year and 3 years after HTx. Diagnosis included acute cellular and antibody-mediated rejection (immunohistochemistry for C3d and immunoglobulins), Quilty (subendocardial infiltration of lymphocytes) and microvasculopathy (luminal radius-to-wall-ratio <1). By nested (RT-) PCR, biopsies and blood samples were evaluated for cardiotropic viruses. Results. Multiple viral infections [Epstein-Barr virus (EBV), human herpes virus-6 (HHV6), parvovirus B19 (PVB19)] were associated with microvasculopathy [33% (22/67) vs. 4% (3/78); p=0.004; RR 10.95, 95% CI 1.38–86.95; p=0.024], antibody-mediated rejection [16% (7/41) vs. 4% (4/104); p=0.058] and Quilty [40% (8/20) vs. 17% (21/125); p=0.026; RR 2.98, 95% CI 1.09–8.18; p=0.034]. EBV was found more frequently in biopsies with acute cellular rejection than in biopsies without [56% (21/37) vs. 32% (35/108); p=0.013] and was associated with poor survival [6.4±0.3 years (95% CI 5.8–7.1 years) vs. 7.2±0.2 years (95% CI 6.9–7.5 years); p=0.041]. Conclusions. Cardiotropic viruses are associated with different alloimmune responses including acute cellular rejection, antibody-mediated rejection, Quilty and microvasculopathy, all of which have been shown to be associated with poor prognosis following cardiac transplantation.

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Abstracts FR-P-120 How effective are heart valve donation from old organ donors? K. Große1, R. Meyer2, C. Wesslau3, D. Bösebeck1, G. Kirste4, R. Hetzer2 Deutsche Stiftung Organtransplantation, Northeast Region, Berlin, 2Deutsches Herzzentrum Berlin, Berlin, 3Foundation of European Tissue Banks, Berlin, 4Deutsche Stiftung Organtransplantation, Frankfurt am Main

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Aims. There is no upper age limit for organ donation and a quarter of all organ donors are 65 years old and older. The Northeast Region of the Deutsche Stiftung Organtransplantation and the Cardiovascular Tissue Bank of the Deutsches Herzzentrum Berlin examined 1.) 1999–2004 whether heart valves from organ donors over the former age limit of 65 years for heart valve donation were morphologically suitable as grafts and 2.) 2005–2009 after raising the age limit to 70 years the clinical acceptance of heart valve grafts from organ donors of 65 to 70 years of age. Methods. 1.) 1999–2004 the heart valves of 100 old organ donors (female/male: 55/45, median age: 71.5) were examined in accordance with the standards of the Bio Implant Services (BIS). To compare the valve grafts above and below the age limit of 65 years, we used data on the aortic and pulmonary valves of 380 organ donors below the age limit in the same time period. 2.) 2005–2009 we harvested 49 hearts from organ donors 65 to 70 years of age for processing heart valve grafts in accordance with the BIS-standards. One aortic and 21 pulmonary valves (25%) of these organ donors (female/male: 11/10, median age: 67) were suitable as grafts. One year after valve replacement we send the recipients’ physicans a form to gather information about valve failure, success of transplantation and current morphological and functional state of the valve graft. Results. 1.) Half of all heart valves above and below the former age limit would have fulfilled the morphological standards as grafts. The great majority (85%) of old pulmonary valves fulfilled the acceptance criteria, 48% even showing good tissue quality. 2.) 2005–2009 the aortic and the 21 pulmonary valve grafts have been allocated to recipients (female/male: 6/15, median age: 32) suffering from late sequelae of congenital diseases with defects of the either native valves or former grafts. Successful treatment without morphological and functional alterations of the grafts 1 year after replacement was reported from the aortic and 13 pulmonary valve grafts. Five further valve grafts were transplanted but follow-up 1 year later is unknown. One recipient of a pulmonary valve graft died postoperatively of left ventricular failure. In 2 cases accepted valve grafts were not transplanted because the surgeons decided intra-operatively on another procedure. Conclusions. The data clearly demonstrate, that heart valves grafts from donors 65 years of age and older can safely be used with good long-term success.

FR-P-121 Change of external surface roughness of vascular implants improves implant tissue integration M. Otto1, J. Kriegsmann1, S. Bertz2 Supra-regional Joint Practice of Histology, Cytology and Molecular Diagnostics Trier – Düren – Düsseldorf, Medical Health Center for Histology, Cytology and Molecular Diagnostics, Trier, 2University of Erlangen, Institute of Pathology, Erlangen

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Aims. Today, one of the most important problems in implant medicine is an optimal designed biointegration of implants. A controlled biointegration of vascular implants is essential for an optimal biofunction as well as for biosafety. The fast fixation of the vascular prosthesis reduces the risk of infection as well as thrombosis, two major events which lead to rapid functional loss. Implants with a silver coating, used to inhibit implant infection were modified at the outer surface to accelerate tissue integration. Methods. Silver coated vascular implant material based on expanded polytetrafluorethylen (ePTFE, expanded Teflon) was modified by application of a microwave procedure. Two different protocols of microwave application induce remodeling of the implant surface. At the first step,

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the material is implanted subcutaneously in a defined sheep model. The tissue integration of the implant was analyzed after 6 weeks by interposition of the implants at the Arteria carotis in a sheep model. We used 8 cm long routinely used but surface modified implants with a diameter of 6 mm. We used two implants, one with highly and one with slightly modified surface roughness. The implants were histomorphologically evaluated using qualitative parameters as well as semiquantitative parameters of vascularization, inflammation, peri-implant fibrous reaction and giant cell induction. The tissue reaction was compared to the standardized, clinically used and FDA accredited SilverGraft prosthesis. Results. After an implantation period of 6 weeks the thrombogenicity of implants was not significantly changed. Formation of fibrotic neointima as well the endothelialization of the inner implant surface was changed. Other histological parameters – lymphocytic infiltration, granulocytic infiltration and giant cell density – did not show any alteration by implant surface modification. The slightly modified implants show a stronger vascularization and mildly increased thickness of peri-implant fibrosis. The highly modified implants show no significant change in vascularization but a minimal increase of thickness of peri-implant membrane. Conclusions. The change of implant surface roughness effectively improves the integration of new developed vascular implant devices by boosting integration into the peri-implant connective tissue without any change of typical parameters correlating with biosafety of the vascular implant material. The highest biofunctionality is found in those implants with slightly increased surface roughness.

FR-P-122 Change of inner surface of ePTFE vascular implants by immobilization of heparin and heparan sulfate M. Otto1, J. Kriegsmann1, S. Bertz2 Supra-regional Joint Practice of Histology, Cytology and Molecular Diagnostics Trier – Düren – Düsseldorf, Medical Health Center for Histology, Cytology and Molecular Diagnostics, Trier, 2University of Erlangen, Institute of Pathology, Erlangen

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Aims. Today, one of the most important problems in vascular implant medicine is thrombosis. Complete or incomplete occlusion of vascular implants by thrombotic masses is the most unfavorable event in vascular endoprosthesis and results in insufficient biofunctionality of the implants. During the last decade several implant coatings were used to reduce thrombotic events. Methods. The inner implant surface of ePTFE vascular implants (ePTFE, expanded Teflon) was modified by a coating of polyurethane with covalent bounded heparin as well as heparin sulfate. To analyze the thrombogenicity of these modified materials vascular implants were applied in a sheep model by interposition into the A.  carotis. Routinely clinically used but surface-modified implants with a diameter of 6mm and a length of 8 cm were applied. Morphology of the implants was analyzed after 6 weeks of implantation by morphologic evaluation of qualitative parameters, especially thrombotic changes at the material surface. Further parameters were semiquantitative analysis of inflammation, periimplant reaction and giant cell induction. The tissue reaction was compared to the standardized, routinely used and FDA accredited ePTFE- as well as GoreTex-prosthesis. Results. After an implantation period of 6 weeks implants with heparin and heparin sulfate coating show macroscopically more and stronger thrombotic events compared to the uncoated ePTFE and GoreTex-controls. The morphological analysis of the thrombotic material shows in the heparin group an early thrombosis of the implants, often with luminal fibrotic organization. On the other hand, the heparan sulfate group shows late thrombosis without any organization effects. The rate of morphologically demonstrable thrombotic events in the heparin group (37.5%) and the heparin sulfate group (75%) exceed the control rate of 10% by far. The heparin sulfate group shows a considerable reduced rate of

fibrotic neointma development as well as a reduced endothelialization whereas in the heparin group a significant difference is not demonstrable. All other estimated parameters did not show any differences between controls and modified implants. Conclusions. In the current setting the covalent binding of heparin and heparin sulfate induce an increased rate of thrombotic events, which counteract the original purpose of the surface modification. The morphological changes may be interpreted as an effect of polyurethane, used as a partner for the covalent binding of the antithrombotic materials.

FR-P-123 Individual risk assessment in AAA – the value of biomarkers and their correlation to statin treatment B. Mühling1, T. Barth2, K.-H. Orend1 1 University of Ulm, Department of cardiothoracic and vascular Surgery, Ulm, 2 University of Ulm, Institute of pathology, Ulm Aims. In patients with infrarenal aortic aneurysm the aneurysm diameter determines the indication for operative repair. An individual marker would be interesting in order to assess the individual rupture risk or in order to control medical treatment, e.g with statins. Hence the activity of known metalloproteinases (MMP2/9), inflammatory cytokines (Osteoprotegerin, Interleukin 6/10) and resistin, an adipokin, and C reactive protein (CRP) in patients with AAA were measured and correlated to aneurysm diameter and statin therapy. Methods. In 63 patients with AAA from 4–9 cm with and without statin therapy serum activity of MMP 2 and 9, Osteoprotegerin (OPG), IL-6 and IL-10, resistin and CRP levels were measured prior to aneurysm repair. The expression pattern of resistin was also analyzed using immunohistochemistry in tissue specimen of the aortic wall. As for age, gender history of coronary artery disease, hypertension and smoking patient groups were similar. The results obtained were correlated to aneurysm diameter and statin therapy. Results. As for CRP and IL 10 levels we found a significant correlation to aneurysm diameter (r=0.38 and. r=0.42). IL-6, MMP 2 and 9, OPG and resistin were not correlated. Patients under regular statin therapy showed significantly lower levels of resistin and CRP (7.73 vs. 11.04 ng/ ml, p=0.005 resp. 1.8 vs. 6.7  mg/ml, p=0.007). Immunohistochemistry of aneurismal tissue showed in part close co-localization of resistin and CD 68 positive cells. Conclusions. The investigated markers are not able to serve as biomarker for individual risk assessment in AAA patients; however they underscore the inflammatory nature of AAA pathogenesis. CD 68 positive cells may mediate this inflammation. Statins have the potential to slow down this inflammation and should be prescribed in the conservative medical management of the disease.

FR-P-124 Risc factors for prosthetic vascular graft infection L. Höller1, M.K. Schilling1, M.R. Moussavian1 1 Saarland University Medical Center and Saarland University Faculty of Medicine, Department of General, Visceral and Vascular Surgery, Homburg Aims. Vascular prosthetic graft infections (VGI) are rare, but are associated with a high risk of limb loss, re-infection as well as a high mortality. Furthermore they lead to high economic costs. In this retrospective analysis predictive factors for VGI were analyzed. Methods. Out of a prospective SAP based database with 270 datasets, 206 patients/data sets were studied. All 206 patients were operated between 2001 and 2010 and had a re-operation within the same hospital stay. This cohort was divided into 3 groups: A: Aortal operations B. Arteriovenous fistulas for dialyses and C. Femoropopliteal bypasses. Patients were studied for the primary end point infectious complications with and without prosthetic graft infection. Infection was verified microbiologi-

cally of bacterial growth at the graft. Beside demographic data a clinical follow-up examination photo documentation of the affected limb was performed in all patients. Results. In group A, neither groin incision nor drainage insertion increased the risk for infection. However, a preoperative low hemoglobin (12.9±0.9 vs. 10.2±0.4 95% CI 9.4–11.0 vs. 11.4–14.6; p<0.05) was associated with graft infection. Furthermore blood loss (646±142  ml vs. 1300±410 ml) was more common in the infection group. Patients with graft infection stayed significantly longer in hospital (12±2.1 vs. 39±9.8 95% CI 7.4–15.5 vs. 20.9–61.4; p<0.05). Expectedly grafts were removed often in the group of infected grafts (79% vs. 21% p<0.05) and in-hospital mortality was higher (11% vs. 8%). Conclusions. Groin incision and drainage insertion were no risk factors in aortic operations. Graft infection caused a prolonged hospital stay, with increased health care costs. Low preoperative hemoglobin is a risk factor for graft infection. Altogether the collective of complicated vascular graft operation is small, prospective data registries are needed for verify these results.

FR-P-125 Vascular pathology in the healthy aging thymus and thymomas F. Pfister1, S. Busch1, K. Wolk1, P. Ströbel1, A. Marx1 University of Heidelberg, University Medicine Mannheim, Mannheim

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Aims. The vascular system of the human thymus is complex and various types of vessels can be distinguished according to their location, diameter and the presence or absence of a specialized thymus-vascular barrier. Age- and tumor dependent changes in the vascular architecture of the thymus are not fully investigated yet. In this study, the vascular architecture was examined in aging human thymi and in different types of thymomas. Methods. Normal human thymi (n=30), aged between birth and 8th decade, were collected from autopsies and 30 cases with thymoma classified from a pathological point of view according to the requirements of World Health Organization (A, AB, B1, B2, B3 and carcinoma) were analyzed. Vessels of healthy thymic cortex, medulla, corticomedullary junction (CMJ), perivascular space (PVS) and pathological thymic tumor vessels in thymomas were studied using quantitative morphometry of immunhistochemically stained sections for fat- and connective tissue, CD34, CD31, SMA, PDGFRb and Desmin. Details of microvascular changes were examined in whole mount digest preparations of thymus vasculature and by immunofluorescence staining. Results. We show that capillaries are predominant in the cortex. In the medulla, the cmJ and the PVS, blood vessels of different diameters, partially with features of a functional transit pathway are present. Age-dependent involution is associated with a reduction of thymus cortex and cortical capillaries and widening of PVS and a relative increase of arterioles and venules in the remaining thymus parenchyma. Accumulation of connective tissue and fat tissue along PVS in trabecules and the cmJ of the thymus leads to a separation of thymic parenchyma and thus to a breakdown of the complex vascular system of the thymus. Examination of the vasculature of thymoma subgroups revealed differences in the architecture, size and expression of vascular markers in tumor vessels. Type  A, AB and lymphocyte-rich B1 and B2 thymoma showed higher vascularization by small capillaries whereas B3 thymoma and thymus carcinomas showed sparsely distributed vessels of different diameters and presence of PVS. Conclusions. In conclusion, we show that thymus vasculature shows compartment specific features and age-dependent involution of the thymus is associated with a dramatic disturbance of the complex functional vascular system. Furthermore, we provide evidence that thymoma subtypes and thymus carcinomas differ in their vascularization, indicating potential for diagnosis and therapy.

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Abstracts FR-P-126 Mild hypothermia induced by surface cooling reduced cerebral cortex lesions after prolonged cardiac arrest in a pig model S. Högler1, F. Sterz2, A. Janata2, W. Weihs2, P. Schmidt1 University of Veterinary Medicine Vienna, Department for Pathobiology, Wien, Austria, 2Medical University of Vienna, Department of Emergency Medicine, Wien, Austria

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Aims. A surface cooling system to induce mild therapeutic hypothermia was tested in a pig model for prolonged cardiac arrest and type and extent of cerebral cortex lesions were assessed in comparison to a control group without cooling. Methods. Experimental ventricular fibrillation cardiac arrest for 10 min was induced in 22 large white pigs (35–45 kg). After 3 min of basic life support and 5 min of advanced life support the animals were defibrillated and randomized into the hypothermia or the control group. Swine in the hypothermia group were cooled to a core temperature of 33°C by the LRS™ ThermoSuit System, which is pumping a thin layer of ice water over most of the skin surface, and were kept at that temperature for 14 h. Rewarming was started 16 h post arrest. Control animals were kept at a constant core temperature of 38.5°C. At day 9 post arrest animals were deeply anesthetized and the brain was perfused with 4 L of saline and 1  L of paraformaldehyde. Coronary brain sections were embedded in paraffin and stained with HE. Frontal, parietal, temporal, occipital and insular cortices were examined by a semi-quantitative method. Type and extent of lesions were evaluated in each region and assessed. For group comparisons the Mann-Whitney-U-test was used. Results. Restoration of spontaneous circulation and subsequent randomization was achieved in 16 animals. The target temperature in the cooling group was reached after 9 (5.3–11.9) min. All animals survived until the endpoint 9 days post arrest. In frontal, parietal, temporal and occipital cortex statistically highly significant differences (p<0.001) were detectable between the two groups. All animals of the control group showed edema, hypereosinophilic neurons, reabsorbed neurons and gliosis to moderate to severe extent in these regions. Furthermore malacia was evident in 6 out of 8 control animals in at least one region. In the hypothermia group lesions were present to a lesser extent and malacia was not detectable in any region in any animal. In both groups comparatively mild lesions were present in the insular cortex. However, the hypothermia group still showed a statistically significant better outcome (p<0.05) compared to the control group. Conclusions. The LRS™ ThermoSuit System rapidly induced mild therapeutic hypothermia and led to significantly reduced lesions in cerebral cortex after prolonged cardiac arrest in this pig model. Further studies directly comparing this method to invasive cooling methods are desirable.

FR-P-127 Combining the Beecher and the stabilization body system to construct paraffin tissue microarrays of superior quality U.F. Vogel1 Eberhard-Karls-University, University Hospital, Institute of Pathology, Tübingen

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Aims. Paraffin tissue microarrays (PTMAs) became an essential technique in translational pathology in the last years. The most used device to construct a PTMA is probably the Beecher tissue microarrayer (Beecher Inc., USA) by which paraffin tissue core biopsies (PTCBs) are installed in punched holes in a paraffin recipient block, the later PTMA. However, unless using the paraffin sectioning aid system (Instrumedics Inc., USA) there might be some rolling and folding of PTCBs at sectioning and staining due to a missing contact between the PTCBs and the surrounding paraffin of the recipient block. This rolling and folding of PTCBs reduces the efficiency of the PTMA technique and can be prevented by a whole

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melting process of the PTMA after construction. Such a whole melting process can be achieved by using a stabilization body made of agar. Methods. 4  mm thick plates of 2% agar were constructed using waste material as molds (lids of disposable pipette boxes) and cut into 30×25×4  mm3 large pieces which fit into a standard embedding steel mold. Those pieces made of agar were paraffinized in a standard tissue processor and embedded into routine paraffin blocks. The blocks were fixed in the block holder of a Beecher tissue microarrayer. Having completed the PTMAs the paraffin blocks were put on a hot plate at 65°C and completely melted. After resolidification the PTMAs were cut and the sections stained using standard procedures. Results. Paraffinized stabilization bodies made of agar can be easily produced with standard laboratory equipment. Those agar bodies can serve as recipient blocks in PTMA construction using the Beecher tissue microarrayer. The agar bodies allow a one step whole melting procedure by stabilizing the PTMAs especially when more than one PTCB are installed in one hole of the PTMA. By melting the PTMA the PTCBs get into a strong contact with the surrounding paraffin thereby minimizing the rolling and folding of PTCBs during sectioning and staining. Conclusions. By combining the Beecher technique with the stabilization body technique it is possible to construct PTMAs of superior quality.

FR-P-128 Detection of VEGFA gene amplification in different neoplastic entities using four different technologies M. Andreozzi1, V. Vuaroqueaux2, A. Ackermann2, S. Schneider1, L. Tornillo1, C. Ruiz1, H.H. Fiebig2, S. Eppenberger1, L. Terracciano1 1 University of Basel, Institute for Pathology, Basel, Switzerland, 2Institute for experimental Oncology, Oncotest, Freiburg Aims. The VEGFA protein plays an important role in the induction of angiogenesis during tumor initiation and progression. The aims of the study were to investigate the presence of VEGFA gene amplification by FISH analysis on a human multi-tumor tissue microarray (MTMA) and to validate these findings on an independent human multi-tumor tissue in mouse implanted microarray (MTMA) by aCGH, SNP6 microarrays, FISH analysis and real-time PCR. Methods. The VEGFA gene status was evaluated on a human MTMA (n=2,957 tissue samples) including 132 tumor entities and 31 normal tissue types. Furthermore, we reconfirmed the presence of VEGFA gene amplification as detected by FISH analysis on a second independent MTMA including 195 different tissue samples from nude mice xenograft models of 24 different human tumor types using three additional genomic techniques: array-CGH, SNP6 microarrays and real-time PCR. Results. VEGFA gene amplification was detected across different tumor types. The following incidence were observed: 5.3% in colorectal carcinoma, 3.2% in endometrial carcinoma, 2.8% in gallbladder adenocarcinoma, 4.2% in renal carcinoma, 1.5% in hepatocellular carcinoma, 4.5% in pancreas carcinoma, 4.8% in stomach carcinoma samples. Additionally, some tumors were characterized by a high polysomy. In the second independent MTMA VEGFA gene amplification was detected by FISH analysis in four samples, including one non-small cell lung cancer, one sarcoma, one gastric and one breast cancer, previously classified as amplified by aCGH (100%) and SNP6 microarrays, though this last technique revealed 1 supplementary amplified sample. All results correlated with real-time PCR (p<0.05). Conclusions. We detected VEGFA gene amplification in different tumor types, on human MTMA and we confirmed our findings in an independent MTMA of human tumors implanted in mice. We demonstrated the occurrence of VEGFA gene amplification in several tumor types with an incidence of up to 5% and also we demonstrated the feasibility of four technologies for reliable VEGFA gene copy number detection. Comparison of the four technologies revealed high concordance (p<0.05).

FR-P-129 Alpha-1-antitrypsin-PiZ-antibody ATZ11 recognizes von Willebrand factor (vWF): diagnostic and pathogenetic aspects K . Hiththetiya1, H . Zhou1, S . Steiner1, H .J . Hertfelder2, H .-P . Fischer1 University Hospital Bonn, Institute of Pathology, Bonn, 2University Hospital Bonn, Institute of Experimental Hematology and Transfusion Medicine, Bonn

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Aims. Antibody ATZ11 which is directed specifically against the mutated Alpha-1-antitrypsin PiZ will be tested on further specific binding sites independent of PiZ. The identity of the underlying ATZ11-binding proteins will be analysed. Diagnostic und pathogenetic relevance of this specific cross-reaction will be discussed. Methods. Comparative immunhistochemical analysis, imunoelectronmicroscopic analysis, Western-blots of cultivated human vitelin cord endothelial cells and thrombocyte aggregates. Results. ATZ11-specific epitope is found immunohistochemically on megakaryocytes, platelets, endothelial cells of arteries, veins and some capillaries of normal human tissues. More extensive staining is found in portal vessels of end-stage liver cirrhoses. Neoexpression is found in sinusoidal endothelial cells of actively fibrosing liver diseases, especially in strongly progressive alcoholic steatohepatitis. Microvascular bed of hepatocellular carcinomas and hepatocellular adenomas remains unstained. The staining reactivity corresponds to that of von Willebrand factor (VWF) and P-Selectin. It is independent from the presence of hepatocellular AAT deposits of PiZ type and AAT genotype. The epitope can be detected in Weibel-Palade bodies (WPB) of human vitellin cord endothelial cells (HUVEC) and in the cytoplasm of patelets by immunoelectronmicroscopy. ATZ11-Western blotting of HUVEC and patelets visualizes a 240 kD molecule which is in the spectrum of the molecular weights of multimeric VWF. VWF deficient serum samples lack this ATZ11-binding molecule. Conclusions. Apparently ATZ11 binds to a conformation-dependent epitope of VWF which might reflect a special functional state of this protein. ATZ11 expression in sinusoids of actively fibrosing steatohepatitis highlights the possible role of VWF in sinusoidal occlusion by locally activated coagulation.

Poster: Pneumopathologie FR-P-130 MAdL- a new specific marker for adenocarcinomas of the lung H . Schultz , S . Marwitz , B . Baron-Lühr , G . Zissel , C . Kugler , K .-F . Rabe , P . Zabel1, E . Vollmer1, J . Gerdes1, T . Goldmann1 1 Research Center Borstel, Borstel, 2University of Freiburg, Department for Pneumology, Freiburg, 3Hospital Großhansdorf, Großhansdorf 1

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Aims. Although the common immunohistochemical markers are well suitable for sub-differentiation a fraction of indistinct cases of NSCLC still remains, demanding upgrades of the panel by new markers. Methods. Here we report the generation and evaluation of a new monoclonal antibody denoted by “Marker of adenocarcinomas of the lung” (MAdL), which was raised against the cytoplasmatic fraction of primary isolated human alveolar epithelial cells type II. Results. Upon screening, one clone was identified as a marker for alveolar epithelial cells type II, alveolar macrophages and particularly adenocarcinomas of the lung. With an optimized staining procedure for formalin fixed tissues this antibody was evaluated together with the established markers TTF-1, SP-A, pro SP-B and Napsin A in a large-scale study on a series of 362 lung cancer specimens. MAdL displays a high specificity for adenocarcinomas of the lung together with a sensitivity of 76.5% and is able to provide independent additional diagnostic information to the established markers.

Conclusions. We conclude that MAdL is a new lung specific marker for adenocarcinomas, which expands sub-differentiation in a notable portion of non-small cell lung cancers.

FR-P-131 Pulmonary haptoglobin (pHp) can be used as a new specific marker for adenocarcinomas of the lung M . Abdullah1, S . Marwitz1, D . Kähler1, H . Schultz1, C . Kugler2, P . Zabel3, E . Vollmer1, T . Goldmann1 1 Research Center Borstel, Clin . & Exp . Pathology, Borstel, 2Hospital Großhansdorf, 3Research Center Borstel, Borstel Aims. There are novel chemo-therapeutic approaches which recently have been developed for NSCLC, known as a largely chemo-resistant tumour. Substantial differences have been shown between adenocarcinomas and squamous cell carcinomas with regard to the adequate therapeutic regimens. Therefore, sub-differentiation of NSCLC is currently getting more into focus and increasingly turning out to be a central element within therapeutic decisions. In this study we analyzed the expression of pulmonary haptoglobin (pHp) in human lung cancer tissues and compare it to common markers of adenocarcinomas. Methods. Immunohistochemistry and heat-induced antigen-retrieval were conducted. For detection, a one-step polymer-system was applied. 119 formalin-fixed, paraffin-embedded tumour tissues were immunohistochemically analyzed for expression of php, TTF-1, SP-A, SP-B and Napsin. Results. Pulmonary haptoglobin was expressed in 35 of 72 (48.6%) adenocarcinomas of the lung, [TTF-1 (79.1%), SP-A (51.3%), SP-B (48.6%) and Napsin (84.1%)]. Expression of pHp in squamous cell carcinomas (N=47) was absent. A proportion of cases exclusively express either pHp and Napsin (pHp+/Napsin+: 4.7%) or pHp and TTF-1 (pHp+/TTF-1+: 3.1%). 7.8–14% of the samples would have caused difficulties if the expression of pHp would not have been addressed. Conclusions. SP-A and SP-B are specific markers for adenocarcinomas with a limited sensitivity compared to TTF1; the same holds true for pHp. Therefore, the inclusion of pHp as an additional marker in the panel has serious impact on diagnostics and therapy.

FR-P-132 The diagnostic value of cytokeratin 5/6, 14, 17, and 18 expression in human non-small cell lung cancer Y . Chen1, T . Cui1, L . Yang1, M . Mireskandari1, T . Knösel1, Q . Zhang1, M . PacynaGengelbach2, I . Petersen1 1 University Hospital Jena, Jena, 2University Hospital Charité, Berlin Aims. The constitution and expression patterns of cytokeratin filaments in human epithelial neoplasms are complex and distinctive. The aims of this study were analysis of the expression of cytokeratins and evaluation of their diagnostic application in human non-small cell lung cancer (NSCLC). Methods. mRNA expression of CK5, CK6, CK14, CK15, CK17, and CK19 was analyzed by Northern blotting. Protein expression of CK5/6, CK7, CK14, CK17, and CK18 was evaluated by immunohistochemistry on tissue microarrays. Results. Northern blotting showed that CKs were highly expressed in human bronchial epithelial cells and/or small airway epithelial cells. In NSCLC cell lines, the expression pattern of CKs was heterogeneous. In the survey of protein expression of CKs in 95 primary lung tumors, we found that CK5/6, CK14, and CK17 proteins were highly expressed in squamous cell carcinoma compared to adenocarcinoma (p=0.001, p=0.030, and p=0.001, respectively) and higher expression is significantly linked to lower grading (p=0.006, p=0.002, and p=0.001, respectively), while increased expression of CK7 and CK18 was observed in adenocarcinoma (p=0.001, respectively). Der Pathologe Suppl 1 · 2012  | 

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Abstracts Conclusions. Our data suggest that CK5/6, CK7, CK14, CK17, and CK18 have diagnostic value in subclassification of NSCLC.

FR-P-133 Malic enzyme expression in non-small cell lung cancer correlates with acetyl citrate expression and is associated with mediastinal lymph node metastasis A. Csanadi1, C. Otto1, N. Hörter1, A. Oser1, M. Donauer1, T. Plönes2, B. Passlick2, M. Werner1, G. Kayser1 1 Institute of Pathology, University Hospital Freiburg, Freiburg, 2Department of Thoracic Surgery, University Hospital Freiburg, Freiburg Aims. In contrast to normal human tissues malignant tumors utilize glucose mainly for the production of reductive equivalents and basic modules of nucleic acid, protein- and cell membrane synthesis, i.e. ribose, aminoacids and fatty acids. This metabolic shift results in pronounced production of lactate, known as the Warburg effect. Malic enzyme (ME) and acetyl citrate lyase (ACL) are two key enzymes involved in glucose metabolism and its linkage to fatty acid synthesis. We here analyzed the expression of these two enzymes in non-small cell lung cancer (NSCLC) and their association with local and systemic tumor disease. Methods. Protein expression in tissue multi arrays with a core diameter of 2 mm of 258 NSCLC patients was evaluated by immunohistochemical staining with ME (clone 3H5) and ACL (Cell Signaling 4331S). An H-score calculated by multiplication of the percentage of positive tumor cells with the predominant staining intensity (score 0 to 3) was used for nonparametric statistical analyses. Results. Expression of ME and ACL showed highly significant positive correlation (p<0.001). Analysis of local tumor stages revealed a correlation of ACL (p=0.004) but not of ME (p=0.686), whereas ME (p=0.008) but not ACL (0.693) was significantly correlated with mediastinal lymphonodal metastasis. ME was furthermore differentially expressed in adenocarcinomas compared to squamous cell carcinomas (p=0.004). Conclusions. Our results further elucidate the metabolic shift in NSCLC with a strong positive correlation of ME and ACL while the apparent different expression of ME not only underlines the biological difference of adeno- and squamous cell lung carcinomas. ME may also be involved with more aggressive tumor invasiveness most probably in the resulting higher amount of lactate as shown by the association with mediastinal lymph node metastasis.

FR-P-134 Pyruvat kinase M2 expression is strongly correlated with the presence of Glucose transporter 1 and only high expression of both proteins is of prognostic significance in non-small cell lung cancer patients A. Csanadi1, C. Otto1, U. Fehrenbach1, C. Küenzlen1, M. Donauer1, T. Plönes2, B. Passlick2, M. Werner1, G. Kayser1 1 Institute of Pathology, University Hospital Freiburg, Freiburg, 2Department of Thoracic Surgery, University Hospital Freiburg, Freiburg Aims. Like normal tissues malignant tumors strongly depend on a sufficient glucose supply but utilization of this basic energy source is mainly confined to aerobic glycolysis. Here the citric acid cycle covers the gap for supply of basic elements for cellular growth, i.e. ribose for nucleic acids, non-essential amino acids for protein synthesis and fatty acids for membrane synthesis. Glucose transporter 1 (GLUT1) is the major glucose transporter in normal as well as neoplastic tissues. Pyruvat kinase M2 (PKM2) converts phosphoenopyruvat into pyruvat, the last step of aerobic glycolysis. It is typically expressed in proliferating cells. The aim of our study is to elucidate the interrelation between PKM2 and GLUT1 expression and their possible impact in patient’s prognosis.

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Methods. Tissue multi arrays (TMA) were constructed of tumor tissue of 256 patients suffering from non-small cell lung cancer (NSCLC). GLUT1 and PKM2 expression were measured by immunohistochemical staining (GLUT1: SPM498; PKM2: D78A4). For analysis the percentage of positive cells as well as the H-score (percentage of positive cells multiplied by staining intensity) were used. Statistical analysis included non parametric tests as well as a Kaplan-Meier survival analysis. Results. Both PKM2 (p=0.001) and GLUT1 (p<0.001) were significantly higher expressed in squamous cell carcinoma (SCC) compared to adenocarcinoma (LAC). Although both proteins show a significant positive correlation (p=0.039) only GLUT1 was significantly associated with pT(p<0.001) and pN- (p=0.042) and the resulting UICC-stage (p=0.020). Using the median as cut-off for the survival analysis, a statistical trend could be observed for high GLUT1 expression as an indicator for poor survival (p=0.082) but only if both GLUT1 and PKM2 were highly expressed statistical significance was reached (p=0.049). Conclusions. GLUT1 and PKM2 are differentially expressed in LAC and SCC giving further evidence of the biological difference of these two histological entities. Although both proteins show a significant correlation in protein expression, an association with local and systemic tumor stage is only evident for GLUT1. Furthermore, GLUT1 is a prognostic marker in NSCLC but only with concomittant high PKM2 expression.

FR-P-135 BDNF/NT4-TrkB signaling in non small cell lung carcinomas A. Warth1, C. Schoewe2, C. Castrupp2, B. Eul3, J. Wilhelm4, P.A. Schnabel1, G. Kwapiszewska5, L. Fink6 1 University Clinic Heidelberg, Department of Pathology, Heidelberg, 2University Clinic Giessen, Department of Internal Medicine II, Giessen, 3University Clinic Giessen, Department of Internal Medicine V, Giessen, 4Max Planck Institute, Bad Nauheim, 5University of Graz, Ludwig Boltzmann Institute for lung vascular research, Graz, Austria, 6UEGP, Institute of Pathology and Cytology, Wetzlar Aims. The neurotrophic tyrosine kinase receptor TrkB and its ligands BDNF and NT4 have been implicated in the pathogenesis of human lung cancer. The role of this signaling cascade was studied in primary human non-small cell lung carcinomas, and A549, a cell line derived from a human lung adenocarcinoma. Methods. A tissue array carrying about 1200 non-small cell lung carcinomas was applied for immunohistochemical staining with anti-TrkB, anti-BDNF and anti-NT4 (Santa Cruz). In A549 cells, mRNA and protein of TrkB, BDNF and NT-4 were detected by RT-PCR, western blot and immunofluorescence, respectively. In colony assays the effect of the receptor inhibitor K252a on cell survival and colony formation ability was determined. Additionally, phosphorylation of p38 and Akt was assessed by western blot. Results. In a pilot study of a tissue array subset (300 tumours) about 36% of the carcinomas showed immunohistochemically protein expression of TrkB, while 34% were positive for Nt-4 and 26% for BDNF. In A549 cells, TrkB RNA transcript expression and high protein levels of TrkB were found. BDNF/NT-4 mRNA and protein were detected as well. Stimulation of A549 cells by BDNF did not result in a (further) proliferation activation, but application of the inhibitor K252a diminished the ability of the cells to grow in soft agar. K252a reduced Akt and p38 phosphorylation in A549, regardless of BDNF presence. Conclusions. TrkB signalling cascade is active in a subset of non small cell lung carcinomas. The data suggest that in this subset application of the inhibitor K252a may be a valid anticancer therapy.

FR-P-136 The expression of central cell cycle regulators in non-small cell lung cancer (NSCLC) has therapy dependent prognostic impact J. Cortis1, A. Warth1, M. Meister2, T. Muley2, H. Hoffmann3, A. Stenzinger1, P.A. Schnabel1, P. Schirmacher1, W. Weichert1 1 University Hospital Heidelberg, Institute of Pathology, Heidelberg, 2 Thoracic Hospital Heidelberg, Heidelberg, 3Thoracic Hospital Heidelberg, Department of Thoracic Surgery, Heidelberg Aims. The relevance of function and expression of central cell cycle regulators in NSCLC has been discussed for years with controversial and non-conclusive results. To address this topic, we evaluated expression patterns of central cell cycle regulators in a very large NSCLC cohort with the goal to ultimately clarify their clinical-prognostic role. Methods. Expression of the G1-phase cell cycle regulators CDK4, cyclin D1 and p16 were analysed by immunhistochemical staining and quantitative evaluation on tissue microarrays comprising 1045 completely resected NSCLC. The data were correlated with clinical-pathological factors, patients’ survival and response to therapy. Results. Loss of expression of CDK4 (p=0.017), cyclin D1 (p=0.001) and p16 (p=0.039) were associated with higher UICC stages. In general, high expression of all cell cycle regulators was associated with better clinical outcome in a slightly variable way, depending on protein and prognostic parameter investigated [CDK4, overall survival (OS): p=0.457, disease specific survival (DSS): p=0.110, disease free survival (DFS): p=0.011; cyclin D1, OS: p=0.037, DSS: p=0.009, DFS: p=0.111; p16, OS: p=0.204, DSS: p=0.484, DFS: p=0.004]. These effects were prominent in adenocarcinomas, adenosquamous carcinomas and in pleomorphic carcinomas, but less pronounced in squamous cell carcinomas. Interestingly, after stratification for therapy, the positive association of protein overexpression with survival was only seen in adenocarcinomas without adjuvant radio- and chemotherapy whereas the association in tumors with adjuvant irradiation and chemotherapy was switched in the opposite direction. Conclusions. The expression of CDK4, cyclin D1 and p16 in NSCLC has prognostic impact depending on therapy. High expression of all three proteins was associated with improved clinical outcome in completely resected NSCLC without adjuvant therapy.

FR-P-137 The FUSE-binding proteins (FBPs) represent essential regulators responsible for tumor cell proliferation, migration and invasion in non-small cell lung cancer M. Malz1, M. Bovet1, J. Samarin1, E. Herpel1, A. Warth1, S. Singer1, T. Muley2, M. Meister2, H. Hoffmann2, P. Schnabel1, P. Schirmacher1, K. Breuhahn1 1 University Hospital Heidelberg, Institute of Pathology, Heidelberg, 2 University Hospital Heidelberg, Thoracic Hospital, Heidelberg Aims. The single-strand nucleic acid binding far upstream element (FUSE)-binding proteins (FBP)-1, FBP-2, and FBP-3 represent a multifunctional protein family, regulating transcriptional and post-transcriptional processes as well as microRNA biogenesis. Elevated expression and pro-tumorigenic functions of all FBPs have been described for human liver cancer. Moreover first data indicated that FBP-1 affects microtubule dynamics through regulation of MT-destabilizing factors in non-small cell lung cancer (NSCLC). Therefore we aimed to analyze expression and functional relevance of FBPs in NSCLC. Methods. The expression of FBPs was analyzed at the transcript (qPCR) and protein level (Tissue Microarrays [TMA], Western Blotting) in primary human NSCLC tissue samples. Using gene-specific siRNAs the expression of FBPs was inhibited in different NSCLC cell lines (Calu-1, Calu-6, and A549). Functional consequences of reduced protein expression on viability (MTT-Assay), proliferation (BrdU-Assay), apoptosis (FACS-Assay; PARP-cleavage), migration (two-dimensional scratch assay), and invasion (sprouting assay) were analyzed.

Results. The expression of FBP-1 and FBP-2 was significantly elevated in human NSCLCs (>60%) in comparison to non-tumorous specimens. In vitro, transient inhibition of FBP-1 in NSCLC cells (Calu-6) was associated with decreased tumor cell viability (−76%), proliferation (−83%), and increased apoptosis (2.8-fold). In contrast, transient inhibition of FBP-2 predominantly reduced tumor cell migration (−62%) and tumor cell invasion (−81%), suggesting that both FBP isoforms facilitate distinct tumor-supporting effects. In addition, FBP-2 inhibition increased FBP-1 expression at the transcript and protein level in A549 cells, demonstrating that FBP-1 may compensate the loss of FBP-2. Accordingly, the FBP1/-2 double-knockdown led to a significant reduction of cell viability (−69%). Conclusions. In summary, this study provides evidence that overexpression of FBP-1 and FBP-2 is frequently detectable in NSCLC tissues and that both proteins are essential factors for tumor growth and NSCLC cell dissemination. Furthermore FBP-2 negatively regulates FBP-1 expression, indicating a functional compensation.

FR-P-138 Interobserver variability in the application of the novel IASLC/ ATS/ERS classification of lung adenocarcinomas A. Warth1, A. Stenzinger1, A.-C. von Brünneck2, B. Goeppert1, J. Cortis1, I. Petersen3, P.A. Schnabel1, W. Weichert1 1 University Hospital Heidelberg, Institute for Pathology, Heidelberg, 2 Charité University Hospital Berlin, Institute for Pathology, 3 University Hospital Jena, Institute for Pathology Aims. Recently, an international consensus classification for adenocarcinomas (ADC) of the lung has been published. The cornerstone of this new classification is the quantification of different growth patterns. However, data on the reproducibility performance of this classification in the routine diagnostic setting are lacking. Methods. We selected 100 constitutive cases of conventional lung ADC resection specimens from our archives. All tumor slides were classified independently by five experienced pulmonary pathologists from three institutions and by two pathologists in training according to the recommendations of the IASLC/ATS/ERS. Results. The most frequent predominant pattern in our cohort was solid (37%), followed by acinar (35%), lepidic (20%), papillary (5%) and micropapillary (3%). kappa values for the denomination of a predominant pattern revealed a substantial agreement for pulmonary pathologists (0.44—0.72) but only fair agreement for pathologists in training (0.38– 0.47). Interobserver variability was significantly higher in cases with higher slide numbers (p=0.028) and was considerably reduced in a second evaluation round after the initiation of a training session. Intraobserver variability was low (kappa=0.79–0.87). Papillary and micropapillary patterns were the most complicated patterns to evaluate, while evaluation of lepidic and solid tumor growth was straightforward. The acinar pattern ranged in between. Conclusions. Our data imply that the novel classification of pulmonary ADC is applicable with adequate interobserver variability if performed by specifically trained pathologists. However, additional efforts are needed to harmonize the application of this novel and clinically important classification scheme of pulmonary ADC.

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Abstracts FR-P-139 Intraoperative subtyping of pulmonary adenocarcinomas according to the IASLC/ATS/ERS classification: a feasibility study P.A. Schnabel1, H. Hoffmann2, E. Herpel1, B. Goeppert1, H. Dienemann2, P. Schirmacher1, W. Weichert1, A. Warth1 1 University Clinics Heidelberg, Institute of Pathology, Heidelberg, 2 University Clinics Heidelberg, Thoracic Clinic Heidelberg, Heidelberg Aims. The IASLC/ATS/ERS proposal for a new classification of pulmonary adenocarcinomas has important impact on prognosis. It may provide the basis for surgical decisions in operative therapy of multifocal, multiple, or recurrent adenocarcinomas as well as of adenocarcinomas in patients with restricted lung function. Methods. Intraoperative frozen sections from 25 adenocarcinomas were analyzed covering the complete central lamella of the tumor (one to four, mean two frozen sections per patient). The results obtained by two independent investigators from the frozen sections were compared to those after paraffin embedding of the whole tumor. Results. In the frozen sections, the 25 tumors showed the following predominant patterns: acinar 8/25, solid 7/25, micropapillary 4/25, lepidic 4/25, and papillary 2/25. After paraffin embedding two predominant patterns were different: one changed from solid to acinar, and one from acinar to lepidic. In both cases these patterns had been reported as the second frequent occurring pattern. Two patterns were reported, if occurring at all: the solid pattern in 10/25, and the micropapillary pattern in 7/25. Conclusions. Subtyping of pulmonary adenocarcinomas according to the IASLC/ATS/ERS proposal for a new adenocarcinoma classification can be reliably applied in frozen sections. These results are encouraging and indicate that the classification system may be translated into the intraoperative setting. This may have implications for surgical strategies and may eventually allow tissue sparing resections, e.g. of predominant lepidic adenocarcinomas. The study will be continued. As in 2/25 tumors the second frequent pattern found in cryosections turned out to be the predominant pattern after paraffin embedding, all patterns should be reported semi-quantitatively.

FR-P-140 Clonality of multifocal non-small cell lung cancer: implications for staging and therapy A. Warth1, S. Macher-Göppinger1, J. Cortis1, T. Muley2, M. Thomas3, H. Hoffmann4, P.A. Schnabel1, R. Penzel1, P. Schirmacher1, S. Aulmann1 1 University Hospital Heidelberg, Institute for Pathology, Heidelberg, 2Thoraxklinik Heidelberg, Translational Research Unit, 3Thoraxklinik Heidelberg, Oncology, 4Thoraxklinik Heidelberg, Thoracic Surgery Aims. Non-small cell lung cancers (NSCLC) display a variety of morphological and molecular features. Accurate subtyping of NSCLC has been shown to predict patient survival as well as response rates and toxicities of specific drugs. Assessment of multifocal lung tumors and the distinction of synchronous primary tumors from intrapulmonary metastases represent an important problem as this decision significantly influences tumor staging and subsequent treatment strategies. Methods. In order to provide a basis for evidence-based treatment decisions in those patients, we analyzed the clonal relationship of multifocal NSCLC with indistinguishable histomorphology in a series of 78 patients by allelotyping (using polymorphic short tandem repeat markers) as well as KRAS and EGFR mutation testing. Results. Our data demonstrate a common clonal origin indicative of intrapulmonary metastases in almost two thirds (~62%) of the cases, while ~36% of multifocal NSCLC displayed unique molecular profiles suggesting separate primary tumors. Divergent KRAS and/or EGFR mutations were observed in approximately 8% of all cases. Conclusions. With the increased availability of EGFR-targeted therapy options, non-resectable, multifocal NSCLC with diverging KRAS and/

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or EGFR mutations are likely to show different treatment responses underlining the need to separately analyze multifocal tumors. Obviously, this also holds true for further, novel molecular predictors of targeted therapies.

FR-P-141 Localized nodular amyloidosis and adenocarcinoma of the lung: a rare association R. Kurth1, M. Scharpf1, F. Fend1 University of Tübingen, Institute of Pathology, Tübingen

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Aims. The lung is frequently involved in generalized amyloidosis, whereas amyloid localized to the respiratory tract is an uncommon finding. Nodular amyloidosis of the lung is a rare condition with formation of tumours containing eosinophilic amyloid deposits and a lymphoplasmacytic infiltrate and usually presents as incidental radiologic finding in asymptomatic patients. Methods. Histology, immunohistochemistry, molecular analysis. Results. We describe the case of a 74-year-old man with a history of smoking (30PY). Due to a history of cough, a chest X-ray was performed and revealed an intrapulmonary tumor mass in the left inferior lobe 4.1 cm in diameter. Bronchoscopy and mediastinoscopy were performed, but the biopsies of lung and lymph nodes were non-diagnostic. Subsequently a video-assisted thoracoscopic resection of the lung lesion was performed and revealed adenocarcioma of the lung in intraoperative frozen section. Thus, a lobectomy and lymph node dissection was performed. Histological examination of the tumor revealed extensive nodular amyloidosis of the lung with accompanying lymphofollicular infiltrates, giant cell reaction as well as calcifications and ossifications. Within the amyloid deposits, a moderately differentiated papillary, non mucinous adenocarcinoma of the lung with a size of 1.1 cm was found. Immunohistochemical and molecular examination of the lymphoid component failed to demonstrate evidence for a B-cell neoplasm or clonality, respectively. All lymph nodes were free of tumor. Conclusions. To our knowledge, the association of nodular amyloidosis and pulmonary epithelial malignancies of the lung is a very rare condition. It is not clear whether this represents a pure chance co-occurrence, or whether there is a causal relationship between the two lesions as the result of a chronic inflammatory process.

FR-P-142 CD34+ fibrocytes in neoplasia of the lung and pleura F. Wötzel1, P.J. Barth1 University Hospital Münster, Institute of Pathology, Münster

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Aims. CD34+ fibrocytes are a cell population that is found abundantly in the human connective tissue. Not only do they play a decisive role for the matrix synthesis but also may appear as antigen-presenting cells. They are particularly important concerning chronic inflammatory diseases of the lung as well as systemic and localized fibrosis. Invasive carcinomas induce a change in the phenotype from CD34+ fibrocytes to SMA+ myofibroblasts. The significance of CD34+ fibrocytes in chronic inflammatory diseases of the lung has been analysed thoroughly. However, there is still a lack of data about CD34+ fibrocytes located in the stroma of primary bronchial carcinomas, metastasis of the lung and pleural mesotheliomas. Methods. 30 primary bronchial carcinomas (10  adenocarcinomas, 10  squamous cell carcinomas, 10  small-cell carcinomas), 10  metastasis and 10  pleural mesotheliomas were analysed immunohistochemically aiming at the expression of CD34, SMA (smooth muscle actin) and SMM (smooth muscle myosin). Each case was compared to pulmonary tissue free of tumor. Results. Pulmonary tissue free of tumor displays CD34+ fibrocytes with slender bipolar cytoplasmic projections especially in the bronchial mu-

cosa and in the periarterial tissue. These cells do not display an expression of SMA or SMM. All analysed malignant tumors demonstrate a loss of stromal CD34 expression and a phenotype change from CD34+SMASMM- fibrocytes to CD34-SMA+SMM+ myofibroblasts. Conclusions. Primary and secondary malignant tumors located in the lung and pleura display a constant stromal phenotype change from CD34+SMA-SMM- fibrocytes to CD34-SMA+SMM+ myofibroblasts. This phenomenon is stereotypical for all organs (i.e. pancreas, mamma, cervix) analysed this far. Furthermore the comparison with in situ carcinomas indicates the major role of the loss of stromal CD34 expression in local tumor invasion.

FR-P-143 Cytology-based diagnosis of malignant mesothelioma on behalf of the German Social Accident Insurance Institution S. Biesterfeld1 1 Heinrich Heine University, Department of Cytopathology, Düsseldorf Aims. Histology usually represents the gold standard for the diagnosis of malignant mesothelioma. However, sometimes cytological material, mainly as pleural fluid, is available solely. Here, we discuss those four cases which were investigated in our institution in 2010 during the estimation procedure on the reduction in earning capacity according to the occupational diseases ordinance (No. 4105: “asbestos-induced malignant mesothelioma”), ordered by the German Social Accident Insurance Institution. Methods. In addition to routine cytology, we applied immunocytochemistry (calretinin, berEP4, HEA125, TTF-1), DNA image cytometry, AgNOR-analysis and FisH (at the region 9p21) and interpreted the results considering the clinical and occupational history. Results. In one of the four cases, a malignant effusion due to metastatic lung cancer was diagnosed. In two cases, the diagnosis of an epitheloid malignant mesothelioma was made. The fourth case in our opinion revealed reactive changes only, and manifest tumor cells of a malignant mesothelioma could not be detected. In all four cases, our interpretation has been agreed by the Statutory Accident Insurance Funds. The first three cases were accepted as asbestos-associated occupational diseases, while the forth case was rejected initially. However, this patient meanwhile has died, and the autopsy findings, taken in another institution, led to the acceptance of asbestos-associated malignant mesothelioma. Conclusions. Cytology-based tumor diagnosis is a useful and reliable approach in those cases of asbestos-associated tumors in which no histology can be obtained. The acceptance of cytological diagnoses by the German Social Accident Insurance Institution enables to come to a conclusive during the remaining lifetime of those patients by shortening the procedure to estimate the reduction in earning capacity.

FR-P-144 Transcriptome analyses and validation of the targets reveal impact of the TGF-β pseudoreceptor BAMBI for COPD S. Marwitz1, D. Drömann2, J. Rupp3, K. Rohmann3, S. Osbahr3, A.-J. Ulmer1, K. Röschmann1, M. Abdullah1, H. Schultz1, E. Vollmer1, P. Zabel2, K. Dalhoff2, T. Goldmann1 1 Research Center Borstel, Leibniz Center for Medicine and Biosciences, Borstel, 2University Clinic Schleswig-Holstein Campus Lübeck, Medical Clinic III, Lübeck, 3University Clinic Schleswig-Holstein Campus Lübeck, Institute of Medical Microbiology and Hygiene, Lübeck Aims. Transforming Growth Factor beta (TGF-β) signaling events control a variety of different cellular reactions and are involved on both, physiologic and pathologic processes. Epithelial as well as cells of the immune system respond to TGF-β signals throughout the body. The influence of TGF-β signals in non-malignant lung diseases are up to date critically discussed and infection-triggered tissue inflammation as well

as remodeling seems to inhabit a central role in the exacerbation and pathogenesis of chronic obstructive pulmonary diseases (COPD). Infection-triggered tissue inflammation and remodeling requires tight regulatory events in the interplay of epithelial and immune cells and TGF-β is one of the major cytokines involved. Methods. Ex vivo infected human lung tissues with Non-Typeable Haemophilus influenzae (NTHI) were subjected to transcriptome analysis. Infection of lung tissue was verified by RNA in situ hybridization targeting NTHI mRNA. Pathway analysis of different TGF-β members was conducted on RNA and protein level and compared to COPD tissues. Expression and secretion of pro-inflammatory molecules (IL-8, TNF-α) were analyzed by means of western blotting and ELISA. Results. 38% of COPD patient samples showed positivity for NTHI on RNA level in contrast to 0% of controls. Transcriptome based pathway analysis showed no significant changes of TGF-β receptors as well as cytokines in contrast to a strong up regulation of Bambi in ex vivo infected lung tissues as well as in COPD tissues. Bambi was found to be expressed on alveolar macrophages as well as alveolar epithelial cells. Conclusions. Here we present the TGF-β pseudoreceptor BMP and Activin Membrane-Bound Inhibitor (Bambi) as a new modulator of TGF-β signaling which might play a potent role in controlling the tissue homeostasis and involvement in infection triggered pathogenesis.

FR-P-145 The contribution of p120-catenin modulated NF-kappa B activation in airway inflammatory responses X. Wang1 1 Department of Pathology, Tongji Medical College Huazhong University of Science and Technology, Wuhan, China Aims. The purpose of this study is to investigate the role of p120-catenin (p120) modulated nuclear factor-kappa B (NF-kappa B) activation in airway inflammatory responses, and further to explore the molecular mechanisms. Methods. In this study, human bronchial epithelial cells (BECs) were treated with LPS to establish an airway inflammation model in vitro. Using confocal immunofluorescence imaging, Western blot, isolation of cytoplasmic and nuclear proteins, we examined the localizations and expressions of p120, NF-kappa B, IkappaB alpha and RhoA. Immumoprecipitation was used to confirm the direct interaction of p120 and RhoA. The RhoA activity was examined by G-LISA method. Then we detected the expressions of interleukin-8 (IL-8) by fluorescence quantitative PCR and enzyme-linked immunosorbent assay. Luciferase reporter analysis was used to detect the activity of NF-kappa B. Finally, transient transfection and small interfering RNA (siRNA) were used for p120 overexpression or knock-down, and then the effects of p120 on NF-kappa B signaling pathway were detected. Results. In the present study, we first confirmed that p120 expression was significantly reduced after LPS stimulation in BECs, the nuclear translocation of NF-kappa B p65 subunit was promoted, IkappaB alpha was phosphorylated and degraded, and NF-kappa B activity was rapidly induced. After LPS stimulation, although the total RhoA and p120-binded RhoA were unchanged, the RhoA activity is increased. Moreover, the expression level of IL-8 increased after LPS treatment. Overexpression of p120 attenuated LPS-stimulated NF-kappa B reporter gene expression and IL-8 mRNA expression and protein synthesis. On the contrary, transfection with p120 siRNA significantly elevated LPS-stimulated NF-kappa B transcriptional activity, p65 nuclear translocation and IL-8 expression. Conclusions. Collectively, these results indicate an anti-inflammatory effect of p120 in BECs, through its modulation of NF-kappa B signaling in a RhoA dependent manner.

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Abstracts FR-P-146 Pneumocystis jirovecii: value of a standardized diagnostic procedure with molecular pathology combined with cytochemistry T . Zeiske1, A . Schad1, T . Wehler2, E . Springer1, A .J . Ullmann2, C .J . Kirkpatrick1, T . Hansen1 1 University of Mainz, Institute of Pathology, Mainz, 2University of Mainz, Third Department of Internal Medicine, Mainz Aims. Pneumocystis jirovecii (PJ) is a frequent cause of pulmonary infection in patients with the acquired immunodeficiency syndrome and other immunosuppressive conditions. Often PJ pneumonia represents a life-threatening disorder requiring a sensitive and fast diagnosis. We report on our experience on a combined diagnostic procedure for the detection of PJ. Methods. A total number of 602 cytological specimens were studied between 2008 and 2010 at the University Medical Center Mainz. As a standard approach, all specimens have been routinely analyzed by Grocott’s silver stain and nested-polymerase chain reaction (PCR) simultaneously. The frequency of positive PJ results revealed by both methods was then compared with respect to two different sampling procedures [bronchoalveolar lavage (BAL) and sputum specimen]. For a subgroup of 18 patients, we analyzed the clinical course over a one-year period. Results. Our cohort included 441 BAL (73.3%) and 161 sputum specimens (23.7%). In BAL, we found 22.45% of cases positive for PJ (n=99), with 24.2% of these diagnosed by both cytochemistry and PCR, whilst 75.8% were detected by PCR alone. In sputum specimens, 33 PJ-positive cases could be found (20.5%), most of them (28/33) being detected by PCR alone. In about 24% of all specimens evaluated, cytochemistry revealed various types of fungi such as candida and aspergillus. In the subpopulation examined for the clinical course, we found 14/18 patients requiring ventilation by respirator. In that PJ group, the large majority (i.e. 11/14) was detected solely by PCR. Conclusions. For the diagnosis of clinically relevant cases with PJ, the combination of PCR with cytology/cytochemistry is mandatory. It remains to be investigated, whether additional detection of fungi (by cytochemistry) influences the clinical outcome of PJ patients.

Poster: Hämatopathologie FR-P-147 Bone marrow biopsies of patients with haematopoietic and lymphoid disorders – epidemiology, chromosomal aberrations and molecular pathology S . Hehne1, S .M . Schulze1, P . Richter1, C . Geier1, Y . Chen1, A . von Deimling2, I . Petersen1 1 Jena University Hospital, 2Heidelberg University Hospital, Institute of Neuropathology Aims. Bone marrow biopsy of the iliac crest is the first and most important step in the diagnostics of haematopoietic disorders. Methods. The biopsies of the years 2006 and 2007 from the institute of pathology of the Jena university hospital were retrospectively analyzed for clinicopathological parameters. In addition, the Mitelman database was retrieved for chromosomal aberrations. Results. The analysis of 2820 reports from 1185 patients revealed that lymphomas, plasmocytoma and acute leukaemia were most frequent. Males predominated in myeloproliferative neoplasms and lymphoma subtypes, particularly CLL, except for plasmocytoma and acute leukaemia. A peak incidence was seen between 61 and 70 years of age with a varying pattern for single entities. The database search revealed that ALL, AML, CLL and cmL were mainly diploid while Hodgkin lymphoma, mature B-cell lymphoma and multiple myeloma mostly carried hyperdiploid chromosome numbers. Numerical aberrations like chromosome 8 gains

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in hyperdiploid cmL were prominent in specific subgroups. Molecular testing is exemplified in cmL, plasma cell myeloma and hairy cell leukaemia. Conclusions. The study highlights typical clinicopathological characteristics and new genetic findings in haematopoietic and lymphoid neoplasms with relevance for the new WHO classification and beyond. We hope that it may help in the differential diagnosis of bone marrow biopsies.

FR-P-148 Clonally related nodular lymphocyte-predominant Hodgkin lymphoma and classical Hodgkin lymphoma occurring as a collision lymphoma M . Szczepanowski1, N . Masqué-Soler1, I . Oschlies1, W . Schmidt2, A . Lück3, W . Klapper1 1 University Hospital Campus Kiel/Institute of Pathology, Section Hematopathology, House 14, Kiel, 2Pathology Practice, Rostock, 3Practice for Internal Medicine, Hematology and Oncology, Rostock Aims. Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) with the typical lymphocyte-predominant (LP) cells, and classical Hodgkin lymphoma (cHL), characterized by Hodgkin and Reed-Sternberg (HRS) cells, are considered two distinct diseases whose co-occurrence in one patient is extremely rare. We report on clonal relatedness in a case of concurrent NLPHL and cHL, residing in one lymph node in a 48-yearold male patient. Methods. We diagnosed synchronous NLPHL (CD20+, CD30−, LMP1−) and cHL (CD30+, CD20−, LMP1−) in formalin-fixed paraffin-embedded (FFPE) tissue sections of one lymph node in a 48-year-old male patient. CD20 or CD30 labeled neoplastic cells were separately collected by laserassisted microdissection (LCM). Immunoglobulin heavy chain (IGH) gene rearrangements were pre-amplified by standard multiplex polymerase chain reaction (PCR) and subjected to fragment analyses and Sanger sequencing. Results. In the frame work (FR) 3 multiplex PCR, fragments of 120/121 and 128 base pairs (bp) in LP cells and 120/121 bp in HRS cells were obtained. The 120/121 bp fragment shared by both LP and HRS cells was re-amplified by VH1 and JHrev primers as a clear 121  bp fragment. Sequencing analyses of the shared 121  bp product revealed an identical rearrangement consisting of IGHV1-2*3/IGHD4-17*01/IGHJ4*02 with identical VH-DH junction (5’-GCCAT-3’) and DH-JH junction (5’-CCTCTCTTTTGACTG-3’) sequences and a mutation rate of 5.6% in both entities. Consequently, PCR with allele-specific oligonucleotides (ASO) yielded identically sized fragments in both LP and HRS cells. Conclusions. We conclude that both approaches, sequencing of VH1/ JHrev PCR products and amplification of identically sized ASO products, are highly indicative for a clonal relationship of the concurrent NLPHL and cHL. The findings point to a common precursor B-cell of germinal center origin for both, the cHL and the NLPHL, in analogy to well documented clonal relations in cases of concurrent or sequential Hodgkin lymphoma (HL) and non-HL.

FR-P-149 Combination of Castleman disease and Langerhans cell histiocytosis in a supraclavicular lymph node H. Geddert1, A. Dimmler1, U. Götschin2, M. Schatz3, G. Faller1 1 St. Vincent Hospital, Institute of Pathology, Karlsruhe, 2St. Vincent Hospital, Department of General Surgery, Karlsruhe, 3St. Vincent Hospital, Department of Hematology and Oncology, Karlsruhe Aims. A 51-year-old female patient presented to our hospital with a 5 cm measuring mass in the right supraclavicular fossa. Because of known nodular goiter an ectopic thyroid nodule was suspected. The patient was in good clinical condition without B symptoms. To exclude malignant lymphoma the whole mass was excised. Methods. During intraoperative frozen section suspicion of Castleman disease was raised, although a second cellular component remained unclear. Results. After routine tissue processing diagnosis of Castleman’s disease of hyaline-vascular subtype in a lymph node was confirmed. The second component emerged as Langerhans cell histiocytosis. No signs of multicentric Castleman disease or its associated conditions were found. Systemic Langerhans cell histiocytosis was excluded clinically. Conclusions. One year after diagnosis the patient is still in good condition without any sign of relapse. To our knowledge, this is the first report of a combination of Castleman disease and Langerhans cell histiocytosis in a single lymph node.

FR-P-150 Notch2, possible a crucial gene in pathogenesis in lymphoma X.-x. Zhang1, Q. Lai1, R. Zhou1 Zhejiang University, School of Medicine, Hangzhou, China

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Aims. In mammals, there are four kinds of Notch transmembrane receptors (Notch1 to 4); and there are many kinds of Notch target genes include Hes family, nuclear factor kappaB (NF-κB), cyclin D1, c-myc, p21, p27, etc. In recent, researches show that Notch2 signaling may be an important regulator of B cell lymphocytes activation and effect cell differentiation. However, the functions of Notch2 in lymphoma remain poorly understood. In our study, we try to investigate the role which Notch2 plays in the pathogenesis of lymphoma, especially on B cell lymphoma. Methods. First, Notch2 was detected in 95 B cell lymphoma samples through PCR, and DNA sequencing was used to see if there were mutations or not. Then the pEYFP-n1, which contains the Notch2 gene or not, was transfected into the lymphoma cell lines (include raji, ramos, daudi, jurkat and molt4) and 293T cell by electro-transfection. And then total RNA and protein were extracted from the transfected cells, and then the expression levels of targeted genes, such as c-myc, cyclinD1, p21, p27, bcl6, p50 and p65, were detected by real-time PCR or Western blot. Statistical analysis was done by the Student’s t-test between the test groups and the control group. A P-value of <0.05 was considered as statistically differences. Results. In 95 B cell lymphoma samples, we found 3 cases with Notch2 mutaion had mutations in exon34, and all of them had A-G dual-peak at 7618 and G-A dual-peak at 7564, which made tryptophan turn to termination codon. In all lymphoma cell lines, the expression level of bcl6 and P65 is down-regulate, especially in Ramos cell (p<0.05), which is a kind of Burkitt lymphoma cell lines. However, the expression level of P65 is up-regulate in 293T cells. Conclusions. In B cell lymphomas, Notch2 receptors are easy to have mutations in exon34, which can make the PEST domain of Notch2 activation by Ubiquitination. The function of Notch2 in lymphoma cells and normal cells may be different, and bcl6 is likely to be regulated by Notch2. We infer form these results that Notch2 may signify good prognosis.

This work was supported by grants from the Natural Science Foundation of Zhejiang Province (Grant No. Y2090167) and Research Foundation of Scientific and Technical Bureau in Zhejiang (Grant No. 2009C33039).

FR-P-151 Pyrosequencing of BRAF V600E in routine bone marrow samples of hairy cell leukemia identifies CD5+ hairy cell leukemia variant that lacks BRAF V600E J.K. Lennerz1, B. Klaus1, R. Marienfeld1, P. Möller1 University Ulm, Institute of Pathology, Ulm

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Aims. Tiacci et al. identified that 100% of hairy-cell leukemias (HCL) carry the BRAF V600E mutation (NEJM 2011; 364). Based on the frequency and availability of BRAF inhibitors there is an implicit understanding that BRAF-testing could become one element of the laboratory diagnosis of HCL. During the implementation process of this test, we performed validation experiments using pyrosequencing. Here, we report the diagnostic performance measures in synopsis with reported data as well as the unexpected finding of a variant HCL that was BRAF wild-type. Methods. We reviewed our files, microdissected tissues, extracted DNA and performed pyrosequencing of BRAF V600E. Mutant peak heights and density of the leukemic infiltrate were assessed and correlated (Spearman with p<0.05 considered significant). The overall diagnostic performance in our series was reviewed in conjunction with cases in the COSMIC database and all 6 published reports (status Nov.15, 2011). Results. We identified a consecutive series of 18 HCL-patients (median age: 56; range: 40–74 years; 16 males). Immunolabeling showed CD20+, DBA-44+, CD11c+ whereas CD23 or cyclin-D1 was negative. Using decalcified and paraffin-embedded bone marrow samples, in one case the DNA-quality was insufficient and 16/17 informative cases were BRAF V600E mutant (analytical and diagnostic sensitivity 94%). Mutant peak heights and infiltrate density correlated significantly (r=0.66; p=0.006). The one case without BRAF V600E was that of a 74-year-old Caucasian man with HCL, which, in addition to CD20/CD11+ and cyclin-D1-, showed strong CD5+, diagnostic of so-called variant HCL; all other BRAF V600E cases were CD5-negative. Currently, a total of >2000 lymphoid/ myeloid neoplasms have been reported and even when counting variant HCL (n=20+1 reported here) as false negatives, the Youden index for BRAF V600E as diagnostic of HCL is 0.93 with excellent specificity (97.6%) and sensitivity (87.9%). Conclusions. Pyrosequencing for BRAF V600E offers a quick, reliable and cost-effective option for clinical testing. Albeit lack of BRAF V600E in variant HCL has marginal influence on the overall excellent diagnostic performance, these findings are an important component of the implied topic whether BRAF status will become a definitional requirement for HCL; especially in light of the high efficacy of current therapies. Ultimately, cost-benefit analysis will determine whether BRAF testing will become part of the laboratory diagnosis of HCL.

FR-P-152 Relapse of angioimmunoblastic T-cell lymphoma after 11 years of complete remission P. Lohneis1, M. Hummel1, K. Jöhrens2, I. Anagnostopoulos2 1 Charité Medical University Berlin/Institute of Pathology, Institute of Pathology, Charité Campus Mitte, Berlin, 2Charité Medical University Berlin, Institute of Pathology, Charité Campus Mitte, Berlin Aims. To analyse the clinical features, histopathological and clonality data of a patient with angioimmunoblastic T-cell lymphoma (AITL) with an unusual long course. Methods. Immunhistochemistry and T-cell receptor (TCR) gamma chain gene rearrangement analysis. Results. In 2000 the diagnosis of AITL was established in a 74 years old female presenting with involvement of cervical, axillary and mediastiDer Pathologe Suppl 1 · 2012 

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Abstracts nal lymph nodes and splenomegaly. Investigation for TCR gamma gene rearrangement revealed the presence of a dominant amplificate. After 8 cycles of standard cytotoxic chemotherapy according to the CHOP regimen a complete remission was achieved. Eleven years later, the patient presented with cervical and axillary lymphadenopathy and splenomegaly. Histological and immunohistological examination of an excised lymph node revealed a T-cell lymphoma with the characteristic features of AITL. Clonality analysis for TCR gamma chain genes revealed a clonal population with a different amplificate size that those from the initial manifestation. Conclusions. To our knowledge, this is the longest recurrence free period in AITL reported so far. Most interestingly the patient exhibited a novel T-cell clone at relapse not identifiable even among the minor clones at initial presentation.

FR-P-153 Severe central nervous system (CNS) graft versus host disease (GVHD) in a patient without any other GvHD symptoms after allogeneic stem cell transplantation C. Schrader1, R. Stingele2, W. Brück3, I. Metz3, C. Riedel4, N. Schub1, A. Humpe1, T. Valerius1, G. Deuschel2, M. Gramatzki1, A. Günther1 1 University Hospital of Kiel, 2nd Department of Medicine, Kiel, 2UKSH, Campus Kiel, Department of Neurology, 3University of Göttingen, Department of Pathology, 4UKSH, Campus Kiel, Department of Neuroradiology Aims. Although graft versus horst disease (GvHD) is the most relevant complication of allogeneic stem cell transplantation (SCT), it rarely affects the central nervous system. Recently, a consensus conference proposed criteria of diagnosis for cerebral GvHD including the obligatory manifestation of chronic GvHD at other organs [Grauer et al., Brain, 133: 2852, 2010]. We observed a 41-year-old women, who developed spastic paralysis and fell into coma 2.5 years after having an allogeneic peripheral blood stem cell stem cell transplantation (PBSCT) for acute myeloblastic leukemia from an unrelated HLA 9/10-matched donor. The patient presented with a history of several month of headache supposed to be caused by migraine. She had a history of acute GvHD stage III (skin and intestinal) but no signs of chronic GvHD. In addition she had no history of an independent autoimmunopathy or migraine prior to SCT. Methods. MRI scan was performed, cerebrospinal fluid was analyzed to exclude CNS relapse and infectious agents, and finally CNS biopsy was obtained by open brain surgery. Results. MRI scan showed disseminated severe leucencephalopathy without established sign of CNS relapse, lymphoma or typical infection. The cerebrospinal fluid analysis was normal. Toxoplasmosis and viral infection such as HSV, VZV, ADV, EBV, cmV, HHV-6 and HIV was excluded by PCR. The blood lymphocyte subset showed lymphopenia, however with normal CD4/CD8 ratio. Finally CNS biopsy revealed T-cells close to blood vessels – a pattern typical for cerebral GvHD. Immunosuppressive treatment was started with high dose steroids in combination with mycophenolatmofetil (MMF). She recovered rapidly and became completely awake within one week. After 9 months of immunosuppression the patient is competent in activities of daily living. Conclusions. GVHD of the central nervous system (CNS) is a rare disease after allogeneic stem cell transplantation. The absence of lymphocytes in the cerebrospinal fluid and normal CD4/CD8 ratio in peripheral blood does not exclude GvHD of the CNS. CNS biopsy should be considered to confirm the diagnosis. This case demonstrates that CNS involvement can be the only manifestation of chronic GvHD. Immunosuppressive therapy may provide an impressive benefit in these patients.

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FR-P-154 Tuberculous lymphadenitis and mycobacterium-negative granulomatous disease in patients receiving Imatinib mesylate (Glivec) treatment for gastrointestinal stromal tumors (GIST) A. Agaimy1, A. Stegmann2, A. Mackensen3, N. Meidenbauer3 1 Friedrich-Alexander University of Erlangen, Institute of Pathology, Erlangen, 2 Friedrich-Alexander University of Erlangen, Department of otorhinolaryngology, head and neck surgery, Erlangen, 3Friedrich-Alexander University of Erlangen, Medical Clinic 5, Erlangen Aims. Imatinib mesylate (IM) represents the standard treatment for patients with BCR-ABL-positive chronic myelogenous leukemia (CML) and is the first line adjuvant and palliative treatment modality for those with disseminated or inoperable gastrointestinal stromal tumor (GIST). IM is not known to be associated with an elevated risk for tuberculosis, since only five patients have been reported to date who developed tuberculosis during or after IM treatment for cmL (n=4) and GIST (n=1). Methods. We describe our experience with 3 patients (45–79 yrs of age) who developed necrotizing (caseating) granulomatous disease during IM treatment for GIST. Mean duration of treatment with Imatinib was 9.5 (range: 9–48) months. Results. In 3 patients, enlarged lymph nodes with increased metabolism in Fludoxyglucose-Positron emission tomography (FDG-PET)-computer tomography-examinations were detected and resected. Affected sites were subcarinal/mediastinal (1), mediastinal/supraclavicular (1) and periparotideal cervical (1) lymph nodes. Histologic examination revealed necrotizing granulomatous disease suggestive of infection with M. tuberculosis or sarcoidosis. Sputum examination was negative for acid fast bacilli in all patients and DNA was negative for M. tuberculosis and other mycobacteria in two cases. However, M. tuberculosis was detected by PCR in the lymph node of one patient who was then successfully treated by antituberculous agents. All other patients received no anti-tuberculous therapy and were on complete response or stable neoplastic disease without evidence of progressive lymphadenopathy or lung lesions suggestive of active tuberculosis. Leucocyte and lymphocyte counts have been within normal limits throughout treatment with IM. Conclusions. Our observations underline the necessity to sample enlarged or metabolic active lymph nodes developing during IM treatment for timely diagnosis and appropriate treatment of these rare complications. Follow up without treatment is safe for lesions without detection of M. tuberculosis by PCR. More studies are needed to clarify the potential causal relationship to IM treatment and to sufficiently explore the pathogenesis of lesions that were negative for M. tuberculosis.

FR-P-155 Therapeutic Polo-like kinase 1 inhibition results in mitotic arrest and subsequent cell death of leukemic cells in acute myeloid leukemia C. Münch1, A.M. May1, A.V. Pfister1, K. Thurig1, M. Lübbert2, R. Wäsch2, T. Taube3, S. Lassmann1, M. Werner1 1 University Freiburg Medical Center, Institute of Pathology, Freiburg, 2University Freiburg Medical Center, Department of Hematology and Oncology, Freiburg, 3Boehringer Ingelheim Pharma GmbH & Co. KG, Clinical Research Aims. The mitotic kinase Polo-like kinase 1 (Plk1) is an important cell cycle regulator which is frequently overexpressed in acute myeloid leukaemia (AML). Our previous examination of AML blasts in pre- and posttreatment bone marrow biopsies of AML patients treated with the small molecule PLK1 inhibitor BI2536 revealed an increase of aberrant mitotic figures and apoptotic cells 24  hours after administration. The aim of this study was to extend this examination by investigating the effects of therapeutic PLK1 inhibition on AML cell lines and lymphoblastoid cell lines (LCLs) from healthy controls. Methods. Five AML cell lines (HL-60, KG1, OCIM2, NB4 and THP1) and two LCLs (LCL1 and LCL2) were cultured for 24 and 48 hours with

or without different concentrations of BI2536 (10 nM, 50 nM, 100 nM, 200 nM and 500 nM). Cell cycle analysis was performed after 24 and 48  hours of culture using FACS (PI-staining). Western blot analysis was conducted after 24 (H3Ser10ph) and 48 hours (cleaved caspase-3) of BI2536 treatment. Immunofluorescence staining of cells was done using antibodies detecting Plk1 (24 h, 100 nM BI2536), cleaved caspase-3 (48 h, 200 nM BI2536) and alpha-tubulin (24 and 48 h). Results. Cell cycle analyses of AML cells after 24 h of treatment revealed an accumulation of mitotic cells (4N) and a decrease of cells in G0/ G1 phase (2N) of the cell cycle. Furthermore, elevated protein levels of the mitosis specific phosphorylated histone 3 (Ser10) were detected by western blot after 24 h. Immunofluorescence staining of PLK1 and alpha-tubulin showed an increase of mitotic figures exhibiting monopolar spindles without Plk1 localization at centrosomes or kinetochores of prometaphase chromosomes. A strong increase of cleaved caspase-3, which was used for the detection of apoptotic cells, was visible after 48 h by western blot and immunofluorescence analyses in inhibitor treated cells. Furthermore, the less proliferative LCLs arrested in mitosis to a lower extent and showed fewer apoptotic cells after 48 h compared to the analyzed AML cells. Conclusions. Our data confirm the sensitivity of leukemic cells to Plk1 inhibition by BI2536. Treatment with variable doses of BI2536 results in prometaphase arrest and a pronounced cell death after 48 h in AML cell lines. In addition, our experiments with lymphoblastoid cell lines indicate that less proliferative hematopoietic non-leukemic cells show a weaker response to therapeutic Plk1 inhibition by BI2536.

FR-P-156 Analysis of BCL6 and MYC coexpression in diffuse large B-cell lymphoma L. Culemeyer1, C. Stuhlmann-Laeisz1, W. Klapper1 UKSH Campus Kiel, Institute of Pathology, Kiel

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Aims. The transcription factor BCL6 is the master regulator of the germinal centre reaction. cmYC is a transcription factor, which is often overexpressed in diffuse large B-cell lymphomas (DLBCL), but is not expressed in the germinal centre under physiological conditions. Herein we aimed to quantify the unphysiological co-expression of BCL6 and cmYC in DLBCL. Furthermore, functional effects of this unphysiological coexpression of BCL6 and cmYC will be estimated by evaluating BCL6 target gene expression. Methods. Fluorescence multi-stainings using the combination of BCL6 and cmYC were quantified by digital image analysis (Tissuequest©). Results. Unphysiological co-expression of BCL6 and cmYC in lymphoma cells was detected in DLBCL. This co-expression occurred in DLBCL with and without BCL6 or cmYC translocations and influences BCL6 target gene expression. Conclusions. The unphysiological co-expression of transcription factors of the B-cell differentiation can be detected in DLBCL without genetic alterations affecting these genes. We suggest that this co-expression interferes with the activation or inhibition of the respective target genes. We consider this mechanism to be a reason for the oncogenic potential of transcription factors, which are also expressed under physiological conditions.

FR-P-157 Conditional PHD2 deficiency leads to non-lethal erythrocytosis and alters the hematopoietic stem cell fate B. Wielockx1 TU Dresden – Pathology, Dresden

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Aims. Hypoxia is a prominent feature during development and physiological as well as pathological conditions in adults. An oxygen-sensing machinery is therefore very important to help the cells adapt instantaneously to any inacceptable O2 level. Such a system relies on the oxygen dependent HIF-prolyl hydroxylases (PHD1-3), enzymes that can inactivate the alpha subunit of the hypoxia inducible transcription factor (HIF). In case of low oxygen availability, PHDs lose their functionality and allow the HIF complex to promote biochemical and physiological changes including anaerobic glycolysis, angiogenesis and hematopoiesis. Results. Our research unit produced a mouse line that lacks PHD2 in a broad spectrum of cell types (e.g. hematopoietic cells, epithelial cells) which resulted in an unexpected hematologic phenotype. The mice display strongly elevated hematocrit levels together with high EPO concentrations in the blood although mice don’t show any premature lethality. Moreover, we found that the hematopoietic stem cell (HSC) compartment in the bone marrow was significantly altered compared to WT mice. Conclusions. Indeed, detailed FACS analyses demonstrate that cKO mice contain much more multipotent progenitors (MPPs). Moreover, under stress conditions in vivo, cKO HSCs are pushed towards self-renewal. Double deficient cKO mice with one of the two HIFα revealed that the erythrocytosis phenotype is exclusively driven by HIF2α, whilst HIF1–α is responsible for the HSC/MPP phenotype.

FR-P-158 Tumor infiltrating T-cells in high risk chronic lymphocytic leukemia (B-CLL): a clinicopathological study of CD3, CD8 and FOXP3 expression C. Schrader1, C. Pflüger1, S. Stilgenbauer2, H. Döhner2, M. Ritgen1, J. Claasen1, P. Dreger3, W. Klapper4 1 University Hospital of Kiel, 2nd Department of Medicine, Kiel, 2University of Ulm, Department of Hematology, 3University of Heidelberg, Department of Hematology, 4UKSH, Campus Kiel, Department of Pathology Aims. The prognostic value of the mutation status of the immunoglobulin heavy chain variable region (IgVH) and cytogenetic abnormalities in chronic lymphatic leukemia is well known. We investigated the tumour associated reactive T-cell infiltrates in bone marrow and lymph nodes biopsies of patient with high risk B-CLL in correlation to other clinical, biological and genetic markers including age, sex, binet stage, bone marrow involvement (infiltration pattern and grade), β2 microglobulin level, leucocytes account, lymphocytes double time, thymidinkinase level, cytogenetic aberrations (e.g. 11q deletion), IgVH mutations status and ZAP 70 expression Methods. Bone marrow (n=51) and lymph node biopsies (n=8) from 59 untreated patients (43 men and 16 women) were investigated immunohistochemically with monoclonal antibodies against CD20, CD5, CD23, CD3, CD8, FOXP3 and ZAP70. Cells with clear positive staining were counted and the percentage was calculated. Molecular analysis of IgVH mutation and FISH analysis was done from fresh peripheral blood tumor cells. Results. In 58 biopsies the CD 3 staining, 57  cases of the CD 8 and in all 59 cases the FOXP staining was evaluable. The CD3 staining had a range of 0.4% to 35.2% with a median of 9.3% and a mean of 11.2%. In lymph node tissue a significant higher number of CD3 cells than in bone marrow biopsies (p=0.0054) was found. Similar results were found in the CD8 (p=0.0052) and FOXP3 (p=0.0087) stainings with higher T-cells account. In the analysis of the bone marrow biopsies the nodular pattern/ involvement showed a significant higher number of CD3 cells (p=0.013) Der Pathologe Suppl 1 · 2012 

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Abstracts than the diffuse pattern. Similar results could be found concerning the FOXP3 cells and the infiltration pattern. The nodular pattern had significant more reactive FOXP3 positive cells than the diffuse pattern (p=0.018). High number of CD8 cells were found in patients with lower leucocytes counts (p=0.049) and higher lymphocytes double time (more than 12 months, p=0.048). All other correlation between clinical, biological and genetic correlation with T-cells accounts shows no significant differences. Conclusions. Lymph node biopsies with B-CLL had a significant higher account of T-cells than bone marrow biopsies. Patients with B-CLL and a nodular bone marrow involvement had a significant higher number of CD3+ and FOXP+ T-Cells. High numbers of CD8+ T-cells cells might have a positive influence on the lymphocytes double time and leukocytes level at the time of diagnosis.

FR-P-159 Collision lymphomas in the bone marrow – a diagnostic pitfall A .M . May1, L . Morawietz2, W . Dietrich3, A . Lindemann4, G . Faller5, P . Fisch1, M . Werner1 1 University Freiburg Medical Center, Institute of Pathology, Freiburg, 2 Klinikum Stuttgart, Institute of Pathology, Stuttgart, 3Klinikum Stuttgart, Stuttgart, 4Oncologic Practice, Ettlingen, 5St . Vincentius-Kliniken, Institute of Pathology, Karlsruhe Aims. The simultaneous co-existence of two distinct lymphoma entities in one bone marrow trephine biopsy (BMB) is rare and can be easily missed, especially in cases with a high density of infiltration. We present the cases of two patients with a compact infiltrate in the BMB, which turned out be composed of two different kinds of lymphoma. Methods. The distinct lymphoma entities in these two patients’ BMB were extensively analyzed, using immunohistochemistry and a PCR-based clonality analysis of immunoglobulin heavy chain gene rearrangements. Results. One patient’s BMB was infiltrated by hairy cell leukemia (infiltration density: 75%). In a lymph node biopsy that had been examined separately, due to a conspicuous abdominal lymphadenopathy, mantle cell lymphoma was diagnosed. Further studies provided a minimal presence of mantle cell lymphoma cells in the BMB as well as a discrete population of hairy cells in the lymph node biopsy. Clonality analysis of the immunoglobulin heavy chain gene identified two distinct clonal B-cell populations. In the other patient’s BMB an unusual gigantocellular variant of B-lymphoblastic lymphoma was diagnosed. The immunohistochemical analysis indicated a diagnosis of cALL. Flow cytometric analyses found two separate, but only discrete B-cell populations in the peripheral blood and bone marrow. One of these populations was immature; the other population was suspicious of mantle cell lymphoma. Subsequent immunohistochemistry showed a minute additional presence of mantle cell lymphoma in the BMB. Conclusions. In case of clinically unusual presentation, additional examinations, such as more extensive immunohistochemistry, molecular methods and flow cytometric analyses, need to be included into the diagnostic spectrum of lymphomas, to be able to identify rare cases of collision lymphomas.

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FR-P-160 Primary gastric ALK-negative anaplastic large cell lymphoma: a  clinicopathologic analysis of four cases C . Liu1, Y . Lai1, X . Wang1, X . Huang1, M . Li1, Z . Gao1 1 Peking University Health Science Center, Beijing, China Aims. To evaluate clinicopathological and immunophenotypic features of primary gastric ALK-negative anaplastic large cell lymphomas (ALCLs). Methods. Formalin-fixed, paraffin embedded tissue blocks of 4 patients diagnosed with primary gastric ALK-negative ALCL were obtained. Hematoxylin and Eosin-stained slides were used to evaluate histological changes and the immunophenotypic features were detected by immunohistochemistry. In addition, the abnormality of ALK gene was determined by interphase fluorescence in situ hybridization (FISH). Results. The cases were comprised of three men and one woman, with a median age of 58.5 years. All the four cases presented with epigastric discomfort with or without gastrorrhagia. Endoscopic examination revealed solitary gastric ulcer in all cases. Endoscopic biopsy from one patient and surgical specimens from three patients were available. Morphologically, the normal architecture of gastric wall was effaced by the diffuse infiltration of tumor cells, in which the characteristic hallmark cells were easily identified in all cases. The tumor cells of all cases showed a consistently strong expression of CD30 but lack of the expression of ALK1. Moreover, the tumor cells demonstrated varying expression of T cell markers such as CD3 (3/4) and CD43 (1/1), and negative for B-cell markers CD20 and PAX5. Chromosomal rearrangement involving ALK gene was not detected by FISH. Three of the four cases underwent total or partial gastrectomy followed by chemotherapy and till the last followup; none of them developed a relapse or progression. The rest one patient refused surgery and chemotherapy, and died 20 months after diagnosis. Conclusions. Here we reported four ALK-negative ALCLs occurred in the stomach. Although it’s a rare condition, we should keep in mind to avoid misdiagnosis especially of the gastric carcinoma. The current results suggested that ALK-negative ALCL may have a good prognosis with surgery combined with chemotherapy.

Poster: Autopsie/Fallstudien/Sonstiges FR-P-161 Postmortem CT in clinical autopsy S . Westphal1, J . Apitzsch2, T . Penzkofer2, A . Perez-Bouza1, B . Sellhaus3, A . Mahnken2, R . Knüchel-Clarke1 1 RWTH Aachen University, Institute of Pathology, Aachen, 2RWTH Aachen University, Department of Interventional and Diagnostic Radiology, Aachen, 3 RWTH Aachen University, Institute of Neuropathology, Aachen Aims. While autopsy rates in clinical pathology are declining for the past decades, and forensic sciences are using virtual autopsy techniques increasingly in daily routine, pathologists are not yet tapping the potential of modern imaging techniques. To assess the value of virtual autopsy techniques in clinical pathology, post- mortem imaging was performed before autopsy. Methods. In 29 autopsy cases, a full-body high-definition pmCT (postmortem computed tomography) scan was performed prior to autopsy. Images were analyzed by experienced radiologists. Autopsy was performed following a standard protocol, taking special care of macroscopical findings, detected in the CT scan previously. Digital macroscopical pictures of the organs and of their histology were taken. We compared pmCT and classical autopsy findings regarding cause of death and death-related diagnoses, reconstruction of the key pathomechanism leading to death and side diagnoses.

Results. In 18 cases (79%) the cause of death (e.g. hemorrhage), was diagnosed correctly by pmCT. In 11 cases (38%) the key pathomechanism of death (e.g. anastomotic leakage from aortic surgical site) was found by pmCT. Side diagnoses found by pmCT improved and complemented those found by traditional autopsy. Especially in the documentation and visualization of bone lesions, CT-imaging was superior to macroscopic examination because of the simplicity and elegance of the method compared to classical bone pathology. Conclusions. pmCT is an elegant and suitable method to complement the macroscopic and microscopic examination of classical autopsy. Our first results are a good basis to continue by application of contrast enhancement or collection of biopsies during pmCT.

FR-P-162 Speed – all that matters? F. Fronhoffs1, B. Roick1, G. Kristiansen1 University Bonn Medical Center, Institute of Pathology, Bonn

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Aims. After change of the head of Pathology Department of the University Hospital Bonn in spring 2011 we conducted a survey among all our senders in order to evaluate their expectations concerning diagnostics and service. Methods. We sent a questionnaire including 27 topics by mail to all our senders. They could answer using a scale from “0”(“not important at all”) to “10” (“very important”). Results. We sent 2172 questionnaires and received 91 answers (gynaecology n=17, internal medicine n=16, pathology n=9, surgery n=6, general practitioners n=6, radiology n=5, haematology n=5, others n=27). Most important for our senders were “speed of communication of the diagnosis” (906 of 910 possible points), “personal availability of the responsible pathologist by phone” (889/910), and “friendliness of contact” (829/910). Less important were “performing autopsies” (422/910), “24-hours on-call duty”(322/910) and “service on Saturday” (321/910). Conclusions. The survey results illustrate the increasing importance of pathology as an interdisciplinary and cross-linking clinical specialization. In addition to speed and efficiency, a professional and amiable colleague is demanded as pathologists play a key role in tumour boards and personalized therapies. The decreasing importance of autopsies, last but not least a tool of quality control, is regrettable. However, this general development is reflected in decreasing numbers of performed autopsies per annum in our department.

FR-P-163 Retrospective autopsy study of the University of Mainz, 1970–2010 T. Hansen1, M. Dusolt1, S. Höring1, C. Kempe1, F. Rosendahl1, M. Kavciakova1, M. Hechtner2, A. Spriestersbach2, C.J. Kirkpatrick1 1 University of Mainz, Institute of Pathology, Mainz, 2University of Mainz, Institute of Medical Biostatistics, Epidemiology and Informatics, Mainz Aims. In the past, numerous analyses studied several aspects of autopsy, in particular with regard to the decline of frequency. Especially in the last years long-term studies comprising more than one decade are sparsely published. Methods. We analysed the archival data of the Institute of Pathology of the University of Mainz for autopsies performed between 1970 and 2010. We focused on patients at least 14 years old (n=14724) who died in the University hospital. We compared the number of autopsies with the total number of deceased patients and studied several epidemiological aspects with special relevance for the cause of death (COD). Results. In 1970, the autopsy frequency was 62% and fell to 49.1% in 1980. In the following decade, there was a steady state (frequency 53.3% in 1985, and 43.2% in 1990), followed by a remarkable decline between 1995 (30.6%) and 2000 (9.7%), and finally 2010 (5.6%). The overall mean age increased

during the observation period (59.6 yrs in 1970, 67.5 yrs in 2008). Among the COD groups, cardiovascular diseases were predominantly recorded (between 35% in the 1970s and 39% in 1995–2010), followed by infectious diseases (between 20 and 25%). Malignancies represented the third most common COD group, with an increase of frequency from about 10.5% in the 1970s to 17% observed in the last decade. Among the single specific CODs, pulmonary embolism was most often encountered in the 1970s (about 11.5%), while in the following decades, myocardial infarction predominated (up to 15.8% between 1995 and 2010). In the overall period, lung cancer was the single most common malignancy of the CODs (between 2.5 and 3.9%). In addition, a total number of 57 patients were described as suffering from infection by Human Immunodeficiency Virus (HIV). Of this cohort, 31.6% died due to the HIV infection. Concerning tuberculosis, the frequency fell from 7.9% in 1970 to about 3% in the late 1970s and increased again to 6–7% in the subsequent decades, followed by a decrease in 1995–2010 to about 2%. Conclusions. In this study, we were able to analyse autopsy data over a long-term period. Most of our results are in line with previous reports. In particular, these data confirm studies showing that in Germany the autopsy frequency began to remarkably decline in the 1990s (by contrast to several Anglo-American reports on decrease in the 1980s). With the exception of some specific findings, we could not observe a general switch in the COD groups in spite of the dramatically changed autopsy number.

FR-P-164 Introduction of a structured report to quantify additional diagnostic information by autopsies G. Ates1, J. Friemann2 Department of Internal Medicine II, Klinikum Lüdenscheid, Lüdenscheid, 2 Institute of Pathology, Klinikum Lüdenscheid, Lüdenscheid

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Aims. Autopsy proves quality of diagnostic procedures as well as success and adequacy of therapeutic decisions near the end of life. Our investigations were aimed finding a way to quantitatively describe the gain of diagnostic information by autopsy and whether this can be made available as an indicator of quality for inter-institutional analysis. Methods. From 2004 to 2010, pathological-anatomical diagnoses of 322 autopsies in the Institute of Pathology, Märkische Kliniken GmbH, were grouped into 4 different categories on a structured report – regarding to their role in the cause of death and compared to life-time diagnoses (1. clinically known major disease, 2. revealed unknown major disease, 3. important findings, 4. secondary findings). Further, in every autopsy the extent of congruency of clinically suspected and pathological-anatomical assured cause of death was classified (totally congruent, partially congruent, not congruent). Results. In the evaluated period 322 autopsies (~5% of the deceased in hospital) were performed. Predominantly (95%) clinically known major diseases leading to death were confirmed by autopsy. Additionally in 71% of the cases autopsy revealed unknown major diseases missed during the clinical course but significantly contributing to the lethal course. Only in 42% there was total congruency of clinical suspected and pathologicalanatomical assured immediate cause of death. Conclusions. This proposal of grouping pathological-anatomical diagnoses into different categories on a structured report may help to quantify the gain of additional diagnostic information by autopsy and to introduce these categories into the quality reports of German hospitals as an instrument for inter-institutional quality control.

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Abstracts FR-P-165 Potential complications of aortic valve implantation via a transfemoral artery catheter: an autopsy perspective H. Löser1, H.P. Dienes1, J. Fries1 University of Cologne, Institute of Pathology, Köln

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Aims. Degenerative or post-endocarditic destruction of aortic valves with secondary left ventricular hypertension and subsequent cardiac insufficiency is seen more frequently in patients with increasing age. The instable health of many of these patients does not permit a removal of the diseased valve in an open surgical procedure. Instead, an aortic valve implantation is achieved via either a transapical access or a transfemoral artery catheter. In Cologne, the catheter carrying a ballon-expandable stent with a valve of bovine pericardium (Edwards-SAPIEN System) has been used for the last 3 years. While in the majority of cases this procedure was completed successfully, we encountered several autopsy cases, in which unforeseen complications occurred directly related to this type of valve replacement. Methods. All patients were in the age range of 65 to 80 years. Preclinical evaluation had indicated that a conventional surgical approach either by transsternal access or by intercostal/apical access would not be tolerated by the patient and an aortic valve implantation via transfemoral catheter using the Edwards-SAPIEN Systems was performed. Patients had died within hours of valve implantation and a full body autopsy was performed in each case once appropriate consent was given. Results. The observed complications had been: 1. irreversible compression of implanted valve due to cardiac resuscitation, 2. implantation of a small diameter valve not properly anchored due to excessive calcification with paravalvular leak, 3. loss of valve being dislodged before the aortic isthmus, 4. tilted implantation of valve due to calcifications of the aortic ring with pressure necrosis of aortic wall and paraaortic bleed, 5. transmural aortic rupture due to a calcified ring of the aortic valve after balloon dilatation with intramyocardial/pericardial bleeding leading to coronary compression with secondary hemorrhagic infarction. Conclusions. In all cases the preoperative lack of information of the degree of calcification of the aortic valve leaflets was a common denominator for postoperative complications. Future improvements of three dimensional imaging appear necessary to increase the chance of preventing such complications. Until then, autopsy analysis of complications may be the only way to detect potential weaknesses of an otherwise lifesaving, but high risk procedure.

FR-P-166 Situs inversus totalis of twins: an autopsy case report C. Tóth1, M. Jäckel2, K. Bartók2 University Hospital Heidelberg, Institute of Pathology, Heidelberg, 2 Military Hospital, Budapest, Hungary

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Aims. Situs inversus totalis is a rare congenital abnormality resulting in visceral malrotation of the internal organs leading to left-right asymmetry. In certain cases it is associated with clinically defined syndromes e.g. Kartagener’s syndrome. In the case presented after 9 attempts of in vitro fertilization and reduction (from three embryos to two embryos) in the 20th gestation week cesarean section was performed because of acute purulent chorioamnitis with spontaneous rupture of membranes. Methods. The medical history of the mother included primary sterility with 9-times in vitro fertilization with F II 20210 heterozigosity and MFHFR polymorphism with homozigosity. Further, factor Xa thrombophily was also in her family diagnosed. After the operation the placenta and two fetus were macroscopically and histologically examined. Results. Placenta: 390 g with intact two amnoitic sacs with two umbilical cords with central origin and three blood vessels (2 arteries and 1 vein). The cut surface of the placenta shows some grayish areals, the rest is normal. Fetus A: 304 g, 22 cm long male fetus. Ventricular and ependymal bleeding in the 4th ventricle. In the thorax the heart apex lies on the

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right side, the left lung consists of 3  lobes, the right one consists of 2. The cut heart and the great blood vessels run according to a total situs inversus situation. In the abdomen after removing a blood clot normal developed organs with total malrotation can be found. The pelvis organs macroscopically show no abnormality. Fetus B is a 297 g, 23 cm female fetus with some petechial skin bleedings. The central nervous system has no abnormalities. The thoracal and abdominal organs show the same malrotational changes as described above at fetus A. The changes were photographically documented. Conclusions. The cause of spontaneous abortion was an acute purulent chorioamnitis due to a probably ascending infection which caused the spontaneous rupture of the membrane. The autopsy cannot explore the cause of infection. Both of the autopsied fetus showed the rare malrotation changes, the situs inversus totalis without any other developmental disturbances. In the daily autopsy practice we need to know about these rare changes which are important not only in fetopathology but in anatomical pathology as well.

FR-P-167 Analysis and appraisal of the clinical autopsy results of a surgically oriented cardiac center in terms of quality assurance J. Grüning1, R. Meyer1, M. Dietel2, R. Hetzer1 1 Deutsches Herzzentrum Berlin, Berlin, 2Charité, Humboldt-University in Berlin/ Institute for Anatomic Pathology, Berlin Aims. We aimed to analyze the quality of clinicopathologic documentation and communication. Concentrating on the following two questions: 1.) How valuable are the medical documentation of the inspection of the corpse (post-mortem/clinical autopsy) and the content of the autopsy requests and are they adequate for a qualified-autopsy? 2.) Are the autopsy findings and examinations in line with the quality requirements of a cardiac center? Methods. Between 2000 and 2009 an average autopsy rate of 37% of all decedents (range, 28% to 45%) was reached at our institute. During that time 1063 decedents underwent autopsy. In accordance with the above objectives the following were analyzed and assessed: documentation of the inspection of the corpse (causal chain of the causes of death; personal data; formalities), content of the autopsy requests (clinical procedures), autopsy reports (date of autopsy; date of compilation of the autopsy report and granting of access for the clinician have been recorded). The clinical and autopsied underlying diseases and causes of death were coded in accordance with ICD 10. Results. It was evaluated as positive that documentation of the inspection of the corpse, content of the autopsy requests and the autopsy reports themselves are in correct form. Also positive is, that the recorded autopsy results are discussed during weekly medical meetings at the institution. Regrettably, however, the pathologists do not participate in these meetings. The fact that the majority of autopsy reports do not reach the clinician until after 30 days is a serious problem precluding their prompt evaluation and discussion. The clinicians and pathologists need to change for the better communication in relation to analyzing and assessing the autopsies. There have been discrepancies between the clinically determined causes of death and the autopsy causes of death. In particular the clinically determined cause of death of “sepsis” needs to be discussed. The preparation of the autopsy reports has no real influence on the hospital’s cost accounting (DRG). Conclusions. It becomes apparent that the autopsy report constitutes an effective tool for quality assurance. The weekly medical meeting is a suitable forum to look at quality assurance issues. The quantity and quality of the communication between clinician and pathologist leave room for improvement and strengthening.

FR-P-168 Autopsy findings in a 2-year-old boy with EHEC/HUS in the 2011 German outbreak M. Kuhlmann1*, C. Baier1*, J.U. Becker1, C. Hartmann2, F.-C. Bange3, T. Ahlenstiel4, H. Kreipe1 1 MH Hannover, Institute of Pathology, Hannover, 2Ruprecht-Karls University Heidelberg, Institute of Pathology, Department Neuropathology, 3MH Hannover, Department of Medical Microbiology and Hospital Epidemiology, 4 MH Hannover, Department of Pediatric Nephrology Aims. Demonstration of histopathological findings in a 2-year-old boy with EHEC/HUS in the 2011 German outbreak. Methods. Routine autopsy procedure including gross and microscopic examination with histochemical and immunhistochemical stainings (MSB, CD 61, GFAP, CD 68), and review of clinical data. Results. A previously healthy 2-year-old boy was diagnosed with EHEC/ HUS (serotype O104:H4, Shiga-toxin 2 positive) and developed in the clinical course severe acute renal and heart failure as well as neurological complications. Our main histopathological findings: Heart: heart weight was increased above the 90 percentile. Histology revealed rather fresh focal necrosis of heart muscle cells and thrombocyte rich thrombi in cardial capillaries. Kidney: on cross sections the kidneys appeared darkly red and no distinction between renal cortex and medulla was possible. Histologic examination revealed luminal thrombocyte rich thrombi in glomeruli and in afferent arterioles, with negligible amounts of stainable fibrin. Moreover, glomeruli exhibited severe endothelial swelling and severe mesangiolysis with microaneurysms. Brain: supra- and infratentorial multiple hemorrhagic infarctions (stage I and II) with severe brain edema as well as a laminar necrosis of the cortex (stage I) were detectable. Furthermore there was a severe gliosis with multiple reactive astrocytes and activated microglia in the white matter. Within the complete central nervous system no thrombi indicating TMA were found. Conclusions. In the kidneys endothelial swelling was dominant reflecting endothelial damage caused by Shiga-toxin 2. Endothelial damage induced formation of thrombocyte microthrombi without stainable fibrin content in the heart and in the kidneys with resultant ischemia. While in previously published cases the thrombi contained fibrin depositions besides thrombocytes, in this case we only found minimal or no fibrin with MSB stain. Another key feature of our renal histology was a severe mesangiolysis, which is described as a rare finding in typical D+ HUS whereas it is more often seen in D- negative HUS cases. Regarding the cerebral findings the severe gliosis of the white matter without similar changes in the cortex could indicate a prior non-ischemic, possibly toxic damage to the white matter, as is discussed in the literature. Our case with cerebral, myocardial and renal involvement causing failure of all three organs argues against an overly rigid clinical separation of HUS and thrombotic-thrombocytopenic purpura. *These authors contributed equally to the study.

FR-P-169 Synchronous presentation of gastrointestinal stromal tumor of the stomach, ganglioneuroma of the adrenal gland and adenomas of the colon N. Pawlaczyk1, K. Neumann1, J. Knolle1, H. Zühlke2 Institute of Pathology, Dessau-Rosslau, 2Department of General, Visceral and Vascular Surgery, Lutherstadt Wittenberg

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Aims. Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. Its carcinogenesis is driven by activating mutations of the KIT or PDGFRA gene but a small subset of GISTs has a negative mutation status. These wild type-GISTs may develop as part of a multi-neoplastic disease. We present a case with concurrent presentation of GIST of the stomach, Ganglioneuroma (GN) of the adrenal gland and adenomas of the colon. To our knowledge, the synchronous existence of these neoplasias has not yet been reported.

Methods. We describe an 85-year-old male patient with initially acute upper gastrointestinal bleeding. Explorative laparotomy and subsequently pathologic analysis of resected specimen revealed three distinct neoplasias: GIST of the stomach (5,5×3,7×3,8 cm, <5 mitoses/50HPF) as the cause of bleeding, GN of the adrenal gland (3,5×4,3×2,5 cm) and adenomas (low and high grade intraepithelial neoplasia) of the colon. Molecular analysis of all tumors was carried by DNA analysis of four different genes KIT (exon 9, 11), PDGFRA (exon 12, 14, 18), KRAS (exon 2, codon 12, 13) and BRAF (V600E) which are known to play a role in carcinogenesis. Results. GIST diagnosis was supported by positive immunohistochemical staining with CD117 and CD34. Staining for S100, Desmin and Calponin was negative. The GN proved positive for S100, Chromogranin A and GFAP. The ganglion cells showed a positive staining for Synaptophysin. GIST and GN did not harbour amino acid changing mutations. Only one of the two adenomas contained a KRAS mutation (34G>T, Gly12Cys). GNs are rarely seen in patients with neurofibromatosis (NF) type 1. An association between the development of GIST and type 1 NF has also been established. Our patient showed no further clinical features of NF. GISTs arising in the setting of type 1 NF are usually KIT- and PDGFRA-wild type and the tumor suppressor gene Neurofibromin is inactivated. Testing for a potential silencing of Neurofibromin is underway. Conclusions. The synchronous manifestation of GIST and other neoplasms is a common observation. In our case, the co-occurence of GIST, GN and adenomas of the colon could represent a syndromal setting. Molecular analysis revealed no amino acid changing mutations of the KIT and PDGFRA genes what differs from sporadic GISTs. The role of alternative oncogenes or pathways in the carcinogenesis of wild type-GISTs as well as in their presentation in a multi-neoplastic context requires further examination.

FR-P-170 Pleural malacoplakia caused by Rhodoccocus equi infection in a patient after stem cell transplantation because of a T-PLL C.L. Behnes1, S. Neumann2, S. Schweyer1, H.-J. Radzun1 1 University of Göttingen/Institute of Pathology, 2University of Göttingen/ Institute of Onkology Aims. Malakoplakia is a disease especially of the urinary tract with typical plaques most frequently observed in the bladder’s mucosa, consisting of accumulated macrophages. The reason for this disease is an impaired lysosomal degradation of bacteria, especially E. coli. In the context of immunosuppression malakoplakia can also occur in other organs such as the prostate, kidney and lung. To our knowledge, affection of the pleura by malacoplakia has not yet been documented. Methods. Case report: a 60-year-old man was admitted to the hospital because of a generalized lymphadenopathy, hepatosplenomegaly and suspicion of pneumonia. Based on a lymph node biopsy the diagnosis of a T-cell-prolymphocytic-leukemia was confirmed and treated with an allogenic stem cell transplantation. A half year later the patient was admitted to the hospital with retrosternal pain and a reduced state of condition. The clinical examinations revealed a 12 cm in diameter and well circumscribed focus within the right upper pleura. Results. The macroscopical examination of the upper lobe of the right lung showed a 12 cm in diameter tumor adherent to the pleura, displacing the lung and showing a gray-white cut surface with central necrotic, disintegrated areas. The microscopical examinations revealed a tumor consisting of a monomorphic cell population, which was predominantly composed of macrophages. Granulomas, multinucleated giant cells, significant cellular atypia or increased proliferation could not be observed. The immunohistochemical examinations revealed numerous Ki-M1P/ CD68 positive macrophages with intracellular PAS positive deposits, which could be identified as calcifications by Kossa staining (MichaelisGutmann-Bodies) being pathognomonic for Malacoplakia. A microbio-

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Abstracts logical examination of tumor tissue during surgery could demonstrate Rhodococus Equi. Conclusions. To our knowledge this case represents the first pleural malacoplakia associated with a Rhodococus equi infection. Extravesikal malacoplakia is an important differential diagnosis in immunosuppressed patient especially in case of a proved Rhodococus equi infection.

FR-P-171 Testicular primitive neuroectodermal tumor – molecularpathological analysis and discussion of developement S. Brandt1, B. Lohe2, A. Vogetseder1, T. Rüdiger2, H. Moch1, P. Bode1 1 Universitiy Hospital Zurich, Zürich, Switzerland, 2Städtisches Klinikum Karlsruhe, Pathologisches Institut, Karlsruhe Aims. The occurrence of a testicular primitive neuroectodermal tumor (PNET) is a rare event in malignant transformation of a teratomatous component in testicular germ cell tumors. Based on morphological, immunohistochemical and molecularpathological findings these tumors resemble central PNETs, as otherwise only seen in children and do not show a rearrangement of the EWS gene on chromosome 22. We describe a case of a PNET occurring in a testicular germ cell tumor. Methods. Routine immunohistochemistry (AE1/AE3-Cytokeratin, S100, Synaptophysin, CD99, GFAP, Oct-3/4 and CD30) was performed as well as Fluorescence in situ Hybridization (FISH) and RT-PCR. Results. Immunohistochemistry showed focal expression of AE1/AE3Zytokeratin, S100, Synaptophysin, GFAP and CD99 and no expression of Oct-3/4 and CD30 in the tumor cells. No translocation of t(11;22) (EWSR1/FLI1) and t(21;22) (EWSR1/ERG) could be shown by FISH and RT-PCR. Conclusions. The occurrence of testicular primitive neuroectodermal tumor is a rare event. It is believed to originate from malignant transformation of a teratomatous component in testicular germ cell tumors. Identification of a PNET component in a testicular germ cell tumor is of clinical relevance since studies have shown that these tumors do not respond to conventional cisplatin based chemotherapy in comparison to usual germ cell tumors. Some authors recommend PNET specific chemotherapy, as well as retroperitoneal lymph node dissection.

FR-P-172 Complicated Malaria tropica – two typical disease patterns I. Klempert1, P. Lohneis1, W.D. Schmitt1, M. Dietel1 Charité University hospital, Institute of Pathology, Berlin

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Aims. Malaria – the second most common infectious disease of the world is rare in Germany. Pathologists are seldom tasked with diagnostics, but if demanded it is generally presented by fulminant development and with impressive findings. Methods. On the basis of two autopsies, which had a fulminant, following the lethal course of the disease the typical histological pattern and the correlative medical findings will be demonstrated. The microscopic slides were prepared with the Hematoxylin-eosin-stain and additionally examined by the polarizing microscope to differentiate between the double refracting malaria pigment and stain dependent artefacts. Results. Anamnestic: Case 1: The patient had a fever, diarrhea and vomiting, with kidney failure as symptoms of Malaria (tropica) during a residence in Sierra Leone. Due to the limited therapeutic possibilities locally, the patient tried to return to his home. The flight back home was interrupted by a forced landing, because the patient collapsed. He was brought to the emergency room of the Charité Berlin. The plasmodiumdensity was very high (>30%). The next day an emergency Splenectomie by laparotomie was necessary, followed by a secondary haemorrhage. The cardiovascular system was stable, the plasmodium-density came down to >1%. Six days later the patient died, caused by a multi organ failure with a leading liver insufficiency. Case 2: The patient came to his

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family doctor, because he felt ill and had slurred speech. A few days before, he had returned from a journey to Cameroon. Within the next few hours he drifted off more and more and showed increasing intracranial pressure. He died the next day, caused by a central cardio-vascular system failure. The density of the plasmodia was 10%. Histologic: Case 1: Expanded intra- and pericapillary deposits of hemozoin (iron free malaria pigment) inside the liver, myocardium, kidney and the alveolus of the lung. Agglutinate erythrocytes were found inside the hepatic sinus and some vital, adipose hepatocytes were present. Case 2 shows analogous to case 1 extensive manifestation at the organs. Additionally a massive edematous brain with plasmodia and hemozoin inside the erythrocytes of the intracerebral capillaries was found. Conclusions. The rare disease pattern of a malaria infection appears with extensive histopathological findings that can be demonstrated by simple histological methods in correlation with the clinical development.

FR-P-173 Orbital epithelioid sarcoma: a case report T. Berg1, J. Knolle1, S. Knipping2, C. Kneifel3, K. Stock4, I.F. Ciernik5, T. Mentzel6 Dessau City Hospital, Institute of Pathology, Dessau, 2Dessau City Hospital, Department of Otorhinolaryngology, Dessau, 3Dessau City Hospital, Department of Ophthalmology, Dessau, 4Dessau City Hospital, Department of Radiology, Dessau, 5Dessau City Hospital, Department of Radiation Oncology, Dessau, 6Dermatopathologie Bodensee, Friedrichshafen 1

Aims. Epithelioid sarcoma (ES) is a rare and aggressive soft tissue neoplasm most prevalent in the distal extremities of young male adults. The proximal type of ES is thought to be the morphological progression with predominance for somewhat older patients and a higher propensity for metastasis than classic ES. Methods. We report on a 30-year-old patient who presented with proptosis of the right eye. He complained of metamorphopsy and visual impairment. Diagnostic imaging was suspicious for sarcoma. Preoperative biopsy was performed followed by histological and immunohistochemical analysis. Antibodies against Cytokeratins (clone MNF116, CK 5/6, CK 8/18, CK 7, CK 20), Vimentin, S100-protein, CD34, CD31, CD10, Desmin, Aktin, p63, PHH3 and TTF-1 were used. Wide local tumor excision with curative intent followed. Results. Histological examination of the biopsy revealed a nodular growth pattern of atypical eosinophilic epitheloid and spindle cells with 25 mitoses per 10 high-power-fields. By immunohistochemistry, the tumor was positive for vimentin, cytokeratins and EMA while there was no reaction for CD34 and INI-1. The diagnosis of proximal-type ES was established. The patient underwent orbital exenteration with negative surgical margins. Largest tumor diameter was 27  mm. Eight months after diagnosis metastatic progression with multiple metastases of the vertebral column and the base of the skull was noted. Conclusions. ES (proximal variant) develops predominantely in the pelvis, perineal region, trunk, mediastinum and genital tract. It is exceedingly rare in the orbital fossa. To our knowledge only three cases have been reported. Differential diagnoses include carcinoma, amelanotic malignant melanoma, epithelioid malignant peripheral nerve sheath tumor (MPNST), epithelioid angiosarcoma and myoepithelioma. ES has been associated with an unfavourable prognosis, early and frequent metastasis and requires adequate surgical treatment at an early stage with proper assessment of surgical margins. Postoperative radiation oncology and/or adjuvant systematic treatment might be considered according to the presentation.

FR-P-174 MPGN-like glomerulonephritis with intracapillary IgM thrombi in Waldenström’s macroglobulinemia D. Kratochvil1, K. Amann2, H. Bruck1, M. Büttner2 1 University Hospital Essen, Internal Medicine, Essen, 2University Hospital Erlangen, Institute of Pathology, Erlangen Aims. Lymphoproliferative disorders causing paraproteinemia can be associated with various kidney injuries. In the context of a case report earlier reports in literature and differential diagnoses of IgM-associated glomerulonephritides (GN) are discussed. Methods. Case report including the results of clinical, light microscopical, immunohistochemical and electron microscopical investigations. Literature search and discussion of earlier descriptions of glomerular monoclonal IgM deposits in lymphoproliferative diseases. Results. A 73-year-old female patient with a history of rheumathoid arthritis and Waldenström’s disease was admitted to hospital for acute renal failure with a mild proteinuria and hematuria. An MPGN-like GN with intracapillary IgM thrombi was diagnosed. The patient reported episodes of palpable purpura reminiscent of cryoglobulinemia. Despite repeated analyses, however, no cryoglobulines could be detected. The renal function recovered before the beginning of the immuno-modulatory therapy. Conclusions. In contrast to M.  Waldenström-associated GN described by Maroger-Morel in the present case a prominent glomerular hypercellularity was found. A strict distinction between cryoglobulinemic and non-cryoglobulinemic GN appears difficult taking previous reports in literature into account.

FR-P-175 c-Met in undifferentiated pleomorphic sarcomas and fibroblastic/ myofibroblastic tumors C. Wölfel1, T. Knösel1, T. Liehr2, S. Hauke3, A. Altendorf Hofmann4, D. Katenkamp1, I. Petersen1 1 Jena University Hospital, Institute of Pathology, Jena, 2Jena University Hospital, Institute of Human Genetics, Jena, 3ZytoVision GmbH, Bremerhaven, 4 Jena University Hospital, Department of General Surgery, Jena Aims. Undifferentiated, pleomorphic sarcomas (UPS), formerly known as malignant, fibrous histiocytoma (MFH) are defined as a group of high-grade sarcomas in which any attempt to disclose their line of differentiation has failed. These undifferentiated, pleomorphic sarcomas tend to occur in the extremities of elderly patients as a deep-seated, enlarging mass. The tumors frequently show an aggressive, rapid growth which contrast with the limited therapeutic options consisting mainly of surgery and radiotherapy while chemotherapy is usually not effective. c-MET inhibition is recently evolving as a promising new target in cancer therapy. C-Met is a proto-oncogene that encodes the hepatocyte growth factor receptor which possesses tyrosine-kinase activity. An abnormal c-Met activation in cancer triggers tumor growth, angiogenesis and encourage metastasis leading to a poor prognosis for the patient. We aimed to analyze the gene in soft tissue tumors. Methods. The tumor collective consisted of 327 fibroblastic/myofibroblastic tumors including 203 undifferentiated, pleomorphic sarcomas, 42 low grade sarcomas and 82 pseudosarcomatous tumors of the fasciitis family. It was analysed for c-Met expression by immunohistochemistry and cMet amplification by FISH. This was done on TMA sections (3 µm). One TMA of fasciitis nodularis tissues was used as control. For immunhistochemistry we applied a polyclonal rabbit anti-c-Met antibody with the dilution 1:50 at pH 6.1. Furthermore, we used FISH probes located at the c-Met region at chromosome 7q31.3 and as reference centromere probes for chromosome 7. The slides were evaluated by fluorescence microscopy. Results. Immunohistochemically, we found 53  sarcomas (16%) with a high c-Met expression. In contrast, the fasciitis cases revealed no or only low expression in all cases. For FISH analyses, 105 samples could so far

be investigated. They showed chromosomal aberrations in 37 cases (39%). Many cases carried a polysomy of chromosome 7. In addition, 9 cases (8.6%) revealed a selective amplification of the c-Met locus which, however, was only rarely associated with a clear-cut c-met overexpression. Conclusions. Conclusion: c-MET may represent an interesting therapeutic target in a subset of undifferentiated pleomorphic sarcoma which needs further evaluation.

FR-P-176 Cement spacers with MicroSilver (Bio-Gate) show decreasing inflammation without hints for detrimental effects in histological study of periprosthetic membranes S. Söder1, T. Bechert2, P. Steinrücke2, R. Ascherl3, A. Hartmann1 Erlangen-Nürnberg, Institute of Pathology, Erlangen, 2Bio-Gate AG, Nürnberg, 3Klinik für Wechselendoprothetik, Chemnitz

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Aims. Following hip replacement an infection rate of 2% was observed in this procedure. So far Gentamycin-spacers are commonly used to control periprosthetic infections. However especially in the presence of multiresistant bacteria like MRSA they are often only of limited effect. Silver has already proven to be effective in vitro. In a randomized prospective clinical study by Bio-Gate AG, Gentamycin-spacers with additional Microsilver component were compared with conventional Gentamycinspacers. In addition to a comprehensive panel of clinical and microbiological tests the periprosthetic membranes were studied histologically. Methods. We received blinded samples from 17  different patients with hip implant infections. Each patient received 2-stage revisions with 2  different cement spacers (Gentamycin and Gentamycin with added Microsilver) following a randomization. Specimens were formalin fixed, paraffin embedded, sectioned and hematoxylin-eosin stained. Results. No silver particles could be identified in any sample, even though occasionally wear particles were found in both groups. There was no indication of allergic or toxic effects in the histological examination. Typically a decline in inflammation was found in the course of the treatment. Periprosthetic membranes from spacers with added Microsilver showed typically a stronger or at least equal reduction in inflammation than spacers with Gentamycin alone. Conclusions. Histological examination gave no hints for adverse effects of adding Microsilver to conventional spacers which is in accordance with preliminary in vitro and animal studies. Furthermore a stronger effect on inflammation activity was observed.

FR-P-177 GNAS1 mutations in tumorigenesis B. Walther1, I. Walther2, Y. Chen1, I. Petersen1 Jena University Hospital, 2Jena University Hospital, Institute of Pathology

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Aims. The GNAS-1 gene is located on chromosome 20 and encodes for the alpha-subunit of ubiquitary existing stimulatory G-proteins. The two mutation hotspots R201H and R201C were evaluated which consists of the replacement of arginine by histidine or cysteine, respectively. These mutations have been previously reported in intramuscular myxomas representing benign soft tissue tumors that are often found in near-trunk muscle tissue of women. Furthermore, the McCune Albright syndrome (MAS) being characterized by Café-au-lait spots, precocious puberty, polyostotic fibrous dysplasia (FD) and other endocrine abnormalities is related to GNAS-1 mutations. In MAS, thyroid adenomas and carcinomas, adrenocortical hyperplasia and adenomas and pituitary tumors do occur. Moreover mutations were detected in isolated fibrous dysplasia (FD) or in combination with intramuscular myxomas in the context of the so called Mazabraud’s syndrome. The aim of the study was to evaluate the prevalence of these mutations in intramuscular myxomas and to verify the usefulness of mutations analysis in the differential diagnosis of soft tissue and bone lesions. Der Pathologe Suppl 1 · 2012 

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Abstracts Methods. Tumor-DNA was extracted from 4–10 µM thick specimen slides of formalin-fixed and paraffin-embedded tissue after micro dissection. These probes were amplified with conventional PCR and checked with agarose-gel-electrophoresis. The mutation status was assessed by direct DNA sequencing. Results. 61 intramuscular myxomas were examined and a mutation rate of 36.07% (22 cases) was detected which correlate with data from published studies. 75.41% of these were found in women. Both hotspots were equally affected. Furthermore 26 other tumor entities (angiomyxomas/fibromas, fibromyxoid sarcomas, chondrosarcomas, liposarcomas, FD, lipomas and neurothekeomas) were analyzed. In 5 out of 8 FDs (62.5%), mutations in codon 201 were discovered. In all other entities including 7 atrial myxomas and 31 gastroenteropancreatic-neuroendocrine tumors (GEP-NET’s) no GNAS-1 mutations were detected. Conclusions. Our results indicate that the GNAS-1 mutation analysis can be helpful to differentiate between FD and unspecific bone lesions (e.g. cystic or inflammatory conditions). It may be particular useful in the differential diagnosis of myxoid tumors. GNAS-1 mutations were never detected in any sarcomatous lesions while carrying a considerable prevalence in intramuscular myxoma. Mutation-positive patient might be screened for bone lesions compatible with FD to exclude Mazabraud’s syndrome.

FR-P-178 Valproic acid stimulation induces downregulation of IRAK-1 protein in a progressive thyroid carcinoma cell line S. Schwertheim1, S.-Y. Sheu-Grabellus1, K. Worm1, K.W. Schmid1 University Hospital of Essen, University of Duisburg-Essen, Institute of Pathology and Neuropathology, Essen

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Aims. Valproic acid (VPA) is a drug in clinical phase 2 for the therapy of advanced/poorly differentiated thyroid cancer with poor prognosis. miRNA-146a/b has been proved to be deregulated in thyroid carcinoma. To clarify if miRNA-146a/b plays a role in cells influenced by VPA we treated the highly progressive thyroid cell line BHT-101 with various doses of VPA (0, 1.0, 1.5 and 3.0 mM) and analyzed the expression levels of these miRNAs. As it is documented that miRNA-146/b is associated with regulation of NF-κB activity we also examined VPA-treated cells on mRNAs and proteins modulated by NF-κB. Methods. Cells were seeded in 6-well plates at 60–80% confluence and incubated with VPA for 48  h; conditioned medium was used for treatment containing 0.2% FCS, 1% Penicillin and 0.1% Amphotericin  B. miRNA and mRNA expression levels were detected by RT-PCR using Taq Man miRNA- and Gene Expression-Assays (Applied Biosystems). Protein analysis was performed by Western blotting. Results. miRNA-146a/b was upregulated at a concentration of 1  mm (foldchange 2.35) and 1.5  mM (foldchange 3.43) VPA and decreased at 3.0 mM (foldchange 2.26). VPA significantly and dose-dependently impaired NF-κB activity, reducing expressions of IRAK-1 (miRNA-146a/b target gene), phospho-IκBα and p50 protein. Remarkably, 1  mm VPA treatment induced upregulation of IL-6 and IL-8 mRNA levels, following reduced expressions at 3.0 mM; examination of IL-8 protein levels confirmed this. Conclusions. Our results suggest that miRNA-146a/b and IRAK-1 levels play a crucial role in VPA’s mechanisms of action and might be promising tools to regulate the therapy of advanced thyroid cancer.

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FR-P-179 Dysregulation of miRNA expression in normal thyroid tissue adjacent to tumor cells S. Schwertheim1, S.-Y. Sheu-Grabellus1, K. Worm1, K.W. Schmid1 University Hospital of Essen, University of Duisburg-Essen, Institute of Pathology and Neuropathology, Essen

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Aims. The miRNAs 146a, -146b -181b, -21, -221, -222, 30d, -125b, -26a, -30a5p, and -let7c have been proved to be deregulated in thyroid carcinoma. To clarify if miRNAs can be used to evaluate tumor progression we analyzed the expression of these miRNAs in 5 normal thyroid tissues adjacent to highly progressive thyroid carcinomas (4 poorly differentiated, 1 anaplastic thyroid carcinoma) and compared the results with expression levels in 4 normal thyroid tissues of individuals without any clinical thyroid disease. Methods. Paraffin embedded tissues were laser microdissected (PALM Laser-Micro Beam System, P.A.L.M., Bernried) for RNA analysis. miRNA expression levels were detected by RT-PCR using Taq Man miRNA assays and relative quantification of miRNA expression was calculated with the 2-ΔΔCt method. Results. We found significantly (p<0.032) increased expression levels of 6 miRNAs (146b, -21, -222, -26a, -30a-5p and -let7c) in normal tissue adjacent to thyroid carcinomas versus normal thyroid tissue of individuals without any clinical thyroid disease. Conclusions. Our results suggest a dysregulation of certain miRNAs in normal thyroid cells adjacent to tumor in terms of a dynamic tumor progression process. This implicates that histologically unaffected thyroid cells close to tumor cells are somewhat influenced by the tumor itself. Therefore miRNA analysis may be a useful tool to evaluate tumor progression and the necessity of additional treatment.

FR-P-180 Expression of chromosome 18 related tumor suppressor proteins in ileal neuroendocrine tumors T. Henopp1, J. Brix1, J. Sperveslage1, M. Anlauf2, K. Petersen1, G. Klöppel3, C.P. Gerlach2, P. Rexin4, T.M. Gress5, R. Moll4, B. Sipos1 1 University Hospital Tübingen, Institute for Pathology and Neuropathology, Tübingen, 2University Hospital Düsseldorf, Institute for Pathology, Düsseldorf, 3Technische Universität München, Institute of Pathology, München, 4 University Hospital Giessen and Marburg, Institute for Pathology, Marburg, 5 University Hospital Giessen and Marburg, Department of Internal Medicine, Marburg Aims. The genetic alterations in ileal neuroendocrine tumors (iNETs) are poorly characterized. The most frequent chromosomal aberration is the loss of one chromosome 18 in iNETs, however, the relevance of this alteration is unclear. In this study we investigated the status of chromosome 18 and the expression of chromosome 18 related tumor suppressor proteins in stage stratified cohorts (N0M0, N1M0 and N1M1) of iNETs, in order to identify tumor progression factors. Methods. The loss of chromosome 18 was examined using fluorescence in situ hybridization in 77 primary iNETs. The expression of tumor suppressor proteins Smad2, Smad4, Maspin and DCC was assessed by immunohistochemistry. Tumor suppressors were also stained in normal endocrine cells by immunofluorescence double labelling. Results. Chromosome 18 was lost in 10 of 17 (58%) N0M0, 21 of 31 (68%) N1M0 and 12 of 20 (60%) N1M1 iNETs, respectively. 5  cases showed mosaicism for chromosome 18. Smad2 and DCC were expressed in all iNETS, while Smad4 protein was absent in two cases. Maspin was not expressed. Conclusions. Loss of chromosome 18 in iNETs is probably related to tumor initiation but not tumor progression. Smad2, Smad4, DCC and Maspin seem to play no significant role in iNET tumorigenesis. Thus additional chromosome 18 associated tumor-related factors have to be explored.

Poster: Gynäkopathologie und Mammapathologie I SA-P-005 HER2 status in breast cancer remains stable with FISH (fluorescence in situ hybridisation) but is highly variable with IHC (immunohistochemistry) methodology in view of 10 years experience Z . Varga1, C . Ramach2, B . Padberg3, H . Moch1, A . Noske1 1 University Hospital Zurich, Institute of Surgical Pathology, Zürich, Switzerland, 2County Hospital St . Gallen, Institute of Pathology, St . Gallen, Switzerland, 3Institute of Pathology, County Hospital Graubünden, Chur, Switzerland Aims. Gold standard methodology in HER2 status determination in breast cancer is still a debated issue. Advantage of IHC analysis over timeconsuming and experience-requiring FISH methodology can be strongly influenced by pre-analytical differences and interpretational-issues. Methods. We analysed 6000 consecutive HER2-FISH tests in breast cancer in 10 years (2001 to 2011) and compared stability of amplification-rate with immunohistochemical 3+ positivity. Four years long (2001–2004) FISH tests were performed in all 3+ and 2+ cases and in some of the 1+ and negative (0) cases. For 6 years (2005–2010) HER2 status was determined with “only FISH” testing. For one year (2011) all cases were tested with both IHC and FISH. Results. Between 2001 and 2004, 61% of 3+ IHC was amplified with FISH (amplification-rate varied from 50%, 67%, 53% and 77%). In 2011, 86% of 3+ IHC cases were amplified with FISH. FISH amplification rate varied between 15–17% in 2005–2008 and 11–13% in 2009–2011 (due to modified ASCO criteria of HER2/CEP17 ratio from <2.0 to <2.2). Among 800 double HER2 tests in 2011, IHC 3+ varied from 10–16%, and FISH was amplified in 11–13%, when data were analysed in quarterly-periods. 3–9% of 1+ and negative IHC cases were amplified by FISH. Conclusions. IHC 3+ frequency and 3+ IHC/FISH concordance showed a huge variability over the years. Relevant improvement in amplificationrate of IHC 3+ cases (from 61% to 86%) was achieved in 2011. FISH “only” amplification rate however remained stable during the last 10 years (11 to 17%). Standardisation of pre-analytical procedures and profound expertise in HER2 IHC/FISH interpretation can contribute to better performance in HER2 testing.

SA-P-006 Evidence for individual laboratory specific cut-offs for Ki-67? H . Bürger1, T . Decker2, C . Focke2, J . Packeisen3, B . Hinrichs4, K .-H . Berghäuser5, W . Meinerz6, E . Korsching7 1 University of Münster/Utrecht, Institute of Pathology, Paderborn, Paderborn, 2Dietrich Bonhoeffer-Klinikum, Institute of Pathology, Neubrandenburg, 3Institute of Pathology, Osnabrück, 4Institute of Pathology, Köln, 5 Institute of Pathology, Saalfeld, 6St .Vinzenz Hospital, Clinics of Gynecology, Paderborn, 7University of Münster, Medical Faculty, Institute of Bioinformatics, Münster Aims. A widely differing interlaboratory reproducibility of KI-67 immunohistochemistry has been shown repeatedly. This has been attributed mainly to different staining protocols, varying tissue fixation and differing evaluation guidelines. Due to this well known fact, several cut-off values have been proposed. According to the actual St Gallen guidelines, a threshold of 14% may distinguish breast cancer patients with a good and a worsened prognosis. However, since this cut-off has been shown in one study only, and due to the fact of the above mentioned methodological uncertainties, it may be possible that a prognostic cut-off-levels may differ between several laboratories. Methods. In order to verify this hypothesis we investigated a series of more than 400 invasive breast cancer cases, treated at the St. Vinzenz-

Frauenklinik, Paderborn, in the years 1997–2003 by means of Ki-67 immunohistochemistry, using tissue microarrays. For all breast cancer cases the long term follow-up was evaluated. The slides were stained in different laboratories under routine conditions and quantitatively evaluated for the percentage of Ki-67 positivity. Laboratory specific cut-off levels were evaluated by the use of Kaplan-Meier survival curve analysis. Results. We were able to show that regarding all breast cancer cases the frequency of Ki-67 positive cells differed within the different, participating laboratories. The use of a fixed 14% cut-off resulted in different significance levels regarding the overall survival. Using Kaplan-Meier survival curve analysis it became obvious that the interlaboratory variation in Ki-67 positivity resulted in different optimized cut-off levels for Ki-67. Conclusions. Our results revealed a large interlaboratory variation in Ki-67 immunohistochemistry. Since Ki-67 immunohistochemistry is an integral part of a breast cancer specimen workup, a reliable protocol is mandatory. However, recent efforts for standardization did not show the requested success. Therefore the definition of a laboratory-specific cut-off value might be an alternative in order to guarantee an optimal therapy prediction.

SA-P-007 Expression of Ki-67 correlates with intermediate and high risk Oncotype DX recurrence-score in breast cancer G . Richter1, D . Jahn2, H . Hüfner1, T . Noesselt2 Institute of Pathology Dr . Richter, Hameln, 2Department of Gynecology and Obstetrics, Sana Klinikum Hameln-Pyrmont

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Aims. Recent advances in understanding the molecular pathology of breast cancer offer significant potential to identify patients who may benefit from adjuvant therapies. To date, few of these advances are utilised in a routine setting. The only widely used breast cancer molecular assay is in situ hybridisation for Her2 gene amplification. In the Plan-B study the multigene expression array Oncotype DX was used. We compare the Oncotype Dx recurrence-score with immunohistochemical detection of Ki-67, estrogen- and progesteron-receptor. Methods. In 2010 and 2011 the Sana Klinikum Hameln-Pyrmont took part in the Plan-B study. Every patient with unilateral breast cancer, aged 18–75, after RO-resection (T1–4, N0) with Her2 negative cancer was offered a place in the Plan-B study. In the Plan-B study an Oncotype DX assay was conducted. Moreover immunohistochemical assessment against Ki-67, Her2, estrogen- and progesterone-receptor were routinely carried out by using the ventana benchmark. Results. 32 breast cancer patients took part in the Plan-B study and an Oncotype DX multigene expression assay was conducted. In comparing the immunohistochemistry and Oncotype DX the correlation for the estrogen-receptor and Her2 was 100%. In 6 of 32 (19%) cancer specimen the progesteron-receptor was detected immunohistochemically positive and negative in the Oncotype DX. In 6 of 32 (19%) cases we detected Ki-67 positive cells in 25% and more. In these specimens the Oncotype DX recurrence-score was in two cases of intermediate risk (18–30) and in four cases of high risk (30+). Conclusions. In breast cancer ER/PgR, Her2 and Ki-67 are well known biological markers. In addition, multigene expression arrays like the Oncotype DX could be used for possible better predicting prognosis and making effective treatment decisions. There is a high correlation of immunohistochemical assessment and the Oncotype DX respective estrogen-receptor and Her2. All cases of high Ki-67 (25% or more) correlated with the intermediate or high risk in the Oncotype DX.

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Abstracts SA-P-008 Correlation of anti-PHH3 positive mitoses to pathological response in neoadjuvantly treated breast cancer S. Timme1, M. Becker2, E. Stickeler2, P. Bronsert1, L. Reischuck2, L. Bogatyreva3, D. Hauschke3, A. zur Hausen4, M. Werner1 1 Institute of Pathology, University Medical Center, Freiburg, 2Department of Obstetrics and Gynecology, University Medical Center Freiburg, Freiburg, 3 Institute of Medical Biometry and Medical Informatics, University Medical Center, Freiburg, 4Department of Pathology, GROW, School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, Netherlands Aims. We evaluated breast cancer (BC) core biopsies taken before neoadjuvant chemotherapy (NACT) by immunohistochemistry using anti-PhosphohistoneH3 antibody (PHH3) to determine the mitotic count. The data were correlated to clinicopathological parameters including intrinsic subtypes as well as the histopathological regression of resected tumour specimens after NACT with Epirubicin/Cyclophosphamide (EC) and Taxotere. Methods. 72 patients with either triple negative (21/72) or luminal type (51/72) BC obtained NACT with EC and Taxotere. Thereafter, tumour regression was analyzed in resection specimens using a semiquantitative score from 0 (no effect) to 4 (no cancer cells) according to Sinn et al. (1994). Pathological complete response (pCR) was defined as no residual invasive carcinoma (score 3+4). In 16/72 cases (22.2%) pCR occurred; 9/16 (56.25%) were TNBC and 7/16 were luminal type BC (ER and/or PrR+/ HER2−). In the pre-treatment biopsies immunohistochemical stainings with PHH3 were performed and mitotic figures were evaluated in 10 high power fields (HPF). The number of mitoses detected by PHH3 was correlated to different clinicopathological parameters determined before treatment (intrinsic subtype, WHO-type, grading, tumour size and nodal stage) and to regressive changes/pCR after therapy by univariate statistical analyzes (using SPSS v 18). Results. The number of PHH3-detected mitoses correlated significantly with the tumour grading (p=0.001), but there was no correlation with WHO-type, tumour size or nodal stage. PHH3/10  HPF differs significantly between the two intrinsic subtypes (p=0.003). PHH3 expression alone was no predictor for pCR (p=0.399). But tumours with 11 or more mitoses/10 HPF achieved significantly more often pCR than those with under 11 mitoses/10 HPF (p=0.031). Furthermore, luminal type BC with 11 or more mitoses/10  HPF and also TNBC had significantly more frequent pCR than luminal type BC with under 11 mitoses/10 HPF (p=0.016). Conclusions. Strong proliferating BC (11 or more mitoses/10 HPF) have significantly more pCR compared to low proliferating tumours (under 11  mitoses/10  HPF) after NACT with EC and Taxotere. Furthermore, mitoses detected by PHH3 are a discriminator for luminal type BC to predict pCR, because luminal type BC under 11  mitoses/10  HPF rarely reach pCR. Thus, the determination of mitoses by PHH3 may be recommended especially for luminal type BC before NACT with EC and Taxotere.

SA-P-009 Transitions between flat epithelial atypia and low-grade ductal carcinoma in situ of the breast S. Aulmann1, F. Mietzsch1, R. Penzel1, P. Schirmacher1, H.P. Sinn1 1 University of Heidelberg, Institute of Pathology, Heidelberg Aims. Flat epithelial atypia (FEA) of the breast typically is a localised alteration involving only few, neighbouring terminal ducto-lobular units (TDLUs). However, occasionally there are cases with extensive FEA and morphological evidence of direct transitions between FEA and classical ductal carcinoma in situ (DCIS). Methods. To investigate the relationship of FEA and DCIS in these cases, we microdissected multiple foci of the respective lesions in a series

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of 10 cases and performed comparative allelotyping using a panel of 14 LOH markers. In addition, phylogenetic tree models were calculated on the basis of mitochondrial DNA sequencing to visualise the clonal relationship of the different lesions. Results. FEA and lg-DCIS shared the majority of chromosomal imbalances, loss of diverging alleles was not detected in any of the 10 cases. mtDNA sequencing and phylogenetic tree clustering revealed direct transitions between FEA and lg-DCIS in all 10 cases. However, in three patients, additional foci of FEA were present which were not directly related to the rest of the FEA and the lg-DCIS. Conclusions. In conclusion, our data demonstrate the presence of direct transitions between FEA and lg-DCIS and support the interpretation of foci of FEA as part of the lg-DCIS in those unusual cases in which multiple areas of FEA are interspersed with areas of classical lg-DCIS and direct transitions of both lesions are apparent.

SA-P-010 Different cytoplasmic and nuclear PARP expression patterns in hereditary and non-hereditary breast cancer M.-L. Klauke1, N. Hoogerbrugge1, J. Budczies2, P. Bult1, J.H.J. van Krieken1, C. Denkert2, B.M. Müller2 1 Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands, 2 Charité Hospital, Institute of Pathology, Berlin Aims. High cytoplasmic poly (adenosine diphosphate-ribose) polymerase (PARP) expression in non-hereditary breast cancer correlates with an aggressive tumor pattern and a poor long-term prognosis. Our study was designed to compare cytoplasmic and nuclear PARP expression between hereditary and non-hereditary breast cancer. Methods. Tissue micro arrays containing 39 familiar and 39 non-hereditary formalin-fixed, paraffin embedded breast cancer tumor samples were immunohistochemically analyzed for cytoplasmic (cPARP) and nuclear (nPARP) PARP expression. Stained slides were digitized and evaluated using the VM Slide Explorer. The intensity and percentage of positive tumor cells were used to calculate an immunoreactive score (IRS), which was divided into three subgroups defined as low (IRS 0–2), intermediate (IRS 3–4) and high (IRS 6–12) expression. The mean age at diagnosis in patients with familiar breast cancer was about 45±11 years and in patients with non-hereditary breast cancer about 65±12.1 years. Results. cPARP and nPARP expression patterns were significantly different in hereditary and non-hereditary breast cancer. cPARP expression in hereditary breast cancer was low in 33.3%, intermediate in 25.6% and high in 41.0% of cases. In contrast, cPARP in non-hereditary breast cancer was low in 59.0%, intermediate in 25.6% and high in 15.4% of cases. Statistical analysis showed that cPARP expression was significantly higher in hereditary compared to non-hereditary breast cancer (p=0.008, χ2-test for trends). Hereditary breast cancer showed a significant lower intermediate nPARP expression (23.1%) than non-hereditary breast cancer (59.0%; p=0.005, χ2-test). Conclusions. PARP expression in hereditary and non-hereditary breast cancer differs significantly, which might be related to the higher frequency of BRCA1 or 2 mutations in hereditary breast cancer and is compatible with a role of PARP-1 in DNA repair and genomic stability. Because patients with hereditary breast cancer have a poor long-term prognosis and show very often an aggressive tumor pattern the overall higher cPARP expression found in this subgroup suggests that high cPARP expression might be correlated with an aggressive tumor pattern and a poor longterm prognosis.

SA-P-011 Genetic aberrations of predictive factors are rare in triple-negative breast cancer T. Grob1, M. Choschzick1, G. Sauter1, A. Lebeau1 1 University Medical Center Hamburg-Eppendorf, Institute of Pathology, Hamburg Aims. In the absence of estrogen as well as progesterone receptors and the lack of HER2 amplification, women with triple-negative breast cancer TNBC do not benefit from endocrine therapy or trastuzumab and are left with chemotherapy as their only option. To reduce the elevated risk of disease progression in these patients, better treatment options are needed that are less toxic and are more targeted to this patient population. Therefore, we performed a comprehensive analysis of potential targetable genetic aberrations affecting the EGFR/HER2/MAPK pathway that are observed at higher frequencies in adenocarcinomas of other organs. Methods. 65 consecutive TNBCs were studied by sequence analysis for HER2, EGFR, KRAS, BRAF mutations. TP53 sequence analysis was included to control DNA quality and tumor cell content. A tissue microarray (TMA) representing two samples of each tumor was constructed to search for EGFR gene copy gain and EML4-ALK fusion by FISH. Triple negative status was confirmed by immunohistochemistry (IHC) and FISH on TMA sections. EGFR and CK5/6 IHC were added for identification of the basal-like phenotype. Results. Sequence analysis revealed HER2 gene mutation in one patient (heterozygous missense mutation in exon  19: p.L755S). No mutations were found in EGFR, KRAS and BRAF. High polysomy of EGFR was detected in 5 of 62 informative cases by FISH. True EGFR gene amplification accompanied by strong membraneous EGFR protein expression was observed in only case. No rearrangement of the ALK gene was detected. Basal-like phenotype was identified in 38 of the 65 TNBCs (58.5%). TP53 mutations were detected in 36 of the 63 (57.1%) informative tumors. Conclusions. Targetable genetic aberrations in the EGFR/HER2/MAPK pathway occur rarely in TNBC. Nonetheless some patients might benefit from HER2/EGFR targeted therapy.

SA-P-012 Luminal B breast cancers are not the end stage of a stepwise dedifferentiation of luminal breast cancers H. Bürger1, B. Schymik2, W. Meinerz2, E. Korsching3 1 University of Münster/Utrecht, Institute of Pathology, Paderborn, Paderborn, 2St.Vinzenz Hospital, Clinics of Gynecology, Paderborn, 3University of Münster, Medical Faculty, Institute of Bioinformatics, Münster Aims. Recent molecular data pointed towards the possibility of a stepwise dedifferentiation in a subgroup of invasive breast cancer (BC) cases. In detail, it was hypothesized, that estrogen receptor positive (ER+) grade 3 ductal invasive BC’s are the end stage of a dedifferentiation process of luminal BC. A progression of luminal A towards luminal B breast cancers, associated with a “progression through grade” seemed the obvious explanation. Methods. In order to verify this hypothesis on a morphological and immunohistochemical level we investigated 865 invasive breast cancer cases. All cases were reviewed for the presence of intratumoural heterogeneity in the invasive cancer and the presence of associated ductal carcinoma in situ (DCIS). With the use of tissue microarrays the molecular subtype was determined and correlated with clinicopathological features. Results. We were able to show that regarding all breast cancer cases the frequency of ER-positivity decreased with gain of tumour size. In detail, the frequency of luminal A breast cancer decreased, whereas the number of luminal B breast cancers remained constant. A constant increase of the frequency of basal, HER2-driven and triple negative breast cancers could be seen. Only in 1 out of 865 breast cancer cases a grade 1 and a grade 3 invasive cancer component within the same breast cancer was

detectable. In 2 cases a ductal invasive grade 1 carcinoma was associated with a poorly-differentiated DCIS. The frequency of cylinder cell lesions was evenly distributed between ductal invasive grade 3 carcinomas, irrespectively of the ER-status. Conclusions. Our results show that a morphological recognizable “progression through grade” is a very rare event in the natural course of invasive breast cancer, including luminal breast cancer. If a progression through grade occurs, this step is more likely located on the stage of ductal carcinoma in situ or other suspected precursor lesions.

SA-P-013 Identification of a tissue-based protein signature associated with triple negative breast cancer by imaging mass spectrometry (MALDI Imaging) C. Schöne1, S. Rauser1, S. Englert1, S. Artmeier2, K. Schulenburg2, B. Balluff1, M. Elsner1, S. Maier1, S. Meding1, M. Schmitt2, H. Höfler3, A. Walch1 1 Helmholtz Zentrum Munich, Institute of Pathology, Neuherberg, 2Klinikum rechts der Isar of the Technische Universität München, Klinische Forschergruppe of the Frauenklinik, Munich, 3Technische Universität München, Institute of Pathology, Munich Aims. The goal of this study is the identification of a tissue-based protein signature associated with triple negative breast cancer for the purpose of reliable classification and to identify potential new biomarkers. Methods. A collective of 25 frozen triple negative and 44 non-triple negative breast cancer samples was analysed in this study. Protein signatures of the tissue samples were generated using MALDI Imaging with a lateral resolution of 70  µM and a mass range of 2,500 to 25,000 Da. Afterwards the cases were separated into a discovery set, containing 15 triple negative and 15 non-triple negative breast cancer cases, and a validation set composed of the other cases. The mass spectra of the discovery set were compared to each other to identify significantly differentially expressed proteins. The resulting protein signature was then tested on the validation set using different classification algorithms (divisive hierarchical clustering, random forest or support vector machine). Results. We were able to identify a signature composed of 10  different proteins that could differentiate between triple negative and other breast cancer samples with an accuracy of over 80%. Three of these proteins were already encountered in a study dealing with HER2-overexpressing breast cancer and are of special interest for identification. Conclusions. MALDI Imaging made it possible to identify protein signatures associated with triple negative breast cancer. The protein signature may have potential for classification approaches and contains proteins that may be of interest as potential biomarkers.

SA-P-014 Expression of RAD23B in invasive breast cancer. Immunohistochemical study using tissue microarrays K. Friedrich1, A. Linge2, F. Goerl1, G. Baretton1 1 University Hospital “Carl Gustav Carus” Dresden, Institute of Pathology, Dresden, 2National Institute for Cellular Biotechnology, Dublin, Ireland Aims. RAD23B is part of the nucleotide excision repair complex (NER) and involved in repair of DNA damage caused by UV light exposure and the chemotherapeutic drug cisplatin. The role of RAD23B expression in invasive breast cancer is still unclear. Thus, the purpose of the study was to analyse the RAD23B expression in correlation to clinicopathological characteristics, proliferation and outcome of patients. Methods. The expression of RAD23B, Ki67, HER-2/neu, estrogen and progesterone receptor was analysed in 164 formalin fixed, paraffinembedded specimens of invasive breast carcinoma using tissue microarrays. All staining results were scored semiquantitatively. The mitotic count was performed on H&E sections as was histopathological grading Der Pathologe Suppl 1 · 2012 

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Abstracts according to Elston & Ellis. The statistical analysis was done by Chisquared test, t-Test according to student, Kaplan-Meier estimation and multivariate discriminant analysis. Results. Nuclear expression of RAD23B was observed in all analysed cases, ranging from 5–90% of tumor cells with different intensities. RAD23B expression was correlated with age, histopathological grade and mitotic activity in univariate analyses. Patients with a disease manifestation after the age of fifty showed a lower RAD23B expression than younger patients. A histopathological grade 1 or 2 was associated with a higher RAD23B expression. The mitotic activity was lower in cases with high RAD23B expression. The mitotic activity was lower in cases with high RAD23B expression than in cases with low RAD23B expression. The multivariate analysis revealed mitotic activity and Ki67 expression as significant markers. There was no correlation between RAD23B expression and the other clinicopathological markers or the outcome of disease. Conclusions. The correlation of RAD23B expression to proliferation, especially to mitotic activity may be associated with the function of RAD23B and the binding of RAD23, XPC and Centrin 2 in DNA damage recognition complex. Its role in repair of cisplatin-damaged DNA suggests that RAD23B may be used as a predictive marker for response to cisplatin based chemotherapy.

SA-P-015 Up-regulation of Kindlin-2 promotes progression of human ­breast cancer cells by increasing their proliferation, drug resistance, genomic instability, and tumorigenesis W.-g. Fang1, T. Zhao1, H.-q. Zhang1 1 Peking University, Health Science Center, Beijing, China Aims. Kindlin-2 has been confirmed as an essential element of bidirectional integrin signaling. In recent years, the relationship between Kindlin-2 expression and cancers has been a focus of interest. Our previous studies have shown that Kindlin-2 expression was up-regulated in several types of human cancers, and a strong correlation between Kindlin-2 expression and clinical outcome of breast cancer patients was found. However, the functional role of Kindlin-2 in breast cancer has not been studied. This study was designed to investigate the role of Kindlin-2 in the progression of human breast cancer cells. Methods. Firstly, Kindlin-2 expression at protein level was detected by Western blot in several breast cancer cell lines. Two luminal-like breast cancer cell lines, MCF-7 and T47D, expressed low level of Kindlin-2. Two basal-like breast cancer cell lines, MDA-MB-231 and HS578T, expressed moderate levels of the protein. Then, Kindlin-2 gene was overexpressed by transfected into MCF-7 cells. In comparison, short hairpin RNA (ShRNA)-mediated knockdown of Kindlin-2 was performed in HS578T cells. Vector controls were also done in the same cell lines. Ki67 Li, FCM cell cycle, anchorage-independent colony formation (in vitro tumorigenesis), and in vivo tumorigenesis in NOD/SCID mice were observed. Apoptotic cells were labeled by fluorescent annexin V assay and quantified by FACS. Array CGH analysis and spectral karyotyping were performed to detect the genomic instability of these cells. Results. The growth rate of Kindlin-2-transfected MCF-7 cells was much quicker than that of the controls. The proportion of G2-M phase cells, clone formation and tumorigenicity were significantly higher than these of the controls. The change of Kindlin-2-ShRNA transfected cells was just the reverse. Moreover, Up-regulation of Kindlin-2 can also reduce the rate of apoptosis induced by the chemotherapy drugs, and these cells showed much more genomic instability compared with the controls. Conclusions. These findings suggested that up-regulation of Kindlin-2 promotes the progression of human breast cancer cells by increasing their proliferation, drug resistance, genomic instability, and tumorigenesis.

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SA-P-016 GPR30: a predictive marker for Tamoxifen resistance in breast cancer T. Kalinski1, A. Roessner2, S.-D. Costa2, A. Ignatov2 Otto-von-Guericke-University/Department of Pathology, Magdeburg, 2 Magdeburg

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Aims. Tamoxifen is the gold standard in the therapy of hormone-dependent breast cancer. However, the development of Tamoxifen resistance is a frequent problem in Tamoxifen-responsive tumors during treatment. The aim was to investigate the role of the new estrogen receptor GPR30 in the development of Tamoxifen resistance. Methods. Mechanisms of Tamoxifen resistance were investigated in Tamoxifen-resistant breast cancer cells and wild type cells. The expression of GPR30 was further analyzed in breast cancer specimens and correlated with clinical data. Results. The results proved the important role of GPR30 in the development of Tamoxifen resistance in breast cancer. GPR30 expression in breast cancer specimens was associated with Tamoxifen resistance and negatively correlated with relapse free survival in patients treated with Tamoxifen. Conclusions. GPR30 plays an important role in the development of Tamoxifen resistance in breast cancer cells. GPR30 expression is a predictive marker for the development of Tamoxifen resistance in breast cancer specimens.

SA-P-017 CD34+ fibrocytes in the stroma of ductal carcinoma in situ (DCIS) of the breast P.J. Barth1, F. Wötzel1 University Hospital Münster, Institute of Pathology, Münster

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Aims. Im Stroma normalen Brustdrüsengewebes finden sich überwiegend CD34+-Fibrozyten, die eine große Rolle in der Matrixsynthese und bei der Aufrechterhaltung der Integrität des mammären Stromas spielen. Zudem spielen CD34+ Fibrozyten eine Rolle als Antigen präsentierende Zellen. Das Stroma invasiver duktaler Karzinome zeigt einen kompletten Verlust der stromalen CD34-Expression und einen Phänotypwechsel der Stromazellen von CD34+SMA−Fibrozyten zu CD34−SMA+-Myofibroblasten. Bisher wurden keine Studien zur CD34 Expression im Stroma von DCIS durchgeführt. Methods. Wir untersuchten DCIS unterschiedlichen Kernmalignitätsgrades immunhistochemisch bezüglich der stromalen Expression von CD34, SMA (Glattmuskel-Aktin) und SMM (Glattmuskel-Myosin). Es wurden nur DCIS untersucht, die nicht mit einem invasiven Karzinom assoziiert waren. In jedem Fall lag tumorfreies Brustdrüsengewebe zum Vergleich vor. Results. Tumorfreies Brustdrüsengewebe zeigte periduktal und periazinär dicht gelagerte CD34+ Fibrozyten mit bipolaren zarten Zytoplasmafortsätzen. SMA+ Stromazellen wurden in tumorfreiem, normalem Brustdrüsengewebe nicht beobachtet. DCIS niedrigen und mittleren Kernmalignitätsgrades zeigten eine erhaltene Population von CD34+ Fibrozyten, SMA+ Zellen wurden im Stroma nicht beobachtet. DCIS hohen Kernmalignitätsgrades zeigten einen Verlust periduktaler CD34+ Fibrozyten; an ihrer Stelle zeigten sich CD34-SMA+ Myofibroblasten, die konzentrisch um die vom DCIS befallenen Ductus angeordnet waren. Conclusions. Diese Untersuchung zeigt, dass es bei DCIS hohen Kernmalignitätsgrades zu Veränderungen des Stromas kommt, die auch bei invasiven Karzinomen gefunden wurden. Der Verlust der CD34+ Fibrozyten führt zu einer Störung der Integrität des Stromas, die Voraussetzung für die Entstehung eines invasiven Karzinoms sein kann. Zudem kann der Verlust der CD34+ Fibrozyten, bei denen es sich um Antigen präsentierende Zellen handelt, zu einer Störung der gegen Tumorzellen gerichteten Immunkontrolle des Organismus führen. Beides sind

Mechanismen, die eine Progression des DCIS zum invasiven duktalen Mammakarzinom begünstigen.

SA-P-018 Elevated expression of LSD1 (Lysin-specific demethylase 1) during tumour progression from pre-invasive to invasive ductal carcinoma of the breast N . Bektas Serce1, A . Gnatzy1, S . Steiner1, J . Kirfel1, R . Büttner2 1 University of Bonn, Institute of Pathology, Bonn, 2University of Cologne, Institute of Pathology, Köln Aims. Lysin-specific demethylase  1 (LSD1) is a nuclear protein which belongs to the aminooxidase enzymes playing an important role in controlling gene expression. It has also been found highly expressed in several human malignancies including breast carcinoma. Our aim was to detect LSD1 expression also in pre-invasive neoplasias of the breast. In the current study we therefore analyzed LSD1 protein expression in ductal carcinoma in situ (DCIS) in comparison to invasive ductal breast cancer (IDC). Methods. Using immunhistochemistry we systematically analyzed LSD1 expression in low grade DCIS (n=27), intermediate grade DCIS (n=30), high grade DCIS (n=31) and in invasive ductal breast carcinoma (n=32). SPSS version 18.0 was used for statistical analysis. Results. LSD1 was differentially expressed in DCIS and invasive ductal breast cancer. Interestingly, LSD1 was significantly overexpressed in high grade DCIS versus low grade DCIS. Differences in LSD1 expression levels were also statistically significant between low/intermediate DCIS and invasive ductal breast carcinoma. Conclusions. LSD1 is also expressed in pre-invasive neoplasias of the breast. Additionally, there is a gradual increase of LSD1 expression within tumour progression from pre-invasive DCIS to invasive ductal breast carcinoma. Therefore upregulation of LSD1 may be an early tumour promoting event.

SA-P-019 Female adnexal tumor of probable Wolffian origin – case report and review of literature for epidemiology, course of disease and differential diagnosis K . Friedrich1, M . Toma1, J . Wienold2, G . Baretton1 1 University Hospital “Carl Gustav Carus” Dresden, Institute of Pathology, Dresden, 2Hospital Weißeritztal Kliniken Freital Dippoldiswalde, Deparment of Gynecology and Obstetrics, Freital Aims. Clinical history: 35-year-old female with lower abdominal pain on laparoscopy showed a hematosalpinx and a tumor close to the right fallopian tube, which was removed laparoscopically. The following laparoscopic staging did not revealed any further tumors. Methods. Pathology: Gross examination showed a tumor (7 cm diameter) with close contact to the fallopian tube, partially covered by serosa with nodular and focally light gray, glistening pale yellow sectioned surface. Microscopically, the tumor showed variable histological architecture with solid, tubular and sieve-like pattern formed by low cuboidal, attenuated or spindle-shaped cells without atypia and only few mitoses. The tumor cells expressed cytokeratin 8/18 and 19, CD10, CD99, vimentin and – at least focally – inhibin and calretinin. EMA, estrogen-, progesterone receptor, cytokeratin 7 and 20, CD34 and S100 were not detectable. Based on the histomorphology and the immunohistochemical expression profile, a female adnexal tumor of probable Wolffian origin (FATWO) was diagnosed. Results. Epidemiology, course of disease and differential diagnosis: FATWO are very rare tumors of female adnexal region. A total of 72 cases have been thus far documented in the literature. The age of reported patients ranged from 15 to 83 years, most patients are between 40 and 45 years old. Remnants of the Wolffian duct, especially the rete ovarii,

are thought to be the origin of this tumor. The broad ligament is the most frequent location, but the tumor may also occur in the serosa of the fallopian tube, the ovary, the mesoalpinx, retroperitoneum and peritoneum. Conclusions. Most cases are benign, but ten of the reported patients developed local recurrences, lung or liver metastases. Three patients died of their tumor. Atypia and a high proliferation activity may predict an aggressive behaviour. But tumors without these criteria showed recurrences, too. The differential diagnoses are sex-cord stroma tumors (Sertoli-Leydig cell tumors, granulosa cell tumors), endometroid adenocarcinomas (of the fallopian tube), adenomatoid tumors, mesotheliomas and metastases.

Poster: Gynäkopathologie und Mammapathologie II SA-P-020 T lymphocytic infiltration in serous ovarian carcinoma: expression and survival analysis S . Scheil-Bertram1, H .-C . Bösmüller2, A . du Bois3, F . Heitz3, P . Harter3, M . Oppitz1, N . Ewald-Riegler4, R . Hils4, A . Fisseler-Eckhoff1 1 Institute of Pathology &Cytology, Wiesbaden, 2Institute of Pathology, Linz, Austria, 3Dept Gynecology & Gyn . Oncology, Essen, 4Clinic for Gynecology & Gyn . Oncology, Wiesbaden Aims. The lymphocytic infiltration of carcinomas should be a prognostic marker of antitumoral immunoreactivity and be associated with prolonged overall survival in ovarian carcinomas. Using CD3 and CD8 antibodies, we analysed immunreactive T-lymphocyts in a cohort of 78 serous ovarian carcinoma (OC), and their correlation with patients’ survival. Methods. We used the CD3 (clone SP7) and CD8 (clone C8/144B) antibodies (both NeoMarkers). Immunohistochemistry was performed on multi tissue microarrays (78  patients; median age at diagnosis 64  years). The stromal and intraepithelial T-cell (CD3 and CD8) density was quantified, counting the average number of cells per high power field (HPF=400x) and reviewing a total of 10 HPF for each microarray. The immunoreactivity was analyzed in a double blind fashion by two pathologists. Results. The survival of patients with a CD3/CD8 ratio above 1 or 2 respectively was significantly shorter than with a CD3/CD8 ratio below 1 (17.3 or 15.1 month versus 22.2 month; p=0.0154; hazard ratio: <1 vs. 2 was 3.5206; 1 vs. 2 was 3.8029). Conclusions. Our study of a collective of advanced OC confirms the prognostic relevance of intratumoral lymphocytes on overall survival, pointing out the significance of increased numbers of cytotoxic CD8 lymphocytes.

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Abstracts SA-P-021 The G-protein coupled estrogen receptor 1 (GPER) is differentially expressed in healthy human ovaries, accompanies progression into non malignant ovarian diseases, and predicts prognosis in ovarian cancer patients S. Heublein1, M. Lenhard2, J. Schöpfer3, C. Kuhn1, K. Friese1, A. Makrigiannakis4, U. Jeschke1, D. Mayr5 1 Ludwig-Maximilians-University of Munich – Campus Innenstadt, Department of Gynecology and Obstetrics, Munich, 2Ludwig-Maximilians-University of Munich, Department of Gynecology and Obstetrics – Campus Grosshadern, Munich, 3Ludwig-Maximilians-University of Munich, Department of Legal Medicine, Munich, 4University of Crete, Department of Obstetrics and Gynaecology, Heraklion, Greece, 5Ludwig-Maximilians-University of Munich, Department of Pathology, Munich Aims. In the ovary deregulation of estrogen signalling is tightly linked to impaired fertility as well as to benign and malignant ovarian diseases. However several of these estrogen mediated effects are clearly independent of the classical DNA binding estrogen receptors. Thus to understand which role the G-protein coupled estrogen receptor 1 (GPER) plays within these processes might define implications for estrogen or anti-estrogen based therapies. Therefore we examined GPER in healthy human ovaries, human folliculogenesis, benign ovarian diseases as well as in epithelial ovarian cancer (EOC) specimens in a large patient cohort (n=282). Methods. Immunohistochemistry, double immune fluorescence and TaqMan® real time PCR were employed to determine GPER expression. Ovaries of 32 pre- and 10 postmenopausal women were compared to 84 women affected by follicle cysts (n=14), serous (n=16) and mucinous (n=18) cystadenofibroma, endometriosis (n=26) or reactive inflammatory diseases (n=10). We further evaluated how GPER correlates with grading, FIGO stage and prognosis in 156 EOC patients. Finally different ovarian cell lines were used to test these interactions functionally. Results. In healthy follicles GPER is abundantly present in oocytes and theca cells followed by granulosa cells. In contrast to controls a pronounced stroma expression of GPER was observed in endometriosis and inflamed tissue which stresses its immune responsiveness to glucocorticoids and galectin found in vitro. GPER is highly expressed in the ovarian outer surface epithelium and down regulated in some entities of benign neoplasias. In EOC low tumour grade and advanced prognosis were predicted by elevated GPER. Furthermore here GPER showed positive correlation to gonadotropine receptors, Galectin-3 and downregulation of p53. Conclusions. GPER is detectable in highly specialized cells throughout human folliculogenesis and thus might be functionally involved in follicle maturation. Progression into benign and malignant ovarian diseases is accompanied by GPER, which we found is regulated by immune modulators in different cell lines. Thus we conclude that GPER is a valuable tool to further investigate human ovarian (patho-)physiology and that it might be interesting for therapeutic interventions in EOC patients.

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SA-P-022 PDGF-C expression in ovarian cancer A.-K. Zimmermann1, A. Noske1, H. Li2, U. Ericsson2, A.M. Neville3, R. Caduff1, H. Moch1, D. Fink4, G. Kristiansen5 1 University Hospital Zurich, Department of Surgical Pathology, Zürich, Switzerland, 2Ludwig Institute for Cancer Research, Stockholm Branch, Sweden, 3Ludwig Institute for Cancer Research, University of Oxford Branch, United Kingdom, 4University Hospital Zurich, Department of Obstetrics and Gynecology, Zürich, Switzerland, 5University Hospital Bonn, Institute of Surgical Pathology Aims. Platelet-derived growth factors (PDGF) and its receptors (PDGFR) promote tumor growth and angiogenesis and are therefore interesting targets for anticancer therapies. So far, little is known about the expression and prognostic role of PDGF-C in ovarian cancer. Methods. We investigated a cohort of 131  invasive ovarian carcinomas and 31 borderline tumors for PDGF-C expression by immunohistochemistry (using a specific antibody against this ligand) and compared expression data with clinicopathological findings and patient overall survival. Results. We observed an epithelial and stromal expression of PDGF-C in 64 (49%) and 62 (48%), respectively of the ovarian carcinomas but did not find any association with clinicopathological features or overall survival. In a subgroup analysis of serous carcinomas, we observed a trend of high PDGF-C levels with higher tumor grade (p=0.047). An epithelial and stromal expression was also observed in borderline tumors in 65% (22/34) and 26% (8/31), respectively. Conclusions. This study shows that PDGF-C is overexpressed in a subset of ovarian carcinomas but not associated with traditional prognostic pathological factors or patient survival. However, PDGF-C has potential as a therapeutic target and in context with the literature it might have a predictive function in anti-VEGF-treatment.

SA-P-023 AGO VISION-1: Improved utilisation of resources and optimization of quality of care through internet-based second opinion pathology – standardized in an optimized ovarian cancer network S. Kommoss1, J. Pfisterer2, J. Diebold3, S. Lax4, D. Schmidt5, A. Staebler6, A. du Bois7, F. Kommoss5 1 University of British Columbia, Department of Pathology, Vancouver, Canada, 2Klinikum Solingen, Dept of Gynecology, Solingen, 3Luzerner Kantonsspital, Institute of Pathology, Luzern, Switzerland, 4LKH Graz West, Institute of Pathology, Graz, Austria, 5Institute of Pathology, A2,2, Mannheim, 6 University of Tübingen, Institute of Pathology, Tübingen, 7Kliniken Essen Mitte (KEM), Dept Gynecology & Gyn. Oncology Aims. Based on the literature as well as on own results from a prospective study one may assume that a considerable number of patients in clinical trials of ovarian carcinoma have diagnoses in conflict with inclusion criteria. Subsequently clinical trials may be biased through unintended disregarding of histological inclusion criteria. To avoid the latter limitation, specialized pathology review should become standard procedure in study protocols prior to randomization. To facilitate specialized second opinion pathology prior to randomization and/or treatment decisions the process of pathological case review has to be completed within a few working days. We hypothesize that our new, internet-based high throughput infrastructure will be capable of providing specialized second opinion pathology within 10 working days. Methods. Patients scheduled for the AGO OVAR17 trial have to be registered for a central pathology review, which has to be performed through the translational subproject termed “AGO VISION-1”. Having provided written informed consent, the patients will be registered for pathology review at a central office. Original slides will then be requested from the outside pathologists in order to be scanned and uploaded to a secured

internet server. A network of internationally recognized gynecological pathologists is connected to the server through a custom-designed software platform. If necessary, immunohistochemistry is available through a collaborating pathology lab. Results. The new internet-based high throughput infrastructure has been set up successfully. Five gynecopathologists from Austria, Switzerland and Germany will provide specialized review of all cases scheduled for inclusion in the AGO OVAR17 trial for all study centers of the AGO study group. A centralized office plays a key role in the logistics of the complex course of action in central pathology review. Conclusions. Preliminary data suggest that the use of a new internet-based infrastructure may allow for specialized case review prior to patient randomization in the AGO OVAR17 trial. This approach might not only help to avoid disregarding of clinicopathologic inclusion criteria but also to further improve the quality of patient care through minimization of overtreatment with chemotherapy of patients with ovarian borderline tumors and inadequate treatment of patients with ovarian metastases.

SA-P-024 Metastatic sebaceous ovarian cancer H. Geddert1, A. Dimmler1, M. Rauchholz2, O. Tomé2, G. Faller1 St. Vincent Hospital, Institute of Pathology, Karlsruhe, 2St. Vincent Hospital, Department of Gynecology and Obstetrics, Karlsruhe

cancer, we investigated HDAC 1 expression and T-cell density in a large series of high-grade serous ovarian carcinomas. Methods. Primary tumors of 141 patients with SOC in FIGO Stage II–IV were studied by immunohistochemistry on tissue microarrays containing 6 cores per case. HDAC1 expression was assessed by applying the semiquantative IRS score. The stromal and intraepithelial T-cell (CD3 and CD8) density was quantified, counting the average number of cells per high power field (HPF=400x) and reviewing a total of 10 HPF for each core. Results. Strong nuclear HDAC1 expression (score 8, 9, 12) was associated with poor overall survival (OS, median 25 vs. 40 months, p=0.008), but not disease free survival (DFS median 23 vs. 27 months, p=0.378). Increased intraepithelial CD3+ lymphocytes (p=0.012) and stromal CD8+ lymphocytes (p=0.026) were associated with favourable OS, whereas DFS was only significantly associated with stromal CD8 density (p=0.019). Strong HDAC 1 expression correlated with increased cytotoxic lymphocyte density (p=0.021, Pearson-correlation). Conclusions. Our study of a large collective of advanced SOC confirms the prognostic relevance of intratumoral lymphocyte density as well as high HDAC1 expression. The observed correlation between HDAC1 overexpression and intraepithelial CD8+ T-cell density points to a potential role of HDAC1 as a tumor specific antigen in SOC.

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Aims. A 47-year-old patient complained of abdominal tenderness and pain. Methods. Ultrasound as well as thoracal, abdominal and pelvic computer tomography demonstrated a heterogenic tumor of the left ovary measuring 14 cm. A singular lesion suspected of metastasis was identified as well in the liver and the lung. A core biopsy from the pulmonary lesion was collected preoperatively. Laparotomy with total abdominal hysterectomy, bilateral salpingo-oophorectomy, infragastric omentectomy and liver lesion biopsy was performed. During operation, no macroscopic sign of peritoneal carcinosis was described. Results. Histological examination of the ovarian tumor proved a sebaceous carcinoma. No features of a teratoma were identified in the completely sampled mass. Hepatic and pulmonary metastases were diagnosed in the core biopsies. Unfortunately, the patient showed a rapid progress postoperatively with increasing number and size of liver and lung metastasis. Therefore, the patient was given chemotherapy with Carboplatin and Paclitaxel. Conclusions. To our best knowledge, this is the first reported case of a sebaceous ovarian carcinoma with synchronous hepatic and lung metastasis.

SA-P-025 Histone-deacetylase-1 expression and intratumoral lymphocyte density are prognostic parameters for predicting overall survival in serous ovarian carcinoma H. Bösmüller1, J. Peper2, B. Gückel3, T. Fehm3, C. Bachmann3, D. Wallwiener3, S. Stefanovic2, D. Pham4, F. Fend4, A. Staebler4 1 Krankenhaus Barmherzige Schwestern, Dept. of Pathology, Linz, Austria, 2 University of Tübingen, Dept. of Immunology, Tübingen, 3University of Tübingen, Dept. of Gynecology, Tübingen, 4University of Tübingen, Dept. of Pathology, Tübingen

SA-P-026 Effects of MDM2 and MDMX splice variants on p53 pathway in ovarian carcinomas S. Hammer1, S. Pelka1, A. Wolf1, A. Böhnke1, S. Mahner2, G. Ott3, S. Hauptmann4, F. Bartel1 1 University of Halle-Wittenberg, Institute of Pathology, Halle/Saale, 2University Medical Center Hamburg-Eppendorf, Department of Gynecology, Hamburg, 3Robert-Bosch-Hospital, Institute of Clinical Pathology, Stuttgart, 4 Insitute of Pathology Allgäu-Oberschwaben, Wangen Aims. Many human tumors show inactivation of p53 pathway e.g. due to overexpression of the negative regulators MDM2 and MDMX. Both, MDM2 and MDMX, are spliced alternatively and aberrantly in many tumor cell lines. So far there are no studies about the expression and effects of MDM2/X splice variants in ovarian carcinomas. Methods. Therefore, we screened 33 frozen ovarian carcinoma samples for the occurrence of MDM2/X splice variants. We detected beside the known variants MDM2-B, MDMX-S and MDMX-211 new variants such as MDM2-p53NLS, MDM2-ARZ, MDMX-A, MDMX-Z and MDMXZR containing different domains. Subsequently, the ovarian tumor cell lines OAW-42 and ES-2 were transfected with these variants and treated with both cisplatin and taxol in order to analyze their effects on the p53 pathway after DNA damage. Results. Overexpression of MDM2-p53 and MDM2-p53NLS resulted in stabilization of p53 but not in induction of p21 expression. In contrast MDM2-AZR and-B did not change p53 level but slightly increased p21 expression after cisplatin. Both MDMX-A and MDMX-211 stabilize MDMX which caused a transcriptional inactivation of p53 after cisplatin treatment. Conclusions. Our results clearly show, that MDM2 and MDMX splice variants affect p53 pathway which may had an impact on tumor formation and/or chemoresistance.

Aims. High level expression of histone deacetylases 1, 2 and 3 (HDAC) are associated with progressive disease and poor prognosis in high-grade serous ovarian carcinoma (SOC) and also are markers for potential treatment with histone deacetylase inhibitors. Intratumoral lymphocyte density is a parameter of antitumoral immunoreactivity and is associated with prolonged overall survival. Since we recently identified HDAC1/2 by HLA ligandome analysis as HLA class I associated antigen in ovarian Der Pathologe Suppl 1 · 2012 

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Abstracts SA-P-027 Primary serous peritoneal carcinoma (PPC) with and without serous tubal in-situ carcinoma (STIC) L.-C. Horn1, K. Leonhardt2, K. Kellner1, R. Scherling2, J. Einenkel2 1 University of Leipzig, Institute of Pathology, Leipzig, 2University of Leipzig, Department of Obstetrics and Gynecology (Institute of Trier), Leipzig Aims. Evaluating the frequency of serous tubal in situ carcinoma (STIC) in cases of primary peritoneal carcinoma. Methods. The present study evaluates immunohistochemically (Ki-67 and p53-staining) the presence of STIC in completely embedded Fallopian tubes of 35 consecutive cases meeting the clinicopathologic criteria of primary high-grade serous carcinoma. Results. p53 signature was seen in four cases (11%) and STIC in seven patients (20%). All STIC occurred at the fimbriated end of the Fallopian tube and in one case a bilateral involvement was seen. These precursor lesions were missed during the initial routine screening. In two cases repeated staining for p53 was negative. Conclusions. STIC and p53-signature as precursor lesions of pelvic serous cancer are seen in some but not all cases of primary serous peritoneal cancer. Further studies are required to identify the source of serous cancer in cases without STIC-lesions.

SA-P-028 KPNA2 protein expression is an independent adverse predictor of survival in patients with endometrial carcinomas K. Ikenberg , A. Noske , R. Caduff , A. Dellas , H. Moch , P.J. Wild 1 University Hospital Zurich, Institute of Surgical Pathology, Zürich, Switzerland, 2University of Basel, Institute of Pathology, Basel, Switzerland 1

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Aims. To analyze rates of expression of karyopherin alpha 2 (KPNA2), an important mediator of nucleocytoplasmic transport, in different endometrial cancer subtypes, and to evaluate its prognostic properties for patients with primary endometrial cancer. Methods. Tissue microarrays (TMA) contained 527 formalin-fixed, paraffin-embedded endometrial cancer tissue cores from two different institutes of pathology. TMAs were stained immunohistochemically for KPNA2 and p53. Results. KPNA2 expression was significantly upregulated in carcinomas of the endometrium and was significantly associated with higher tumor grade, higher FIGO stage, and overexpression of p53. KPNA2 expression was not associated with different subtypes of endometrial carcinomas. Positive nuclear KPNA2 immunoreactivity in at least 10% of nuclei was identified as a novel predictor of survival, and was independent of the well-established prognostic factors age, grade, FIGO stage, histological type, and p53 immunoreactivity in Cox regression analyses (hazard ratio=1.7, 95% CI 1.13–2.56, p=0.01). Conclusions. KPNA2 is a novel independent prognostic marker for survival after hysterectomy. This may allow identifying patients who need more aggressive treatment.

SA-P-029 Loss of ARID1A/BAF250a expression in endometriosis – a new molecular mechanism for transformation into cancer? A. Noske , N. Samartzis , M. Rechsteiner , H. Moch , P. Imesch 1 University Hospital Zurich, Institute of Pathology, Zürich, Switzerland, 2 University Hospital Zurich, Dept. of Gynecology, Zürich, Switzerland 1

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Aims. Mutations of the tumor suppressor gene ARID1A are frequently found in endometriosis-associated ovarian carcinomas, and correlate with expression loss of the coding protein BAF250a. We hypothesize that deficient ARID1A/BAF250a expression may contribute in transformation of endometriosis into cancer.

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Methods. We evaluated ARID1A/BAF250a protein expression in 74 endometriosis, and 30 eutopic endometrium samples by immunohistochemistry. Further, an analysis of ARID1A/BAF250a and p53 expression in a cohort of 129 primary ovarian carcinomas was performed. To classify the ovarian carcinomas in type I and type II, the mutational status of p53, KRAS, and BRAF will be determined by deep-sequencing technology. Results. There was a loss of ARID1A/BAF250a expression in three endometriomas and one deep-infiltrating endometriosis, but in none of the peritoneal endometriosis and eutopic endometrium samples. Lack of expression was found in 22.5% of the ovarian carcinomas and was significantly associated with the endometrioid histological subtype and wild-type p53 protein. Conclusions. The deficiency of ARID1A/BAF250a expression in few endometriosis lesions may indicate an early event in malignant transformation of endometriosis. In context with the literature, the data support an association of ARID1A and p53 in ovarian cancer.

SA-P-030 Detection of micrometastasis in paraaortic lymph nodes in patients with carcinoma of the uterine cervix after negative frozen section analysis L.-C. Horn1, K. Kellner1, R. Scherling2, M. Höckel2, J. Einenkel2 University of Leipzig, Institute of Pathology, Leipzig, 2University of Leipzig, Department of Obstetrics and Gynecology (Institute of Trier), Leipzig

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Aims. Previous studies considered the presence of micrometastases (MM) in pelvic lymph nodes as clinically relevant prognostic indicator and reported a 2.4 to 3.2 greater risk for recurrent disease and dead of the disease when compared to nodal negative patients. There is limited information about the value of (immunohistochemical) ultrastaging in negative para-aortic lymph nodes (PAN). Methods. Frozen section analysis was performed in all cases with PALNE because of clinical requirements. From all FS-blocks, routinely three step sections were performed. After FS-examination nodes were examined by one H&E-stained slide. All nodes without metastatic disease after frozen section and permanent section examination were subject of the present study. 43 patients and 418 PAN were enrolled and immunohistochemical staining using two cytokeratin-cocktail antibodies (AE 1/AE 3 and KL-1) was performed. MM were defined as foci of tumor cells ranging from 0.2 mm to no more than 2 mm in size; isolated tumor cells (ITC) are defined as single tumor cells or small clusters of cells less than 0.2 mm in size within the subcapsular sinuses of the lymph node. Results. In one case, one single node showed micrometastasis, representing an incidence of 2.3% of the studied cases and 0.23% of the examined lymph nodes. In three cases benign endosalpingiosis was seen. The patient with MM is alive without evidence of disease 96 months after surgery. ITC were not observed. Conclusions. The frequency of MM in PAN is very low. There are only limited data regarding their prognostic impact within the literature. After careful examination of all removed PAN using H&E-staining (and step sectioning), immunohistochemical ultrastaging cannot be recommend for routine use.

SA-P-031 Mismatch repair protein expression of cervical adenocarcinoma B. Helmchen1, R. Brand2, M. Kurrer3, P. Komminoth1, H.-P. Sinn2 Community Hospital Triemli, Dept. of Pathology, Zürich, Switzerland, 2 University of Heidelberg, Institute for Pathology, Heidelberg, 3 Enge Institute of Pathology, Zürich, Switzerland

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Aims. Charakterisierung des immunhistochemischen Expressionsprofils der Mismatch-Repair-Proteine (MMRP) MLH1, MSH2, MSH6 und PMS2 an primären zervikalen Adenokarzinomen (ZAK) in Abgrenzung zu Karzinomen des unteren Uterinsegmentes (UUSK).

Methods. HE-gefärbte Schnittpräparate und pathologische Befunde von 42 archivierten Hysterektomie-Präparaten von Tumorfällen mit der Diagnose eines ZAK, wurden bezüglich zervikaler Vorläuferläsionen (ZVL) und Befall des unteren Uterinsegments (UUS) reevaluiert. An ZVL−/UUS+ Fällen wurde eine PCR-Analyse für HPV-DNA durchgeführt. Fälle mit ZVL+/− und UUS-, und UUS+/ZVL+ Fälle, sowie UUS+/ZVL−/HPV+Fälle wurden als primäre ZAK klassifiziert. UUS+/ ZVL−/HPV-Fälle wurden als UUSK reklassifiziert. Repräsentative Tumorschnitte wurden mittels standardisierter, automatisierter Methoden für MLH1, MSH2, PMS2 und MSH6 gefärbt. Vollständig fehlende Expression eines MMRP in Tumorzellen bei positiver interner Kontrolle im Normalgewebe wurde als Verlust der Proteinexpression gewertet. Expressionsprofile primärer ZAK und UUSK wurden verglichen und korreliert (Fisher-Exakt-Test). Zusätzlich wurden alle Tumoren für ER, Vimentin, CEA und p16 gefärbt. Results. 37 Fälle wurden als primäre ZAK klassifiziert (37/42; 88.1%), davon waren 27 UUS−, 8 UUS+/ZVL+ und zwei UUS+/ZVL−/HPV+Fälle. Fünf UUS+/ZVL−/HPV−- Fälle (5/42; 11.9%) wurden als UUSK reklassifiziert. Die MMRP Expression war in sämtlichen primären ZAK erhalten (37/37; 100%). In vier von fünf UUSK (4/5; 80%) zeigte sich ein Expressionsverlust von mindestens einem MMRP (p<0,005). Verlust von MSH2 und MSH6 wiesen ein weitgehend undifferenziertes Adenokarzinom und ein gemischt serös/ endometrioid differenziertes Adenokarzinom auf. Ein isolierter Verlust von PMS2 war in einem muzinös differenzierten UUSK nachweisbar. Ein gemischt serös/endometrioid differenziertes UUSK zeigte Verlust von PMS2 und MLH1. Von den ZAK zeigten 21 Fälle (21/37; 56,8%) ein typisches Immunprofil (ER-/Vimentin−/CEA+, p16+). Keines der UUSK wies ein ZAK-typisches Immunprofil auf (0/5; 0%). Conclusions. Ein Verlust der MMRP-Expression ist ungewöhnlich für primäre Adenokarzinome der Cervix uteri. Zur sicheren Abgrenzung zwischen ZAK und Karzinomen des unteren Uterinsegments sollten in Zweifelsfällen immunhistochemische (ER, Vimentin, CEA und p16) und molekulare (HPV-PCR) Untersuchungen hinzugezogen werden. Karzinome des unteren Uterinsegments zeigen häufig einen Verlust der MMRP-Expression.

SA-P-032 Role of CCND1 amplification and expression in squamous cell carcinomas of the vulva T.S. Clauditz1, P. Tennstedt1, F. Holst1, F. Gieseking2, S. Mahner2, L. Woelber2, R. Simon1, G. Sauter1, M. Choschzick1 1 Institute for Pathology, Hamburg, 2Gynäkologie, Hamburg Aims. CCND1 belongs to the family of D-type cyclins involved in cell cycle progression, transcriptional regulation and cell migration. CCND1 was found to be amplified and overexpressed in a variety of cancers, including some vulvar carcinoma cell lines. The aim of our study was to analyze CCND1 copy number changes and CCND1 protein expression in a large set of vulvar cancers with clinical follow up data. Methods. The vulvar cancer tissue microarray used for this study contains a total of 183 formalin fixed, paraffin-embedded tumors. Dual-color FISH analysis of the CCND1 copy number was performed by using Vysis CCND1/CEP 11 FISH Probe Kit CE (Abbott Laboratories, order# 3N8820). A tumor was considered amplified if the ratio of Gene/centromere was ≥2.0. Standard indirect immunoperoxidase procedures were used for detection of CCND1 protein according to the recommendations of the manufacturer (DAKO, clone EP12). The CCND1 staining intensity and the fraction of stained tumor nuclei were recorded for each tissue spot. A final 2-step scoring result was made, distinguishing low and high CCND1expression levels. Results. CCND1 amplification was observed in 32 (22.4%) vulvar carcinoma specimens and was statistically related to the presence of regional lymph node metastases (p<0.001). There was no association between CCND1 amplification and patient age, tumor stage, histopathological

subtype, grading and HPV status of vulvar carcinomas (p>0.05 each). Detectable CCND1 expression was found in 139 (83.2%) vulvar carcinomas and 76 (45.5%) tumors exhibited a high expression level according our predefined criteria. Increased levels of CCND1 expression were significantly related to higher patient age (p=0.013), positive pN category (p=0.004) and negative HPV status (p<0.001). Basaloid as well as verrucous, warty-type and mixed vulvar carcinomas showed lower CCND1 expression levels than keratinizing or non-keratinizing tumors (p<0.001 and p=0.032, respectively). Elevated CCND1 expression levels and amplification of the CCND1 gene were closely connected in the present analysis (p<0.001). Patient prognosis was independent from CCND1 amplification status and expression level (p=0.57 each). Conclusions. CCND1 is amplified and overexpressed in a substantial proportion of vulvar carcinomas and associated with the occurrence of locoregional lymph node metastases, especially in HPV negative tumors. Our results suggest that CCND1 plays a role in tumor cell motility and development of metastasis in cancer progression.

SA-P-033 Tubulo-squamous vaginal polyp: Case report and focus on histogenesis T. Hansen1, D. Macchiella2, C.J. Kirkpatrick1 1 University of Mainz, Institute of Pathology, Mainz, 2University of Mainz, Department of Obstetrics and Gynecology Aims. Tubulo-squamous vaginal polyp has been recently characterized as a specific entity with characteristic histological features. Until now, less than 30 cases have been recorded in the literature. This benign lesion is commonly localized in the upper third of the vagina of postmenopausal woman. In addition, it is believed to derive from misplaced paraurethral Skene’s glands, which are the female equivalent of prostatic glands in men. We report on a further case of tubulosquamous vaginal polyp with special emphasis on the histogenesis. Methods. We describe a 77-year-old female patient presenting with a polyp causing no specific symptoms. Biopsy was taken and routinely processed. Immunohistochemistry was applied according to standard protocols using antibodies against cytokeratins (CK) 5/6 and 7, as well as prostate-specific antigen (PSA), prostate-specific acidic phosphatase (PAP), and androgen receptor (AR). Results. Histological examination revealed a polyp, which was covered by normal squamous epithelium, but showed prominent epithelial nests in a spindle cell-rich stroma. The nests were predominantly squamous in type with small tubules at the periphery. By means of immunohistochemistry, the tubules were positive for CK7, PAP and AR, but negative for CK5/6 and PSA. The solid areas were positive for CK5/6 and most interestingly, also expressed AR, but not CK7, PAP or PSA. The stroma also revealed strong staining for AR, but not for the other markers applied. Conclusions. Tubulo-squamous polyp is a rare, but distinct entity with an established morphology. This is the first report on AR expression in this benign polyp. Our finding confirms the view of a derivation from paraurethral Skene glands.

SA-P-034 Radius metastasis in a patient with endometrial cancer M. Gajda1, H. Schimmel1, R. Friedel2, O. Camara3, J. Herrmann4, I. Petersen1 1 Institute of Pathology, Jena University Hospital, 2Department of Trauma, Hand and Reconstructive Surgery, Jena University Hospital, 3Department of Gynecology and Obstetrics, Jena University Hospital., 4Department of Gynecology and Obstetrics, Apolda Hospital Aims. Unlike other non-gynecologic solid tumors, such as breast cancer, lung cancer, metastasis to bone from endometrial carcinoma is rare. Bone metastases are usually located in axial skeleton. Metastasis to bones of the extremities is quite rare. Der Pathologe Suppl 1 · 2012 

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Abstracts Methods. Detailed clinical and histopathologic review of a clinical case and review of the literature using PUBMED. Results. We report on a 70-year-old woman with FIGO Stage 1A Grade II (pT1a pN0 L0 V0 R0) endometrial adenocarcinoma who presented 2 years following total abdominal hysterectomy, bilateral salpingo-oophorectomy and lymphonodectomy with left radius bone metastasis. A left forearm amputation was performed. The tumor measured 7.1×6.9×6.5 cm. The histological diagnosis was a poorly differentiated (G3), endometrioid endometrial carcinoma. Both carcinomas were oestrogen and progesterone receptor negative, vimentin, OSCAR and AE1/AE3 positive. Immunohistochemical stains for S-100, desmin, a-smooth muscle actin and CD 34 were negative. Conclusions. This case highlights the rare and unusual presentation of endometrial cancer. For this reason a review of the literature is also provided for all cases with evidence of bone metastasis at presentation of the disease and as a recurrence. Its diagnosis requires immunohistochemistry and awareness of its possible existence.

Poster: Molekularpathologie I SA-P-035 Identification of uncommon PIK3CA-mutations in lung cancer by using pyrosequencing V . Schildgen1, J . Lüsebrink1, J .D . Appel1, C . Wübben1, W . Engel-Riedel1, E . Stoelben1, O . Schildgen1, M . Brockmann1 1 University Witten/Herdecke, Kliniken der Stadt Köln gGmbH, Köln Aims. Phospatidylinositol-3-kinases (PI3K) play an important role in various cell processes. Oncogenic mutations in the PIK3CA gene which codes for the catalytic subunit have been identified in various malignancies and activate the PI3K/AKT/mTOR pathway which is a critical driver of tumorigenesis. Methods. We tested 41 tumor samples with known KRAS, BRAF, and EGFR mutation status for the occurrence of mutations in the PIK3CA gene using a new commercial pyrosequencing kit. Results. Pyrosequencing revealed 2 mutations (4.9%) in the PIK3CA gene, one in exon 9 and one in exon 20. Both mutations have not yet been identified in lung tumor tissue. Conclusions. The screening of our small patient cohort by pyrosequencing identified two mutations (4.8%) in PIK3CA, on in exon 9 (Q546H) and on in exon 20 (M1043T). Both mutations have not yet been described in lung tumours and seem to be rather uncommon mutations. Future screening of large patient cohorts with pyrosequencing may contribute to the detection of more mutations in lung cancer due to the low limit of detections of this method and may contribute to a better understanding of the interaction of mutations and tumorigenesis.

SA-P-036 Detection of EML4-ALK fusion oncogenes in NSCLC using a commercial three-color FISH assay (ZytoLight SPEC TriCheck) B . Schmitt1, H . Schultz1, F . Stellmacher1, E . Vollmer1, T . Goldmann1 1 Borstel Research Center, Clinical & Experimental Pathology, Borstel Aims. About 5% of non-small cell lung cancers (NSCLC) harbor an inversion of the short arm of chromosome 2 that causes a rearrangement of the ALK and EML4 genes. The resulting fusion oncoprotein comprises a constitutively active tyrosine kinase. Those patients will benefit substantially from treatment with crizotinib, a Met-/tyrosine kinase inhibitor, highlighting the need for appropriate tests allowing one to reliably detect such fusions in biopsy specimens. Existing techniques such as immunohistochemistry, multiplex PCR, or 2-color FISH using break apart-probes each possess specific strengths and weaknesses. By design, a novel

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commercial 3-color FISH assay offers several theoretical advantages. However, its actual performance in research and diagnostic settings has not been tested. Methods. We analyzed formalin-fixed and paraffin embedded (FFPE) tissue sections (pulmonary adenocarcinomas) and tissue microarrays (TMA; assembled from adenocarcinomas, squamous and large cell carcinomas) for EML4-ALK rearrangements by 3-color FISH assay using ZytoLight SPEC TriCheck probes (Zytovision). Stained slides were evaluated multiply by pathologists and molecular biologists according to standardized criteria. Cancers exhibiting a characteristic fluorescence pattern in at least 15% of tumor cells were taken as EML4-ALK fusion positive. Results. High quality signals were obtained in FFPE tissues, in individual sections as well as in TMAs. Interobserver variability was negligible for wild type identification. Discrimination between FISH signals from wild type vs. EML4-ALK fusions in cancer tissue was clear and binary owing to probe design, and without the problematic intermediate results observed with break apart-probes in case of a small split. Evaluation was more laborious and less reproducible, however, when more non-tumor tissue was interspersed and tumor cells were harder to identify. Assay hands-on time and total turnover time were less than for multiplex PCR. Conclusions. Detecting EML4-ALK fusions based on a 3-color FISH assay using the “ZytoLight SPEC TriCheck”-probes requires specialized equipment, familiarization of the observer and reliable distinction of tumor cells from non-tumour cells. The approach does not require assessment by multiple observers, unlike assays using break-apart probes. Overall, this approach appears particularly suited for clinical studies and basic research in that it will also identify rare and putative novel fusion variants, as well as deletions and amplifications.

SA-P-037 Systematic quantitative cross-validation and content analysis of the 450k methylation array from Illumina J . Rößler1, O . Ammerpohl2, J . Gutwein2, U . Lehmann1 Medical School Hannover, Institute of Pathology, Hannover, 2University Hospital Schleswig-Holstein, Institute for Human Genetics, Kiel

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Aims. The relationship between the beta-values provided by the newly released 450k methylation array from Illumina for individual CpG dinucleotides was compared with quantitative methylation levels obtained by conventional pyrosequencing. In addition, the representation on the Illumina array of two groups of genes, which are important in tumour biology and display extensive aberrations in DNA methylation in cancer (microRNAs and imprinted loci), was assessed in detail. Methods. High molecular weight DNA was isolated from histologically examined 18 fresh-frozen human breast cancer specimens, 4 normal mammary epithelial fractions and 4 human breast cancer cell lines (IPH-926, HCC1937, MDA-MB-134, PCM42) using standard procedures. DNA was bisulfite treated and analyzed on 450k arrays following the manufacturer’s protocol. For primary data processing and analysis the software provided by Illumina was employed. These analyses were complemented and extended by employing the Omics Explorer from Qlucore and the R package IMA. The beta-values for individual loci were cross-validated using conventional pyrosequencing of the same DNA samples independently treated with sodium bisulfite. Results. The newly released 450k array methylation array from Illumina shows a high concordance with quantitative pyrosequencing if identical CpG sites are analysed by the two different methods in cell lines (Spearman r=0.88, p<0.0001), which is somewhat reduced in primary tumour specimens (Spearman r=0.73, p<0.0001) Concordance is reduced if different CpG sites in the same locus or CpG islands are targeted by the two different methods (r=0.82 and 0.68, respectively). The number of CpG sites analysed per microRNA gene or imprinted locus ranges from 1 to 70 and does not follow coherent rules.

Conclusions. The newly released 450k methylation array from Illumina provides a genome-wide representation of DNA methylation aberrations in a convenient format with direct quantification of methylation levels at each individual CpG site. However, the representation of microRNA genes and imprinted loci is quite uneven and has to be taken into account during data evaluation and derivation of any conclusion concerning these two important classes of genes.

SA-P-038 Luminescent conjugated oligothiophenes: novel optical probes that detect cross beta-sheet conformation of inclusion bodies “in situ” V. Mahajan1, T. Klingstedt2, R. Simon2, P. Nilsson2, A. Thueringer1, K. Kashofer1, H. Denk1, K. Zatloukal1, J. Haybaeck1 1 Medical University of Graz, Institute of Pathology, Graz, Austria, 2Linkoping University, Sweden Aims. Mallory-Denk bodies (MDBs) are cellular hallmarks of protein aggregation diseases such as alcoholic and non-alcoholic steatohepatitis (ASH and NASH). MDBs are also seen in other liver diseases such as hepatocellular carcinoma (HCC) and alpha-1-antitrypsin deficiency. Additionally presence of Intracellular Hyaline bodies (IHBs) is also a common observation in HCC. Despite of advances in technologies highlighting protein conformation, structural characterisation of inclusion bodies remains unresolved. Therefore investigating molecular structure of the major MDB constituents keratin 8 (K8) and 18 (K18), p62 and ubiquitin may provide deeper mechanistic insights in MDB/IHB formation. Methods. Luminescent conjugated oligothiophenes (LCOs), h-HTAA, p-FTAA and PTAA contain swivelling thiophene backbones whose geometry modulates fluorescence properties. LCOs were demonstrated to specifically bind proteins with cross beta sheet conformation. In addition to the older ones the new generation of LCOs were used for in situ investigation of conformational changes in MDBs in human and murine livers. Results. LCOs demonstrated constant presence of cross beta-sheet conformation in human MDBs but not in IHBs, alpha-1-antitrypsin and ground glass inclusions. The spectral signatures collected from MDBs in ASH, NASH and HCC indicated a similar molecular structure of MDBs under the various disease conditions. MDBs induced by 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine-feeding of mice revealed h-HTAA and p-FTAA binding to MDBs in all experimental stages. CHO-K1 cells transfected with various combinations of SQSTM1/p62, ubi and Krt8/ Krt18 showed that K8 was more prone than K18 to lead to generation of cross beta-sheets. Aggregates of p62 alone were reproducibly negative for LCOs. Circular dichroism analysis elucidated intrinsically different conformational nature of purified K8 and K18 polypeptides. Conclusions. The comparative higher tendency of K8 to undergo conformational changes from predominantly Alpha-helical to cross β-sheet explains its essential role in MDB formation. This elucidates why the absence of K8 prevents MDB formation whereas its excess facilitates MDB formation. The nature of keratins to acquire cross beta sheet conformation appears to be dependent on intrinsic factors but may not be the consequence of pathological conditions. With PTAA, LCOs may serve as a multicolour novel diagnostic “in situ” technology that can be applied on formalin-fixed and paraffin-embedded and fresh frozen tissues.

SA-P-039 Comparison of cobas and HC2 HPV testing in a German routine laboratory H. Ikenberg1, C. Börsch1, B. Pittel1, A. Xhaja1, F. Britz2, T. Iftner3 1 Cytomol, MVZ for Cytology and Molecular Biology, Frankfurt, 2Roche Diagnostics, Mannheim, 3University of Tübingen, Institute for Virology, Tübingen Aims. Up to now the Digene HC2 test (Qiagen, Hilden) is regarded the gold standard for human papillomavirus (HPV) DNA testing. Meanwhile several new test systems for HPV are available, among them the cobas HPV assay (Roche Diagnostics, Mannheim, Roche) which allows simultaneous genotyping for HPV 16 and 18. We compared the performance of the cobas HPV with the HC2 assay in cervical samples collected with the PreservCyt collection medium (Hologic, Frankfurt). Methods. 1781 anonymized routine specimens pretested with the HC2 test were available for analysis with cobas HPV. Cases with discrepancies between the two tests were retested with the Linear Array HPV genotyping test (LA; Roche). Partially, histologic diagnoses were available. Results. In 1566 (87.9%) of the cases HPV results were concordant. Of the 215 cases (12.1%) with discrepancies LA results are available in 214 cases. 94 cases were LA-negative: 13 of 105 cobas-pos/HC2-neg and 81 of 99 cobas-neg/HC2-pos cases. 110 cases were LA-positive: 92 of 105 cobas-pos/ HC2-neg and 18 of 99 cobas-neg/HC2-pos cases. 325 cases with histologically confirmed CIN2+ were included. In 261 out of 293 cases (89.1%) where a HC2 result was available this was positive, while 298 out of 318 of the cases (93.7%) tested with cobas HPV were positive. The rate of HPV positivity in cytologically normal cases and in HSIL was slightly higher with cobas HPV, while it was in the same range in ASC-US and in LSIL cases. With increasing severity of the cytologic findings the rate of HPV 16- and 18 positivity increased proportionally. Conclusions. In routine specimens from a German commercial laboratory the cobas HPV test showed similar performance compared to the HC2 test. Preliminary data point to a potentially higher sensitivity and specificity with cobas HPV while adding information by HPV 16 and 18 genotyping.

SA-P-040 Comparison of Abbott RealTime and HC2-HPV testing in a routine laboratory H. Ikenberg1, C. Noppen2, M. Faber1, A. Xhaja1, S. Böhm3 Cytomol, MVZ for Cytology and Molecular Biology, Frankfurt, 2Viollier AG, Basel, Switzerland, 3University Hospital Leipzig, Dep. for Gastroenterology and Rheumatology, Leipzig

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Aims. The Digene Hybrid Capture2 test (HC2, Qiagen, Hilden) is the standard in routine human papillomavirus (HPV) DNA testing. Several new test systems have become commercially available in the past years, among them the automated Abbott RealTime-High-Risk-HPV assay (RealTime-HPV, Abbott Molecular, Wiesbaden). It detects 14 HR-HPV types and simultaneously differentiates between HPV16, HPV18 and 12 non-HPV16/18 HR types in a single test, while HC2 targets the same HR types, except HPV66, without typing. RealTime-HPV amplifies human β-globin in the same reaction. We evaluated RealTime-HPV versus HC2 with cervical specimens from a large German routine laboratory sampled with the PreservCyt collection medium (Hologic, Frankfurt). Methods. 505 anonymized routine specimens referred for cytology and pretested with HC2 were run with RealTime-HPV on the m2000 System (Abbott). Samples with discordant results between both assays were genotyped by Linear Array (Roche, Mannheim), targeting 37 HPV genotypes. Results were correlated with routine histology available for 280 cases (16.8%
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Abstracts were LA-positive for RealTime-HPV/HC2 targeted HPV types: 27 of 33 RealTime-HPV-pos/HC2-neg and 11 of 40 RealTime-HPV-neg/HC2pos cases. 35 cases were LA-negative: 6 of 33 RealTime-HPV-pos/HC2neg and 29 of 40 RealTime-HPV-neg/HC2-pos cases. In 280 histology confirmed cases overall-agreement between RealTime-HPV and HC2 was 82.5%. Detection rates for CIN2+ and CIN3+ were 90.6% and 90.7% with RealTime-HPV and 89.3% and 88.3% with HC2, respectively. A high correlation of HPV16/18 positivity with RealTime-HPV and increasing histological severity was found. Conclusions. Clinical performance of RealTime-HPV in routine specimens was comparable to that of HC2. Discrimination of HPV 16/18 from other HR HPV types provides additional information for risk stratification.

SA-P-041 FISH analysis does neither reveal genomic ETV1 amplification nor break-apart in gastrointestinal stromal tumors E. Wardelmann1, S. Huss1, R. Menon2, F. Göke2, G. Kristiansen2, R. Buettner1, S. Perner2 1 University Hospital Cologne, Institute of Pathology, Köln, 2University Hospital Bonn, Institute of Pathology, Bonn Aims. Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Recently the transcription factor ETV1 was shown to be highly expressed in GISTs both at transcript and protein levels. ETV1 is a member of ETS family of transcription factors. The ETS family members share an evolutionary highly conserved 80-amino-DNA binding domain and individual proteins of this family are capable of regulating gene promoter activity. Chi et al. reported that ETV1 is highly expressed in those ICCs, from which GISTs arise. Taken together, they propose that GISTs originate from ICCs with high endogenous level of ETV1 when an oncogenic activation via KIT or PDGFRA mutation occurs. However, they could not detect any genomic alteration leading to the high ETV1 expression performing FISH analysis on four GIST samples and two GIST cell lines. The present study was accomplished to evaluate in a larger cohort whether an ETV1 amplification or break apart might represent the genomic alteration causing the reported ETV1 expression. Methods. For the ETV1 amplification assay, we used the commercially available Chromosome  7 reference probe CEP 7 (D7Z1), spanning the region 7p11.1–q11.1. The target probe was located on the ETV1 locus at 7p21.2. The target probe was labelled with biotin to produce a red signal using CTD-2220I3 BAC clone. For the ETV1 break-apart assay, we used BAC clones CTD-222013 for centromeric labelling with biotin and RP-11769K2 for telomeric labelling with digoxigenin. BAC clones were obtained from Invitrogen (Invitrogen, CA, USA). Results. We performed FISH analysis on 140 GIST patients using tissue micro arrays. Neither ETV1 amplification nor break apart was detectable. Conclusions. The present study ensures that neither a genomic ETV1 amplification nor a break apart is found in GISTs. We conclude that not genetic aberrations but rather upregulation of ETV1 due to high KIT receptor levels are responsible for the high ETV1 expression in GISTs.

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SA-P-042 A new whole genome amplification method for studying clonal evolution patterns in malignant colorectal polyps D. Hirsch1, J. Camps1, S. Varma2, R. Kemmerling3, T. Ried1, T. Gaiser1 1 Section of Cancer Genomics, Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, United States, 2HiThru Analytics, Bethesda, United States, 3University Hospital Salzburg of the Paracelsus Private Medical University, Institute of Pathology, Salzburg, Austria Aims. The transition of normal colonic epithelium to adenoma and invasive carcinoma is defined by chromosomal aberrations. To identify the genetic drivers involved in this progression in samples from individual patients, we applied array comparative genomic hybridization (aCGH) to 13 formalin-fixed paraffin-embedded (FFPE) samples of early, localized human colon adenocarcinomas arising in high-grade adenomas (so called “malignant polyps”). These lesions are small and hence the amount of DNA limited. Additionally, the quality of the DNA is compromised due to the fragmentation as a consequence of formalin fixation. To overcome these problems, we optimized a newly developed isothermal whole genome amplification system (NuGEN Ovation® WGA FFPE System). Methods. DNA was isolated from the FFPE blocks of 13 malignant polyps, each consisting of areas of adenoma and carcinoma. Starting with 100 ng of FFPE DNA, the amplification system produced 4.01±0.29 µg (mean ± standard deviation) of DNA. The samples were then analyzed using Agilent SurePrint G3 Human CGH Microarrays 4×180K. Results. Genomic imbalances were conserved in this procedure as assessed by comparing amplified and unamplified FFPE DNA using aCGH. The excellent quality of amplified DNA was further indicated by a high signal-to-noise ratio and a low derivative log2 ratio spread. Both, the amount of amplified DNA and aCGH performance were independent from the age of the FFPE block and the associated degradation of the extracted FFPE DNA. We observed losses of chromosome arms 5q and 18q in the malignant polyp samples, while the embedded early carcinomas revealed losses of 8p, 17p, and 18, and gains of 7, 8q, 13, and 20. Aberrations detected in the adenoma were invariably maintained in the embedded carcinomas. Conclusions. In conclusion, this approach demonstrates that using isothermally whole genome amplified FFPE DNA is technically suitable for aCGH. Besides demonstrating clonal origin of the adenoma and carcinoma part within a malignant polyp, the gain of chromosome arm 20q was an indicator for progression from adenoma to carcinoma in malignant polyps.

SA-P-043 Dissimilarities of colorectal (CRAIT) and sinonasal adenocarcinoma (SNAIT) of intestinal type K. Donhuijsen1, I. Kollecker1, H. Hannig1, H.-G. Schroeder2 Academical Hospital Braunschweig, Department of Pathology, Braunschweig, 2Academical Hospital Braunschweig, Department of Otorhinolaryngology, Braunschweig 1

Aims. SNAIT and CRAIT are considered to be nearly identically whereas an uninformed pathologist would diagnose an endonasal metastasis of a CRAIT instead of a primary of the inner nose. However, in a closer look are there discrepancies to detect by morphology, immunohistology and molecular results? Methods. Fifty consecutive cases of SNAIT and CRAIT were compared with regard to morphological criteria as subtype, histological inhomogeneity, mucin production, desmoplastic reaction and vascular invasion. Further the expression of CDX2, CK7, CK20, Synaptophysin, p53, Ki67, were compared and also the molecular status (30 cases) by PCR for KRAS, b-raf, EGFR and p53. Results. SNAIT and CRAIT are histologically quite similar but not identical: adenomatous precursor lesions are absent. Tumor inhomogeneity

and desmoplastic reaction are different. SNAIT revealed more cohesiveness and lower vascular spreading than CRAIT. CXDX2 and CK20 expressions are identically. CK7 and Synaptophysin demonstrated no reliable differences, even though SNAIT are more often positive as CRAIT. Mutations analyses exhibited for 30 SNAIT negative results for EGFR. One case (1/30) only was positive for K-RAS Mutation (Mut g/g 12 Asp.) and only one case for b-rafV600E, whereas about 60% of cases revealed a p53 expression mostly in Exon 5, thrice in Exon 8/9. Conclusions. Subtile studies detect dissimilarities of CRAIT and SNAIT. However, if in a given case the discrimination is problematic, the molecular status can be helpful. Molecular results reflect the different pathway of these similar tumors. Hence it follows, that a morphologic mimicry does not imply biologic relatedness!

SA-P-044 The G protein coupled-receptor GPR81 is upregulated in colorectal carcinomas with KRAS wild type genotype A.-K. Wenke1, K. Balschun1, C. Röcken1 Christian-Albrechts-University Kiel, Department of Pathology, Kiel

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Aims. The KRAS genotype is essential for the response to a targeted therapy in advanced colorectal carcinoma (CRC). Several studies revealed that only patients with wild type KRAS (KRASwt) benefit from a therapy with monoclonal antibodies like cetuximab (Erbitux®) and panitumumab (Vectibix®) which block the intracellular cascade following activation of the epidermal-growth-factor-receptor (EGFR). Therefore, the identification of new therapeutic targets is very important. G protein coupled-receptors (GPCRs) are interesting candidates, since they are known to transactivate the EGFR signaling pathway. The aim of this study was the identification and functional analysis of GPCRs which are differentially expressed in colorectal carcinoma dependent on KRAS genotype. Methods. mRNA expression of KRASwt tumor and non-tumor tissue of eight patients was compared to eight cases with KRASmut tumor and non-tumor tissue using Affymetrix GeneChip® Human Gene 1.1 ST Arrays. Differential gene expression of interesting candidate genes was validated by quantitative RT-PCR and immunohistochemical staining of a validation series of 46 colorectal carcinomas (28 KRASwt, 18 KRASmut tumors). Results. We found 32 differentially regulated GPCRs comparing gene expression of normal and tumor colon tissue. An increased expression of GPR81, NIACR1, NIACR2, GPR143, HTR1D and LGR5 in carcinoma tissue could be confirmed. However, GPR81 is the only receptor being significantly differentially regulated according to the KRAS genotype of the tumor. Conclusions. Our expression analyses demonstrate a correlation of GPR81 expression with the KRAS genotype in CRC. The functional role of GPR81 in colorectal carcinogenesis has to be analyzed in further studies. Additionally, a comprehensive expression analysis on an enlarged patient series of 2000 cases will show if GPR81 could serve as a proper therapeutic target or a potential prognostic marker for colorectal carcinoma.

differences in the polysomy and amplification status exist which could influence targeted therapies. Methods. 54 primary colorectal cancers and 94 metastases of primary colorectal cancers were analyzed. The EGFR gene copy number was evaluated by fluorescence in situ hybridization (FISH). Specimens that have >40% of cells displaying ≥4 copies of the EGFR signal and specimens that showed an EGFR to CEP7 ratio ≥2 over all scored nuclei were counted as EGFR FISH-positive. Also specimens with gene clusters (≥4 spots) in ≥10% of tumor cells and specimens with at least 15 copies of the EGFR signals in ≥10% of tumor cells were counted as EGFR FISH-positive. Results. A high EGFR polysomy was found in 18 of the 54 primary colorectal cancers (33.3%) and in 30 of the 94 evaluated syn-or metachronous metastases in lymph nodes, liver, lung, ovary, peritoneum, skin, brain, urinary bladder and bone (31.9%). In contrast no real gene clusters and real EGFR amplifications were found. 18 of 22 matched metastases from EGFR FISH-positive primary colorectal cancers were also EGFR FISHpositive (81.8%) showing a high accordance but no complete congruence. In addition, 1 case showed an EGFR FISH-negative primary colorectal cancer and a EGFR FISH-positive metastasis. Conclusions. The discrepancy of EGFR polysomy and amplification status found in primary colorectal cancers and matched metastases demonstrate a tumor-metastases diversity, which should be considered in patients’ treatment regime.

SA-P-046 Detection of KRAS and BRAF mutation in Chinese colorectal carcinoma patients by pyrosequencing M. Ye1, J. Chen1, M. Lai1 Zhejiang University, School of Medicine, Hangzhou, China

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Aims. KRAS and BRAF mutation status were reported to be a possible biomarker which response to EGFR antibody chemotherapy in colorectal carcinoma. Recently, pyrosequencing is proved as a powerful and convenient method for KRAS and BRAF mutation detection by a lot of groups in the U. S. and Europe. In China, there are rare labs or groups to setting up pyrosequencing method for molecular clinical diagnosis. Methods. 180 FFPE CRC tissue samples were microdissected to obtain the tumor cells and remove the stroma contents, then the genomic DNA was extracted from these samples by routine method. A pyrosequencing assay based on PCR was designed to characterize KRAS codon 12, 13 and BRAF codon  600 mutation status by Pyromark Q24. The SW620 and HT29 CRC cell lines were used as controls in pyrosequencing. Results. Mutation status of KRAS codon 12 was identified in 65/180 (36.11%) samples, among them,1 sample was 34G>A mutation(0.56%), 2 samples were 34G>C mutation(1.11%) , 2 samples were 34G>T mutation (1.11%), 29 samples were 35G>A mutation (16.11%), 1 was 35G>C mutation (0.56%) and 30 were 35G>T mutation (16.67%). 20 samples were mutant for KRAS codon 13 (11.11%)with the nucleotide change 38G>A for 19 samples (10.56%) and 1 for 38G>A (0.56%). 16/180 tumors harbored a mutation of 1799T>A in BRAF codon 600 mutation (8.89%). Conclusions. The KRAS and BRAF mutant frequencies of the 180 FFPE tissue samples are generally consistent with those recently reported. Although the amount of samples was limited, more CRC patients are needed to be detected in the year future.

SA-P-045 Polysomy and amplification status of EGFR in primary colorectal cancer and in matched metastases C. Giedl1, A. Kiesl1, F. Hofstädter1, W. Dietmaier1 University of Regensburg, Institute of Pathology, Regensburg

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Aims. There is a need for predictive biomarkers that identify patients most likely to respond to targeted treatment. EGFR is an important target for therapies using monoclonal antibodies and tyrosine kinase inhibitors (TKIs). We analyzed the polysomy and amplification status of EGFR in primary colorectal cancers, matched metastases, and looked if Der Pathologe Suppl 1 · 2012 

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Abstracts SA-P-047 Alternative splicing of Daxx-beta is reduced in renal cell and colorectal carcinoma

SA-P-049 Multiciplicity in sporadic inflammatory fibroid polyps and GISTs implicating a field effect

S. Funke1, N. Wethkamp1, S. Heikaus1, K.L. Schäfer1, H.E. Gabbert1, C. ­Mahotka1 University Hospital Düsseldorf, Düsseldorf

S. Huss1, H. Löser1, E. Kleimann2, S. Eidt3, R. Budde4, W. Weichert5, A. Szöke6, C. Röcken7, A. Hölscher8, W. Hartmann1, S. Merkelbach-Bruse1, H.-U. Schildhaus1, R. Buettner1, E. Wardelmann1 1 University Hospital Cologne, Institute of Pathology, Köln, 2St. Franziskus Hospital, Cologne, Department of Surgery, 3St. Elisabeth-Krankenhaus Köln Hohenlind, Institute of Pathology, Köln, 4Institute of Pathology in Cologne – St. Vinzenz-Hospital, 5University Hospital Heidelberg, Institute of Pathology, 6 Institute of Pathology, Cologne – Deuz, 7Christian-Albrechts-University, Kiel, Institute of Pathology, 8University Hospital Cologne, Department of Surgery

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Aims. Death associated protein (Daxx) is an important transcriptional co-repressor for a large number of genes, mostly related to apoptosis. Daxx interacts with p53 and represses its transcriptional activity. Alternative splicing of the Daxx results in the generation of a c-terminally truncated and modified isoform termed Daxx-beta. As we shown before, the new C-terminus leads to a markedly reduced affinity of Daxx-beta to PML and p53. Consequently, Daxx-beta is unable to repress transcriptional activity of p53. Because deregulation of p53 is closely related to carcinogenesis, alternative splicing of Daxx may also participate in tumour development and progression. Here, we examined the in vivo splicing pattern of Daxx-beta during renal and colon carcinoma progression. Methods. Semi-quantitative real-time PCR were performed with flash frozen resected tissue of 43 renal cell carcinomas (RCCs) of the clear cell type and 40 colorectal carcinomas (CCs) from different tumour stages as well as 49 and 38 samples from the corresponding non-neoplastic kidney and colon epithelia, respectively. Results. Statistical calculation revealed a significant reduction of Daxxbeta isoform in both tumour entities compared to the corresponding non-neoplastic tissue. These alterations were detected already at early tumour stages (pT1+pT2) and did not further change in late stages (pT3+pT4). Conclusions. Our results indicate for the first time that splicing of Daxxbeta is reduced in all tumour stages of renal cell and colorectal carcinoma, and that Daxx-beta could be a potential candidate for a new biomarker on transcript level.

SA-P-048 Modulation of Wnt and Hedgehog signaling pathways is linked to retinoic acid-induced amelioration of chronic fibrosing inflammation P.J. Nelson1, S. Porubsky2, C. von Toerne1, J. Bedke2, S. Safi2, N. Gretz2, H.-J. Gröne2 1 University of Munich, München, 2German Cancer Research Center, DKFZ, Heidelberg Aims. Chronic renal allograft damage (CAD) is manifested by a smoldering inflammatory process that leads to transplant glomerulopathy, diffuse interstitial fibrosis and tubular atrophy with loss of tubular structures. Methods. Gene expression pathway analysis was used to detect new inducers of fibrosis. Using a Fischer 344 (RT1lvl) to Lewis (RT1l) rat renal allograft model, transcriptomic profiling and pathway mapping, we have previously shown that dynamic dysregulation of the Wnt signaling pathways may underlie progressive CAD. Retinoic acid, an important regulator of differentiation during vertebrate embryogenesis, can moderate the damage observed in this experimental model of CAD. Results. We show here that subsets of the Hedgehog (Hh) and canonical Wnt signaling pathways are linked to the pathophysiology of progressive fibrosis, loss of cilia in epithelia and chronic dysfunction. Oral treatment with 13cis retinoic acid (13cRA) was found to selectively ameliorate the dysregulation of the Hh and canonical Wnt pathways associated with CAD, and lead to a general preservation of cilial structures. Conclusions. Interplay between these pathways helps explain the therapeutic effects of retinoic acid treatment in fibrosing inflammation, and suggests future targets for moderating chronic fibrosing organ damage.

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Aims. Inflammatory fibroid polyps (IFPs) are rare benign and mesenchymal tumors. Activating PDGFRA mutations play a central role in the pathogenesis of IFPs and have been shown to be a key player in a subset of gastrointestinal stromal tumors (GISTs). Whereas in GIST PDGFRA mutations are considered to transform one of the subtypes of precursor cells of interstitial cells of Cajal, in IFP an hitherto not further identified mesenchymal progenitor cell type of the submucosa is mutated and develops towards a tumour. Methods. The vast majority of IFPs occur as solitary tumors. Here, we report on molecular and clinicopathologic features of three patients presenting with multiple IPFs and syn- or metachronous GISTs. Results. The first case had multiple IFPs in a duodenal segment and developed a metachronous gastric GIST. Both tumors (IFPs and GIST) revealed exactly the same somatic PDGFRA exon 12 mutation (p.Y555C). The same observation of an identical PDGFRA exon 18 mutation (p.D842V) in both a gastric GIST and multiple IFPs in the duodenum was made in a second case. In the third case, multiple IFPs were found in a jejunal segment, all carrying the same somatic substitution mutation in exon 18 of PDGFRA (p.[R841K(+)D842I]). Conclusions. The coincidence of exactly the same sporadic PDGFRA mutation in multiple IPFs and syn- or metachronous GISTs points at closely interrelated precursor cells driving both neoplastic processes. With respect to sporadic multiciplicity, our data suggest a field effect contributing to the pathogenesis of such IFPs and GISTs.

SA-P-050 Quantitative PCR analysis for detection of cmV infection in patients suffering from ulcerative colitis using FFPE material N. Wethkamp1, C. Bersch1, H.-W. Kohlmann2, V. Meister3, M. Respondek1 Institute of pathology, Dr. Respondek, Vechta, 2Praxis f. Pathologie, ­Respondek, Vechta, 3St. Marien-Hospital, Gastroenterology, Vechta

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Aims. Several lines of evidence indicate that cmV infection can be substantially associated with the onset of ulcerative colitis, especially in immunocompromised patients. In order to estimate the impact of cmV infection and monitoring efficacy of antiviral treatment a real-time PCR Assay was developed to quantify cmV viral load in gastric tissue samples. Methods. DNA-samples derived from FFPE material of patients with ulcerative colitis were quantitatively analysed for cmV infection by TaqMan technology. Via two independent PCR reactions cmV DNA and human GAPDH copy numbers were quantified, using a plasmid coding for both target sequences as an external standard. Finally, viral load in the analysed tissue was expressed as cmV copy numbers per 100,000 cells (as calculated by the obtained GAPDH copy numbers). Results. A total of 89 samples from 22 patients were evaluated for cmV infection while varying section numbers (ranging from 1–12) were analysed per patient. In 29 samples (32%) referring to 50% of the patients cmV DNA could be detected. Here, three patients (27%) showed a viral load of <100 cmV/10E5 cells, two (18%) displayed a viral load ranging between 100–1000 and 1000–10,000  cmV/10E5 cells, respectively, three patients (27%) had a viral load between 10,000 and 100,000 cmV/10E5 cells and

one patient revealed more than 9 million cmV/10E5 cells. Histological tissue assessment of the latter patient also revealed the existence of inclusion bodies which are typical for a severe cmV infection. Interestingly, samples with viral loads higher than 8000 cmV/10E5 cells were also positive for cmV detection using immunohistochemistry. By analysing different tissue sections of individual patients major differences in viral loads were notable indicating that cmV infection in ulcerative colitis could be locally quite heterogeneous. From three patients consecutive samples were analysed after initiation of antiviral therapy. In every case a significant reduction in cmV viral load could be detected indicating a positive response to the therapy. Conclusions. Quantitative real-time PCR is useful for detection of cmV infection in tissue samples of patients suffering from ulcerative colitis. Moreover, the assay seems to be capable for follow up monitoring of therapy response during antiviral treatment.

SA-P-051 Ion semiconductor sequencing: a novel technology for analysis of somatic mutations in formalin-fixed and paraffin-embedded tumor biopsies K. König1, U. Koitzsch1, J. Altmüller2, S. Merkelbach-Bruse1, J. Fassunke1, C. Vollbrecht1, L. Heukamp1, P. Nürnberg2, R. Büttner1, M. Odenthal1 1 University Hospital Cologne, Institute for Pathology, Köln, 2Unversity ­Cologne, Cologne Center of Genomics Aims. Somatic mutations in a panel of genes encoding signal transducers that are involved in cell growth, proliferation and differentiation take center stage in molecular pathology due to their impact on tumor prognosis and therapy response. Recently, an ion semiconductor sequencing system, based on the semiconductor determination of proton release after each nucleotide coupling to the DNA strand, was described (Rothberg et al.; Nature 2011). In the present study, we aimed to evaluate this novel sequencing technology for mutation detection in formalin-fixed paraffin-embedded (FFPE) tumor biopsies. Methods. DNA was purified from FFPE biopsies or from macrodissected tumor materials by the M48 platform (Qiagen). PCR amplification of a wide panel of target genes including EGFR exon 18, 19, 20, 21, Kras codon  12, 13 locus, the Braf codon 600  locus, and Pik3Ca was performed according to the routine processing in molecular pathology. Up to 109 molecules of the target amplicons of each patient were pooled, sheared, and adapter and multiple identifier were ligated following the Xpress protocol (ABI life Technologies). Multiplexed libraries were then used for clonal amplification by means of emulsion PCR and applied to ion semiconductor sequencing using the Ion Torrent platform. Results. Amplicons from EGFR exon 18, 19, 20, 21, Kras codon 12, 13 locus, the Braf codon 600 locus, and PikCa exons were successfully sequenced and somatic mutations were detected after bioinformatic analyses. Therefore, the ion semiconductor sequencing technology combined with the Xpress adapter ligation approach revealed that amplicons of well established PCR assays such as assays that are already established in the routine diagnostics, are suitable templates for parallel sequencing. Conclusions. In conclusion, parallel ion semiconductor sequencing is a promising and efficient technology for multiplexed mutation analyses that may be easily linked to existing PCR approaches of molecular routine diagnostics.

SA-P-052 Inactivation of JNK as pathogenic factor in colorectal carcinogenesis upon inflammation K. Reissig1, T. Guenther1, A. Silver2, R. Hartig3, P. Steinberg4, A. Roessner1, A. Poehlmann1 1 Otto-von-Guericke University Magdeburg, Department of Pathology, Magdeburg, 2Queen Mary University London, Colorectal Cancer Genetics, London, United Kingdom, 3Otto-von-Guericke University Magdeburg, Department of Molecular and Clinical Immunology, Magdeburg, 4Institut of Food Toxicology and Analytical Chemistry, Hannover Aims. We have recently developed a cell culture model using human colon epithelial cells (HCEC) and H2O2 to study tumor-initiating molecular events in inflammation-associated colorectal cancer. As we have supposed epigenetic changes that may account for HCEC transformation, we aim to find epigenetic signaling events. As DNA damage checkpoints are important in cancer cell proliferation, their role in HCEC transformation was analyzed. Methods. We simulated acute inflammation by treating HCEC with a pathophysiologic concentration of H2O2 as ROS (reactive oxygen species). Furthermore, we developed transformed HCEC by repeated treatment cycles to mimic chronic inflammation. In order to analyze DNA damage checkpoints, we performed cell cycle analysis using FACS. The function of JNK was investigated using the JNK inhibitor SP600125. The cells were further analyzed by immunoblotting. Results. To prove whether single H2O2 treatment leads to activation of DNA damage checkpoints, cell cycle analysis was performed. Acute ROS activated the G1/S and G2/M DNA damage checkpoints. At the same time, there was prominent activation of JNK signaling. This coincided with periods when cell cycle arrest was observed. As the JNK inhibitor was able to rescue S and G2/M arrest, the observed cell cycle arrest is JNK-dependent. Immunoblotting analysis after JNK inhibition identified p21, an inhibitor of cell cycle progression, as cellular JNK target. Importantly, in line with uncontrolled proliferation of transformed HCEC, we observed altered JNK activation. This down-regulation of activated JNK caused down-regulation of p21 in transformed HCEC. Conclusions. JNK inactivation plays an important role in HCEC transformation and seems to be a pathogenic factor. Subsequent down-stream p21 down-regulation occurs early, seems to be as efficient as p53 mutation, and fits very well with the suggestive role of p21 as a potential tumor suppressor in the colon. We speculate that both inactivation of JNK and p21 down-regulation might cause the cell to turn on a one way street to uncontrolled proliferation as one defining feature of the initiation of the inflammation-carcinoma pathway in the colon.

SA-P-053 Validation of primary antibodies for immunohistochemistry H.J. Grote1 1 Merck KGaA, Histopathology – Biomarker Technologies, Darmstadt Aims. Tissue biomarkers are gaining increasing importance in establishing targeted therapies. In oncology tissue biomarkers are used for target validation and indication seeking, for patient stratification and for pharmacodynamic assays. Immunohistochemistry on formalin-fixed paraffin-embedded (FFPE) tissue (IHC-P) is a key technology in biomarker discovery and development. However, there is increasing evidence that IHC-P is frequently impacted by limited specificity of primary antibodies. This report presents strategies, technologies and examples for improved primary antibody validation. Methods. Antibody validation is a multi-step process. It starts with gathering information on target expression and identification of positive and negative expression controls. Standard laboratory procedures like Western blot, FACS or qRT-PCR may be useful to corroborate or complement published expression data in cancer cell lines. A Western blot may also provide first information about selectivity of a candidate priDer Pathologe Suppl 1 · 2012 

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Abstracts mary antibody which is intended to be used for IHC-P. Subsequently, antibody specificity can be tested on multiple cell lines using a FFPE cell line microarray (CMA). Correlating cmA IHC-P profiles with mRNA gene expression profiling data may be helpful. If the target is ubiquitously expressed, evaluation of antibody specificity may require more sophisticated technologies like siRNA knock down of target in cancer cell lines. The siRNA knock down or a transfection of cell lines with wild type or mutated target generates gene expression modified cell lines which may be assembled to a gene expression modified cmA. Thereby IHC-P can be linked to functional assays. Results. In-depth antibody validation discloses that primary antibodies may be not selective in IHC-P, although the antibody datasheet or published data suggest the IHC-P application. This observation applies not only to polyclonal antibodies but also to monoclonals. Using antibodies that demonstrate a specific staining, IHC-P may correlate remarkably with alternative protein detection methods like Luminex’s multiplex bead-based immunoassay that is limited to fresh samples. Conclusions. The data underline the need for in-house validation of primary antibodies prior to their use as analytic tool in IHC-P. It is likely that the unsatisfactory validation status of published antibodies represents one of the causes for contradictory IHC-P data in the literature. Concerted activities should be considered to improve the current situation.

Poster: Molekularpathologie II SA-P-054 The homeobox gene HOPX is methylated in human lung cancer, and the methylation status of HOPX is a diagnostic marker for patients with lung adenocarcinoma Y . Chen1, T . Cui1, L . Yang1, N . Posorski2, I . Petersen1 University Hospital Jena, Institute of Pathology, Jena, 2University Hospital Jena, Institute of Human genetics, Jena

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Aims. The homeobox gene HOPX has been considered as a tumor suppressor in various cancers. Our previous studies showed that HOPX is downregulated in lung cancer cell lines and primary lung tumors, and forced expression of HOPX by stable transfection could inhibit tumor cell growth. However, the regulation of HOPX in lung cancer has not yet been well elucidated. Therefore the aim of the study is to investigate the epigenetic regulation, analyse the potential target genes of HOPX, and evaluate the clinical relevance of HOPX in lung cancer. Methods. HOPX protein expression was analysed by western blotting. Demethylation test by 5-aza-2-deoxycytidine (DAC), bisulfite sequencing (BS), and methylation-specific-PCR (MSP) were carried out to analyse the methylation status of HOPX in lung cancer cells and primary lung tumor samples. cDNA microarray analysis was performed to identify target genes of HOPX. Results. In line with decreased expression of HOPX mRNA in lung cancer cell lines, western blotting showed that HOPX protein was downregulated compared to normal HBEC cells. Ten out of 12 lung cancer cell lines restored HOPX expression after treatment with DAC , and the methylation status of HOPX in the promoter region and exon 1 was confirmed by bisulfite sequencing. MSP showed that HOPX was methylated in 64 out of 88 (72.7%) primary lung tumor samples, and methylation of HOPX was significantly associated with lung adenocarcinoma (p<0.001). Additionally, we found that more than 20 genes were at least 6-fold upregulated in HOPX transfectants compared to mock transfectants by cDNA microarray analysis. The validation of cDNA microarray data and the investigation of HOPX target genes are going on. Conclusions. Downregulation of HOPX is caused by DNA methylation, and methylation status of HOPX may be a diagnostic marker for patients with adenocarcinoma.

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SA-P-055 Prospective validation of routine diagnostic SHOX2-methylation testing in lung cancer F . Klauschen1, M . Gerhold1, A . Lehmann1, N . Rudath1, C . Denkert1, M . Dietel1, C . Schewe1 1 Charite, Pathology, Berlin Aims. Epigenetic modifications such as changes in DNA methylation patterns are known to be involved in the pathogenesis of different types of cancers. The hypermethylation of the homeobox transcriptional regulator SHOX2, for instance, is known to be one of the most common genetic alterations in lung cancer with more than 96% of lung tumors affected. Therefore, SHOX2 DNA methylation may be a useful biomarker for lung cancer detection. Here, we evaluate the utility of SHOX2 methylation testing in bronchoalveolar lavage and aspirates in a prospective setting in a cohort of >200 patients. Methods. We performed quantitative real-time PCR to analyse the DNA methylation of the SHOX2 gene compared with beta-actin using the EpiProLung Test (Epigenomics) in >200 patient undergoing routine diagnostics for lung cancer at the Charité in 2011. A dichotomized methylation status was determined by using a ddCT cut-off value of 9.5. Results. From the 239 cases, 173 were SHOX2-, 44 SHOX2+ and 17 undetermined. The cytological analysis yielded 200 cases unsuspicious of cancer (or with only mild atypia) and 34 cases moderately to highly suspicious of cancer. This comparison between SHOX2 and cytology displays a sensitivity of 73% and a specificity of 78.4% (AUC 75.1%, SHOX2 testing benchmarked with cytology). Conclusions. The results demonstrate the utility of SHOX2 methylation testing for routine diagnostics of lung cancer in direct comparison with cytology in lavage samples. Clinical, radiological and histological data will be included upon availability to determine the absolute performance of SHOX2 testing.

SA-P-056 Detection of a heterogeneous BRAFV600E mutation status in a patient with metastasized malignant melanoma R . Marienfeld1, J . Heinrich2, S . Jung2, P . Möller1, K . Scharffetter-Kochanek3, M . Huber3, T . Menzel4, L .-A . Schneider3, T . Barth2 1 University of Ulm, Institute of Pathology, Ulm, 2University of Ulm, Institute of Pathology, 3University of Ulm Medical Center, Department of Dermatology and Allergology, 4Dermatophathology Friedrichshafen Aims. Targeted tumor therapy is expected to lead to a breakthrough in the treatment of advanced stage melanoma. By end of this year Vermurafenib® (Roche PLX4032) is expected to be licensed as the first BRAF pathway inhibiting substance for metastasised melanoma carrying a V600E BRAF mutation. Up to 50% of all melanoma patients are estimated to carry a mutation in the codon 600 of the BRAF gene. It is therefore in the focus of scientists and clinicians since targeted therapy is supposedly less harmful and more effective than conventional chemotherapy. Mutational analysis of the codon  600 of the BRAF gene is strictly required to determine the eligibility of the patient for a targeted therapy with Vermurafenib®. The goal our study is to determine the reliability of a BRAF mutational analysis on the basis of a single tissue specimen. Here, we present the BRAF mutational analysis of four different specimens of a patient with metastasized melanoma. Methods. Microdissection of FFPE tumor tissue. DNA isolation and PCR amplification of the BRAF gene segment. Pyrosequencing of codon 600. Results. The patient is a 38-year-old woman with a pTxpN3pM1 metastasized acrolentigeneous melanoma. We analysed the BRAF mutation status in the primary tumor as well as in three different biopsies from a lung axillary lymph node and brain obtained with a time difference of six years. BRAF mutation has been shown to be an early event in melanoma and thus should be present in the first biopsy. However, whereas the analysis of the first biopsy from lymph node revealed a wild type

situation, the V600E mutation in the BRAF locus was observed in the primary tumor and the later biopsies from the lung and brain implying that different metastases may resemble different tumor subclones which need to be analysed separately. Conclusions. In conclusion, our observations point to a heterogeneous BRAF mutation status i) during the time course of the disease, and ii) in a very small subset of tumor cells in a given sample. These results highlight the need for a critical therapy evaluation in which the this mutational analysis of the BRAF gene target needs to be embedded in the overall clinical setting rather then simply rely on a yes-or-no result of the mutational test of a single biopsy. Thus, the whole setting has to be taken into account by the pathologist, molecular biologist and clinician.

SA-P-057 Integrin expression in primary tumor and their brain metastasis A. Vogetseder1, S. Thies1, M. Weller2, P. Schraml1, H. Moch1 1 UnversitätsSpital Zürich, Clinical Pathology, Zürich, Switzerland, 2 UnversitätsSpital Zürich, Neurology, Zürich, Switzerland Aims. To determine the expression of avb3, avb5 and avb8 integrins in breast, lung and kidney cancer as well as in malignant melanoma and their brain metastases. Methods. Novel subtype specific rabbit monoclonal antibodies for avb3, avb5 and avb8 integrins. Immunohistochemistry on tissue microarrays. Results. Integrin avb5 is predominantly expressed in all primary tumours and their metastases. Negative or weak integrin avb3 and avb8 expression in primary lung, renal and breast cancers. Significant increase of avb3 and avb8 expression in brain metastases. Primary malignant melanoma and their brain metastases show varying amounts of avb3 and avb8 expression. Conclusions. Rabbit monoclonal antibodies allow the analysis of specific integrin isoforms. The avb3 and avb5 inhibitor cilengitide could also, like in glioblastoma treatment, be effective in patients with brain metastases of melanomas as well as lung, breast and kidney carcinomas.

SA-P-058 Activating PDGFRA mutations in inflammatory fibroid polyps occur in exons 12, 14 and 18 and are associated with tumour localization S. Huss1, E. Wardelmann1, D. Goltz2, W. Hartmann1, E. Binot1, H. Löser1, S. Merkelbach-Bruse1, R. Buettner1, H.-U. Schildhaus1 1 University Hospital Cologne, Institute of Pathology, Köln, 2University Hospital Bonn, Institute of Pathology, Köln Aims. Inflammatory fibroid polyps (IFP) are mesenchymal tumours of the gastrointestinal tract. This study was performed to broaden the base of evidence of the pathogenic role of PDGFR mutations in IFP. Methods. A total of 38 IFP, extracted from our consultation files, were included in this study. Clinicopathological features were collected and mutational analysis was carried out. Results. Activating mutations in three different exons of PDGFRA were found in 25 IFP. For the first time we report two cases with PDGFRAexon  14 mutations (p.N659K; p.[N659K(+)T665A]). The results of our study and cases reported earlier in the literature clearly indicate that there is a localization specific pattern: exon 12 mutations predominate in the small intestine, while exon 18 mutations frequently occur in the stomach (p<0.001). Codons 567-571 of PDGFRA represent an IFP specific mutational hot spot and are most frequently affected by deletions. Furthermore, in our series IFP of the stomach share common features. Incontrast to intestinal IFP, gastric tumours occur at higher age, show heavy inflammation and tend to be smaller. IFP located in the small intestine are frequently associated with intussusception.

Conclusions. We conclude that there is a “small bowel” and a “gastric” phenotype of IFPs which are associated with exon 12 and exon 18 PDGFRA mutations, respectively.

SA-P-059 Papillary renal cell carcinoma of three sisters in a Chinese family carry trisomy 7 and 17 and MET mutation J. Xu1, C. He2, M. Jin1, Z. Yang2, D. Hong2, A. Huang1, R. Zhou2, E. Haralambieva3, A. Rosenwald3, Z. Mao2 1 Department of Pathology, Sir Run Run Shaw Hospital of Collage of Medicine, Zhejiang University, Hangzhou, China, 2Department of Pathology and Pathophysiology, Collage of Medicine, Zhejiang University, Hangzhou, China, 3Institute of Pathology, Wuerzburg University, Wuerzburg Aims. To observe the morphology characteristics and the change of chromosome7/ 17 of PRCC of three sisters in a Chinese family, compare the mutation of MET either in tissues or in peripheral blood samples of three patients, and in the meantime, test the blood samples of their next generation to find clues for early prediction of PRCC. Methods. The detection of chromosomal 7 and 17 has been performed by fluorescence in situ hybridization (FISH). Polymerase chain reaction (PCR) and direct sequencing of exon 14, 16–19 has been used to analyze the mutation status of MET, and the expression of CK7, CKpan, Vimentin, CD10, p53, p16, ki67 has been evaluated by immunohistochemical staining. Results. Trisomy 7 and 17 both are found in tumor tissue of three sisters. The 3522 point mutation (G-A) of MET exon16 with amino acid from valine to isoleucine (V1110I) exists in either tumor cells or normal kidney tissue and blood samples of three PRCC patients, and tumor tissue is positive with CK7, CKpan and p16, negative with p53 and low proliferation (Ki67) . Four of seven people of their next generation carry the same germline mutation of MET. Conclusions. Trisomy 7 and 17 and a missense mutation in exon  16 of MET in a Chinese three sisters PRCC family indicates the molecular oncogenesis of PRCC and may lead to early predictions of PRCC.

SA-P-060 High resolution genomic profiling reveals unexpected molecular heterogeneity in angiosarcomas K. Mößinger1, E. Hartmann2, E. Leich2, K. Simon-Keller1, A.-L. Bohlender1, P. Hohenberger3, A. Rosenwald2, A. Marx1, P. Ströbel1 1 University Mannheim, Institute of Pathology, Mannheim, 2University of Würzburg, Institute of Pathology, Würzburg, 3University Mannheim, Surgical Oncology and Thoracal Surgery, Mannheim Aims. Angiosarcomas (AS) are rare vascular tumors that arise either de novo as primary tumors or secondary to irradiation or chronic lymph edema. The cytogenetics of angiosarcomas are poorly characterized. Methods. To investigate the genes recurrently affected by chromosomal disorders in AS, we analysed Affymetrix array 6.0 SNP and CGH microarray data from 13 AS samples (3 primary and 10 radiation-associated). Results. The genome-wide single-nucleotide polymorphism (SNP) array analysis reveals a majority of gains against very few losses of chromosomal materials in all AS samples. We classified 4 subgroups of AS based on the quantity of their copy number alterations (CNA) and their origin. This study shows the known myc high level amplification of radiation associated AS as a distinct subgroup. In addition, radiation associated AS without myc amplification segregate in 2 subgroups with more than 20 CNAs and less than 10 CNAs, respectively. Primary AS showed very low CNAs with recurrent gains on chromosome 20. Conclusions. Our results revealed unexpected molecular heterogeneity of AS. In spite of their identical morphology, secondary AS are genetically different from primary AS. Among radiation induced AS, myc amDer Pathologe Suppl 1 · 2012 

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Abstracts plification identifies a separate subgroup. The identification of distinct molecular subgroups of AS forms a basis for the identification of novel pathways that may help to better understand the biology of AS and may eventually lead to the development of more specific treatments.

SA-P-061 Molecular analysis of COL1A1/PDGFB translocation and PDGFB amplification in patients with Dermatofibrosarcoma protuberans K. Walluks1, S. Hauk2, C. Wölfel1, Y. Chen1, T. Cui1, L. Yang1 1 Universitätsklinikum Jena, Institute of Pathology, Jena, 2Zytovision, Bremen Aims. Dermatofibrosarcoma protuberans (DFSP) is a dermal and subcutaneous tumor of intermediate malignancy. The most remarkable cytogenetic feature of DFSP is the chromosomal translocation t(17;22) (q22;q13), causing a fusion of the platelet-derived growth factor beta chain (PDGFB) gene on 22q13 and the collagen type 1 alpha 1 (COL1A1) on 17q22. The aim of the study was to analyze the molecular characteristics of DFSP Methods. We performed Fluorescence in situ hybridization (FISH) and multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to detect chromosomal translocation and fusion gene transcripts in 16 formalin-fixed, paraffin-embedded DFSP samples. Additionally, we analysed the gene copy number of PDGFB in the 16 samples by real-time PCR. Results. Eleven out of 16 samples (68.8%) showed fusion transcripts by multiplex RT-PCR analysis. Various exons of the COL1A1 gene were fused with PDGFB gene, among them, exon 25 was found to be more frequently involved. It turned out that, PDGFB copy numbers in the DFSP samples was slightly higher than in normal skin tissues (p=0.007). Conclusions. Our results suggest that the COL1A1/PDGFB fusion transcripts and amplification of PDGFB may be diagnostic markers for patients with DFSP, and PDGFB could be a potential target for treatment of patients with DFSP.

SA-P-062 PHD3 silencing leads to increased tumor growth accompanied by enlarged tumor vessels A. Kettelhake1, M. Rezaei2, A. Kuzmanov1, D. Poitz3, B. Wielockx1, G. Breier1 1 Technical University of Dresden, Department of Pathology, Dresden, 2Technical University of Dresden, Department of Pathology, Dresden, 3Technical University of Dresden, Department of Internal Medicine and Cardiology, Dresden Aims. The fast growth of solid tumors causes the development of hypoxic areas that are very often marked by the upregulation of the hypoxia-inducible factor (HIF). HIF enables the tumor cells to survive under hypoxic conditions for example by increasing the production of angiogenic factors. HIF is regulated by prolyl hydroxylase domains proteins (PHD1, 2, 3, 4). However, the function of PHD3 during tumor progression and angiogenesis has not been investigated in detail so far. It is our aim to address these open questions. Methods. We stably silenced PHD3 in a murine osteosarcoma cell line (LM8) using a lentiviral transduction system and analyzed the cell clones by quantitative real-time PCR and Western blotting. To investigate the behavior of these clones in vivo we injected them subcutaneously on the back of C3H mice and measured the tumor size every 2 days for a period of 14 days. Perfusion of tumor tissue, immunohistochemical staining as well as immunofluorescent staining was performed to analyze tumors. Results. Surprisingly, downregulation of PHD3 does neither affect the protein level of HIF nor the expression levels of known HIF-target genes. The other PHD isoforms (PHD1, PHD2, PHD4) as well as factor inhibiting HIF-1 (FIH) show equal mRNA levels in sh-PHD3 clones compared to controls. However, transforming growth factor α (TGF-α) and platelet-derived growth factor c (PDGF-C) are markedly upregulated, whereas the angiopoietin 2 (Ang2) mRNA level is decreased in the sh-PHD3

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clones. In vivo experiments show the development of significantly larger tumors compared to control tumors. Interestingly, the density of tumor vessels is decreased but the vessels are enlarged as evidenced by PECAM staining. α-smooth muscle actin (αSMA) immunofluorescence staining reveals that more vessels in the PHD3 silenced tumors are covered with αSMA-positive cells. We found no difference in vessel perfusion or leakiness between shPHD3-tumors versus control tumors. Conclusions. Our data suggest that PHD3 plays an important role as tumor suppressor because knocking it down leads to accelerated tumor growth. Unexpectedly, this function seems to be HIF-independent. The structure of the shPHD3-tumor vasculature is completely altered indicating that PHD3 is involved in tumor angiogenesis as well. At the moment we are determining which factors are responsible for the observed phenotype. For this, we are using clones simultaneously knocking down PHD3 and one of the differentially expressed growth factors.

SA-P-063 Ubiquitin, SH2 and UBA domains of p62 regulate interaction of p62 with K8/18 and aggregation properties V. Mahajan1, C. Stumptner1, A. Thueringer1, T. Klingstedt2, P. Nilsson2, K. Metchler3, K. Kashofer1, H. Denk1, K. Zatloukal1, J. Haybaeck1 1 Medical University of Graz, Institute of Pathology, Graz, Austria, 2Linkoping University, Sweden, 3Medical University of Vienna, Institute of Molecular Pathology, Vienna, Austria Aims. Steatohepatitis and other liver diseases are characterized by presence of cytoplasmic protein aggregates termed Mallory-Denk bodies (MDBs) and Intracellular Hyaline Bodies (IHBs. Sequestosome 1/p62, ubiquitin, Keratin 8 (K8) and Keratin 18 (K18) are the major constituents of MDBs. We investigated whether interaction of p62 with K8/18 depends on a) ubiquitination, b) phosphorylation, c) conformational alterations, or d) structural domains of p62. Methods. The SH2/PB1, ZIP/PB1 and UBA domains of p62 were deleted. Specific phosphorylation sites (S24 and S152) of p62 were mutated. The phosphorylation and deletion constructs were co-transfected along with K8 and K18 in presence or absence of ubiquitin. We used Luminescent conjugated oligothiophenes (LCOs) to investigate conformational changes of p62 deletion and phosphorylation mutants. Results. Deletion of the SH2 domain or partially of the PB1 domain leads to loss of the filamentous ultrastructure of p62 but resembles an IHBlike aggregation pattern. Deletion of the ZIP domain or the remaining PB1 domain lead to irregularly shaped intracytoplasmic aggregates whereas UBA deleted p62 displayed a diffuse distribution pattern but only a partial loss of filaments ultrastructure and did not interact with K8/18 anymore. CHO-K1 cells transfected with various combinations of SQSTM1/p62, ubi and Krt8/Krt18 demonstrated that SH2 domain deleted p62 co-localizes with K8 in the absence of ubiquitin. The phosphorylation sites S24 and S152 do not seem to regulate the interaction of p62 with ubiquitinated keratins but mutation at S24 created a diffuse distribution pattern of p62. LCO analysis demonstrated the presence of cross beta-sheet conformation in SH2 domain deleted p62 and K8. Additional oxidative stress may interfere with MDB components but not with their interaction. Conclusions. These findings explain the observation that SH2 and UBA domains govern the aggregation property of p62 and influence the interaction patterns with K8/K18. Thus it is clear that filamentous assemblies of type I MDBs are predominantly due to p62 while type II MDBs may need p62 and ubiquitin. Alternatively the amorphous granular nature of type III MDBs results from insoluble K8/K18 aggregates. K8 and SH2 deleted p62 can undergo conformational changes from predominantly Alpha-helical to cross β-sheet structures which allow their interaction even in the absence of additional ubiquitin. Therefore the SH2 domain might regulate the interaction between K8 and p62wt.

SA-P-064 Evolution of molecular pathology at the Institute of Basel M.P. Bihl1, S. Hoeller1, A. Foerster1, R. Chaffard1, S. Schneider1, A. Rufle1, L. Terracciano1, L. Tornillo1 1 University of Basel, Institute of Pathology, Basel, Switzerland Aims. Molecular genetics in pathology is a very young field. It began with analysis of haematological diseases or inherited disorders, but in the last decade, also many of solid tumors have been found to be related to specific somatic mutations. These mutations can give a hint to drug sensitivity in a given tumor and therefore are urgently required in modern oncology. Here we show how this field has developed in the recent years using the example of the activity of the Institute of Pathology during the last 6 years. Methods. The number of PCR based analysis increased from 206 (in 2006) to over 800 analyses (in 2011). Results. In the beginning only CKIT/PDGFRA mutation and EGFR mutation analysis and clonality analysis were performed. However, the overall percentage of analysed sites remained stable with 50% from the lung, 10–20% from the blood or lymph node and 10–25% from colorectal or gastrointestinal sites. Recently, new mutations are also routinely tested like (IDH 1 and 2, BRAF and CTNNB1) and therefore new organs were included like brain (glioma), skin (melanoma) and liver (adenomas). From three available assays in 2006 the spectrum of our analysis expanded to 20 different assays today. The number of performed FISH analysis stayed stable, while the number of HER2 hybridisations dropped, but new assays were introduced into the routine panel like 1p19q and ALK translocation analysis for glioma and adenocarcinoma of the lung, respectively. Therefore, the dynamic changes in molecular pathology are due to new tumor classification systems, the development of new tumor specific drugs and the increased knowledge of drug sensitivity in cancer patients harboring specific somatic mutations. Conclusions. In the upcoming years molecular based prognostic markers and mutation specific therapies will even more expand the spectrum of molecular testing in pathology. Its role is crucial to improve and optimize the diagnosis and therapy of tumors and is essential in modern oncology. Adaptation of the increasing knowledge of pathogenetic pathways will be a major issue for molecular laboratories in the future.

SA-P-065 The amyloid precursor protein (APP) is a novel biomarker for transformed human pluripotent stem cells V. Venkataramani1, K. Thiele2, C.-L. Behnes2, G.G. Wulf1, P. Thelen3, L. Opitz4, G. Salinas-Riester4, O. Wirths5, T.A. Bayer5, H.-J. Radzun2, S. Schweyer2 1 University Medicine Göttingen, Department of Hematology and Oncology, Göttingen, 2University Medicine Göttingen, Department of Pathology, Göttingen, 3University Medicine Göttingen, Department of Urology, Göttingen, 4 University Medicine Göttingen, DNA Microarray Facility, Göttingen, 5University Medicine Göttingen, Division of Molecular Psychiatry, Göttingen Aims. There is no doubt that the amyloid precursor protein (APP) and its proteolytically derived Aβ species significantly contribute to the pathogenesis of Alzheimer disease. However, the normal physiological role of this ubiquitously expressed protein has remained largely unknown. In the current study, we characterized APP expression in a panel of human testicular germ cell tumors (TGCT) of different histological origin. Furthermore, we analysed whether histone deacetylase (HDAC) inhibitors effectively induce cell differentiation and impact stem cell signature and APP protein levels in embryonal carcinoma (EC) cell lines. These analyses were also performed in a physiologically relevant in vivo setting using an established xenograft mouse model. Methods. Paraffin-embedded tissue blocks from orchiectomy specimens were used for tissue microarray construction consisting of 173 cases of pure and mixed TGCTs as well as eight randomly selected normal testicular tissues. Following TGCT cell lines were used: NCCIT, NTera-2 (EC

cell lines) and TCam-2 (seminoma cell line). Cellular differentiation was analysed by cell proliferation and cytotoxicity assays, cell morphology via fluorescence microscopy and expression analyses of stem cell genes and lineage-specific differentiation markers were determined using microarray analysis, qRT-PCR and Western blot analysis. APP expression was selectively down-regulated using target-specific siRNA duplexes. Xenografts inoculated with NTera-2 were orally treated with the HDAC inhibitor VPA and tumor growth as well as APP protein levels were compared to vehicle treated animals. Results. APP is exclusively expressed in pluripotent germ cell cancer subtypes (EC and seminoma). Differentiated TGCTs (e.g. teratoma) only presented low or lack of APP expression. APP knock-down induced the expression of lineage-specific differentiation markers. HDAC inhibitor treatment induced cell differentiation, accompanied by down-regulated APP protein levels and stem cell genes. Moreover, GRP78 could be identified as a key factor that specifically triggers proteasomal degradation of APP. Oral administration of VPA significantly suppressed tumor growth and depleted APP protein levels in vivo. Conclusions. Our results indicate that APP behaves as a reliable biomarker for transformed human pluripotent stem cells and also shed light on the significance of APP as a novel molecular target and furthermore broaden the therapeutic potential of HDAC inhibitors in the clinical treatment of TGCT.

SA-P-066 Thymoquinone lowers toxicity and increases efficacy of 5-fluorouracil C. El-Baba1, S. Morgenthal2, M. Ocker3, H. Gali-Muhtasib4, R. Schneider-Stock1 University of Erlangen-Nuremberg, Institute of Pathology, Erlangen, 2Christian-Albrechts-University, Institute of Pathology, Köln, 3Phillips-University of Marburg, Institute of Surgical Research, Marburg, 4American University of Beirut, Department of Biology, Beirut, Lebanon

1

Aims. Colorectal cancer is a non-negligible cause of mortality worldwide. 5-fluorouracyl (5-FU) has proved to be one of the most effective chemotherapeutics for colorectal cancer but an inactivated p53 status leads to a resistance to 5-FU. We have shown that thymoquinone (TQ), a bioactive compound extracted from Nigella sativa (black seed) exerts promising anti-apoptotic effects independent of the p53 status of tumor cells. Our aim is to investigate if TQ is able to sensitize colorectal cancer cells to 5-FU to minimize its cytotoxic side-effects in the clinical setting. Methods. Human colon cancer cells HT29 (mutant p53), HCT116 (p53+/+) and normal intestinal cells HCEC were treated with TQ and/or 5-FU. Cell viability was measured via crystal violet, mitochondrial activity and colony formation assays. Cell cycle distribution and apoptosis were assessed by flow cytometry via propidium iodide (PI), Annexin-V staining, and Western blotting. A mouse xenograft study was conducted for a better assessment of potential in vivo effects. Results. HCT116wt cells were more sensitive to TQ than HT29 cells. HCEC cells were highly resistant to TQ treatment, which indicates that TQ has an effect mainly on cancerous cells. In HT29 cells, the combination TQ/5-FU was equally efficient as the treatment with ten times higher concentration of 5-FU alone. Annexin-V, propidium iodide-cell cycle analysis, as well as Caspase 3 and Caspase 9 cleavage along with the increase of Bax to Bcl2 ratio, revealed a higher apoptosis induction when the cells are treated with both drugs in comparison to either TQ or 5-FU. In vivo study showed that the relative tumor size of mice co-treated with TQ and 5-FU was significantly reduced in comparison with the tumors of control mice or mice treated with either drug alone. Conclusions. TQ when combined with the antineoplastic agent 5-FU was increasing apoptosis in colon cancer cells. The combination therapy could overcome the resistance to 5-FU in the p53 mutant tumor cells without showing dramatic cytotoxicity on normal colon cells. Combined with further clinical studies this approach might be promising for the improvement of colorectal cancer treatment. Der Pathologe Suppl 1 · 2012 

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Abstracts SA-P-067 Overexpression of anti-apoptotic CIAP2 is typical of thymic squamous cell carcinoma but not thymomas – hints to functional relevance D . Belharazem1, B . Huang2, L . LI3, P . Stroebel1, A . Marx1 University Medical Center Mannheim; University of Heidelberg, 2Institut of Pathology; University of Würzburg, 3Medical Research Center, Medical Faculty Mannheim 1

Aims. Thymomas and thymic carcinomas (TCs) are epithelial tumors of the thymus. Their molecular oncogenesis is poorly understood. Recent extensive sequencing of a broad spectrum of receptor and non-receptor kinase genes in thymomas and TCs (Girard N et al, Clin Cancer Res 15:6790, 2009) failed to identify recurrent mutations except for known activating KIT mutations in <10% of thymic squamous cell carcinomas (TSCCs), Therefore, we asked whether other tumor-promoting mechanism, e.g. anti-apototic properties, might be operative in TSCC and comparably aggressive thymomas. Methods. Based on chip-derived gene expression data, we investigated two independent sets of 16 TSCC and thymomas (23 B3 and 13 A thymomas) for expression of a spectrum of anti-apoptotic genes, using real time RT-PCR and western blotting for cryopreserved material and immunohistochemistry (IHC) for formalin-fixed, paraffin-embedded tissue. The functional relevance of the expressed anti-apoptotic cIAP2 gene was proven in a thymic carcinoma cell line by an siRNA approach, followed by FACS analysis for apoptosis quantification. Immortalized HaCat kerationycte served as control. Results. Both the original set (set I, studied by cDNA chip) and the validation set (set II) showed overexpression of cIAP2 mRNA in TSCCs carcinomas but not in type A- (p<10-4 and p<10-4) and B3- (p=0.0062 and p<10-4) thymomas. By contrast, mRNA levels of other anti-apoptotic proteins (AIPs) tested, i.e. cIAP1, XIAP and cFLIP were not differentially expressed in any thymic tumor type and cell line. Corresponding results were obtained on the protein level by western blot and IHC. Transient down-regulation of cIAP2 by siRNA in a thymic carcinoma cell line strongly sensitized tumor cells to various inducers of apoptosis. Conclusions. The anti-apoptotic protein cIAP2 is differentially expressed on the transcriptional and proteomic level in TSCCs compared to both aggressive and indolent thymomas (i.e. B3 and A types, respectively), complementing previous observations that genes are expressed in TCs (e.g. CD5 and CD117) but not thymomas. The functional results suggest that overexpressed cIAP2 might play a role in the pathogenesis of TSCCs, and deserves future in vivo testing as a new therapeutic target. The results challenge the current clinical strategy to treat aggressive B3 thymomas and thymic carcinomas by virtually identical adjuvant therapeutic protocols.

SA-P-068 cFLIP supports the maintenance of resistance to apoptosis in thymic carcinoma cell line 1889c D . Belharazem1, P . Stroebel1, A . Marx1 1 University Medical Center Mannheim; University of Heidelberg Aims. Cellular FLICE-like inhibitory protein (cFLIP) has been identified as a protease-dead, procaspase-8-like regulator of death ligand-induced apoptosis by binding to the molecule DISC (death inducing signaling complex) and prevents thereby the transmission of the apoptotic signal. This makes cFlip Molecule as a potent inhibitor of apoptosis and an important regulator of the cell death signaling. It is already shown, that cFLIP overexpression correlates with the progression of different tumors. Thymomas and thymic carcinomas are thymic epithelial tumors, in which the regulation of apoptosis is still unknown. The aim of this study was to investigate the role of cFLIP in regulating the viability of thymus carcinoma cell line 1889c and to investigate the mechanisms by

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which it exerts its effects by using a cFLIP RNA Interference. The immortalized keratinocyte cell line HaCat was used as a control. Methods. A stabile cFLIP RNA interference (RNAi) was established by cloning a designed siRNA in pIRES/PURO shRNA expression vector and a transfection in the 1889c and HaCat cell lines was performed. The transfected cells 1889c-shcFlip and HaCat-shcFlip were selected with puromycin. CFLIP suppression and its Effect on the others pro and antiapoptotic molecules were analyzed using q-PCR and Western blot. Apoptosis was quantified with Annexin/PI using the flow cytometry. Results. The 1889c-shcFlip, but not the shcFlip HaCat, showed sensitivity to TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), accompanied by an overexpression of both the pro-apoptotic protein Noxa (p<10-4) and the anti-apoptotic proteins BIRC2, BIRC3 and XIAP compared to the non-transfected cells (p<10-4). Conclusions. cFLIP shRNA seems to induce apoptosis in the thymus carcinoma cell line. Simultaneously antiapoptotic proteins (AIPs) BIRC2, BIRC3 and XIAP were up regulated in order to protect the cell from apoptosis. Could these AIPs provide the way to escape the cell death? AIPs Selective inhibitions could represent a promising therapeutic approach for malignant thymic carcinomas.

Poster: Paidopathologie SA-P-069 Hepatobiliary rhabdomyosarcoma in a 2-year-old: a case report K . Wieczorek1, G . Fitze2, R . Knöfler3, G . Hahn4, G . Baretton1 1 University Hospital “Carl Gustav Carus”, TU Dresden, Department of Pathology, Dresden, 2University Hospital “Carl Gustav Carus”, TU Dresden, Department of Pediatric Surgery, Dresden, 3University Hospital “Carl Gustav Carus”, TU Dresden, Department of Pediatric Oncology, Dresden, 4University Hospital “Carl Gustav Carus”, TU Dresden, Department of Radiology, Dresden Aims. Although representing the most common sarcoma in the pediatric patient, rhabdomyosarcomas of the liver account for only 0.8% of all rhabdomyosarcomas, and 1% of all liver tumors. Unless they present in the characteristic setting of a botryoid mass in the biliary tree, they are difficult to diagnose, as they share many histomorphologic similarities with undifferentiated embryonal sarcomas of the liver. This case report summarizes clinical data and histomorphologic and immunohistochemical features of a hepatobiliary rhabdomyosarcoma in a 2-year-old boy. Methods. A 2-year-old boy presented to an external hospital with severe jaundice and hepatomegaly. Initially, an infectious process was suspected, and the boy was referred to the pediatrics department of the Dresden university hospital. MR imaging revealed a very large liver tumor and multiple metastases in the lung and bone. The gallbladder and the biliary tree were initially not visible due to the size and partially cystic appearance of the tumor. A biopsy of the liver was taken. Results. Liver biopsy showed a malignant mesenchymal neoplasia consisting of primitive round to spindled cells with multiple mitoses. A zonal architecture was noticed around small bile ducts. Very few (<1%) tumor cells showed myogenic differentiation with eosinophil cytoplasm. Immunohistochemistry confirmed the suspected diagnosis of a hepatobiliary rhabdomyosarcoma with expression of desmin and myf4. The tumor-free liver parenchyma showed severe cholestasic cirrhosis. After neoadjuvant chemotherapy, left-sided hemihepatectomy was performed, including resection of the left and common hepatic duct and the gallbladder. Conclusions. Distinction between hepatobiliary rhabdomyosarcoma and undifferentiated embryonal sarcoma is difficult based on histomorphology alone. As both prognosis and therapy differ between the two entities, a thorough histomorphologic and immunohistochemical analysis of pediatric malignant mesenchymal liver tumors is necessary. Myoge-

nic markers such as myf4 and MyoD1 are a helpful and sometimes indispensable diagnostic tool for the correct diagnosis of rhabdomyosarcoma.

SA-P-070 Angiofibromyxoma of the umbilical cord A.M. Müller1, U. Gembruch2, M. Vogel3 1 University Bonn, Department of Pediatric Pathology, Bonn, 2University Bonn Medical Center, Dept. of Obstetrics and Prenatal Therapy, Bonn, 3University of Leipzig, Institute of Pathology

Conclusions. Pregnancy luteoma is a rare ovarian lesion which can macroscopically be misinterpreted as malignancy. Awareness of this entity can avoid unnecessary adnexectomy in young patients as pregnancy luteomas regress spontaneously. Although hyperandrogenism can be associated with IURG it is hardly probable that in the present case fetal hypotrophy was due to pregnancy luteoma because the level of secreted androgens was not high enough to cause manifest hyperandrogenism. Instead IUGR in our case is more likely attributable to placental insufficiency and/or beta thalassemia major.

SA-P-072 Infantile digital fibromatosis (IDF) – A case report and review of the literature

Aims. Tumours of the umbilical cord are extremely rare. Differential diagnosis includes hemangiomas, teratomas, abdominal wall defects and vascular lesions like hematoma and thrombosis. Hemangiomas are by far the most common umbilical cord tumours. Methods. In a 35-year-old woman in the 25th week of gestation a tumour of the umbilical cord was ultrasonographically diagnosed. Ultrasound displayed a significantly broadened umbilical cord with hyperechogenic areas and increased whartons’ jelly and arterio-venous fistulas. Apart from a slight cardiomegaly fetal heart was regular. In week 38 a healthy girl was born by cesarean. Results. Histologically the placenta showed several chorangiomas. The umbilical cord displayed an angiofibromyxom with – taking into consideration ultrasound findings – intratumoral arterio-venous malformations. Conclusions. Angiofibromyxoma with intratumoral arteriovenous malformations is a very rare subtype of hemangiomatous lesion of the umbilical cord. Umbilical cord is formed between the sixth and eighth week of gestation by the approximation of the omphalomesenteric duct resp yolk sac and the allantoic duct within the body stalk. The two arteries and one vein derive from the allantoic vessels. Hence cord hemangiomas are recognized as arising from omphalomesenteric or allantoic vessels. As they often occur together with the more common chorangiomas – like in our case – an underlying congenital predisposition to vascular neoplasms has to be discussed.

Aims. We report on a 7 months old male infant who received excisionbiopsy of a rapidly growing dermal tumor from the 2nd toe of the right side. Methods. Histological, immunohistological and ultrastructural examination revealed typical findings of an infantile digital fibromatosis (WHO: inclusion body fibromatosis). Results. Infantile digital fibromatosis (IDF) is a rare, distinctive benign fibroblastic/myofibroblastic tumor of infancy typically arising in the digits of the hands or feet. Characteristic morphological findings in the proliferating spindled cells are characteristic rounded eosinophilic paranuclear inclusions. Conclusions. Etiology of IDF remains uncertain. Despite of its benign behavior, local recurrence was seen in up to 60% of cases after surgical therapy. Local installations of corticosteroids do not reduce the size of these lesions. Current management of IDF recommends avoiding surgical intervention, as spontaneous involution is the rule.

SA-P-071 Pregnancy luteoma – an uncommon ovarian tumor: association with intrauterine growth retardation (IUGR)?

SA-P-073 Male fetus with ectrodactyly ectodermal dysplasia clefting (EEC) syndrome

C. Jayasinghe1, T. Ameziane2, C. Auerbach2, A.M. Müller3 1 Gummersbach Hospital, Institute of Pathology, Gummersbach, 2 St. Elisabeth Hospital Bonn, Department of Obstetrics and Gynecology, Bonn, 3University Bonn, Department of Pediatric Pathology, Bonn

F. Fronhoffs1, S. Detering2, S. Gerlach1, C. Berg3, M. Born4, A.M. Müller1 1 University Bonn Medical Center, Institute of Pathology, Bonn, 2University Bonn Medical Center, Institute of Pathology, Bonn, 3University Clinic of Cologne, Department of Prenatal Medicine and Ultrasound, Köln, 4University Bonn Medical Center, Institute of Radiology, Bonn

Aims. Pregnancy luteomas are rare benign lesions of the ovary induced by hormonal alterations during pregnancy. They present as unilateral or bilateral tumorous masses and are usually incidental findings during imaging or caesarean section. Most of the patients are asymptomatic. However, virilization of the mother occurs in one third of cases and virilization of the female fetus is found with two thirds of virilized mothers. Pregnancy luteomas regress postpartum spontaneously, therefore conservative treatment is sufficient. Nevertheless pregnancy luteomas are challenging, particularly for clinicians, as they can mimic malignant ovarian tumors. Methods. We present the case of a 30-year-old primigravida with beta thalassemia major who underwent caesarean section at week 37 because of pathological CTG. A hypotrophic girl was delivered with normal Apgar score. Both mother and daughter showed no endocrine abnormalities. Intraoperative both ovaries were enlarged and multicystic. One ovary was resected for histopathologic examination. Results. Histomorphology of the ovary displayed nodules of luteinized cells and muliple luteinized ovarian cysts within an edematous stroma, typical histological findings of a pregnancy luteoma. Placental examination revealed signs of insufficiency.

U. Titze1, R. Rödl2, G. Köhler1 University Hospital Münster, Gerhard-Domagk-Institute for Pathology, Münster, 2University Hospital Münster, Department of General and Tumor Orthopedics, Münster

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Aims. Characteristics of the ectrodactyly ectodermal dysplasia clefting (EEC) syndrome, first described by Rüdiger et al in 1970, are ectrodactyly, dysplasia of skin, its adnexal structures and/or teeth as well as orofacial clefts. Its exact prevalence is unknown. Until now, about 300 cases have been reported in literature. In more than 90% of all cases, a missense mutation in the gene TP63 can be detected. Methods. 33-year-old mother, gravida 1, para 1. Proof of bilateral complete cleft of lip and palate and ectrodactyly of both feet and hands and tentative sonographic diagnosis of complex cloacal persistence and malformation. Confirmation of EEC-syndrome by genetic testing. Feticide in 24th+4 week of gestation. Results. Male fetus, appropriate for gestational age, with bilateral complete cleft of lip and palate accompanied by deformation of the nasal apex. Ectrodactyly of both feet and hands. Right hand with five metacarpals (I, III–V regular, II shortened) and agenesis of phalanges II and III. At the left hand only a rudimentary anlage of digitus II but regularly formed digitus I and III–V. Right foot with five metacarpals but shortened metacarpal II. Left foot with five regularly shaped metacarpal bones, but only four phalanges (I and III–V), i.e. missing second toe. Time-adequate development of the nails. Histologically, in the skin biopsy only very few Der Pathologe Suppl 1 · 2012 

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Abstracts perspiratory and sebaceous glands. Very scarce scalp hair. Additionally, a megacystis with pseudodiverticulum and dilatated and sidled ureters. Conclusions. This fetus presented classic findings of the EEC syndrome. Because of the additional urogenital anomalies the diagnosis was expanded to ectrodactyly ectodermal dysplasia syndrome with urinary tract pathology (EECUT).

SA-P-074 Femur fibula ulna (FFU) complex A.M. Müller1, S. Detering2, U. Gembruch3 1 University Bonn Medical Center, Institute of Pathology, Bonn, 2University Bonn Medical Center, Institute of Pathology, Bonn, 3University Bonn Medical Center, Dept. of Prenatal Medicine and Obstetrics, Bonn Aims. Femur fibula ulna (FFU) complex is a sporadic, non-lethal malformation characterized by typical unilateral combination of defects of the femur and fibula and contralateral defect of the ulna. Methods. We present a fetus of 23 weeks of gestation of consanguine parents. Results. Macroscopically the fetus showed a short collar, hypertelorism, slightly down sloping palpebral fissures, short, flat nose, small lips and high arched palate and dysmorphic ear concha. In comparison to the right side, left sided upper and lower leg were significantly shortened, the foot appeared as clubfoot. Furthermore, in comparison to the left side, the right forearm was shortened, the right hand displaying four fingers and an aplasia of the thumb. Conclusions. Etiology of the sporadically occurring FFU complex is unknown. Up to now there are no signs a paternal age effect or an association with consanguinity. Neither could chromosomal studies reveal any abnormalities. Furthermore there is no evidence for an infectious or teratogenic cause. Children show normal mental development and normal life expectancy. As – dependent on the number of involved malformed limbs – the FFU complex is grouped in four groups (I–IV) this case can be assigned to type II.

SA-P-075 Fetal manifestation of the Peters’ plus syndrome associated with lenticular ectopia and occipital meningocele in one of the cases K. Schoner1, J. Kohlhase2, J. Steinhard3, R. Bald4, A. Schwan5, P. Wieacker6, H. Rehder1 1 Philipps University Marburg, Institute of Pathology, Marburg, 2Praxis of Human Genetics, Freiburg, 3Department of Obstetrics, Münster, 4Clinic of Gynecology, Leverkusen, 5Division of Human Genetics, Dortmund, 6Institute of Human Genetics, Münster Aims. Fetal pathology aims to recognize syndrome specific patterns of malformations and dysmorphic features for goal directed mutation analyses, genetic counselling of the parents and early prenatal molecular diagnoses in consecutive pregnancies. Here we report on four fetuses with Peters’ plus syndrome from two distinct families. Methods. We performed fetal autopsies after prenatal ultrasound diagnoses of malformations and termination of pregnancy and carried out molecular genetic investigations on fetal and parental DNA. Results. Four fetuses of 16 to 22 gestational weeks presented with multiple malformations and dysmorphic signs in addition to Peters’ anomaly of the eyes. The latter comprised central sclerocornea, absence of the posterior corneal stroma, and a variable degree of iris and lenticular attachments to the central posterior cornea in association with microphthalmia and lenticular ectopia. Additional features concerned hydrocephaly, a characteristic round face with cleft lip and palate, hypertelorism and prominent front, short stature, brachydactyly, and also cardiac, renal, genital and cerebral malformations including occipital meningocele. Peters’ plus syndrome was confirmed by sequence analysis of the B3GALTL gene revealing homozygosity for the common 660+1G>A donor splice

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site mutation in intron 8 in all four cases and heterozygosity for this mutation in the Caucasian, non-consanguineous parents. Conclusions. The four affected fetuses show a characteristic facial aspect that in association with the accompanying malformations should enable the diagnosis of a Peters’ plus syndrome. Peters’ anomaly of the eyes, representing an evolutive feature, is already evident at 18 weeks of gestation. However, manifestation of the disorder is variable. Occipital meningocele is a novel finding in Peters’ plus syndrome.

SA-P-076 Massive ovarian edema (MOE) V. Sailer1, S. Huss1, F. Fronhoffs1, E. Wardelmann1, A.M. Müller1 1 University Clinic of Bonn, Institute of Paidopathology and Institute of Pathology, Bonn Aims. Massive ovarian edema (MOE) is a very rare benign tumor-like condition found in young women resulting from accumulation of fluid mostly due to partial or intermittent torsion of the ovary or secondary to a pre-existing ovarian lesion. Methods. We report a case of a 13-year-old girl that presented with an ovarian mass measuring 16  cm in diameter. Ultrasound and CT-scan revealed a multilobulated cystic mass. CA-12-5  levels were increased. Concerns regarding underlying malignancy lead to unilateral salpingooophorectomy. Results. Pathological evaluation revealed a MOE and multiple thromboses of ovarian veins. Conclusions. Differentiation MOE from malignant tumor is crucial to prevent unnecessary surgery potentially resulting in hormonal dysfunction and infertility. Conservative treatment is possible and may be more appropriate in cases when histology on frozen section supports a benign lesion.

SA-P-077 Infantile myofibroma of the thyroid gland A. Agaimy1, D. Schmidt2, P. Klein3, R. Carbon4, W. Holter5 1 Friedrich-Alexander University of Erlangen, Institute of Pathology, Erlangen, 2 Friedrich-Alexander University of Erlangen, Department of Nuclear Medicine, Erlangen, 3Friedrich-Alexander University of Erlangen, Department of Surgery, Erlangen, 4Friedrich-Alexander University of Erlangen, Department of Surgery, Erlangen, 5Friedrich-Alexander University of Erlangen, University Children‘s Hospital, Erlangen, Erlangen Aims. Spindle cell lesions of the thyroid gland are rare and may thus be diagnostically challenging. They encompass a heterogeneous group of reactive mesenchymal lesions, and a variety of benign and malignant neoplasms of epithelial and mesenchymal origin. Methods. A 5-year-old girl presented with a rapidly growing firm nodular cervical mass localized to the right thyroid lobe associated with bilateral lymphadenopathy. Because of symptoms and concern about malignancy, an open surgical biopsy was performed followed by resection of the right lobe and biopsy of the cervical nodes. The patient is alive with no evidence of recurrence 18 months after surgery. Results. The specimen contained a 3.8  cm firm tan circumscribed nodular mass surrounded by a thin rim of thyroid tissue. Histological examination displayed a moderately cellular lesion composed of alternating fascicles of eosinophilic myoid spindled cells and primitive looking small rounded cells with hemangiopericytoma-like vascular pattern and a prominent myointimal proliferation at the periphery of the lesion. The myoid cells expressed strongly alpha-smooth muscle actin but were negative for desmin, h-caldesmon, epithelial membrane antigen, pankeratin, CK7, thyroglobulin, TTF-1, protein S100, TLE1, ALK-1, beta-catenin, CD31, CD34 and CD99. The lymph nodes showed reactive florid hyperplasia without evidence of tumor.

Conclusions. To our knowledge, this case represents the first report of solitary myofibroma presenting as a thyroid mass. Awareness of this differential diagnosis is necessary to avoid misinterpretation as a sarcoma with the sequelae of unnecessary over-treatment.

Poster: Uropathologie I SA-P-078 Rearrangement of the ETS genes ETV-1, ETV-4, ETV-5 and ELK-1 is a clonal event during prostate cancer progression M . Braun1, Z . Shaikhibrahim1, P . Nikolov1, D . Böhm1, V . Scheble2, R . Menon1, F . Fend3, G . Kristiansen1, N . Wernert1, S . Perner1 1 University Hospital Bonn, Institute of Pathology, Bonn, 2University Hospital Tübingen, Division of Hematology and Oncology, Tübingen, 3University Hospital Tübingen, Institute of Pathology, Tübingen Aims. ETS gene rearrangements are frequently found in prostate cancer (PCa). Several studies have assessed the rearrangement status of the most commonly found ETS gene, ERG, and the less frequent genes, ETV-1, ETV-4, ETV-5 and ELK-4 in primary PCa. However, the frequency in metastatic disease is still not well investigated. Recently, we have assessed the ERG rearrangement status in both primary PCa and the corresponding lymph node metastases, and observed that ERG rearrangement in primary PCa transfers into lymph node metastases, suggesting it to be a clonal expansion event during PCa progression. As a continuation, we investigated in this study whether this observation is valid also for the less frequent ETS rearranged genes ETV-1, ETV-4, ETV-5 and ELK-4. Methods. Using dual color break-apart FISH assays, we evaluated the status of all the less frequent ETS gene rearrangements for the first time on tissue microarrays (TMAs) constructed from a large cohort comprised of primary PCa and the corresponding lymph node metastases. Additionally, we evaluated the rearrangement status of all these ETS genes in a second cohort comprised of distant metastases. Results. ETV-1, ETV-4, ETV-5 and ELK-4 rearrangements were found in 8/81 (10%), 5/85 (6%), 1/85 (1%) and 2/86 (2%) of the primary PCa, respectively, and in 6/73 (8%), 5/85 (6%), 4/72 (6%), 1/75 (1%) of the corresponding lymph node metastases, respectively. Rearrangements of ETV-1 and ETV-5 were not found in any of the distant metastases cases, whereas ETV-4 and ELK-4 rearrangements were found in 1/25 (4%) and 1/24 (4%) of the distant metastases, respectively. Conclusions. Our results suggest that rearrangement of the less frequent ETS genes is a clonal event during prostate cancer progression. Our findings provide insights into potential clonal expansion events during PCa progression and may have significant implications in understanding the molecular basis of the metastatic cascade of PCa.

SA-P-079 ERG protein expression and genomic rearrangement status in primary and metastatic prostate cancer – a comparative study of two monoclonal antibodies M . Braun1, D . Goltz1, Z . Shaikhibrahim1, W . Vogel1, D . Böhm1, V . Scheble2, K . Sotlar3, F . Fend4, A . Dobi5, G . Kristiansen1, N . Wernert1, S . Perner1 1 University Hospital Bonn, Institute of Pathology, Bonn, 2University Hospital Tübingen, Division of Hematology and Oncology, Tübingen, 3University Hospital Munich, Institute of Pathology, München, 4University Hospital Tübingen, Institute of Pathology, Tübingen, 5Uniformed Services University of the Health Sciences, Center for Prostate Disease Research, United States Aims. Overexpression of the ERG protein is highly prevalent in prostate cancer (PCa) and most commonly results from gene fusions involving the ERG gene. Recently, an N-terminal epitope targeted mouse and a C-terminal epitope targeted rabbit monoclonal anti-ERG antibody have

been introduced for the detection of the ERG protein. Independent studies reported that immunohistochemical (IHC) stains with both monoclonal anti-ERG antibodies (ERG-MAbs) highly correlate with the underlying ERG gene rearrangement status. However, a comparative study of both antibodies has not been provided so far. Here, we are the first to compare the mouse ERG-MAb to the rabbit ERG-MAb for their concordance on the same PCa cohort. Furthermore, we assessed if the ERG protein expression is conserved in lymph node and distant PCa metastases, of which a subset underwent decalcification. Methods. We evaluated tissue microarrays of 278 specimens containing 265 localized PCa, 29 lymph node, 30 distant metastases, and 13 normal prostatic tissues. We correlated the ERG protein expression with the ERG rearrangement status using an ERG break-apart fluorescence insitu hybridization (FISH) assay and IHC of both ERG antibodies. Results. ERG protein expression and ERG rearrangement status were highly concordant regardless of whether the mouse or rabbit ERG-MAb was used (97.8% versus 98.6%, respectively). Of interest, both ERG antibodies reliably detected the ERG expression in lymph node and distant PCa metastases, of which a subset underwent decalcification. Lymphocytes revealed immunoreactivity using the rabbit ERG-MAb, but not using the mouse ERG-MAb. If an ERG protein expression was present in localized PCa, we observed the same pattern in the corresponding lymph node metastases. Conclusions. This is the first study to comprehensively compare the two available ERG-MAbs. By demonstrating a broad applicability of IHC to study ERG protein expression using either antibody, this study adds an important step towards a facilitated routine clinical application. Further, we demonstrate that the clonal nature of the ERG rearrangement is not restricted the genomic level, but proceeds in the proteome. Together, our results simplify future efforts to further elucidate the biological role of ERG in PCa.

SA-P-080 MicroRNA miR-205 is down-regulated in prostate cancer depending on Gleason score and tumour size B . Verdoodt1, M . Vogt1, V . Kuhn1, A . Tannapfel1, A . Mirmohammadsadegh1, M . Neid1 1 Ruhr-University Bochum, Institute for Pathology, Bochum Aims. miR-205 plays a role in the repression of the epithelial to mesenchymal transition in different epithelial tumours, and targets anti-apoptotic and cell cycle regulating genes. We studied the expression of miR205 and its relation to clinical parameters in archival samples of prostate cancer with different Gleason scores. Methods. miRNA was isolated by micro-dissection from 84 formalin fixed/paraffin embedded prostatectomy specimens with prostate cancer of Gleason score 3+3 (n=12), 3+4 (n=32), 4+3 (n=30), and 4+4 (n=10), and from corresponding benign prostate tissue. miR-205 levels were determined by quantitative real time PCR in comparison to RNU6B (U6 small nuclear RNA 2) as reference gene. Data was correlated with Gleason score, size, and extraprostatic tumour extension. Results. Expression of miR-205 was lower in tumour tissue than in benign tissue from the same patient in 76 of 84 cases (90.5%, median expression 18%). It decreased depending on Gleason score, with median 0.303 in tumours with Gleason score 3+4, median 0.150 in tumours with Gleason score 4+3, and median 0.087 in tumours of Gleason score 4+4 (p<0.05). Gleason 3+3 samples though had a median expression of 0.168fold of the benign samples. miR-205 level was inversely correlated to tumour size (p<0.05). In addition, miR-205 expression was typically lower in tumours with extension beyond the prostate (pT3) in comparison to those confined to the prostate (pT2). Conclusions. miR-205 expression is strongly decreased in prostate cancer and its expression correlates inversely to Gleason grade and tumour size.

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Abstracts SA-P-081 Significance of Gleason Grading in a clinical setting that considers active surveillance as a therapeutic option of prostatic low grade cancer B. Helpap1, G. Kristiansen2, J. Köllermann3, U. Oehler1, C. Fellbaum1 1 HBH-Hospital, Dept. Pathology, Singen, 2University of Bonn, Dept. Pathology, Bonn, 3HKS-Wiesbaden, Dept. Pathology, Wiesbaden Aims. Active surveillance has become an increasingly accepted clinical strategy to handle patients with presumably insignificant carcinomas by observant waiting, without endangering the curative intent. Aim of this analysis was to evaluate the diagnostic value of nuclear features in addition to Gleason grade in the prediction of non-aggressive disease in a large and representative prostate cancer cohort. Methods. A cohort of 968 prostatectomy specimens with matching preceding biopsies (12 cores) was morphologically analysed. Architectural features according to Gleason and cytological grading were recorded and compared. Results. The combination of architectural and cytoplogical features as incorporated in the Helpap Grading increase the rate of agreement of grading between the biopsy and the prostatectomy specimens especially GS 6 up to 90%. The parallel use of Gleason and Helpap grading allows for a better prediction of organ confined disease (pT2) following prostatectomy. Conclusions. By adding cytologic features to Gleason grading, an increased diagnostic accuracy in the identification of low grade carcinomas, which may be treated by active surveillance, can be achieved.

SA-P-082 The Microtubule-associated Protein 2 (MAP2) is frequently expressed in prostate cancer and its precursor lesions M. Majores1, E. Krappe1, N. Wernert1, J. Ellinger2, G. Kristiansen1 1 University of Bonn, Department of Pathology, Bonn, 2University of Bonn, Department of Urology, Bonn Aims. The microtubule-associated protein 2 (MAP2) is involved in microtubule assembly and plays a crucial role for nucleation and stabilization of microtubules. MAP2 is frequently expressed in mature neurons and tissue with neuroendocrine differentiation. Methods. We incidentally revealed that MAP2 is expressed in prostate cancer (PCA) and have evaluated the immunohistochemical characteristics of MAP2 expression in 107 PCA specimens in comparison to adjacent normal and dysplastic tissue. Results. MAP2 expression was strikingly focused on high-grade PIN lesions and invasive tumour glands: moderate or strong immunolabelling was found in 92% of high-grade PIN (n=61/68) and in 58% (n=62/107) of low-grade PIN lesions. In contrast, normal glands or hyperplastic epithelia of the periurethral zone stained weakly. Invasive carcinoma was MAP2-positive in 86% of Gleason pattern (GP) 3 glands (n=89/103), in 78% of GP 4 (n=28/36) and in 75% of GP 5 areas (n=6/8). In several cases, MAP2 was expressed in high-grade PINs with continuous transition to invasive carcinomas. Conclusions. Our preliminary findings support MAP2 as a promising new immunomarker for PCA and PIN lesions and moreover point to MAP2 as an “interface marker” in the progression from in-situ lesions to invasive carcinomas. We currently conduct correlations between MAP2 expression and clinicopathological features including patient survival times.

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SA-P-083 CyclinD1 expression indicates possible lymph node metastasis in invasive bladder cancer J. Rokahr1, E. Herrmann2, M. Bögemann2, S. Bierer2, E. Eltze3 1 University of Muenster, Institute of Pathology, 2University of Muenster, Department of Urology, 3Institute of Pathology Saarbrücken Rastpfuhl, Saarbrücken Aims. Overexpression of proteins involved in antiapoptosis or proliferation is often associated with tumour progression and treatment resistance. Cyclin D1 and BCL2 play a potential role in progression of bladder cancer like Ki67 and TP53. Methods. Immunhistochemical stainings were performed for Cyclin D1, BCL2, TP53 and Ki67 on TMA containing paraffin embedded tissues of 219 invasive bladder cancer patients who had undergone radical cystectomy between 1987 and 2004. The results were correlated with clinicopathological parameters and overall and recurrence-free survival. Results. Expression of the BCL2 was found in only 19 tumours, and only moderately in 6 of these. Overexpression of BCL2 correlated with a low proliferation rate (Ki67< 10%, p=0.067) and a low grade (p=0.004). No correlation could be found to pTstage or TP53 expression or survival data. Cyclin D1 expression of correlated significantly with pN1 (p=0.031), whereas no correlations to tumour stage, grade, TP53 expression or proliferation rate could be detected. Kaplan Meier analysis showed a significant shorter overall survival for patients with Cyclin D1 expression tumours (p=0.022). Conclusions. The correlation between Cyclin D1 expression and lymph node metastasis and a poor prognosis in invasive bladder cancer after cystectomy indicates a role in mediating invasion and metastasis of cancer cells.

SA-P-084 High IMP3 protein expression is a negative prognostic factor in advanced urothelial carcinoma of the bladder H. Reis1,2, F. vom Dorp3, C. Niedworok3, C. Niedworok4, A. Melchior-Becker4, J.W. Fischer4, D. Gödde1, B.B. Singer5, A. Bankfalvi2, I. Romics6, K.W. Schmid2, S. Störkel1, S. Ergün5, H. Rübben3, T. Szarvas3,7 1 University of Witten/Herdecke, Institute of Pathology, Wuppertal, 2University of Duisburg-Essen, Institute of Pathology and Neuropathology, Essen, 3 University of Duisburg-Essen, Department of Urology, Essen, 4University of Duisburg-Essen, Institute of Pharmacology and Clinical Pharmacology, Essen, 5University of Duisburg-Essen, Institute of Anatomy, Essen, 6Semmelweis University Budapest, Department of Urology, Budapest, Hungary, 7 Medical University of Vienna, Department of Urology, Wien, Austria Aims. The identification of the prognostic influence and interactions of the Insulin-like growth factor mRNA-binding protein 3 (IMP3) in advanced urothelial carcinoma of the bladder (UCB). Methods. A total of 224 urothelial bladder carcinoma cases were studied regarding IMP3 expression by immunohistochemistry, real-time PCR and Western blot analyses. The molecular targets of IMP3 – CD44, IGF2 and its receptor IGF1-R – were also investigated. Expression levels were correlated with clinical follow-up data in univariate and multivariate analyses. Results. In high-stage and high-grade UCB both IMP3 protein and mRNA levels were significantly elevated. In muscle-invasive cancer IMP3 protein but not gene expression proved to be an independent predictor of disease-specific (HR=2.58, 95%CI 1.28–4.56, p=0.004) and overall survival (HR=2.07, 95%CI 1.12–3.82, p=0.020). IGF2 and CD44 expression showed no correlation with that of IMP3. Conclusions. Identification of high IMP3 protein levels in UCB might aid in the selection of patients at high risk for disease progression and in the stratification to groups with more intensive therapy or strict follow-up. No tumor promoting effect of IMP3 in its regulatory action on IGF2 and CD44 expression was observable.

SA-P-085 Expression of the eukaryotic translation initiation factor 3a in urinary bladder cancer R. Spilka1, A.K. Mehta2, J. Haybaeck2, P. Obrist1 1 Pathologylab Dr. Obrist & Dr. Brunhuber OG, Zams, Austria, 2 Institute of Pathology, Medical University of Graz, Austria Aims. Urinary bladder cancer (UBC) is a frequent and aggressive urinary tract cancer, of transitional cell type, with high mortality rates. One obstacle in defining novel treatment approaches is that the cancer’s aetiology and genetics are not yet completely understood. eIF3a, the largest subunit of eukaryotic initiation complex eIF3, is up-regulated in many cancers including breast, cervix, colon, esophagus, lung and stomach and its suppression leads to inhibition of tumour cell proliferation in vitro. We are therefore aiming at evaluating the translation initiation factor eIF3a in UBC to gain insight in the pathogenesis of these tumors and to further determine the role of eIF3a in cancer development and progression. Methods. eIF3a expression was determined by immunohistochemistry on paraffin embedded tissues from 178 UBC patients. Protein expression levels of eIF3a were analysed in five UBC cell lines by western blotting. Furthermore by manipulating eIF3a levels in tumor cell lines, HT1197 and RT4-31, we want to explore whether eIF3a expression levels can directly influence global as well as specific translation and proliferation. We are therefore generating an inducible shRNA mediated eIF3a-knockdown construct for lentiviral transfection. Results. eIF3a is upregulated in UBC when compared to normal tissues, similar to the cancer entities where eIF3a was described to be overexpressed before. We have successfully tested two lentiviral shRNA constructs for the inducible knockdown of eIF3a. The knockdown construct generated proves efficient in all tested cell lines and first results show an association of eIF3a knockdown with growth retardation of tumor cells. Conclusions. Overexpression of eIF3a seems to be tumor-associated over a wide range of tumor entities, with a potential as prognostic marker. The knockdown of eIF3a leads to reduced proliferation rates, indicative of subcellular changes arising probably not exclusively from altered translational profiles.

SA-P-086 A case of clear cell renal carcinoma arising in a renal angiomyolipoma A. Dellmann1, A. Vandromme2, P. Hammerer2, K. Donhuijsen1 1 Academical Hospital Braunschweig, Department of Pathology, Braunschweig, 2Academical Hospital Braunschweig, Department of Urology, Braunschweig Aims. Renal angiomyolipoma (AML) is a mesenchymal tumor of the kidney that usually shows a benign course. MRI usually enables reliable detection of fat, which is typical for angiomyolipoma, and allows the differentiation to a renal cell carcinoma. We present a rare case of a renal cell carcinoma appearing in AML, thus showing that clear cut diagnosis of AML in MRT in not always possible. Methods. We report about a case of a renal cell carcinoma appearing in AML in a 77-year-old male. Pretherapeutic radiologic imaging of the tumour was fitting for AML. Histological examination including immunohistochemistry was performed. Results. In this case of an AML appearing in the right kidney the pretherapeutic imaging was typical. Histologic examination showed a combined tumour with features of an AML and of renal cell carcinoma within. Immunohistochemical studies showed typical results for AML on the one hand and for RCC on the other. Chromosomal aberrations will be examined by FISH. Conclusions. Angiomyolipoma is a combined mesenchymal tumour of the kidney with a usually benign course. Tumour can be associated with tuberous sclerosis. Although MRI usually allows the differential diagno-

sis to a renal cell carcinoma, in our case a RCC was found within the AML. The possibility of RCC appearing in AML has to be kept in mind.

SA-P-087 Proteomic analysis of renal cell carcinoma and adjacent normal fresh, snap-frozen tissue by MALDI Imaging mass spectrometry B. Häupl1, C. Recktenwald1, D. Berger2, H.-J. Holzhausen2, F. Bartel2, B. Seliger1 1 University of Halle-Wittenberg, Institute of Medical Immunology, Halle/ Saale, 2University of Halle-Wittenberg, Institute of Pathology, Halle/Saale Aims. Renal cell carcinoma (RCC) is the most common renal malignancy among the tumors that are highly resistant to systemic therapy. However, specific biomarkers for this tumor entity are not well defined. In order to understand the biology of the tumor and to detect features which allow the distinction between tumor and adjacent renal tissue and thus may serve as specific diagnostic biomarkers, in situ-proteome profiling of matched tumor and normal kidney tissue sections was carried out. Therefore, respective biopsy samples were dissected and analyzed via MALDI Imaging mass spectrometry (MALDI-IMS). Methods. Cryosections (thickness 8 μm) of 28 fresh frozen tumor samples and the respective adjacent kidney tissue were mounted onto conductive glass slides and coated with sinapinic acid using an automated spraying device (Bruker ImagePrepTM) after the removal of salts and lipids by several washing steps in graded ethanol solutions. Subsequently the samples were subjected to mass spectrometric measurement with a MALDI-TOF MS device (Bruker ultrafleXtremeTM) in linear positive detection mode operating at the following parameters: i) lateral resolution of 100 μm, ii) mass range between 2 to 20 kDa and iii) 500 laser shots per measurement position. MS data was further processed using a software application specialized for MALDI-IMS analysis (Bruker flexImaging) and the Bruker Clinprotools software package. Results. Data analysis led to the generation of specific average spectra for tumor and normal renal tissue. Although the spectra show a quite similar peak pattern, tumor and normal specific features could be detected. The significance of these features, which subsequently will be subjected to mass spectrometric identification, is currently analyzed by different algorithms such as Support Vector Machine, Genetic Algorithm or Quick Classifier. Conclusions. Tissue proteome profiling via MALDI-IMS is able to detect differentially expressed features and thus allows the identification of biomarkers for improved diagnosis that may be further used as targets for immunotherapy of RCC.

SA-P-088 Is the mucinous tubular and spindle cell carcinoma of the kidney a distinct entity or not? I. Kollecker1, A. Dellmann1, P. Hammerer2, K. Donhuijsen1 1 Academical Hospital Braunschweig, Department of Pathology, Braunschweig, 2Academical Hospital Braunschweig, Department of Urology, Braunschweig Aims. Mucinous tubular and spindle cell carcinoma (MTSCC) is a rare, low grade renal epithelial neoplasm included into the WHO since 2004 as a distinct entity. However, with rising numbers of such cases the histologic pattern is more and more expanding. The discrimination from papillary renal cell carcinoma (PRCC) on the one hand and the undifferentiated renal cell carcinoma on the other can be problematic. The question arises whether it is really a distinct tumor entity or an new hotchpotch of renal cell carcinoma. Methods. We report about a little series of cases with tubular and mucinous pattern suspect for the diagnosis of MTSCC. The cases were selected from urologic specimen with 108 non-clear cell carcinoma out of the last five years. The tumors were analysed on paraffin slides stained Der Pathologe Suppl 1 · 2012 

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Abstracts by H&E, Pas-Alcian and immunohistochemical staining by CK7, CK 19, EMA, AMACR, Vimentin, NSE, Chromogranin, Synaptophysen, CD 10, CD 15, CD 57 and Ki-67. A FISH-analysis was done for chromosom 7 and 17. Results. Classic cases of MTSCC mainly show a relative homogeneous tubular architecture with low grade morphology and typical mucinous stroma. The latter react pale for Pas and contains in Acian-blue-reaction acid mucopolysaccids. In most cases more or less areas of papillary, plexiforme or glomeruloid growth pattern exists. In parts with very close packed tubuli the cells become a spindle shape character because of compression of the tubulus but with predominant low grade atypia. Immunohistochemistry react positive for CK 7, Ck 19, EMA, AMACR and Vimentin. Neuroendocrine markers show different pattern. CD 10 reacts negative. FISH-analysis brought different results for PRCC and MTSCC. Conclusions. The main part of the MTSCC represents the described tubular architecture with low grade atypia and typical mucinous extracellular stroma component in variable dimension. Despite the classic pattern different extent of other differentiation pattern like papillary, spindle shaped, plexiforme or glomeruloid occur in the cases. Only in exceptions tubular growth pattern was displaced by last named differentiations. Especially papillary growth pattern make it difficult to distinguish MTSCC from other renal cell carcinomas especially PRCC. The demarcation to the other types is important because of prognostic differences. Immunohistochemistry could help with CD 10 negativity but this reaction is however non-specific. Against this background molecularpathologic methods are due to help, but also with limitations.

SA-P-089 Solitary fibrous tumor of the kidney M . Gajda1, S . Harz1, H . Wunderlich2, K . Junker2, M . Grimm2, D . Katenkamp1, I . Petersen1 1 Institute of Pathology, Jena University Hospital, 2Department of Urology, Jena University Hospital Aims. Solitary fibrous tumors (SFT) are rare spindle cell neoplasms usually arising in the pleura. They have, however, also been reported at extrapleural locations. Urogenital localization is rare and to our knowledge, only 40 cases of SFT of the kidney have been described. Methods. Detailed clinical and histopathological review of a clinical case and review of the literature. Results. We report the case of a typical SFT of the right kidney in a 71-year-old woman. As part of a CT investigation, a large mass of the right kidney with suspicion thrombembolic compression and occlusion of the inferior vena cava was discovered. She underwent radical nephrectomy and lymphadenoctomy, adrenalectomy and interaortocaval lymph node dissection. The gross specimen included the kidney (12.8×6.6×7.5  cm), ureter and adrenal gland. Cut section showed a circumscribed pale brown mass measuring 9.6×7.4×5.2 cm tumor. The tumors consisted of bland-looking spindle cells arranged in short, ill-defined fascicles and storiform pattern with characteristic hemangiopericytoma-like blood vessels. Immunohistochemistry showed reactivity for vimentin, CD 34, BCL-2 protein and CD99. Immunohistochemical stains for cytokeratin, S-100, desmin, a-smooth muscle actin, RCC, EMA and CD 10 were negative. Ki67 labelling index exceeded 10%. Conclusions. The possibility of SFT should be considered in the differential diagnosis of a renal mass which consists of benign-looking spindle cells and hemangiopericytomatous blood vessels. Its diagnosis requires immunohistochemistry and awareness of its possible existence.

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Poster: Uropathologie II SA-P-090 Topotecan sensitizes renal cell carcinomas towards ABT-263 induced apoptosis S . Heikaus1, V . Nitsche1, S . Funke1, H .E . Gabbert1, C . Mahotka1 1 Heinrich-Heine University Hospital, Institute of Pathology, Düsseldorf Aims. Renal cell carcinomas (RCCs) exhibit a marked resistance towards conventional chemotherapeutic regiments, thus making new therapeutic approaches necessary. So-called “targeted therapies” could be one strategy to overcome this broad resistance. Whereas “targeted therapies” with Kinase-Inhibitors like Sunitinib or Sorafenib are established in RCCs, therapies directly targeting apoptotic pathways are not validated. In this context, the BCL-2 inhibitor ABT-263 might be a promising new agent, aiming at the mitochondrial pathway of apoptosis, which is impaired in RCCs. We therefore examined the apoptotic effects of ABT-263 alone and in combination with Topotecan on the apoptosis of RCC cell lines. Methods. Cell culture, cell count, quantitative real-time detection PCR, Western Blot, Fluorescent Immunohistochemistry, Ribonuclease Protection Assay. Results. 1.) Expression of pro- and antiapoptotic BCL-2 family members was analysed in 7  RCC cell lines. Surprisingly, all cell lines expressed not only multiple antiapoptotic but also important proapoptotic BCL2 family members of all functional groups on the mRNA and protein level. 2.) ABT-263 and Topotecan induced apoptosis but not autophagy in RCCs. 3.)  Sensitivity towards ABT-263 induced cell death inversely correlated with the expression of MCL-1. 4.) Topotecan downregulated MCL-1 protein expression in RCCs; in contrast, ABT-263 upregulated MCL-1 protein expression, whereas the expression of most of the other BCL-2 family members remained unchanged. 5.)  Topotecan pretreatment weakly sensitized RCC cell lines towards ABT-263 induced apoptosis by synergistically activating the mitochondrial pathway of apoptosis. Conclusions. Inhibition of antiapoptotic BCL-2 family members by BCL2 inhibitors like ABT-263 might be a promising new approach in terms of a “targeted therapy” in RCCs. Furthermore, a combination with other chemotherapeutic agents can obviously enhance their proapoptotic effects.

SA-P-091 Cadherin expression in different histological types of papillary renal cell carcinoma: a diagnostic tool C .L . Behnes1, B . Hemmerlein1, A . Strauss2, H .-J . Radzun1, F . Bremmer1 1 University of Göttingen, Institute of Pathology, 2University of Göttingen, Institute of Urology Aims. Cadherins constitute a family of transmembrane glycoproteins and include a variety of subtypes, of which E-cadherin has been widely studied. Functionally, the cadherins belong to the adhesion molecules and form cell-cell contacts. Furthermore they also play a role in the development of different organs and in tumorigenesis. The papillary renal cell carcinoma (RCC) is a rare tumor and is divided, based on histological criteria, into two subtypes, from which the type II papillary RCC do have a worse prognosis. There are no immunohistochemical markers for the distinction of these subtypes. Therefore we have examined the expression of four different cadherins in both subtypes of papillary RCC in order to find any diagnostically relevant differences. Methods. The expression of N-, P-, E- und KSP-Cadherin was deternined in 22 papillary RCC of histological type I and 18 papillary RCC of histological type II (n=40). The intensity of membranous and cytoplasmic immunhistochemical staining was semiquantitativly evaluated by a score from 0 to 3. To compare the data for significant differences we used the Wilcoxon-Test.

Results. The papillary RCC type  II were all positive for membranous N-Cadherin staining, whereas type  I did not show any membranous positivity for N-Cadherin. The E-Cadherin staining showed a stronger membranous as well as cytoplasmic expression in type II than in type I RCC. A relevant expression of KSP- and P-Cadherin could not be demonstrated. Conclusions. Our data show that all papillary RCC type II are characterized by a membranous N-Cadherin expression. In contrast the papillary RCC type I do not express N-Cadherin membranous at all. Thus N-Cadherin represents the first immunhistochemical marker for a clear differentiation between papillary RCC type I and type II.

SA-P-092 Myoglobin expression in renal cell carcinoma is regulated by hypoxia C.L. Behnes1, J. Bedke2, A. Strauss3, F. Bremmer1, H.-J. Radzun1 1 University of Göttingen, Institute of Pathology, 2University of Tübingen, Institute of Urology, 3University of Göttingen, Institute of Urology Aims. Myoglobin (Mb) is a member of the hemoprotein superfamily, including in addition hemoglobin, neuroglobin and cytoglobin. The functions of the cytoplasmic localized Mb are the prevention of hypoxia and radical scavenging, well known from the myocytes of skletal and myocardic muscles. In case of hypoxia Mb acts as an oxygen reservoir by binding O2 and improving the diffusion of oxygen into the cell. It could be shown that some cancers do express Mb preventing hypoxia. In this study we investigated whether Mb also plays a role in renal cell carcinoma (RCC) and a potential influence of hypoxia on the expression. Methods. Four different RCC cell lines were cultivated under hypoxic conditions and the expression of Mb was evaluated by real-time PCR. For the correlatin between microvessel density and Mb expression tissue microarrys (TMAs) with 42 different RCCs were immunhistochemically stained with a myoglobin- as well as CD31-antibody. Results. We could show that RCC do express Mb. Immunhistochemical examinations of RCC tissues show a reverse relationship between Mbexpression and capillary density. Especially in clear cell RCC a significant relationship between decrease in capillary density and increased expression of Mb could be shown. Furthermore, the expression of Mb in all analyzed RCC cell lines increased under hypoxia up to 60-fold. Conclusions. Mb especially known as a marker for myogenic differentiation is expressed in RCC and RCC cell lines under hypoxia. A significant reverse correlation between capillary density and Mb expression exist in clear cell RCC. Thus, Mb might be a marker for hypovascularized tumor entities/tumor areas.

SA-P-093 Impact of early ultrastructural damage after ABO-incompatible and ABO-compatible kidney transplantation V. Broecker1, A. Pfaffenbach1, C. Bockmeyer1, M. Dämmrich1, S. Immenschuh2, A. Schwarz3, H. Kreipe1, F. Heinemann4, J.U. Becker1 1 Hannover Medical School, Institute for Pathology, Hannover, 2Hannover Medical School, Institute for Transfusion Medicine, Hannover, 3Hannover Medical School, Department of Nephrology, Hannover, 4Essen University, Institute for Transfusion Medicine, Essen Aims. Following ABO-incompatible kidney transplantation (iABO), c4d in peritubular capillaries (ptc) is frequently positive without further indicators of humoral rejection. Using electron microscopy (EM), we investigated the presence and prognostic relevance of early endothelial damage to glomerular and ptc in transplant kidneys with c4d positivity under different circumstances. Methods. In 20 iABO patients (57 biopsies), 16 ABO-compatible (cABO) patients (26  biopsies with c4d-positivity of ptc) and 14  transplant patients (14 biopsies) without c4d and signs of humoral rejection (controls)

were included. 10 different EM parameters were semiquantitatively evaluated: loss of foot processes (FF), lamina rara interna widening (LRI), swelling of glomerular and ptc endothelial cells (GES and ptcES), inflammatory cell adhesion to glomerular and ptc endothelial cells (GIS and ptcIS), glomerular basement membrane double contours (DK), glomerular and ptc basement membrane lamellation (LG and Lptc), loss of glomerular endothelial fenestration (FE). Status of donor specific antibodies (DSA) was available for 35/50 patients. Kidney function (GFR) at biopsy and follow up (51.8±31.74 months) was available for all patients. Results. 3/10 EM parameters were significantly less severe in iABO versus cABO (FF, FE, ptcES); 1/10 significantly more severe in cABO versus controls (FE). No differences were seen between iABO and controls. GFR at biopsy and follow up was significantly lower in cABO versus iABO, although the decline (GFR/time) was not different. GFR at biopsy and follow up correlated with 2/10 EM parameters in iABO (LRI, FE), 2/10 in cABO (GIS, FF). None of the EM parameters correlated with GFR decline. Patients with positive DSA (6/35) had significantly lower GFR at biopsy and follow up, although, GFR decline was not different between DSA positive and negative patients. The presence of DSA correlated with 6/10 EM parameters (FF, LRI, GES, ptcES, DK, FE). Conclusions. Long term outcome in iABO patients is favourable despite c4d positivity of ptc. In this context the value of EM in the detection of early endothelial damage, although more pronounced in cABO patients with signs of humoral rejection, is limited and lacks prognostic relevance. In contrast, the presence of DSA correlates with ultrastructural signs of endothelial alteration and is associated with impaired kidney function.

SA-P-094 Large scale in vivo isolation of glomerular podocytes by cationic colloidal silica-coated ferromagnetic nanoparticles A. Blutke1, R. Wanke1 1 Ludwig-Maximilians-University Munich, Institute of Veterinary Pathology at the Centre for Clinical Veterinary Medicine, München Aims. Podocyte homeostasis plays a crucial role for maintenance of the physiological glomerular function and podocyte injury is regarded as a major determinant of development and progression of various renal diseases. Since cultured podocytes cannot completely reflect the complex properties of podocytes in the glomerular environment in vivo, analyses of the molecular processes occurring during podocyte development and injury require appropriate methods for preparation of fresh podocyte isolates. Methods. A novel, fast, antibody-free and most cost-efficient method for reproducible large scale isolation of fresh podocytes from mouse kidney glomeruli is described. Briefly, cationic silica-coated colloidal ferromagnetic nanoparticles were utilized to bind to negatively charged cell surfaces of podocytes in preparations of isolated glomeruli. After enzymatic and mechanical dissociation of the glomerular cells, nanoparticle-coated podocytes were isolated in a magnetic field. Results. Podocytes isolated with this method displayed characteristic phenotypical and ultrastructural features. Protein and mRNA expression abundances of marker-molecules of podocytes, endothelial or mesangial glomerular cells indicated a significant enrichment of podocytes in the generated isolates. Conclusions. The described method offers a great potential for different downstream transcript- and protein profiling analysis technologies, which might contribute to an improved understanding of podocyte biology and of the molecular mechanisms involved in podocyte injury in vivo.

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Abstracts SA-P-095 Can fibrils induce a humoral immune reaction? A case report and review of literature S. Porubsky1, C. Boldt2, V. Kliem2, H.-J. Gröne1 1 DKFZ Heidelberg, Heidelberg, 2Nephrologisches Zentrum Niedersachsen, Hann. Münden Aims. Fibrillary glomerulonephritis is a rare disease of later adulthood and is characterized by randomly arranged Congo-red negative fibrillary deposits with a diameter of 10–20 nm in glomeruli. Due to sclerosis of glomeruli and tubulointerstitium the disease leads usually to chronic kidney failure. We describe a 50-year-old female patient whose medical history was negative with exception of smoking and a febrile bronchitis a few days before admission. The patient presented with proteinuria (3.8 g/d), active urine sediment, hypertension and acute kidney failure. The laboratory findings revealed solely an ANA-titer of 1:160. ANCAand ASLO-titers as well as hantavirus serology were negative. Complement levels were normal. Methods. Light-microscopical, immunhistochemical and ultrastructural investigations of the kidney biopsy and correlation of the findings with the current literature. Results. The biopsy showed 15 glomeruli of which 5 were characterized by a segmental necrosis and extracapillary proliferation. 2 glomeruli showed a global and 4 a segmental sclerosis. There was moderately severe chronic interstitial damage. Immunohistochemically IgG, IgA, IgM C1q and C3 depositions could be detected in mesangium and along the basement membrane of all glomeruli. Congo-red stain was repeatedly negative. In mesangium and within the glomerular basement membrane, electron microscopy visualized randomly arranged fibrils with a diameter of 10–20 nm. The diagnosis of a fibrillary glomerulonephritis was made. Conclusions. Although seldom crescents have been observed in idiopathic fibrillary glomerulonephritis, a necrotizing form of this disease has not been described in detail. The simultaneous occurrence – with a “full house” pattern – of immunoglobulin, complement factors and fibrils is usually seen in fibrillary glomerulonephritis. Necrosis points to the potential of fibrillary deposits to induce a pronounced complement activation and to cause a necrotizing lesion. This is contradictory to the current paradigm that fibrils are immunologically inert. Furthermore it demonstrates that glomerulopathies with organized deposits are to be considered upon a diagnosis of necrotizing glomerulonephritis.

SA-P-096 Pathology of resolving polyomavirus nephropathy T. Menter1, M. Mayr2, H.H. Hirsch3, M.J. Mihatsch1, S. Schaub4, H. Hopfer1 1 Institute of Pathology, Basel, Switzerland, 2Medical Outpatient Department, Basel, Switzerland, 3Institute of Medical Microbiology, Basel, Switzerland, 4Clinic for Transplantation Immunology and Nephrology, Basel, Switzerland Aims. Polyoma virus nephropathy (PVN) is a common complication after renal transplantation, affecting up to 20% of patients. The standard therapy is reduction of immunosuppression, which leads to an effective virus control in more than 90% of cases. So far, the morphology of resolving PVN has not been investigated. We compared the morphological findings in protocol biopsies of PVN patients prior to viremia, during increasing and decreasing viremia as well as after virus clearance. Methods. The study included 101 transplant biopsies of 34 patients transplanted between 2005 and 2010 and diagnosed with PVN which were treated by reduction of immunosuppression only. Biopsies were scored according to Banff-criteria, the extent of inflammation and interstitial fibrosis was estimated as% of renal cortex, and the number of tubular cross sections with SV40+ cells per mm of biopsy length was counted. Biopsy findings were correlated with virus load in the serum and cli-

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nical follow-up data, and grouped as pre-, increasing, decreasing, and post-viremia. Results. We found a significant increase in interstitial inflammation (median decreasing viremia 10% vs. increasing viremia 0.3%, p<0.005) as well as in the tubulitis-score (median decreasing viremia score 2 vs. increasing viremia score 0, p<0.005) during the phase of decreasing viremia. Both could be observed even 2 months after peak viremia and to a lower extent persisted after virus clearance from the serum. The number of SV40+ tubuli correlated with the virus load in the serum, but SV40 was frequently negative (33/55 cases), especially if the viremia was below 106 geq/ml. Conclusions. The cellular immune response against polyoma virus is essential for its clearance. During resolution the morphological pattern in the transplant kidney is that of a self limiting acute interstitial nephritis, which is often SV40 negative. This is important because the diagnosis of acute interstitial rejection depends on the same morphological criteria. Therefore, an acute interstitial rejection cannot be diagnosed with certainty during polyoma viremia.

SA-P-097 Adrenal-renal fusion tissue with adrenal cortical adenoma: a pitfall in frozen section A. Jakubzik1, A. Vandromme2, P. Hammerer2, K. Donhuijsen1 1 Academical Hospital Braunschweig, Department of Pathology, Braunschweig, 2Academical Hospital Braunschweig, Department of Urology, Braunschweig Aims. Adrenal tissue as heterotopia is not unusually and should be diagnosed even in a frozen section. However, a much more difficult situation is given in the very rare case of a fusion from kidney and adrenal gland. Methods. A 64-year-old man clinically presented with pneumonia. A computer tomography done to exclude a lung cancer revealed accidentally a tumor suspicious nodule of 2 cm diameter in the upper pole of the left kidney. Consequently, the clinical diagnosis of a renal cell carcinoma was done and the patient underwent organ-sparing surgical resection of the upper part of the left kidney and neighbored tissue with a 2.7 cm enlarged nodule to an intraoperative diagnostic procedure on frozen sections. Results. Macroscopically the renal tissue showed no normal architecture of cortex and medulla and was diffuse grey. Adrenal tissue was immediately neighbour the kidney without capsular border and indistinct to renal tissue. In addition the adrenal tissue exhibit a yellow nodule of about 2 cm. Microscopically adrenal cortical cells were spread as single cells or small nests between normal glomeruli and tubuli of the renal cortex. The frozen section diagnosis was adrenal tissue but no renal cell carcinoma and was confirmed on paraffin embedded tissue. The final diagnosis was adrenal cortical adenoma in adrenal-renal fusion. Conclusions. The spectrum of differential diagnoses of tumor like lesions of the kidney has to respect the possibility of an adrenorenal fusion with an adrenal cortical adenoma. A correct diagnosis in frozen section can avoid an unindicated nephrectomy.

SA-P-098 Male infertility: assessment of juvenile testes dysfunction and risk for malignancy in cryptorchic boys based on resin semithinsection evaluation J. Schröder1, W. Rösch2, F. Hofstädter1 1 University Regensburg, Pathology Dept., Regensburg, 2 University Regensburg, Clinic St. Hedwig, Regensburg Aims. Infertility may become more a man’s than a woman’s problem: new data shows both were level pegging – 40% of cases are linked to women, 40 to men, and 20 to joined problems. Failure in congenital testes descends (cryptorchidism) is the most frequent genital malformation affecting approx. 1% of 1-year-old mature birth boys. Untreated maldescensus testis impairs the germ cell development and reduce significantly the fertility capacity in adults. Additionally there is an increased risk for testicular cancer. We report how the pathologic biopsy examination of juvenile cryptorchic testes can assess infertility and malignancy risk. Methods. Biopsies of ectopic or cryptorchic testes were immersed in Karnovsky-fixative, postfixed in osmium-tetroxide, dehydrated in graded ethanols and routinely embedded in epon resin (EmBed812, LYNXautomated EM-tissue processor). Semithin sections (1 µM) were prepared by ultramicrotomy using a diamond knife, mounted on a glass slide and double stained with toluidine blue/basic fuchsine as well as triple stained according to Laczko [1975] for intracellular glycogen detection. Results. A semithin resin section provides an excellent structure preservation of the testicular tissue and is a proofed basis for light microscopic spermatogenesis assessment in the tubuli contorti. The evaluation includes the recognition and counting of different development stages of the germinal cells and detection of specific germinal glycogen-rich TIN-cells (testicular intraepithelial neoplasia) which represents a preneoplastic lesion. The light microscopic biopsy examination allows the cornerstone parameter estimation for the adult fertility, which are the: (1) tubular index of total germinal cell number; (2) tubular index of adult dark (A-dark Spermatogonia) spermatogonia – the stem cell pool of all future spermatozoa [Hadziselimovic, 1983, 1990]; (3) tubular index of primary spermatocytes. Conclusions. The demonstrated assessment of spermatogenesis dysfunction and malignancy risk in juvenile cryptorchic testes in semithin resin section is a crucial step for the right therapy. Today there is consent, that an intrauterine hormonal dysfunction of the hypothalamo-pituitary-gonadal axis is involved in most testicular maldescended cases. In general, late diagnosis of undescended testis will have a poor prognosis for future fertility and increased risk for cancer. In our opinion, an electron microscopy lab is predisposed for processing testicular biopsies for fertility assessment.

SA-P-099 Rete testis invasion by malignant germ cell tumors of the testis A.K. Höhn1, J.-U. Stolzenburg2, L.-C. Horn1 1 University of Leipzig, Institute of Pathology, Leipzig, 2 University of Leipzig, Clinic of Urology, Leipzig Aims. Rete testis is a communicating network of seminal channels in the hilum of the testis. Its invasion is described as a risk factor in German guidelines for the management of malignant germ cell tumors. Tumor size and rete testis invasion (RTI) were considered as prognostic factors for recurrent disease within stage I seminomas (Warde et al. 2002) and was suggested to represent an independent prognostic factor (Vogt et al. 2010). The present study evaluates the documentation of RTI within routine workup and the pattern of involvement. Methods. 100 cases with testicular germ cell tumors were re-evaluated regarding the presence of rete testis within the examined tissue, the documentation of rete testis status and, if present, the pattern of RTI (direct infiltration versus pagetoid) as well as which tumor component was seen in RTI.

Results. The mean age was 38 years (15–75 years). Mean tumor size was 3.45 cm (0.8–14.0 cm). In the originally reports presence of RTI was recognised in 27 and its absence in 25 cases. In 48 cases rete testis status was not reported. After the re-evaluation 51 cases showed RTI. Among these 31 (61%) represented direct invasion and 3 cases (6%) a pagetoid pattern. In 17 cases (33%) a mixed pattern of invasion was found. 34 of 51 cases (67%) showed an invasion by a pure seminoma, 16 cases (31%) showed an invasion by the seminomatous component of a combined germ cell tumor and 1 case (%) showed a pagetoid pattern of invasion in a non-seminomatous germ cell tumor. 42 cases showed no evidence of RTI. In 7 cases the rete testis was not available within the examined, paraffine-embedded tissue of the specimen. Conclusions. In 48% the RTI-status was not documented during routine examination. In case of RTI, the majority of cases represented direct or mixed type pattern on involvement. The status of RTI should be mentioned within the pathology report. In case of missing rete testis recutting with embedding of additional tissue is recommended. Further analyses of the correlation between the tumor size and RTI and the prognostic impact of RTI are required.

SA-P-100 N-cadherin expression in malignant germ cell tumors of the testis F. Bremmer1, S. Schweyer1, B. Hemmerlein1, A. Strauß2, H.J. Radzun1, C.L. Behnes1 1 University of Göttingen, Department of Pathology, Göttingen, 2 University of Göttingen, Department of Urology, Göttingen Aims. Testicular germ cell tumors (TGCTs) are the most common malignancy in young men aged 18–35 years. They are clinically and histologically subdivided into seminomas and non-seminomas. Cadherins are calcium-dependent transmembrane proteins of the group of adhesion proteins. They form cell junctions in various tissues including desmosomes and adherens junction. Furthermore, cadherins play a role in the stabilization of cell-cell contacts, the embryonic morphogenesis, in the maintenance of cell polarity and signal transduction. Ncadherin (CDH1), the neuronal cadherin, stimulates cell-cell contacts during migration and invasion of cells and is able to suppress tumor cell growth. The aim of this study was to determine whether N-cadherin is expressed in TGCT and potentially differentiates between the tumorentities of TGCT. Methods. We investigated 74 TGCT (seminoma n=40, embryonic carcinoma n=14, teratoma n=14, chorioncarcinoma n=3, yolk sac tumor n=5) by immunohistochemistry for the expression of N-cadherin. Furthermore, the tumor cell lines NCCIT and NTERA were analyzed by PCR, Western blot analysis and immunocytochemistry. Results. The studied TGCT showed a distinct membrane-bound N-cadherin expression for seminoma, teratoma, yolk sac tumor and chorioncarcinoma. All examined embryonic carcinoma did not show expression of N-cadherin. In the investigated mixed tumors each of the embryonic carcinoma-components was negative for N-cadherin, whereas the other tumor components were positive for N-cadherin. Both investigated cell lines expressed N-cadherin mRNA, but only NCCIT showed N-cadherin protein expression. Conclusions. In contrast to embryonic carcinomas seminomas, teratomas, yolk sac tumors and shorioncarcinomas express N-cadherin. Ncadherin is able to differentiate embryonic carcinomas from other tumor entities of TGCT also in mixed tumors. Thus, N-cadherin may play a role in tumor progression and in the pathogenesis of TGCT.

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Abstracts Poster: Informatik SA-P-101 How virtual slides save pathologist’s time and inspire students I . Klempert1, K . Schlüns1, P . Hufnagl1, M . Dietel1 Charité University Hospital, Institute of Pathology, Berlin

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Aims. Due to the changing curriculum of the Charité Universitätsmedizin Berlin we have to manage larger groups of students in shorter lessons with a more important focus on the relation to the clinical aspects of the program. Less than a third of the lessons of the institute of pathology are classical histological lessons. We decided to introduce virtual microscopy to overcome these problems and to offer the students unlimited access to comprehensive, exciting and clinical related pathohistology. To realize such an offer, we arranged the slides in cases (as small exercises). Methods. We already use the E-learning system “Blackboard”, but histology was only presentable as still pictures. Additionally, we established the “Slidebox” System to increase availability of virtual slides. This software allows the use of each picture ratio of our slide scanners. The software is password-protected and can be used from every internetaccess point. The number of well documented cases (of diagnostics) is increasing and serves as a basis for case-based learning. Results. The anatomy-histology room allows up to 80 students to work by microscope and/or PC. The computers are connected by a 2.33 GHZ XEON Server via 1 GB LAN. This technical foundation is sufficient for students to work during the lessons. The virtual slides can be annotated and the system allows them to be viewed down to the single cell level. The students are able to use the virtual slides during the lessons (under supervision) or at home. The software works fast and is reliable, the handling aspects are easy to understand and intuitive. The students accepted the improvements to the system very quickly, with positive feedback and good results. Conclusions. Prepared virtual slides or documents are logged within the system for repeated use, allowing them to be used with constant quality or for enhancement. The educator saves time in preparation. The students are well prepared; are capable of comparing the solutions or starting discussions. The use as a part of the academic education is just one possibility. The use in further education of physicians in one location, or between different institutes or universities is also conceivable.

SA-P-102 Integrative, multimedia-based learning with the aid of a virtual microscope C . Brochhausen1, H . Winther1, V .H . Schmitt1, J . Horstmeyer2, C .J . Kirkpatrick1 1 University Medical Centre, Institute of Pathology, Mainz, 2Johann Wolfgang Goethe-University, Academy for Educational Research and Teacher Education, Frankfurt am Main Aims. Virtual microscopy is already established in teaching curricula at many universities. However, the technique is the subject of continuous further development. A variety of features exist in which the individual applications differ. However, which of the numerous features are considered useful, necessary or redundant is unknown. Therefore, we have investigated the needs of the students with a questionnaire and developed a new application for virtual microscopy on the basis of these data. Methods. In cooperation with the Center for Quality Assurance and Development Mainz a questionnaire with a feature-set was created. 216 students assessed the importance of functions such as annotations, additional teaching texts and a broad scope of target devices. Based on this evaluation a new application was constructed. Results. The analysis of the questionnaire revealed that 96.2% of the students consider virtual microscopy as a meaningful learning opportunity and desirable for test preparation. The features annotations

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(87.3%) and additional teaching texts (52.3%) have also been evaluated positively. Furthermore, many students requested a quiz mode in the comments section. In contrast, the establishment of a discussion forum appeared less important to the students (4.2%). These results provided the basis for the development of a novel application, in which technologies were used (including HTML1.1, AJAX) both to satisfy the needs of the students and also to ensure the expansibility (OpenLayers) and sustainability of the application. Hence, proprietary technologies like Flash were abandoned. Conclusions. The survey revealed the requirements of the users, which formed the basis for the development of a new application. In future innovations the applications should be able to run on all modern browsers without proprietary extension. The course of further developments, e.g. detailed depth portrayal or adventure experience microscopy should be determined in further questionnaires.

SA-P-103 A web-based virtual microscopy platform as supplement for histopathology lectures S . Friedl1, G . Oehmke2, T . Wittenberg1, F . Paulsen3, A . Hartmann2, C . Geppert2 Fraunhofer Institute for Integrated Circuits, Erlangen, 2University Hospital Erlangen, Institute of Pathology, Erlangen, 3University of Erlangen-Nürnberg, Department of Anatomy Chair II, Erlangen 1

Aims. In academic education of microscopic histology and pathology, an independent training in the recognition of specimen is an important supplement to core lectures. To limit the effort regarding hardware, rooms, and personnel for such an open microscopy, innovative internet technologies can help to provide flexible and continuous opportunities. A web-based virtual microscopy platform can offer training possibilities at any time without the need of constant administration. Such a platform has been developed and introduced, and the usage and the feedback of the students have been evaluated during the last two academic terms. Methods. To provide specimens for the web-based platform, microscopic slides have been digitalized using a slide scanner at 40-fold magnification. Resulting in image data with up to 5 GB per slide, a conversion of the images is necessary to optimize the transfer over the internet. A tiled pyramid structure can provide selected tiles in defined resolutions directly without additional computation. The user can zoom to any preferred magnification level and pan the slide freely while only the visualized parts and details are transferred to the client. The digital slides are categorized by anatomy and findings, and enhanced by additional descriptions and iconic annotations. Currently approx. 200 specimens are digitalized and provided to the students. Results. With 146 5th-semester students using the platform, an average login count of 180 logins per week was observed. As expected, one week before the exams the login rate went up near to 1000 per week. According to a survey of 6th-semester students, 79% indicated, that lack of time for microscopic examination during the course could be well compensated by the web-based platform. In the evaluation, students favored that this environment offers more possibilities to provide additional information like e.g. attendant macroscopic pictures. Conclusions. A web-based virtual microscopy platform is an innovative and valuable opportunity to enable students an independent and flexible training in the recognition of different specimen. Furthermore, nowadays students are used to multimedia and internet usage and can learn and examine the slides any time at any place. The introduced platform is in use for the second academic term with appropriate access rates and runs stable with a low amount of administration. The internal solution offers possibilities to enhance the education with further features and information according the given lectures.

SA-P-104 Process oriented scientific data management in the Open European Nephrology Science Center T. Schrader1, S. Niepage2, C. Hahn2, S. Hanß3 1 University of Applied Sciences Brandenburg, FB Informatics & Media, Brandenburg, 2Charité – Universitätsmedizin Berlin, Institut für Pathologie, 3 Charité – Universitätsmedizin Berlin, Institut für Medizinische Informatik Aims. The Open European Nephrology Science Center (OpEN.SC) is a center for research related clinical data supported by the German Research Foundation. The reuse of clinical for translational medicine is a cornerstone for the further scientific development. Various projects try to collect data from different resources for scientific purpose. The OpEN.SC platform consists of process oriented tools for data import, management, analysis and presentation and solved the two main problems in scientific data management: storage of data taking to consideration the legal issues (secure patient data) and save the intellectual properties of the diagnostic and therapeutic process by the physicians. Methods. The platform based on a Service Oriented Architecture and consists of various web services as modules. The orchestration of these web services is realized by business process models. At the backend an Apache Geronimo Application Server ensures the availability of the web services. The Open Source database engine PostgreSQL is used to store the data from three different resources. The user interface is realized by OpenSource Liferay Portal. Different tools for retrieval and image analysis are available. The processes were modeled using the standard BPMN (Business Process Modelling Notation by OMG). Results. The Open European Nephrology Science Center is a flexible platform for scientific data management. Flexibility means that data from different resources, with different structures and various file types can be stored at this server and managed related to the resources. Each resource has a complete control and transparency for its own data. Due to the Service Oriented Architecture the platform is scalable. The process oriented modelling offers the opportunity to adapt the system for each specific use case and any type of data management model. The process models can be used for business simulation to evaluate the impact of changes very early. Conclusions. Scientific data management should cover different aspects of data handling: data import, storage, retrieval, access and presentation concerning all legal issues and changing as well as increasing requirements to storage, retrieval and analysis. Flexibility is a cornerstone for management data from different resources and can be realized by application of business process modeling, executing, analysis and simulation.

SA-P-105 CognitionMaster: an open source biomedical image analysis development framework S. Wienert1, D. Heim1, K. Saeger2, C. Denkert1, P. Hufnagl1, F. Klauschen1 1 Charité University Hospital Berlin, Institute of Pathology, Berlin, 2VMscope GmbH, Berlin Aims. Automated microscopic image analysis has been a research focus in medical informatics since many years. The topic has also attracted attention among pathologists who face an increasing demand for a standardized and quantitative evaluation of (immuno-)histological parameters in patient specimens. Here, computerized image analysis may assist pathologists and can also help to more efficiently test hypothesis on the relevance of certain tissue properties. One limitation in this field is the difficulty on one side for computer scientists to efficiently develop and test image analysis algorithms with realistic histological data, and on the other side for (quantitatively-inclined) pathologists to test and optimize the potentially useful analysis software: While developers would usually favour sophisticated software environments that are usually inaccessible to non-computer scientists and do not facilitate easy

testing of realistic image data, pathologists are normally confronted with ready-to-use software applications with no or limited flexibility. Our aim was to design an open and flexible software platform that may support collaboration between pathologists and computer scientists. Methods. The software was implemented in C# for Microsoft .NET. SharpDevelop was used for the integrated editing of C# code. Icons from the Tango! project were use for the graphical user interface. Results. We present an open-source software platform that may be used for a broad spectrum of tasks in the field of medical image analysis: Ranging from algorithm development with an integrated C# compiler to one-click analysis provided through a powerful plug-in interface. A novel object layer structure was designed to handle image objects and their properties and therefore allow high-level formulations of image analysis algorithms. The tool provides flexible and interactive functionality with a variety of image analysis algorithms that may be combined in process chains, an object model editor, and plotting/statistics functions. Conclusions. The platform presented here may help both computer scientists and pathologists to efficiently design and test novel image analysis approaches and quickly obtain a “first guess” on their practical utility. It may therefore foster collaboration in the field of quantitative virtual microscopy and accelerate the integration of novel image analysis approaches into diagnostic applications.

SA-P-106 Computer-assisted histology for the diagnosis of Barrett’s Esophagus – a pilot study C. Dach1, C. Geppert2, S. Friedl1, M. Benz1, C. Münzenmayer1, A. Hartmann2, M. Vieth3, T. Wittenberg1 1 Fraunhofer IIS, Erlangen, 2University Erlangen, Institute for Pathology, Erlangen, 3Klinikum Bayreuth, Institute for Pathology, Bayreuth Aims. Gastroesophageal reflux disease (GERD) is one of the most common and in the frequency increasing diseases in the western world. Intestinal metaplasia, or “Barrett’s Esophagus”, is a precancerous condition and complication of GERD. A large interobserver variation is known in histopathology of Barrett’s Esophagus. Hence, it makes sense to support pathologists with an automated pre-analysis of the images. Goal of this study is the evaluation of possibilities to differentiate three types of tissue automatically, namely Barrett’s Esophagus (BE), normal cardiac mucosa (CA) and normal squamous epithelium of the esophagus (EP). Methods. Histological slides of 86 randomly selected patients with Barrett’s Esophagus have been digitized with a high-resolution whole-slide scanner (3DHistech). 26 data sets were selected in which equally all 3 types of tissue (BE, CA, EP) are depicted. In these data sets 50 rectangular regions for each of the 3 classes were manually labeled. It was evaluated, which type of image-based features, namely color enhanced 2nd order texture statistics and statistical-geometrical features, can be used for a best differentiation of the 3 tissue classes. Furthermore, various parameters for the texture features were evaluated. For the classification step a nearest-neighbor classifier with various parameters was applied. For each experiment all classification rates as well as the confusion tables were computed. Results. Using an n-fold cross validation and a combination of 2nd orders statistical features, a maximum diagnostic classification rate of 90% (BE 88%, CA 84%, EP 100%) could be achieved, denoting the possibility of a correct differentiation of the diagnostic classes with respect to the annotated ground truth. The highest possible classification rate for the discrimination of Barrett’s Esophagus was achieved with 90% on a basis of color co-occurrence matrices and correlates to a total classification rate of 89% over all 3 classes (CA 76%, EP 100%). Conclusions. The results show, that the evaluated classes (BE, CA, EP) can be differentiated quite well on this image data base by applying color-extended texture features, whereas the detection and elimination of EP tissue is possible with a high sensitivity. Hence, the basic criteria Der Pathologe Suppl 1 · 2012 

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Abstracts have been defined, on which experiments can be made in order to differentiate and pre-sort various types of tissue of the esophagus in histological recordings using image analysis methods and possibly detecting conspicuous tissue automatically.

SA-P-107 Computer-based morphological analysis of endomyocardial structure M. Schmauder1, J. Zoschke2, N.E. Hiemann2, R. Hetzer2, R. Meyer2 Realtime Imaging, Berlin, 2German Heart Institute Berlin

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Aims. Histopathological and immunohistological examinations provide important information to characterize myocardial tissue. The aim of computer-based analysis of microscopic biopsy images is to detect and quantify relevant structures of the myocardium by a standardized procedure. The results provide the basis for correlative assessment with clinical results. Methods. The assessment of interstitial changes is done with Sirius redstained tissue sections. Fibrous parts are extracted and automatically measured using an online adjustable, adaptive colour segmentation method. Immunohistochemical CD31 staining is used to determine the size and distribution of microvessels. Here, the image analysis consists of optimized colour segmentation and morphological classification followed by automated evaluation of area ratios and numerical densities of capillaries per normalized area. For the analysis of myocardial cells based on haematoxylin eosin stained samples, a user-guided interactive process was implemented. Results. In an interdisciplinary project, we developed a new system for quantitative morphological image analysis. The system includes three measurement procedures for the analysis of myocardial structure. The results are summarized in a combined record. Mean assessment time is 30–60 min. To date, our preliminary experience has been obtained in endomyocardial biopsy samples from cardiac transplant recipients. Conclusions. Our investigations are a step towards a standardized computer-based analysis of myocardial structure.

SA-P-108 The BMBF Initiative to build up centralized biomaterial banks in Germany: the BioMaterialBank Heidelberg E. Herpel1, R. Kirsten2, J. Berger2, C. Döllinger2, E. Frei3, C. Ulrich3, P. Schirmacher1 1 Institute of Pathology, University Hospital Heidelberg, Heidelberg, 2 BioMaterialBank Heidelberg, University Hospital Heidelberg, Heidelberg, 3 National Center for Tumor disease Aims. The BMBF Initiative aims to foster the assembly of centralized structures for biomaterial banks in Germany, based on site-related strategic concepts. The BioMaterialBank Heidelberg (BMBH) as one of 5 granted centres will merge all on-site high-quality biomaterial collections (comprising both tissue and liquid samples) on an administrative level and integrate them into a consistent project and quality management, with respect to ethical and legal aspects. The overall aim is to provide biomaterials or biomaterial collectives in a comprehensive way for research purpose for researchers in Heidelberg and their cooperation partners. Methods. Core of the project is the tissue bank of the National Center for Tumor Diseases (NCT) Heidelberg, where the essential structures, regulations and procedures are realized and therefore can serve as a template. Moreover, other tissue and liquid banks with focus on special, non-tumorous diseases will be integrated, applying the following measures: – Setting up central BMBH administration, including IT/data and quality management

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– Further developing and integrating liquid biobanking, thereby adapting the existing structural concept of the NCT tissue bank – Further development of a QM assessment program for biomaterials – Setting up a uniform IT solution for all BMBH modules with optimized interfaces to material administration –Completing the biobank technology platform with specific emphasis on improving the generation of derivatives at BMBH –Expanding the NCT tissue bank outreach and training Program Results. The BMBH was initiated in May and started to work in July 2011. The following steps of milestone planning were realized so far: – Assessment of the current state of all processes, regularities and structures – Formal integration of all tissue bank modules into BMBH – Implementation of a consistent IT-structure (STARLIMS) Conclusions. In the following years, standardization of processes will be continued, with a special emphasis on IT- and quality management, and expanded to all other on-site modules. Thereby, we will lay our focus on the implementation of a conform quality management for tissue banking, aiming to accredit all tissue bank modules in year 3 of the grant. Until the end of the grant period (year 5) all processes, regularities and structures will be continued, to finally become a highly professional and sustainable biomaterial bank.

SA-P-109 The BMBF Initiative to build up centralized biomaterial banks in Germany: the Interdisciplinary Bank of Biomaterials and Data Würzburg (IBDW) M. Neumann1, S. Störk2, R. Lohmüller3, S. Kircher4, A. Rosenwald4, R. Jahns3 University of Würzburg, Institute of Clinical Biochemistry and Pathobiochemistry, Würzburg, 2University of Würzburg, Comprehensive Heart Failure Center, Würzburg, 3University of Würzburg, Interdisciplinary Bank of Biomaterials and Data Würzburg, Würzburg, 4University of Würzburg, Institute of Pathology, Würzburg

1

Aims. The Interdisciplinary Bank of Biomaterials and Data Würzburg (IBDW) aims to systematically collect liquid (blood/DNA/urine) and solid biomaterials (BM) from patients and study participants of the Medical Campus. One key challenge is to integrate acquisition of BM for research purposes into clinical routine. Further, BM-collection must comply with the current legal framework and storage conditions with current OECD recommendations. BM are linked with corresponding clinical datasets in accordance with current data protection regulations and ethical principles securing donor’s privacy. Methods. The Medical Faculty holds full responsibility for the IBDW governed by its own steering committee. The IBDW is composed of 3 central units (liquid BM/solid BM/database) and a limited number of decentralized subunits. All units adhere to IBDW standards and rules. The build-up process is split into four partly overlapping phases: (1) build up organisation including patient consent and standard operating procedures (SOP), (2) build up liquid biobank, (3) build up tissue biobank (4), implement unified IT infrastructure and interface to the clinical database. All data within the IBDW will be stored pseudonymized and are protected by an independent gatekeeper. Results. Together with the local ethics committee a patient and a proband consent has been developed. The individual subject donates BM to the IBDW for future research for an unlimited time period. The consent allows collecting specified amounts of BM from an individual subject once within a pre-specified time period. Processing of liquid BM samples is highly automated to ensure a high quality pre-analytic phase. All BM is managed by a Biobank Management System which tracks all handling and processing steps of a sample starting with its acquisition until storage of the sample or its aliquots in the cryo-repository. Quality control algorithms are currently implemented. Several existing BM collections within the University Hospital have been identified to be integrated into the IBDW.

Conclusions. The IBDW offers a unique and professional service to both clinicians and researchers bringing the “two worlds together”. The highly standardized and centralized way of collecting BM and corresponding data secure top quality BM and corresponding clinical data. This will serve as a basis for high quality research within the local Medical Campus and for national and international networking.

SA-P-110 A modular and virtual microscope platform based on Eclipse Technologies M. Flügge1, N. Zerbe1, P. Hufnagl1 Charité – Universitätsmedizin Berlin, Institute of Pathology, Berlin

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Aims. Virtual microscopy is a fast growing field in histology based medical research. The base of every virtual microscopy system is digital microscope software, thus several implementations, free and commercial, are available. The main challenge of most implementations is the lack of customizability. Often the microscopes are closed in there functionality and not design to be extended by a third party. But his in turn is often required especially in research. We demonstrate a new framework that allows extending the behavior of the virtual microscope with new features in an easy and flexible way. The system is rather a base for a new virtual microscope system than a pure microscope with limit functionality. Thus it is designed with the strong focus on extensibility. Methods. To build up this extensible platform we are making an extensive use of Eclipse based technologies expressively the Rich Client Platform (RCP) architecture and the Eclipse Modeling Framework (EMF). Using the plug-in concept allows to provide a simple core which can be extended to more sophisticated applications by simply plug-in in additional functionality. As the platform in written in Java, it can be easily combined with other Java-based technologies or frameworks. Results. We demonstrate an extensible platform which allows adapting new contexts easily to the scope of virtual microscopy. Leveraging the advantages of modularizable OSGI/RCP based applications the components of the platform can be plugged together to provide highly specialized microscopes for different fields of application. This allows building up clean and non-overloaded user interface to simplify the work of pathologists. An extendible layer concept in combination with a fine grained request/notification mechanism ensures that additional functionality (e.g false color visualization) can be easily adopted leveraging automatically build-in support for zooming, panning, rotation and other editor operations. This is available for standard sized images as well as whole slide images (WSI). Image access is hidden behind an open service provider interface which allows to plug-in own implementations. Conclusions. The introduced architecture allows easy and fast integration of new functionality to the basic microscope which allows creating highly customized solutions for special field of applications in term of virtual microscopy. This reduces the costs and increases the speed of implementing such applications.

data and probes that have been collected in compliance with legal and ethical regulations. Methods. Under coordination of popgen, a well established populationbased biobank, P2N will comprise the data and probes of seven collections, namely of popgen itself, the Cancer Centre North, the Department of Neurosurgery, the Institutes of Pathology and Pharmacology, the University Lung Centre North and the Centre of Family Medicine. In order to harmonise, standardise and ease access to these manifold probe types together with the both comprehensive and sensible data attached to them, P2N has performed a survey of the participating biobanks by evaluating infrastructure, workflows, probe and data types, recruitment and consent policies. In addition, P2N has analysed the portfolios of the leading laboratory information management system (LIMS) suppliers. Results. A shared network of peripheral biobanks with centralised access is merely as useful as the consistent and uniform quality of the probes and data collected and provided by the network partners. Due to the broad heterogeneity of the participating probe and data collections the usage of one common efficient albeit flexible LIMS is a crucial ingredient for a properly functioning biobank network. Equally important is a standardised handling of the probes itself and the accompanying data. Ideally, these routines must adequately be supported by the LIMS and a uniform set of SOPs. Conclusions. Within the next 5 years P2N will have set up one of Germany’s largest probe and data collections, comprising approximately probes of more than 800,000 individuals. Standardised IT and quality control infrastructures will facilitate an efficient and data safety-conform access.

SA-P-111 The BMBF Initiative to build up centralized biomaterial banks in Germany: the popgen 2.0-Network U. Nöthlings1, A. Wolf1, A. Rüther1, M. Krawczak1 1 Christian-Albrechts-University Kiel, Kiel Aims. Funded by the Federal Ministry of Education as one of the five facilities supported within the National Biobank Initiative, the popgen 2.0-Network (P2N) will merge seven data and probe collections currently existing at the Medical Faculty of the Christian Albrechts-University (CAU) Kiel. This network will aim towards a uniform and centralised research infrastructure which will enhance scientific usability by means of fast and easy access to vast quantities of quality-controlled

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Autorenregister

Autorenregister A Abdullah M. FR-P-131 Adam P. DO-051 Agaimy A. DO-097, DO-098, FR-P-056, FR-P-059, FR-P-154, SA-P-077, SO-058 Akpalo H. DO-080 Al-Mohamed H. FR-P-115 Andreozzi M. FR-P-128 Anlauf M. FR-003 Arend J. FR-P-055 Ates G. FR-P-164 Aulmann S. SA-P-009 Aumann K. DO-076 Aust D. VO-011

B Baldensperger M. FR-P-048 Bandapalli OR. SO-007 Barros M. DO-062 Barth PJ. SA-P-017 Barth TFE. FR-P-074 Batarello D. DO-020 Bauer K. FR-P-081 Bauer L. DO-018 Beckervordersandforth J. FR-014 Behnes CL. FR-P-170, SA-P-091, SA-P-092 Bektas Serce N. SA-P-018 Belharazem D. SA-P-067, SA-P-068 Benedix F. FR-P-076 Berchtold S. SO-009 Berezowska S. DO-016 Berg T. FR-P-173 Bergmann F. FR-P-097 Bian X-W. SG-136 Biesterfeld S. FR-P-143 Bihl MP. FR-P-060, SA-P-064 Bläker H. VO-003 Blank P. FR-P-078 Blutke A. SA-P-094 Bockmayr M. DO-078 Bockmeyer C. DO-107 Böger C. FR-P-066 Böhm F. FR-P-109 Bombari D. SO-018 Bonzheim I. DO-048 Boor P. FR-017, SO-075 Boruschek U. DO-055 Bösmüller H. SA-P-025 Brachtel E. SO-025 Brandt S. FR-P-171 Braun A. FR-P-057 Braun M. DO-111, SA-P-078, SA-P-079 Bremmer F. SA-P-100 Brochhausen C. FR-P-093, SA-P-102 Brockmoeller S. FR-011 Broecker V. SA-P-093, SO-072 Bronsert P. FR-P-091, SO-023

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Budczies J. FR-030 Burandt E. SO-032, SO-033 Bürger H. SA-P-006, SA-P-012 Bussmann C. VO-013 Büttner M. SA-004

C Cacchi C. FR-002 Calvisi DF. DO-006, SO-012 Carneiro F. VO-024 Cathomas M. FR-P-047 Chakilam S. SO-001 Chen Y. FR-P-132, SA-P-054 Clauditz TS. SA-P-032 Conradi L-C. FR-P-079 Cortis J. FR-P-136 Csanadi A. FR-P-133, FR-P-134 Cui T. SG-P-119, SG-135 Culemeyer L. FR-P-156

D Dach C. SA-P-106 Dämmrich M. DO-108 Datta K. VO-037 FR-013 de Jonge M. Decker T. SO-029 Dellmann A. SA-P-086 Denkert C. SO-031 Dihlmann S. DO-103 Doberenz E. SO-041 Donhuijsen K. SA-P-043 Dreyer J. DO-087

E Ehling J. SO-017 El-Baba C. SA-P-066 Elfimova N. DO-004 Elsner M. FR-P-037, FR-P-042 Erber R. SO-035 Evert M. DO-007

F Fang W-g. SA-P-015, SG-141 Fichter CD. FR-P-041 Floßbach L. DO-056, DO-057 Flügge M. DO-073, SA-P-110 Focke C. SO-027 Franken L. SO-036 Franz M. FR-P-114 Franzen A. DO-034 Frick L. FR-P-107 Friedl S. SA-P-103 Friedrich K. SA-P-014, SA-P-019 Friemel J. FR-P-099 Fritsche R. DO-120 Fronhoffs F. FR-P-162, SA-P-073, SO-054 Fuchs D. FR-P-092 Funke S. SA-P-047

G Gaida MM. DO-081 Gaisa NT. SO-065 Gaiser T. DO-023, FR-019 Gajda M. SA-P-034, SA-P-089 Gaßler N. SG-P-112, SG-128 Gdynia G. SO-006 Geddert H. FR-P-058, FR-P-075, FR-P-149, SA-P-024 Gerlach S. DO-110 Giedl C. SA-P-045 Goeppert B. FR-P-103 Göke A. FR-P-090 Göke F. DO-091, DO-093 Goltz D. DO-109, FR-P-112, FR-P-113 Gradhand E. FR-P-068 Griesser H. SO-019 Griff S. FR-P-065 Grob T. SA-P-011 Gröschl B. DO-123 Große K. FR-P-120 Grote HJ. SA-P-053 Grüning J. FR-P-167 Gündisch S. FR-027 Gutschner T. DO-033

H Haab M. SO-010 Haag J. FR-P-050 Hahn C. DO-069 Haller F. SO-014 Hammer S. SA-P-026 Hämmerle M. DO-003 Hansen T. FR-P-095, FR-P-102, FR-P-163, SA-P-033 Haroske G. DO-002b Haugg A. FR-006 Häupl B. SA-P-087 Hehne S. FR-P-147 Heikaus S. SA-P-090 Heilmann T. SO-045 Heindl S. DO-017 Helmchen B. SA-P-031 Helpap B. SA-P-081 Henopp T. FR-004, FR-005, FR-P-180 Herbach N. SA-001 Herpel E. SA-P-108 Herrmann TS. FR-P-098 Heublein S. SA-P-021 Hiemann NE. FR-P-117, FR-P-118, FR-P-119 Hirsch D. SA-P-042 FR-P-129 Hiththetiya K. Högler S. FR-P-126 Höhn AK. SA-P-099, SO-020 Höller L. FR-P-124 Höller S. SO-039 Horn L-C. SA-P-027, SA-P-030 Horst D. SG-P-114, SG-130 Hui C. FR-P-086

Huss S. Huth S.

SA-P-049, SA-P-058 SG-P-130, SG-144

I Ikenberg H. SA-P-040 Ikenberg K.

FR-012, SA-P-039, SA-P-028

J Jakubzik A. SA-P-097 Jarrin Franco MC. SO-021 Jayasinghe C. FR-P-071, SA-P-071 SO-052 Jiang X. SG-P-132, SG-146 Jie Z. FR-P-087 Jöhrens K. DO-063 Jonigk D. DO-032 Jungbluth A. SO-049 Just A. FR-P-053

K Kalinski T. SA-P-016 Karimi D. FR-P-036 Kayser G. DO-027, DO-068 Keck B. SO-066 Kelterborn J. FR-P-104 Kemter E. SO-069 Kettelhake A. SA-P-062 Kitz J. SO-003 Klauke M-L. SA-P-010 Klaus C. SG-P-113, SG-129 Klauschen F. SA-P-055 Kleine M. DO-112 Klempert I. FR-P-172, SA-P-101 Kloten V. SG-P-129, SG-143 Kluth M. FR-035 Knaup S. DO-115 Knöß P. DO-086 Knüchel-Clarke R. VO-019 Koitzsch U. DO-113 Kollecker I. SA-P-088 Koller K. SO-047 Kommoss S. SA-P-023, SO-022 König K. SA-P-051 Korsching E. DO-077 Kosmidis P. DO-049 Kratochvil D. FR-P-174 Krause M. VO-035 Kriegl L. DO-024 Kristiansen G. VO-020 Krüger U. FR-P-105 Küffer S. DO-125 Kuhlmann M. FR-P-168 Kuhne HP. FR-P-116 Künstlinger H. FR-033 Kurth R. FR-P-141 Küttner B. SG-P-115

L Lasitschka F. DO-046 Lehmann A. FR-031 Lehmann U. DO-117 Lehnen NC. FR-P-094 Leisten I. DO-045 Lenggenhager D. FR-001 Lennerz JK. FR-P-151, SO-042 Lenze D. SG-P-133, SG-147 Li XZ. SG-P-117, SG-133 Linge A. SO-037 Liu C. FR-P-160 Lohneis P. FR-P-152 Longerich T. FR-018 Löser H. DO-124, FR-P-165, SO-040

M Ma Y. SG-P-125, SG-138 Macher-Göppinger S. SO-073 Maegel L. DO-079 Mahajan V. SA-P-038, SA-P-063 Mairinger F. DO-114, FR-032 Majores M. SA-P-082 Malz M. FR-P-137 Marienfeld R. SA-P-056 Märkl B. SA-003 Marwitz S. FR-P-144 Marx AH. FR-P-039 May AM. DO-043, FR-P-159 Mehta AK. FR-P-100, SO-013 Meijer GA. VO-005 Menon R. FR-029 Menter T. DO-059, SA-P-096 Messner I. FR-P-082 Metzig M. FR-P-080 Meyer A-S. FR-026 Michels S. SO-015 Michl P. VO-031 Miehlke S. VO-010 Mietzsch F. DO-118 Mihic-Probst D. SA-002 Moch H. VO-018 Mogler C. FR-P-106 Mollenhauer M. DO-089 Mones D. FR-P-043 Morawietz L. DO-102 Mößinger K. SA-P-060 Muders M. SG-P-126, FR-P-089, SO-061, SG-139, VO-038 Mühling B. FR-P-123 Müller AM. SA-P-070, SA-P-074, SO-51, SO-055 Müller BM. SO-030 Müller S. DO-122 Münch C. FR-P-155 Munding J. DO-021

N Nelson PJ. SA-P-048 Nettersheim D. SO-068 Neumann J. SO-005 Neumann M. SO-043 Neumann M. SA-P-109 Neumann O. SO-011 Niendorf E. DO-064 Nitta H. FR-021

Noske A. SA-P-029 Nöthlings U. SA-P-111

O Ommer KS. DO-001a Omran H. ABS-888, SO-038 Ormanns S. DO-121 Otto M. FR-P121, FR-P-122

P Papadoupoulus N. VO-007 Papadopoulos T. FR-008 Pawlaczyk N. FR-P-169 Pelisek J. DO-101 Pellegrino R. DO-005 Perner S. VO-015 Perren A. VO-039 Petersen M. FR-P-054, FR-P-064, FR-P-101 Pfaltz K. SO-034 FR-P-125 Pfister F. Piscuoglio S. DO-022, FR-P-083 Poehlmann A. SO-002 Poremba C. DO-085 Porubsky S. SA-P-095

Q Quan P.

SO-016

R Rao C. SG-P-123 Rao C. SG-P-116, SG-132 Rau TT. FR-P-069, FR-P-070 Rechsteiner M. FR-024 Reis H. SA-P-084 Reissig K. SA-P-052 Rennspiess D. DO-047 Rezaei M. VO-034 Ribback S. FR-P-110 Richter G. FR-010, SA-P-007 Richter P. DO-094 Riedl E. DO-044 Risio M. VO-001 Röcken C. VO-026 Rokahr J. SA-P-083 Rose M. FR-028 Rößler J. SA-P-037 Roßner M. DO-070 Roth W. VO-033 Rüping K. DO-082 Rupp NJ. SO-063 Rüschoff J. VO-040

S Sailer V. SA-P-076, SO-057 Schaefer I-M. FR-P-061, FR-P-063 Scharbatke EC. FR-P-052 Scharenberg C. DO-092 Scheil-Bertram S. SA-P-020 Schierle K. FR-P-062 Schildgen V. SA-P-035 Schildhaus H-U. DO-035

Schirmacher P. DO-036 Schmauder M. SA-P-107 Schmidt J-A. DO-066 Schmidt R. DO-075 Schmitt B. SA-P-036 Schmitz-Rixen T. DO-099 Schnabel PA. FR-P-139 Schneider N. FR-P-072 Schneider J. DO-084 Schneider-Stock R. FR-007 Schöne C. SA-P-013 Schoner K. SA-P-075, SO-056 Schöpfer A. VO-012 Schrader T. DO-001b, DO-074, SA-P-104 Schrader C. DO-050, DO-053, FR-P-077, FR-P-084, FR-P-085, FR-P-088, FR-P-153, FR-P-158 Schramm M. FR-P-111 Schröck E. FR-022 Schröder J. SA-P-098 Schultz H. FR-P-130 Schuster C. DO-116 Schwertheim S. FR-P-178, FR-P-179 Seitz V. DO-061 Senft E. DO-090 Shaikhibrahim Z. SO-062 Siegmund B. VO-008 Simon E. FR-P-108 Simon F. DO-104 Simon-Keller K. SO-046 Sinn BV. SO-008 Sinn H-P. SO-024 Sipos B. VO-029 Slotta-Huspenina J. FR-P-040 Söder S. FR-P-176 Soltermann A. FR-020 Spilka R. SA-P-085 Steffen JS. FR-P-051 Steiner T. SO-071 Steinhilber J. DO-065 Stenzinger A. DO-088 Sterlacci W. DO-026 Stöhr R. FR-016 SO-064 Stomper J. DO-083 Straub M. Szczepanowski M. FR-P-148

V

T

Xu J. Xu J.

Tannapfel A. VO-030 Teller A. FR-P-045 Thies SA. FR-P-044 Thorns C. DO-054 Tiede C. DO-095 Timme S. DO-015, SA-P-008 Tindall DJ. VO-027 Tischler V. SG-P-131, SG-145 Titze U. SA-P-072, SO-053 Tóth C. FR-P-166 Trapani F. SO-004 Troidl K. DO-100 Tzankov A. DO-058, DO-060

U Ullrich A.

VO-014

Varga Z. SA-P-005, SO-026 Veits L. FR-P-038 Venkataramani V. SA-P-065 Verdoodt B. SA-P-080 Vlajnic T. FR-P-046 Vogel UF. FR-P-127 Vogetseder A. SA-P-057 Vogt M. SG-P-127, SG-140 Vogt N. DO-052 Vokuhl C. SO-048 Vollmer E. DO-025 von Laffert M. FR-015 von Winterfeld M. FR-P-067

W Wachter DL. FR-P-096 Wagner S. FR-034 Walluks K. SA-P-061 Walther B. FR-P-177 Wang L. SG-P-121 Wang X. FR-P-145 Wardelmann E. SA-P-041, VO-017 Warneke V. FR-023 Warth A. DO-028, DO-029, FR-P-135, FR-P138, FR-P-140 Wassilew K. DO-106 Weber A. FR-P-049 Weiler C. DO-096 Weinberg RA. VO-032 Wenke A-K. SA-P-044 Westphal S. FR-P-161 Wethkamp N. SA-P-050 Wieczorek K. SA-P-069, SO-050 Wielockx B. FR-P-157, VO-036 Wienert S. SA-P-105 Winkler J. DO-002a Wirtz R. SO-067 Wölfel C. FR-P-175 Wötzel F. FR-P-142 Wuensch T. FR-P-073 Wulf L. SO-044 Wyler-von Ballmoos L-G. SO-074

X SA-P-059 SG-P-122

Y Yasui W. VO-025 Ye M. SA-P-046

Z Zeiske T. FR-P-146 Zeng Y. SG-P-118, SG-134 Zerbe N. DO-067, DO-071, DO-072 Zhang J. SG-P-124, SG-137 Zhang W. SG-P-120, SG-142 Zhang X-x. FR-P-150 Zimmermann A-K. SA-P-022 Zimpfer A. FR-009

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Rudolf-Virchow-Preis Ausschreibung 2013 Der Preis wird laut Satzung der Rudolf-Virchow-Stiftung und der Deutschen Gesellschaft für Pathologie einem Pathologen unter  Jahren für eine noch nicht veröffentlichte oder eine nicht länger als ein Jahr vor der Bewerbung publizierte wissenschaftliche Arbeit verliehen . Die Verleihung des Preises erfolgt auf der  . Jahrestagung der Deutschen Gesellschaft für Pathologie e . V .  . Zusammen mit einem Lebenslauf und einer Publikationsliste reichen Bewerber ihre Arbeit ein . (Bitte alle Unterlagen in doppelter Ausfertigung sowie elektronisch einreichen!) Abgabetermin: bis zum . Dezember  Einzureichen bei: Prof . Dr . med . Holger Moch Geschäftsführendes Vorstandsmitglied der Deutschen Gesellschaft für Pathologie e .V . UniversitätsSpital Zürich Institut für Pathologie Schmelzbergstrasse  CH –  Zürich

Deutsche Gesellschaft für Pathologie e . V . – Vorstand – Frühjahr 

© neumann-meding

 

Abstracts

Ergänzung zu: 96. Jahrestagung der Deutschen Gesellschaft für Pathologie e. V. Berlin, 31. Mai – 3. Juni 2012

AG Informatik in der Pathologie DO.067 2nd International Scanner Contest in Berlin – First results

DO.068 Mandatory minimum standards for routine virtual microscopy – consensus of the working group IT of the German society of Pathologists in cooperation with the professional association of German Pathologists

N. Zerbe1, F. Klauschen1, A. Alekseychuk2, G. Kayser3, P. Middel4, P. Hufnagl1 1 Charité - Universitätsmedizin Berlin, Institut für Pathologie, 2TU Berlin, Computer Vision and Remote Sensing, 3Universitätsklinikum Freiburg, Institut für Pathologie, 4PATHOLOGIE NORDHESSEN, Institut für Pathologie

G. Kayser1, G. Haroske2, W. Schlake3, M. Werner1, M. Dietel4, P. Hufnagl4 1 Institute of Pathology, University Hospital Freiburg, Freiburg, 2Institute for Pathology, Hospital Dresden Friedrichstadt, Dresden, 3Berufsverband Deutscher Pathologen, Berlin, 4Institute of Pathology, Charite Berlin, Berlin

Aims. The current fast development in Digital Pathology based on availability of high quality images digitalized by slide scanners. Different technologies are used to create Whole Slide Images (WSI). The pathologists‘ requests are a short scanning time, high quality of WSI and the availability of different illumination technologies e.g. fluorescence. From pathologists‘ point of view it is impossible to compare the different scanners and evaluate the functionality, quality of scanning process and post processing tools. During the 2nd International Scanner Contest 2012 scanner of various vendors were tested in different domains to compare the results. The Second International Scanner took place at the Week of Pathology 2012, here in Berlin. Methods. The scanner were evaluated in five different domains: 1. High Throughput - 35 slides should be scanned automatically 2. Quality Scan - 10 slides should be scanned manually to get the best possible image quality. 3. Fluorescence Scan - 3 slides with Quantum Dots should be scanned manually. 4. Image analysis - Algorithms to quantify Ki67-stained breast cancer TMA dots were compared to a manual evaluation 5. Technical - Calibration slides for geometry and color were digitalized and analyzed to calculate differences and compare them. During the contest different technical parameter were measured and compared such as scanning time and power consumption. Results. Different scanners were tested in domains representing requirements in routine pathology, education, and research concerning advantages and weakness of the scanner for special use cases. Pathologists evaluated random selected areas of WSI and rank them and express their opinion about image quality. Conclusions. The International Scanner Contest is an event for vendors as well as for pathologist. Many data were collected and evaluated to specify image quality of WSI. The contest is a good chance to measure and discuss different aspects of image quality. The community of pathologist do not have the same impression about image quality. Due to these differences it is quite important to evaluate images manually by pathologist directly to get data about their preferences and understand their quality criteria.

Aims. Ten years after the first slide scanner became commercially available virtual microscopy is beginning to move into the pathologist‘s routine life. In first trials the feasibility of virtual microscopy has been shown for tissue based diagnostics and companies developing hard- and software are pushing into the market of digital pathology. Nevertheless, minimum standards for digitization of histological slides, the processing of virtual slides and their handling, as well as for the digital graphical display of these have not been set up in national or international associations of pathologists. Methods. To implement a standard to assure the diagnostic quality in digital pathology these standards have to be defined in order to serve the patient and to guide the technology according to the pathologist‘s needs. Issues addressed are needs in actual and optical resolution as well as image processing procedures for example in terms of multi-layer scanning as well as compression algorithms. Results. Thus, we here report the results of our consensus meetings for minimum standards in routine virtual microscopy. Based upon the preconditions of physics, evidence based experiences and the results of the 1st European Scanner Contest prerequisits for routins diagnostic virtual microscopy have been elaborated. Conclusions. We here give a critical review and resulting recommendations of the requirements for virtual microscopy in tissue based digital pathology. These recommendations for minimum standards for routine diagnostic virtual microscopy have been elaborated in conjunction with the German Society of Pathologists and the professional association of German pathologists.

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Abstracts DO.069 An Ontology for Image- and Data Quality for scientific data management C. Hahn1, S. Niepage1, Y. Zhou1, S. Hanß2 1 Charité - Universitätsmedizin Berlin, Institut für Pathologie, 2 Charité - Universitätsmedizin Berlin, Institut für Medizinische Informatik Aims. The Open European Nephrology Science Center (OpEN.SC) as a center for research relevant clinical data discussed the topic of data quality very early. Data quality is a multidimensional entity, represents different aspects such as process or service quality, and depends on use case and purpose. A Data Quality Framework based on a Service Oriented Architecture was suggested including an ontology for image and data quality. Methods. The OpEN.SC platform based on a Service Oriented Architecture with various web services. The backend is an Apache Geronimo-Application Server, the Open Source database engine PostgreSQL is used to store the data from three different resources. The user interface is realized by OpenSource Liferay Portal. The ontolgy developement based on the Delphi-Model. Protege was use to model the ontology. Results. The ontology covers 18 different quality categories with various concrete aspects and measurable characteristic numbers. They are related to use case, application area and responsibilities and tasks of persons who are involved in the data management process. Conclusions. The ontology helps to understand the complexity of quality and can be use to describe and manage quality calculation web services in the Service Oriented Architecture.

DO.070 DICOM PACS integration into Pathowiki - an academic knowledge database M. Roßner1, R. Zwönitzer2, H. Hofmann3, T. Kalinski4 1 University Hospital Essen, Institute of Pathology, Essen, 2Imassense Deutschland GmbH, Berlin, 3University Hospital Magdeburg, Medical Datacenter, Magdeburg, 4University Hospital Magdeburg, Institute of Pathology, Magdeburg Aims. The technical structure of the pathowiki supersedes limitations of traditional media and encourages users to participate in a growing specialized library for pathologists. Like all disciplines in medicine using image-based workflow, there are special requirements that belong to the nature of information handled. In addition to the macroscopic assessment of preparations, the evaluation of microscopic slides presents the major work of pathologists in the routine. The challenge in development of a specialized platform is to recognize the requirements of users in information processing, convert them accurately on a technical level and implement and integrate them into the existing systems. The goal is to achieve an optimal solution based on conventional requirements and advanced technology. Methods. All already existing objects were categorized in a newly created dicom database with object descriptions, relationships and hierarchies. New uploaded objects will be categorized accordingly by the user through a web front end, which is embedded in the wiki. The slides are automatically transcoded into the JPEG2000 format and integrated into the PACS structure. The user retrieves links to the PACS-object(s), which can be inserted into articles of the wiki. Results. The Pathowiki consists of two separate installations in english and german language with over 400 articels and 1026 categorized PACS objects online, which can be easily assigned or retrieved. An PACS integration allows more possibilities for user interaction and comfort with WSIs. PACS objects with assigned information get searchable and can be a ressource for a future extensions like a discussion platform in a separate name space, other academic projects or research groups. The possibility to transfer objects from a routine PACS to the Pathowiki PACS will result in a higher growth rate and more diversity for the users. Open file formats, a distributed network storage at different locations and unique object identifiers are preconditions for a budget-independent, sustainable PACS-Network with a long-term availability of functioning PACS-links and the possibility to balance network load.

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Conclusions. The integration of an independent scientific Pathowiki PACS into the existing mediawiki platform provides users a simplified and more efficient handling of specimens and the ability to use new features that meet the specialized requirements of a modern scientific information system.

DO.071 Virtual Specimen Scout – pre-diagnostic image analysis, quantification, and retrieval in virtual microscopy N. Zerbe1, K. Saeger2, O. Hellwich3, P. Hufnagl1 1 Charité - Universitätsmedizin Berlin, Institute of Pathology, Berlin, 2VMscope GmbH, Berlin, 3Technical University - Berlin, Dept. of Computer Engineering and Microelectronics, Berlin Aims. Image analysis in whole slide images is an upcoming topic in pathology informatics. The virtual specimen scout project is a joint research and technology transfer project of Charité - Universitätsmedizin Berlin, Technical University Berlin, VMscope and Nexus/DIS. We focus on image analysis questions and aim on the creation of a computer assisted diagnosis and content based image retrieval system in histology with breast cancer as a first application field. Methods. Prior to image processing an automatic quality and focus assessment of virtual slides is necessary to identify and reject slides or parts of slides with poor quality. Utilizing parallel processing of image analysis algorithms on whole slide images enables an expeditious quality inspection. A multimodal image registration using feature point extraction and subsequent geometric and texture based validation of correspondences is applied to align sections with different stains from the same tissue block. For texture analysis we employ Haralick- and LBP based methods in low resolution as well as a Texton based approach for high resolution. A subsequent segmentation of nuclei with contour optimization, cluster separation and detection of non-tumor cells is applied to identify diagnostic clues in full resolution. A set of features based on spatial relationships and distribution of histological structures from different stains is used for indexing of reference cases. Results. Several independent applications for quality assessment, registration of multimodal sections from the same tissue block, texture analysis and marker quantification have been developed and results have been reviewed by pathologists. As a first step towards computer assisted diagnosis a content based image retrieval system for breast cancer cases was created and integrated into a web portal solution together with a web based reference case database. Conclusions. The „Virtual Specimen Scout“ supports pathologists in handling of reference cases in the context of a current diagnostic question. The comparison of an actual case to reference cases in the database is convenient especially with respect to an intuitive and real-time alternation of stains/ markers in a virtual microscope. A prerequisite is the a priori registration of all stains from the same block.

DO.072 Block-centric virtual microscopy N. Zerbe, P. Hufnagl, K. Schlüns 1 Charité - Universitätsmedizin Berlin, Institute of Pathology, Berlin Aims. In clinical pathology several use cases exist where viewing of identical histological structures in different slices of the same block is conducive. This requires an alignment of slides, which is currently done manually by the slide reading pathologist as required and just in time. Furthermore, alignment information is not stored permanently. Neither in conventional nor in virtual microscopy an alternation of three or more slides by retaining a relative position is feasible. We address this issue and present a framework to read slides in a more comfortable and intuitive way using computer assisted navigation integrated into virtual microscopes. Methods. Whole slide images contain multiple distortions from different sources in the laboratory and digitalization process. In the proposed WSI interconnection model distortions are described by several layers to provide a normalized representation of tissue. Each layer is associated with a specific distortion and serves as a new level of abstraction. The interconnection model enables a coarse alignment of tissue sections. Further alignment of

sections is done by multi-resolution feature point extraction and subsequent RANSAC filtering. Results are validated using manual inspection and refinement as. Corresponding image locations are saved in a database. Transformations from source locations to destination locations are calculated using the corresponding pairs. Inside the convex hull of all fiducial points a local affine transformation is applied and outside a rigid transformation. Results. The implementation of the interconnection model stack uses REST web services to deliver normalized image data as well as metadata. In addition, a localization service provides a query interface to calculate corresponding image locations for an input location. The services are used in our whole slide image based tumor board meeting system. Conclusions. Computer assisted navigation is a valuable tool to save time in routine pathology. Alignment of histological slides can be preprocessed and it is no longer assigned to pathologists. The proposed framework allows pathologists to examine tissue sections of the same block independent of laboratory and scanner related distortions. Hence, slide reading shifts from a slide centric towards a block centric perspective. Due to the heterogeneity of laboratories, slide scanners, and their integration further standardization with respect to the WSI interconnection model may be useful.

DO.073 S4 –A distributed, cross-platform system for image analysis in histological contexts M. Flügge, N. Zerbe, D. Heim, P. Hufnagl 1 Charité - Universitätsmedizin Berlin, Institute of Pathology, Berlin Aims. Several frameworks for histological image processing and analysis have been developed over the years. Mostly implemented in different languages, like C/C++, Java or C#, they often cannot be combined directly to develop higher level algorithms for analyzing histological images on a specific target platform. Due to the language barriers it is also impossible to combine functionality from these different platforms. Similar limitations exist for semantic reasoning frameworks. To overcome these limitations we developed a platform (S4 , Specimen Scout Service System)which supports sharing algorithms from different platforms in one common system. Thus it allows combining algorithms, which are actually incompatible by their implementation, to build up new functionality. Methods. The primary usage of the system focuses on easy creation of algorithms from different frameworks to allow fast and efficient solutions of tasks in the context of medical research. Instead of re-implementing an algorithm for a primary host platform, the suitability can be tested by simply using an already available algorithm implemented in a different language. To design and develop the architecture we use a wide range of Open Source frameworks and focus on common standards like SOAP, REST and XMI to make the new platform usable with open standards. The system was also designed with a various range of service provider interfaces allowing it to be extended and to be adapted for specific customer needs. Thus it provides an easy integration layer to add additional frameworks to the system to leverage from its platform independent nature. Results. This platform provides web-based access as well as local execution and can be set up flexible to fit the user‘s needs. It also integrates automatic processing of whole slide images (WSI) and parallel algorithm execution to increase the overall performance. To demonstrate the power of this new platform we integrated ImageJ, an Open Source Java-based image processing tool, OpenCV implemented in C, the VMScope #Accessory Imaging Framework which is available in C# as well as Definiens Developer XD which provides access using a SOAP service. Conclusions. Based on the provided system we were able to integrate algorithms which were developed by our project partners into our own digital slide processing environment although we are using actual incompatible systems. Equally our partners were able to integrate our algorithms into their architecture and products.

DO.074 Business Process Monitoring and Analysis in Pathology T. Schrader1, H. Woecht2, S. Krumnow3, R. Pauli4 1 University of Applied Sciences Brandenburg, FB Informatics & Media, Brandenburg, 2Innocon-Systems GmbH, 3Signavio GmbH, 4Städtisches Klinikum Brandenburg, Institut für Pathologie Aims. Concerning current activities of IHE (Integrating the Healthcare Enterprises) Working Group Anatomic Pathology and as result of a European Project COST Action IC0604 „EuroTelepath“ business processes in pathology were described and modeled using the standard BPMN (Business Process Modeling Notation). The main aims of this work were to improve the standardization process in Digital Pathology, increase transparency of all processes in pathology, and establish requisites for implementation of slide scanners into the routine processes. To evaluate standard models with real processes in a pathology department a case tracking system using barcode readers were implemented. Methods. The general diagnostic was modeled in a standard modeling language: Business Process Modeling Notation using the web based modeling tool by Signavio. This model was extended with special data entries to store process data of tracking. The tracking software „nomic“ by InnoCon-Systems manages the tracking data process. During February to November 2011 the diagnostic processes of all cases were tracked. On the the general pathology process 6 points of measurements were defined and barcode reader installed at the working places of the process stage. Results. The data of this observation period allows a deep view inside the pathology processes. Flow information and tracking data were evaluated. Real processes were compared with the model processes of COST Action IC0604. Differences, problems were analyzed to identify reasons and potentials for changes. Conclusions. Business modeling is an useful tool to increase transparency of all involved process. It helps to understand essential process steps, meanderings and the reasons for that. This is an very important information for improving the processes and plan resources in a pathology department. The basic model from COST Action was minimal changed and extended to store real process data. A specialized software for business modeling (Signavio) and tracking (nomic) supports the process monitoring directly. These data can be used for further analysis and simulations of process changes.

DO.075 Centralizing Biomaterial Banks using a computer-based Biobank Management System R. Schmidt1, C. Spreckelsen2, J. Jäkel1, R. Knüchel1, E. Dahl1 1 RWTH Aachen / Institute of Pathology, Aachen, 2RWTH Aachen / Institute for Medical Informatics, Aachen Aims. A centralized Biobank Management System (BBMS) shall be integrated in order to improve biomedical research investigations by providing ubiquitous access to a comprehensive sample research database. Sample collections of pre-existing as well as new biobank projects shall be managed by the centralized BBMS. The samples shall be annotated with prior specified, consented, and well-structured metadata, documenting the individual sample characteristics, such as the donor‘s clinical history or research results. Furthermore, a search interface needs to be realized, allowing complex database queries with respect to the samples‘ metadata annotations. Methods. Semantic Mediawiki serves as central communication medium allowing a consented specification of research projects. The BBMS StarLIMS is deployed, meeting the local requirements of: flexible metadata annotation, complex queries according to metadata characteristics, and projectspecific storage and rights management. An automatism is realized to transform the pre-existing sample collections and its‘ (semi-)structured sample documentations in order to be captured by the StarLIMS database. Results. Biobank WiKi Aachen (BIWIKA) is an adapted Semantic Mediawiki software system extended by several interfaces allowing convenient specification of: research projects, structured documentation parameters, and their characteristics, such as definition or domain. BIWIKA allows consenting discussions on research project aspects and standard operating procedures. StarLIMS has been configured according to the local precondi-

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Abstracts tions and extended by new interfaces allowing convenient sample submission, individual metadata documentation, and complex query submission. The specified and consented projects in BIWIKA are implemented in StarLIMS accordingly. The StarLIMS database comprehends the pre-existing sample collections, project-specific sample collections, and their structured metadata documentation respectively. Complex research investigations and statistical analysis can be performed. Conclusions. Research investigations on biomaterial samples are significantly improved by a centralized BBMS providing a comprehensive view on the characteristics of in parts spatially distributed sample collections. Investigations with respect to the samples‘ prior specified, consented, and structured metadata characteristics can be performed as well as statistical analysis. Hence, long, tedious, and error-prone investigations for qualified biomaterial for research are omitted.

DO.076 Template based synoptic reporting improves the quality of pathology reports of lung resection specimens K. Aumann1, D. Amann1, G. Kayser1, D. Hauschke2, B. Passlick3, M. Werner1 1 University Medical Center, Institute of Pathology, Freiburg, 2University Medical Center, Department of Medical Biometry and Medical Informatics, Freiburg, 3University Medical Center, Department of Thoracic Surgery, Freiburg Aims. Usually, pathology reports are textual with a high degree of variability. In part, they miss some of the information needed, e.g. for therapy decisions. To meet all requirements, it would be helpful to have a tool providing reminders of the necessary data and facilitating the transfer of these data into a pathology information system (PIS). Here, we describe a TNM-adapted toolset including a PIS-integrated structured template that contributes to improving pathology reports of lung resection specimens. Methods. All reports of lung resection specimens of the Institute of Pathology, University Hospital Freiburg, between 01/02 and 04/11 (n=881) were classified into descriptive reports (DR, n=312), structured reports arranged in TNM relevant tumor spread, lymph node and resection status (SR, n=356) as well as template based synoptic reports (TBSR, n=213). They were compared regarding the content of organ specific essential data (ED) proposed by the College of American Pathologists, the Royal College of Pathology and the Association of German Pathologists. The amount of recorded ED was summarized and an essential data score (EDS) was calculated. Statistical analyses were performed by using SPSS 16.0. Results. Median EDS of DR was 8, of SR 9, and of TBSR 10. In total, all ED (EDS=10) were included in only 28.1% of the cases; divided into the report types 3.8% of DR, 16.6% of SR and 83.9% of TBSR obtained an EDS of 10. In detail, statistically highly significant differences could be seen comparing the report types regarding the content of tumor size and location, pleura visceralis invasion, histological tumor type according WHO classification, lymphovascular invasion and additional findings like atelectasis or pneumonitis. Conclusions. SRs, and particularly TBSRs, are advantageous as compared with DRs regarding the content of ED and the clarity of the data layout. The use of TBSRs leads to a reduction in failed data transfer and therefore to an increase in the quality of pathology reports.

DO.077 Development of a parallel version of a dependency search algorithm for protein expression data and application to simulated data and TMA data of invasive breast cancer cases E. Korsching1, F. Boecker1, K. Poos2, W. Nadler3, H. Bürger4 1 University of Münster, Medical Faculty, Institute of Bioinformatics, Münster, 2 University of Münster, Medical Faculty, Institute of Bioinfomatics, Münster, 3 Forschungszentrum Jülich, Jülich Supercomputing Centre (JSC), Jülich, 4University of Münster/Utrecht, Institute of Pathology, Paderborn, Paderborn Aims. The efforts to understand the formation and progression of breast cancer are focused on the complex molecular mechanisms of the cell. Not all factors are known up to now and the knowledge on the function and the specific interaction partners of those which are known is still sparse. The

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given approach here tries to act very directly on a set of protein expression data (from tissue microarray experiments, so the more final steady state outcome of cellular regulation) by making only a few assumptions on the assumed interaction scheme. The far goal is to describe the macromolecular interactions of the cellular system in a pathway plan and in turn be able to engineer functionality in predictable way, to e.g. be able to establish specific molecular therapies. Methods. The interaction scheme is hosted on an abstract level of a similarity measure, means monitoring similarities or dissimilarities in a structure of measurements. Because of the combinatorial nature of this specific approach it is a prime objective to speed up the computing time of the given algorithm. As a main result more complex dependency structures can be calculated thereafter than before. The existing algorithm was ported to Fortran a computing language which is dedicated to fast scientific calculations and parallel computing approaches. This compact program was than optimized for distributed calculations. Results. The approach was applied on a multitude of simulated data sets to a) reveal the theoretical properties of the algorithms in more detail and b) analyse the scalability of the algorithm on multiple computing cores. The scalability could be proved and simulated data sets with a given network structure reproduced the observed structures. The stable procedure was than applied to slightly different breast cancer TMA data sets to analyse the sensitivity of this approach in discriminating these two entities. The sensitivity of the approach is high in dissecting differences but might therefore also show differences in the sample collecting strategies and laboratory procedures. Conclusions. The approach to assemble interactions to represent a cellular development or progression state seems to be feasible. The computing demands of this combinatorial approach can in part be softened by scaling the computations across multiple processing units. This approach bears a lot of optimization potential for future improvements.

DO.078 Identification of differential co-expression patterns in breast cancer M. Bockmayr, F. Klauschen, C. Denkert, J. Budczies 1 University Hospital Charité Berlin, Institute of Pathology, Berlin Aims. Differential expression between subtypes of breast cancer has been widely investigated by microarray data analysis. We propose a novel approach which identifies sets of genes that are differentially correlated between two conditions. In these sets, the patterns of correlation change, while the expression levels themselves need not to be different. The identification of condition specific gene co-expression patterns could help to identify structures which are of prognostic relevance or can be targeted by drugs. Methods. We construct gene co-expression networks using the matrix of Pearson correlations generated from the microarray data. The networks are constructed by connecting two genes with an edge whenever the correlation is above a correlation threshold. We propose an algorithm which identifies sets of genes corresponding to a stable change in the network topology over a wide range of thresholds. Thus, the problem of choosing a particular threshold is avoided. The correlation pattern is studied independently of a particular threshold, and insignificant changes in the correlation structure are filtered out. Results. We applied our method to compare the correlation structure in ER+ compared to ER- and in HER2+ compared to HER2- breast cancer. As result, we obtained sets of genes which show differential co-expression between the molecular subtypes of breast cancer. An overlap analysis with gene ontology (GO) categories was done in order to uncover the biological function of the co-expression modules. Conclusions. Our method reveals groups of genes which show differential correlation patterns between the subtypes of breast cancer. This novel approach may help to identify structures of regulation that could serve as prognostic variables or could be therapeutically targeted.