Abstracts der Deutschen Gesellschaft für Pathologie

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Der Pathologe

Supplement 1 • 2008

2. Woche der Pathologie

Berlin, 15.–18. Mai 2008

Editorial Manfred Dietel

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Abstracts der Vorträge und Poster

2. Woche der Pathologie . Jahrestagung der Deutschen Gesellschaft für Pathologie e.V. und . Bundeskongress Pathologie Berlin des Berufsverbandes Deutscher Pathologen e.V. in Zusammenarbeit mit der Internationalen Akademie für Pathologie, Deutsche Abteilung e.V.

Schwerpunkte der Jahrestagung F F F F F F

Systempathologie Rolle der virtuellen Mikroskopie Pathologie und individualisierte Therapie Molekulare Infektionspathologie Pathologie des Ovars Gewebebanken in der Pathologie

Kongress-/Tagungspräsident und Organisation Vorsitz: M. Dietel, Berlin Organisation: M. Dietel, Berlin H. H. Kreipe, Hannover

Sitzung: AG Gastrointestinale Pathologie Sitzung: AG Informatik in der Pathologie Sitzung: AG Hämatopathologie Sitzung: AG Oralpathologie Sitzung: AG Paidopathologie Sitzung: AG Dermatopathologie Symposium: Molekulare Infektionspathologie Symposium: Systempathologie des Gewebes (I) Symposium: Uro-/Nierenpathologie Symposium: Beste Forschungsbeiträge zur Gynäkopathologie Symposium: Infektionspathologie Symposium: Systempathologie des Gewebes (II) Symposium: Gynäkologische Pathologie State-of-the-art lecture Systems Pathology – The new paradigm for multifactorial diseases Symposium: Pathologie des Ovars Symposium: Aktuelle Habilitationen Symposium: Mammapathologie in Screening und Prädiktion Symposium: Klinische Pathologie des Ovars Symposium: Meet the Expert (I) Symposium: Freie Vorträge zur prädiktiven Pathologie Poster: Hämatopathologie Poster: Kardiopathologie Poster: Endokrine Pathologie Poster: Orthopädische Pathologie Poster: Gynäkologische Pathologie Poster: Lunge Poster: Varia Symposium: Pathologie und zielgerichtete Therapie (I) Symposium: Beste Forschungsbeiträge Symposium: Orthopädische Pathologie Symposium: Pathologie und zielgerichtete Therapie (II) Symposium: Aktuelle Entwicklungen in der virtuellen Mikroskopie Symposium: Freie Vorträge zur Gynäkopathologie State-of-the-art lecture Early lesions of the Breast Symposium: Zielgerichtete Therapie beim Kolonkarzinom Symposium: Biobanking in der Pathologie Symposium: Meet the Expert (II) Symposium: Aktuelle Entwicklungen in der Virtuellen Mikroskopie Symposium: Freie Vorträge zu Varia Poster: oberer GIT und GIST Poster: unterer GIT Poster: Leber Poster: Pankreas/Varia Poster: Urologische Pathologie Sitzung: AG Gynäko- und Mammapathologie

Do-001 - Do-025 Do-026 - Do-038 Do-039 - Do-060 Do-061 - Do-070 Do-071 - Do-081a Do-082 - Do-092 Fr-001 - Fr-006 Fr-007 - Fr-011 Fr-012 - Fr-017 Fr-018 - Fr-026 Fr-027 - Fr-028 Fr-029 - Fr-031 Fr-032 - Fr-034

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Fr-035 Fr-036 - Fr-040 Fr-041 - Fr-044 Fr-045 - Fr-048 Fr-049 - Fr-051 Fr-052 - Fr-054 Fr-055 - Fr-058 Fr-059 - Fr-076 Fr-077 - Fr-092 Fr-093 - Fr-099 Fr-100 - Fr-112 Fr-113- Fr-118 Fr-119 - Fr-128 Fr-129 - Fr-163 Sa-001 - Sa-005 Sa-006 - Sa-012 Sa-013 - Sa-018 Sa-019 - Sa-021 Sa-022 - Sa-024 Sa-025- Sa-028 Sa-029 Sa-030 - Sa-033 Sa-034 - Sa-038 Sa-039 - Sa-041 Sa-042 - Sa-044 Sa-045 - Sa-049 Sa-050 - Sa-062 Sa-063 - Sa-080 Sa-081 - Sa-092 Sa-093 - Sa-104 Sa-105 - Sa-150 So-001 - So-017

32 32 33 34 34 35 35 36 40 44 46 49 51 53 62 63 65 65 65 66 67 67 67 68 68 68 69 73 77 80 84 95

Titelbild: Bildagentur adpic · Urheber B. Leitner

Der Pathologe · Supplement 1 · 2008

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Herausgeber 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 Berufsverbandes Deutscher Pathologen Federführende Schriftleitung / Editor-in-Chief Prof. Dr. K.-M. Müller, Institut für Pathologie, Berufsgenossenschaftliche Kliniken „Bergmannsheil“, Universitätsklinikum Bochum Assistenz der Schriftleitung / Assistant Editor-in-Chief Prof. Dr. C. Kuhnen, Institut für Pathologie am Clemenshospital Münster Schriftleitung/Editors Prof. Dr. L. Bubendorf, Institut für Pathologie, Universität Basel, Schweiz Prof. Dr. H. Denk, Institut für Pathologische Anatomie, Universität Graz, Österreich Prof. Dr. W. Feiden, Institut für Neuropathologie, Homburg/Saar

Prof. Dr. H. Höfler, Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar, München Prof. Dr. D. Katenkamp, Institut für Pathologie, Universitätsklinikum Jena PD Dr. T. Mentzel, Dermatologische Gemeinschaftspraxis Friedrichshafen Prof. Dr. H. Nizze, Institut für Pathologie der Universität Rostock Prof. Dr. W. Saeger, Institut für Pathologie des Marienkrankenhauses Hamburg Prof. Dr. D. Schmidt, Institut für Pathologie, Referenzzentrum für Gynäkopathologie, Mannheim Prof. Dr. A. Schmitt-Gräff, Abt. Allgem. Pathologie und Pathologische Anatomie, Albert-Ludwigs-Universität, Freiburg PD Dr. M. Werner, Institut für Pathologie, Vivantes Klinikum Neukölln, Berlin

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Der Pathologe · Supplement 1 · 2008

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Editorial

2. Woche der Pathologie mit der

92. Jahrestagung der Deutschen Gesellschaft für Pathologie e. V. und dem

8. Bundeskongress Pathologie Berlin des Berufsverbandes Deutscher Pathologen e. V. in Zusammenarbeit mit der

Internationalen Akademie für Pathologie, Deutsche Abteilung e. V. Berlin, 15.–18. Mai 2008

Liebe Kolleginnen und Kollegen, es ist mir eine große Freude, Sie zur 2. Woche der Pathologie hier in Berlin begrüßen zu dürfen und ich freue mich besonders darüber, dass es erneut gelungen ist, die wissenschaftliche und die der Fort- und Weiterbildung sowie der Krankenversorgung verpflichtete Pathologie zusammenzuführen. Dies ist ein der Voraussetzungen, um die Kräfte zu bündeln und unser Fach gemeinsam auf allen Ebenen weiter zu entwickeln. Hier im Abstrakt-Band der 92. Tagung der DGP hat nun insbesondere die Wissenschaft das Wort. Die insgesamt 300 Beiträge legen Zeugnis dafür ab, das im deutschsprachigen Raum die Attraktivität und wissenschaftliche Potenz der universitären Pathologie nach wie vor hoch ist und insbesondere von vielen jüngeren Kollegen als berufliche Chance erkannt wird. Betrachtet und vergleicht man das wissenschaftliche Niveau der Arbeiten mit denen anderer internationaler Kongresse so wird deutlich, dass die deutsche Pathologie einen führenden Platz in der internationalen Scientific Community einnimmt. Eine Tatsache die uns anspornen sollte, diesen Weg zukünftig intensiv weiter zu verfolgen, um international ein noch deutlicheres Profil zu erreichen. Bemerkenswert ist dabei die zunehmende Bedeutung der sog. „Translational Research“ in der, wie kaum in einem zweiten medizinischen Fach, die wissenschaftlichen und diag-

nostischen Aufgaben in gegenseitiger Ergänzung entwickelt werden. Beispiele dafür sind die über einen langen Zeitraum entwickelte in-situ-Hybridisierung zur Detektion der Her-2 Amplifikationen im Mammakarzinom oder – ganz aktuell – die Mutationsanalyse des KRAS-Gens im Kolonkarzinom. Dies sind Prozesse die exemplarisch verdeutlichen, dass sich das Aufgabengebiet unseres Faches aufgrund wissenschaftlicher Aktivitäten ernorm erweitert hat und die Pathologie zunehmend in das Zentrum der diagnostischen Entscheidungen rückt. Dies gilt speziell für den Einsatz der zielgerichteten Medikamente (Targeted Therapy), die als neue Herausforderung für unsere Fachdisziplin angesehen werden kann. Dies ist zweifellos ein ausgesprochen spannendes Aufgabenfeld, welches ganz sicher in der Zukunft sich noch erheblich erweitern wird. Man denke nur an Bronchial-, Plattenepithel- und Urothelkarzinome etc., gegen die in absehbarer Zeit auch zielgerichtete Medikamente vorliegen werden und für die jeweils diagnostische Tests durch die Pathologie entwickelt werden müssen. Ganz wichtig dabei ist, dass die moderne molekulare Diagnostik nach primärer Entwicklung an der Universität, in die breite diagnostische Pathologie getragen wird und dass z. B. in vernetzter Kooperation es gelingt, eine flächendeckende Versorgung sicherzustellen. Hierfür ist von entscheidender Bedeutung, dass die DGP, der Berufsverband sowie die IAP eng zusammen-

arbeiten, damit auf allen drei Ebenen die entsprechende Expertise entwickelt und vermittelt wird. Worauf wir gemeinsam besonders achten sollten ist, dass die neuen molekularen Techniken durch Qualitätskontrollen der Pathologie abgesichert sind. Die forschende Pathologie wird zukünftig als treibende Kraft dieser Entwicklungen eine noch wichtigere Rolle spielen, so dass die wissenschaftliche Ausrichtung gestärkt und die Unterstützung junger, wissenschaftlich interessierter Mitarbeiter intensiviert werden müssen. Ein kurzes Wort zu unserem neuen Publikationsorgan für die Abstracts. Aus verlagsinternen Gründen sah sich unser früherer Vertragspartner nicht mehr in der Lage die Abstracts zu publizieren. Daher hat sich der Vorstand entschlossen, das Angebot des Springer-Verlages anzunehmen und die Beiträge in „Der Pathologe“, dem Organ der deutschen, österreichischen und schweizerischen Gesellschaft für Pathologie sowie der deutschen Abteilung der IAP und des Berufsverbandes Deutscher Pathologen, zu publizieren.

Manfred Dietel Berlin, April 2008

Der Pathologe · Supplement 1 · 2008

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Abstracts

2. Woche der Pathologie mit der

92. Jahrestagung der Deutschen Gesellschaft für Pathologie e. V. und dem

8. Bundeskongress Pathologie Berlin des Berufsverbandes Deutscher Pathologen e. V. in Zusammenarbeit mit der

Internationalen Akademie für Pathologie, Deutsche Abteilung e. V. Berlin, 15.–18. Mai 2008

Sitzung: AG Gastrointestinale Pathologie Do-001 Methylierung in GI-Tumoren H. Hermeking Bochum

Neues in der GI Pathologie Do-002 Was ist neu? Regressionsgrading im GI-Trakt Ösophagus sophagus und Magen S.E. Baldus Köln öln ln Kolorektum D. Aust Dresden Pankreas J. Lüttges üttges ttges Saarbrücken Do-003 Eﬀects of Cathepsins B and X Gene Disruption on Helicobacter pyloriinduced Gastric Inﬂammation S. Krueger, D. Kuester, S. Backert1, T. Reinheckel2, A. Roessner Institut für Pathologie und 1 Institut für Medizinische Mikrobiologie, Universitätsklinikum Magdeburg 2 Institut für Molekulare Medizin und Zellforschung, Universitätsklinikum Freiburg Aims: Cathepsins play important roles in degradation and remodeling of the extracellular matrix, thus contributing to cell migration and adhesion. Cathepsins B, L, K and X are differentially expressed in normal and chronically inflamed gastric mucosa. Cathepsin B was the most abundant cathepsin but was found to be unchanged by H. pylori-infection. Only cathepsin X was strongly upregulated in H. pylori-infected patients. The present study aimed to determine the effects of cathepsin B and X deficiency on H. pylori-induced gastric inflammation. Methods: Wild-type C57BL/6 and cathepsins B and L knockout mice (CB-/and CX-/-) were inoculated separately with H. pylori strains SS1, B128 and the newly described 7.13. Mice were killed after 8–24 weeks of infection. Uninfected WT and cathepsin-deficient mice were used as controls. Levels of H. pylori colonization and gastric mucosal inflammation were determined. In

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Der Pathologe · Supplement 1 · 2008

parallel, human gastric epithelial cells in coculture with macrophages were infected with the H. pylori strains and expression of cathepsins, cellular migration and adhesion were examined. Results: All H. pylori strains induced upregulation of cathepsin X in both human epithelial cells and macrophages resulting in induced adhesion and cell spreeding. Cathepsin B was found to be unchanged. SS1 and B128 showed higher colonization levels in the mice than 7.13 irrespective of the mice genotype. After 12 weeks of infection WT and CB-/- mice had developed more severe gastritis than the CX-/- animals. Conclusions: This comparative model of H. pylori-induced gastritis allowed dissection of both strain and host specific effects, to define specific functions of cathepsin X in H. pylori infection.

Do-004 Modulation of Ghrelin in atrophic gastritis T. Rau Pathologisches Institut, Universitätsklinikum Erlangen Aims: Hormonal regulation of the gastric mucosa has a great impact on inflammatory, metaplastic and neoplastic processes in the stomach. The recently discovered gastrotropic anabolic hormone Ghrelin is a new candidate within this scenery. Methods:: Using immunohistochemistry, rodent primary cell culture models and ELISA techniques from blood samples of patients the role of Ghrelin in atrophic gastritis was investigated. Results: Ghrelin could be established as a gastroprotective factor. Morphologically Ghrelin is reduced in atrophic gastritis. The Gastrin-receptor (CCK2) could be localized on Ghrelin-expressing endocrine cells. In primary cell culture Ghrelin was reduced by artificially adding Gastrin. Conclusions: Less is known about the hormonal regulation of the recently discovered anabolic hormone Ghrelin in the human stomach. The loss of Ghrelin as a gastroprotective factor in atrophic gastritis is another example of hormonal dysregulation within this disease. There’s evidence for Ghrelin being embedded in the Gastrin-cyclus as Gastrin reduces the expression of Ghrelin. Thus hypergastrinemia may play a pivotal role in Ghrelin-depletion in atrophic gastritis.

Do-005 Oxidative stress-induced DNA damage in the Barrett´s carcinogenesis C. Fathke1, V. Neumann1, D. Walluscheck1, A. Agaimy2, W. El-Rifai3, A. Poehlmann1, T. Guenther1,4, P.H. Wuensch2, H.U. Schulz5, M. Vieth6, M. Stolte6, A. Rossner1, R. Schneider-Stock1 1 Institut für Pathologie, Universitätsklinikum Magdeburg 2 Institut für Pathologie Klinikum Nürnberg 4 Institute of Pathology Northwick Park Hospital, London, UK 6 Institut für Pathologie Klinikum Bayreuth 3 Institute of Pathology Vanderbilt University, Nashville, USA 5 Chirurgie, Universitätsklinikum Magdeburg Aims: It is widely accepted that oxidative stress plays a significant role in the pathogenesis of Barrett´s esophagus. In order to investigate if oxidative stress induces DNA-damage, we performed in vivo and in vitro studies. Methods: As a sensitive marker for oxidative damage we investigated immunohistochemically the 8-hydroxy-deoxyguanosine (8-OHdG) level in 50 Barrett´s carcinomas (BCa) and 52 precursor lesions (21 Barrett mucosa-BM, 20 low grade dysplasia-LGD, 11 high grade dysplasia-HGD). In parallel, the base excision repair proteins OGG1 and MYH were analyzed immunohistochemically and by mutation analysis (hot spots: Y165C and G382D) on a pyrosequencer, respectively. In addition, three Barrett´s adenocarcinoma cell lines (SEG1, TE7, BIC1) were treated with 100μM H2O2 for 6, 24, or 48 h and induction of oxidative DNA-damage was measured by 8-OHdG staining, COMET assay, γH2A.X expression and cytochrom c release in Western Bloting. Effects on apoptosis and cell cycle distribution were determined by Annexin assay, caspase 3, 8, and 9 cleavage and FACS analysis. Results: The 8-OHdG score differed significantly between BM and BCa. There was one case with a hereditary heterozygous MYH mutation. H2O2 treatment induced severe DNA-damage as early as after 15 min reflected by a dramatic increase in DNA double strand breaks. Oxidative DNA-damage was accompanied by G2/M arrest and apoptosis induction dependent on the p53 status of the tumor cells. Conclusions: Increased DNA-damage during Barrett´s carcinogenesis is associated with a decrease in antioxidant and repair capacity. Otherwise, oxidative stress inducing DNA-damaging agents might have a therapeutical impact in Barrett´s adenocarcinomas.

Do-006 Enhanced activation of epidermal growth factor receptor caused by tumor-derived somatic E-cadherin mutations A. Bremm1, A. Walch2, M. Fuchs1, J. Mages3, J. Duyster4, G. Keller1, K.-F. Becker1, S. Rauser2, R. Langer1, C.H. von Weyhern1, H. Höﬂer1,2, B. Luber1 1 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München 2 Institut für Pathologie, GSF-Forschungszentrum Neuherberg 3 Institut für Mikrobiologie und 4 III. Medizinische Klinik, Klinikum rechts der Isar der TU München Aims: Mutations of E-cadherin and overexpression of EGFR are among the most frequent alterations associated with diffuse-type gastric carcinoma. The aim was to study the effect of E-cadherin mutations on activation of EGFR. Methods: MDA-MB-435S cells transfected with wild-type or mutant E-cadherin were analysed by Western Blot, immunoprecipitation and FACS analysis. Gastric carcinomas were examined immunohistochemically for expression of mutant E-cadherin and phosphorylated EGFR. Results: We report that somatic mutation of E-cadherin was associated with increased activation of EGFR and its downstream acting signalling components Grb2, Shc and Ras. Reduced complex formation of mutant E-cadherin lacking exon 8 with EGFR compared to the wild-type protein was observed. Mutation of E-cadherin also influenced the endocytosis of EGFR. Moreover, we demonstrate increased activation of EGFR in gastric carcinoma samples with mutant E-cadherin lacking exons 8 or 9. Conclusions: The EGFR signalling pathway is one of the most important cancer-related signalling networks. We describe activation of EGFR by mutant E-cadherin as a novel mechanism in tumor cells that explains the enhanced

motility of tumor cells in the presence of an extracellular mutation of E-cadherin.

Do-007 HER2 Status Evaluation in Gastric Cancer – Validation of a Modiﬁed Immuno Scoring System M. Hofmann, O. Stoss1, D. Shi2, E. Reyes3, R. Büttner4, T. Henkel1, J. Rüschoﬀ Institut für Pathologie Nordhessen und 1 TARGOS Molecular Pathology GmbH, Kassel 2 Cancer Hospital, Fudan University, Shanghai, China 3 Instituto Nacional de la Nutrician, Mexico City 4 Institut für Pathologie, Universitätsklinikum Bonn Currently the value of trastuzumab therapy in advanced gastric cancer is under investigation in a large phase III trial (ToGA). How- ever, data about the prevalence of Her2 overexpression and/or amplification in gastric cancer (GC) are contradictory (4–25%). Aims: Validation of Her2 testing algorithm in gastric cancer. Methods: Resection specimens of 168 gastric cancer samples from different countries (Germany, China, Mexico) were tested by IHC (HercepTest) and FISH (pharmDx). Scoring was initially done according to the breast cancer (BC) criteria and critically re-evaluated by an international consensus panel. Data using a modified IHC scoring system will be presented for the TOGA trial with currently 2431 tested GC resection and biopsy specimens. Results: Her2 positivity rate was 10.7% using the BC-IHC scoring system with 83.3% being intestinal type adenocarcinomas. Concordance between IHC and FISH was 93% with 7.4% FISH positivity. However, 11 samples were FISH+ but IHC 2+ (5) or negative (1+: 2; 0:4). Major reasons for this discrepancy was incomplete membrane staining at the luminal boarder of tumour glands as well as weaker and heterogeneous staining in complete stained cells. Due to these oberservations strong (incomplete) basolateral staining and focal (<10%) staining in biopsies was considered positive (3+) by IHC. Accordingly, the prevalence of Her2 positivity in advanced GC is 22% (ToGA data). Conclusions: Her2 immuno-scoring in GC should be modified in order to determine the optimum patient population for Her2 targeted therapy.

Do-008 Role of DNA polymorphisms in genes related to the tumor microenvironment for response and survival of neadjuvant treated gastric cancer patients G. Stocker1, K. Ott2, K. Becker1, F. Lordick2, J.R. Siewert2, H. Höﬂer1,3, G. Keller1 1 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München 2 Chirurgische Klinik, Universitätsklinikum Heidelberg 3 GSF-Forschungszentrum für Umwelt und Gesundheit, Neuherberg Aims: The aim of our study was to evaluate the role of polymorphisms in genes related to the tumor microenvironment for an association with response and survival of neoadjuvant treated gastric cancer patients. Methods: DNA from blood or non tumorous, paraffin-embedded tissues was isolated from 178 patients treated with a platin/5FU based neoadjuvant chemotherapy. Polymorphisms in the following genes were studied by PCR or PCR-RLFP analysis: VEGF (C936T), TNF☐ (G-308A), interleukin-1β (IL-1β; T-511C), interleukin-1 receptor antagonist (IL-1RN; variable tandem repeat polymorphism) and interleukin-8 (IL-8; A-251T). Results: The genotype distribution among the patients was as follows: VEGF(C936T): 76% CC, 21% CT, 3% TT; TNFα (G-308A): 74% GG, 25% GA, 1% AA; IL-1β (T-511C): 12% TT, 48% TC, 40% CC; IL-1RN: 57% long/long, 34% long/short, 9% short/short; IL-8 (A-251T): 18% AA, 51% AT, 31% TT. None of the single polymorphisms showed a statistically significant association with histopathological response. Patients homozy-gote for the short/short genotype of the IL-1RN demonstrated a statistically significant worse survival (log rank test: p=0.032). Combined analysis of IL-1β and IL-1RN revealed a trend for an association with worse response (p=0.107) and a significant correlation with worse survival for the short/short and IL-1β TT genotype (p=0.006).

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Abstracts Conclusions: None of the single polymorphisms is useful for response prediction. The polymorphism in the IL-1RN gene showed a prognostic significance. In particular the combined analysis of IL-1β and IL-1RN genotypes demonstrated a strong prognostic relevance. Comparison with an untreated group of patients should clarify the role of these findings for the neoadjuvant treatment.

Do-009 Functional characterization of Decoy Receptor 3 in chronic inﬂammatory bowel disease B. Funke1, F. Autschbach1, F. Lasitschka1,2, G. Gdynia1,4, S. Macher-Göppinger1, B. Sido3, S. Meuer2, P. Schirmacher1, W. Roth1,4 1 Pathologisches Institut, 2 Institut für Immunologie und 3 Chirurgische Klinik, Universitätsklinikum Heidelberg 4 Deutsches Krebsforschungszentrum Heidelberg Aims: Epithelial barrier dysfunction and apoptosis resistance contribute to the pathogenesis of Crohn’s disease (CD) and ulcerative colitis (UC). Here, we investigated the involvement of the soluble Decoy receptor 3 (DcR3), an inhibitor of CD95L-induced apoptosis, in chronic inflammatory bowel disease (IBD). Methods: The epithelial fraction of human ileal mucosa samples was obtained by laser microdissection. Expression of DcR3 was examined by global gene expression profiling (Affymetrix), quantitative RT-PCR, immunoblot analysis, and immunohistochemistry. DcR3 concentrations in the serum of patients with CD and UC were measured by ELISA. The effects of DcR3 overexpression in intestinal epithelial cell lines were studied by apoptosis and cytotoxicity assays. Results: DcR3 is over-expressed in the epithelial layer of the ileum in patients with CD. DcR3 serum levels are significantly elevated in patients with CD or UC as compared to healthy controls. Increased DcR3 expression results in activation of NF-βB and in protection of intestinal epithelial cells from CD95L-induced apoptosis. Moreover, up-regulation of DcR3 in intestinal epithelia is paralleled by an increased expression of the intercellular cell adhesion molecule-1 (ICAM-1). Conclusions: DcR3 might play a crucial role in IBD by inhibiting CD95L-induced apoptosis of epithelial and immune cells, by inducing NF-βB activation in epithelial cells, and by up-regulating adhesion molecules such as ICAM-1. Further, DcR3 might be useful as a diagnostic marker in IBD.

Do-010 Molecular classiﬁcation of colorectal carcinoma? F. Prall, C. Ostwald Institut für Pathologie, Universitätsklinikum Rostock Aims: To test if by molecular analyses an unselected, consecutive series of colorectal carcinomas would partition into the predicted classes1). Methods: Using DNA from 130 snap-frozen colorectal carcinoma surgical resection specimens collected ad hoc, tumours were assayed for CpG methylation (CIMP) by MethyLight technology at 6 loci (including MLH1 and MGMT; PMR >4); for microsatellite instability (MSI; Bethesda panel); and for K-ras, B-raf, APC, and p53 gene mutation. For chromosomal instability (CIN) status, LOH was investigated at 4 loci (5q, 9p, 17p, 18q; total of 9 markers). Results: Classification based on these data clearly separated a group of 13 tumours CIMP-H, MSI-H, MLH1+, B-raf+, CIN- (sporadic MSI-H, class 1), and a group of 4 tumours CIMP-, MLH1-, MSI-H, B-raf-, APC-, p53-, CIN(HNPCC, class 5), this was even maintained in an unsupervised hierarchical cluster analysis. 4 tu-mours were CIMP-L, B-raf+, MSS/MSI-L, a class predicted to de-velop from serrated lesions without MSI-H (class 2). Classical mole-cular aberrations of the FAP-type adenoma-carcinoma sequence (class 4; CIMP-, MSS/MSI-L, CIN+, and/or p53+, APC+, K-ras+) were observed in 47 tumours; and an additional 24 tumours, though CIN- with the markers investigated, according to the remaining ab-errations also very likely belonged to these. 13 tumours fulfilled all criteria of a “mixed” class (class 3; CIMP-L, MSS/MSI-L, K-ras+). But for an additional 25 tumours assignment to either class 3 or class 4

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remained uncertain. Serration was a frequent histologic feature only in class 1, and (partial) mucinous differentiation was frequent in classes 1 and 5 Conclusions: By molecular typing, classes 1, 2, and 5 can be delineated with confidence, but distinction between classes 3 and 4 remains problematic in a substantial fraction of tumours. ) Jass JR. Classification of colorectal cancer based on correlation of clinical, morphological and molecular features. Histopathology /–. 

Do-011 The β-catenin target gene FGF-2 is expressed in a small fraction of tumour cells showing characteristics of cancer stem cells in human colorectal adenocarcinomas S.K. Scheel1, B. Das2, T. Brabletz3, F. Hlubek1, C. Knoll4, A. Haynl1, T. Kirchner1, A. Jung1 1 Pathologisches Institut, Universitätsklinikum München (LMU) 2 Institute of Medical Science, University of Toronto, CA 3 Chirurgische Klinik, Universitätsklinikum Freiburg 4 Pathologisches Institut, Universitätsklinikum Erlangen Aims: Tumour cells at the invasive front of human colorectal cancers (CRCs) with nuclear expression of β-catenin indicate poor prognosis and were proposed to be migrating cancer stem cells (CSC). As maintenance of stemness in cell culture is strictly dependent on the presence of FGF-2, we hypothesized that CSC might produce this factor in an autocrine manner. Methods: Immunohistochemistry (IHC), RT-PCR, Western Blot, RNA interference (RNAi), quantitative PCR (qRT-PCR), FACS, mouse xenograft model, luciferase reporter assay, electrophoretic mobility shift assay (EMSA), ChIP (chromatin immunoprecipiation). Results: EMSA and ChIP proved that β-catenin/TCF4 directly bind the FGF-2 promoter/ enhancer and confer transcriptional activity when doing luciferase reporter assays. FGF-2 expression was found in cultured human colorectal tumour cell lines shown by RT-PCR and Western Blot. FGF-2 mRNA-expression decreased after β-catenin specific RNAi. A site population of FACS-sortedβultivated CaCo2 cells displayed strong FGF-2 expression as well as tumour formation in a xenograft nude mouse model in contrast when CaCo2 cells were taken from bulk cultures. Finally, IHC revealed FGF-2 expression at the invasive front of CRCs in cells with nuclear expression of β-catenin. Conclusions: We show that the stemness supporting factor FGF-2 is under the transcriptional control of β-catenin, which is by itself, when expressed in the nucleus, a marker of CSC. Thus, the data presented in this work are another piece of evidence supporting the migrating CSC concept.

Do-012 PDCD4, a new tumor suppressor gene: expression and correlation analysis with molecular and clinico-pathological characteristics in colon cancer C. Vogel, F. Hofstädter, A. Pauer1, W. Dietmaier Institut für Pathologie und 1 Tumorzentrum, Universitätsklinikum Regensburg Aims: PDCD4 (programmed cell death 4) has been reported recently as a new tumor suppressor gene. Reduction or loss of activity of the Pdcd4 gene has been shown in different tumortypes. However, there are rare data about the PDCD4 expression in colorectal cancer (CRC). Methods: Using Affymetrix chip technique and real-time RT PCR we measured the expression of PDCD4 on transcription level in 44 CRC. A quantitative PDCD4 promoter methylation analysis was performed by real-time PCR. Nuclear and cytoplasmic PDCD4 protein expression was examined by immunohistochemistry in 172 stage III colon carcinomas. The expression pattern was correlated with microsatellite status, histopathological data as well as overall survival. Results: We found a 3,9 fold downregulation (median) of PDCD4 in CRC on RNA level. A regulation of PDCD4 expression by promoter hyper-methylation could not be seen. Nuclear and cytoplasmic PDCD4 expression was detected in 10/142 (7,4%) and 101/142 (71,1%), respectively. 40 tumors showed a complete loss of PDCD4 expression. Patients with nuclear PDCD4 expres-

sion had a mean overall survival of 64,0 months compared to 87,9 months in patients with nuclear negativity. Patients with cytoplasmic PDCD4 expression showed a reverse distribution with a mean overall survival of 87,2 months in positive cases compared to 50,5 months in negative cases. The PDCD4 expression pattern correlated significantly with T-stages and age. No correlation of PDCD4 expression with microsatellite instability or chromosomal instability was seen. Conclusions: Loss of PDCD4 expression or a switch from nuclear to cytoplasmic pattern is a common feature of colon cancers, correlates with age and T-stage and may indicate a longer overall survival.

Do-013 MUCDHL Protocadherin Depletes Transcriptionally-active betacatenin A. Soleiman, S. Krieger, C. Mayrhofer, A. Schmatz, R. Kalt, I. Raab, K. NagyBojarszky, M. Stichenwirth1, G. Allmaier2, M. Exner3, D. Kerjaschki Klinisches Institut für Pathologie und 1 Universitätsklinik für Dermatologie, Medizinische Universität Wien 2 Institut für Chemische Technologie und Analytik, Technische Universität Wien 3 Klinisches Institut für Medizinische und Chemische Labordiagnostik, Medizinische Universität Wien Physiological differentiation of colorectal epithelium from proliferative crypt progenitors to differentiated superficial cells relies on regulation of beta-catenin/TCF function. Using a differential interaction screen of normal colon mucosa versus colorectal cancer (CRC) we have identified a novel membrane-bound regulatory assembly that promotes cell differentiation and that is absent in CRC. Its main component, a 100 kDa protocadherin-type protein, MUCDHL, is expressed at the transition from proliferative to differentiated crypt epithelium and shows direct cytoplasmic interaction with the armadillo-type protein beta-catenin. During the „adenoma-carcinoma“ sequence, neoplastic cells express low levels of MUCDHL in colorectal adenoma and completely lack MUCDHL upon transition to invasive carcinoma. Re-expression of MUCDHL in an E-cadherin free SW480 CRC cell model depletes nuclear b-catenin and down-regulates beta-catenin/T-cell factor (TCF) driven target gene transcription. MUCDHL efficiently promotes cellular differentiation without induction of intercellular adhesion and decreases tumor cell proliferation in vitro and in mice xenografts by G1 cell cycle arrest in-vivo. Thus, formation of the MUCDHL /beta-catenin complex triggers a yet unknown cell cycle regulation in the colorectal crypt via beta-catenin/ TCF suppression thereby contributing to the differentiation of colorectal epithelium.

protein levels were higher in MIN- as compared to CIN-type tumours (n=65; p=0.0043). In invasive tumours, AURKA protein levels were associated with tumour cell proliferation (p<0.0001), which was higher in MIN- as compared to CIN-type tumours (p=0.0335). Low level AURKA amplification was detected in 41.4% CIN- and 30% MIN-type tumours and aneuploidy in 67% CIN-, but none (0%) of the MIN-type tumours (p=0.0003). Finally, aneuploidy did correlate with elevated AURKA- (p=0.0001) and CEP20- (p=0.0002) signals. Conclusions: AURKA regulation is different between CIN- and MIN-type sporadic CRC tumours. In particular, whilst AURKA is tightly associated with increased cell proliferation in MIN-type tumours (“normal regulation”), AURKA appears to be regulated independent of cell proliferation in CINtype tumours and to be linked to AURKA DNA copy number changes and aneuploidy.

Do-015 Evidence for a novel intestinal cancer predisposition syndrome linked to chromosome 6q14–22 H. Bläker1, G. Mechtersheimer1, P. Schirmacher1, M. Kloor2 1 Pathologisches Institut und 2 Abteilung für angewandte Tumorbiologie, Universitätsklinikum Heidelberg Aims: To elucidate the genetic background of early age of onset, non-polyposis, non-HNPCC and non inflammatory bowel disease associated intestinal cancer. Methods: 12 small and large intestinal adenocarcinomas of 11 patients with early age of onset carcinoma (age 35 or younger) and absence of known cancer predisposition syndromes were analysed for somatic genetic alterations by a combination of CGH, loss of heterozygosity (LOH) and gene analysis. Results: The microsatellite stable tumors showed similar chromosomal imbalances as compared to late age of onset sporadic carcinomas except for chromosome 6q deletions which were found in 50% of carcinomas. By LOH analysis the critical area of deletion was assigned to a 35mpb area at 6q14–22. One patient with multiple independent tumors showed deletions of identical parental 6q14–22 alleles in 6 of 10 tumors. Conclusions: Our data indicate that alterations affecting chromosome 6q are frequent in early onset intestinal carcinomas. Moreover, deletions of identical parental alleles in a 35 Mbp area at chromosome 6q in multiple independent tumors from one of the patients are compatible with the existence of a novel 6q-associated cancer susceptibility syndrome.

Do-014 Diﬀerential regulation of Aurora-A/STK15 in chromosomal and Microsatellite-unstable sporadic colorectal cancers and correlation to aneuploidy S. Lassmann1, M. Danciu1,2, M. Müller1, R. Weis1, F. Makowiec3, J. SchulteMönting4, U. Hopt 3, M. Werner1 1 Institut für Pathologie, 3 Chirurgische Klinik und 4 Insitut für Medizinische Biometrie, Universitätsklinikum Freiburg 2 Dept. Pathology, University of Medicine and Pharmacy, Lasi, R

Do-016 Stronger activation of the ERK signalling cascade to proliferation in gastrointestinal stromal tumors (GISTs) with KIT exon 11 deletions: Molecular basis for their clinically aggressive phenotype F. Haller, C. Löbke1, M. Ruschhaupt1, H.-J. Schulten, S. Schwager, B. Gunawan, C. Langer2, T. Armbrust3, G. Ramadori3, H. Sültmann1, A. Poustka1, U. Korf1, L. Füzesi Abteilung Gastroenteropathologie, Universitätsklinikum Göttingen 1 Abteilung Molekulare Genomanalyse, Deutsches Krebsforschungszentrum Heidelberg 2 Abteilung Allgemein- und Viszeralchirurgie und 3 Abteilung Gastroenterologie und Endokrinologie, Universitätsklinikum Göttingen

Aims: To investigate Aurora-A/STK15 (AUKRA) at the DNA, mRNA and protein level in sporadic chromosomal- (CIN) and Microsatellite- (MIN) unstable colorectal cancers (CRC). Methods: Archival resection specimens of 71 cases with sporadic CRC were analyzed for AURKA mRNA (Q-RT-PCR) and protein (IHC) expression (n=71) and DNA copy numbers (FISH, n=37) by established techniques. Aneuploidy was assessed by FISH analysis of 5 centromeric regions (CEP7, CEP8, CEP11, CEP13, CEP20). IHC for Ki67 was performed to assess tumour cell proliferation (n=65). Results: Within normal and dysplastic epithelium AURKA mRNA and protein expression was similar between CIN- and MIN-type cases, but AURKA

Aims: KIT signalling is directed through signal transduction molecules binding to distinct phosphorylation sites, and further involves the intracellular signalling cascades ERK (proliferation), AKT (protein synthesis) and STAT (differentiation). GISTs with KIT exon 11 deletion exhibit a more aggressive clinical phenotype compared to GISTs with KIT exon 11 point mutation. Methods: 42 primary GISTs were evaluated for the mRNA and protein expression of key regulators of intracellular signalling cascades using quantitative RT-PCR and reverse phase protein arrays. Results: GISTs with KIT exon 11 deletion exhibited a significantly stronger activation of the ERK signalling cascade to proliferation. Consequently, the negative cell cycle regulator RB was inactivated via hyperphosphorylation, Der Pathologe · Supplement 1 · 2008

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Abstracts and mRNA expression of the cell cycle promoter cyclin D1 as well as protein expression of the cell cycle promoter CDK2 were upregulated. Conclusions: Deletions of KIT exon 11 results in a more effective activation of the ERK signalling cascade enabling increased cell proliferation, compared to KIT exon 11 point mutation. These observations provide a possible basis for their observed clinically aggressive phenotype. Stronger activation of the ERK signalling cascade to proliferation in gastrointestinal stromal tumors (GISTs) with KIT exon 11 deletions: Molecular basis for their clinically aggressive phenotype

Do-017 Beta-adrenergic blockade decelerates periportal ﬁbrosis in ABCB4 knockout mice I. Strack, M. Scheﬄer, E. Konze, K. Wendtland, H. Varnholt, S. Schulte, H.P. Dienes, M. Odenthal Institut für Pathologie, Universitätsklinikum Köln Aims: In order to study different cellular pathways of sympathetic implication in liver fibrosis we used the Abcb4 knock-out model developing periportal, cholestatic fibrosis, which mimicks primary sclerosing cholangitis (PSC). Methods: Liver tissues of Abcb4 (-/-) mice untreated or treated with the βadrenoceptor antagonist propranolol for 6, 12, 20 and 32 weeks were taken to analyse inflammation and fibrosis progression. Chlor acetate esterase and immunochemical stainings for SMA, F4/80 and CD3 were performed and hydroxproline contents were analysed. portal fields (acinar zone I) and parenchymal areas (acinar zone II–III) of 100 μm diameter from each liver tissue of the group of treated and untreated mice were laser-microdissected and used transcriptional analyses of fibrogenic mediators. Results: In Abcb4 (-/-) mice after three months periportal fibrosis has developed, but SMA-immunostaining revealed no sinusoidal and only minor periportal contribution of myofibroblasts. Propranolol treatment of the Abcb4 (-/-) mice resulted in diminished fibrosis and hydroxyproline levels. After 3 month treatment the effect of the β-blockade was most prominent. The transcription levels of collagen I, TGFβ, CTGF and endothelin were significantly reduced in the portal areas of the treated mice compared to untreated Abcb4 (-/-) controls, whereas expression pattern of acinar zone II, III was not or only slightly effected by the β-adrenergic inhibition. Conclusions: In Abcb4 (-/-) mice, which undergo primary sclerosing cholangitis, β-blockade of the sympathicus leads to significant deceleration of periportal fibrogenesis.

Do-018 Activation of the ERK and AKT signalling pathway predicts poor prognosis in hepatocellular carcinoma K.J. Schmitz, J. Wohlschlaeger, H. Lang1,3, G.C. Sotiropoulos1, M. Malago1, K. Steveling, H. Reis, V.R Cicinnati2, K.W. Schmid3, H.A. Baba3 Institut für Pathologie und Neuropathologie, 1 Klinik für Allgemein-, Viszeral- und Transplantationschirurgie, 2 Abteilung für Gastroenterologie und Hepatologie und 3 Westdeutsches Tumorzentrum Essen (WTZE), Universitätsklinikum Essen Aims: The aim of the study was to determine the prognostic relevance of two kinases in hepatocellular carcinoma (HCC) important in the regulation of cell proliferation and apoptosis: AKT and extracellular regulated kinases (ERK1/2). Methods: This study comprised a series of 208 patients incorporating HCCs treated either by surgical resection (n=109) or liver transplantation (n=99). Immunohistochemically demonstrated phospho-ERK1/2 and phospho-AKT was correlated with a series of clinico-pathologically relevant parameters (EGFR, Cyclin-D1, HCV/HBV infection, liver cirrhosis, chronic alcohol abuse), proliferative activity, and apoptosis. Results: Activation of ERK1/2 correlated statistically with the presence of HCV infection. pERK1/2 (P<0.001) and pAKT (P=0.052) expression showed a significant correlation with a decreased overall survival. Cox regression analysis identified pERK1/2 as an independent prognostic parameter in HCC (P=0.028).

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Conclusions: Activation of ERK1/2 in HCC cancer indicates aggressive tumour behaviour and constitutes an independent prognostic factor. Furthermore our data confirm that HCV infection activates the ERK pathway and thereby might contribute to HCC carcinogenesis. Immunohistochemical determination of pERK1/2 status can thus be proposed as a promising candidate for the identification of high risk patients which may benefit from new anticancer drugs targeting the ERK-pathway.

Do-019 Comprehensive genome analysis reveals etiology-dependent and etiology-independent molecular mechanisms in human hepatocarcinogenesis T. Longerich*, C. Schlaeger1*, C. Schiller*, P. Bewerunge2, A. Mehrabi3, R. Eils2, P. Lichter1, B. Radlwimmer1, P. Schirmacher *equal contribution Pathologisches Institut, Universitätsklinikum Heidelberg 1 Abteilung für Molekulare Genetik und 2 Abteilung für Theoretische Bioinformatik, Deutsches Krebsforschungszentrum Heidelberg 3 Chirurgische Klinik, Universitätsklinikum Heidelberg In order to identify host changes related to tumor etiology as well as novel driver genes of human hepatocarcinogenesis in general, we combined highresolution fine-mapping of genomic imbalances with expression and functional analyses. Array-based comparative genomic hybridization (aCGH) of a panel of etiologically well-defined HCCs (n=68) revealed DNA copy number gains of chromosome arm 8q to be significantly less frequent in cryptogenic HCCs compared to other etiologies. Expression and network analyses validated c-myc as the potential target gene of 8q gains. In addition, we were able to fine-map frequent etiology-independent chromosomal imbalances. From several candidates of these regions, we validated MDM 4 (1q32.1) and eEF1A2 (20q13.33) as likely candidate genes using expression and functional analyses. Detailed analyses revealed a complex alteration of the MDM 4-TP53-MDM 2 regulatory network in HCC and furthermore, preliminary functional analyses suggest a connection of eEF1A2 to this network. In conclusion, molecular hepatocarcinogenesis of cryptogenic HCC differs from the other etiological subgroups at least with respect to 8q gain and MYC activation. Furthermore, activation of the oncogenes MDM 4 and eEF1A2 represent an etiology-independent mechanism a subset of HCC by presumably overcoming TP53dependent growth control. Therapeutic approaches targeting MDM 4 may potentially improve the currently poor prognosis of human HCCs.

Do-020 Constitutive occurrence of cis-E:N-cadherin heterodimers in normal hepatocytes, during development and in tumorigenesis B.K. Straub1,2, J. Boda-Heggemann2, U.F. Pape2, R. Zimbelmann2, C. Grund2, C. Kuhn2, P. Schirmacher1, W.W. Franke2 1 Pathologisches Institut, Universitätsklinikum Heidelberg 2 Abteilung Zellbiologie, Deutsches Krebsforschungszentrum Heidelberg Aims: Cadherins, transmembrane glycoproteins of adherens junctions (AJ), play a pivotal role in cell development and tumor formation and progression. Particularly the epithelial (E-cadherin) and neural/mesenchymal (N-cadherin) representative are attributed mutually exclusive roles in development and carcinogenesis. As only E-cadherin was described in AJs of normal hepatocytes, we were surprised to find both E- and N-cadherin in liver. Methods: Normal fetal and adult tissues of inner organs, derived tumors and cell cultures were analysed using immunofluorescence and electron microscopy in combination with proteinbiochemical and molecular biological methods. Results: In normal hepatocytes, N-cadherin-containing AJs were localized near bile canaliculi and in newly identified puncta adhaerentia at the basolateral membranes, in colocalization with E-cadherin, α- and β-catenin, plakoglobin and protein p120ctn. Using immunoprecipitation from protein lysates of normal liver and PLC cells, E:N-cadherin cis-heterodimers were isolated. E:N-cadherin cis-heterodimers were also identified in fetal liver, in hepa-

tocellular adenomas and carcinomas as well as in other endodermal tissue derivatives such as bile and pancreas ducts, as well as in kidney and derived tumors. Using siRNA-mediated abrogation of E- and/or N-cadherin in PLC cells, not only AJs, but also tight junctions appeared to be affected. Conclusions: Cis-E:N-cadherin heterodimers constitute a novel AJ type in normal and malignantly transformed hepatocytes with pivotal importance for hepatocyte homeostasis. The mere existence of these hybrid cadherin structures demands a reassessment of the currently prevailing “yin-yang”hypothesis of cadherins during development and tumorigenesis.

Do-021 A novel function of the FBP-interacting repressor (FIR) in human (hepato)-carcinogenesis M. Malz, A. Weber1, V. Ehemann, P. Schirmacher, K. Breuhahn Pathologisches Institut, Universitätsklinikum Heidelberg 1 Institut für klinische Pathologie, UniversitätsSpital Zürich Aims: The FBP-interacting repressor (FIR) inhibits transcription factor activity of FUSE-binding proteins (FBPs) which have been described to regulate fine-tune expression of the proto-oncogen c-MYC under physiological conditions. Whether FIR reduction or inactivation is involved in aberrant overexpression of c-MYC in liver cancer is currently not known. We analyzed the expression of FIR, its effect on c-MYC expression, and its functional relevance in human hepatocarcinogenesis. Methods: The expression of FIR and c-MYC was analyzed on transcript (realtime PCR) and protein levels (Tissue Microarrays [TMA], Western Blotting) in premalignant lesions (dysplastic nodules) and hepatocellular carcinoma (HCC). Using gene-specific siRNA transfection the expression of all FIR isoforms with exon 2 and/or exon 5 deletions were inhibited in different HCC cell lines. Functional consequences of reduced FIR expression on proliferation (BrdU-Assay), viability (MTT-Assay) and on c-MYC expression were analyzed. Results: All FIR isoforms were not reduced but significantly elevated in human HCCs (33%) as compared to normal hepatocytes and human liver samples. Overexpression of FIR isoforms correlated with dedifferentiation but not with c-MYC expression. FIR isoforms with exon 2 deletions were exclusively detected in HCCs but not in normal hepatocytes. SiRNA-mediated inhibition of FIR isoforms reduced tumor cell proliferation and viability. Conclusions: In human hepatocarcinogenesis elevated FIR expression does not facilitate predicted repressor activity. The pro-tumorigenic properties of FIR are mediated by c-MYC independent mechanisms. Whether distinct FIR isoforms (e.g. containing exon 2 deletions) exert oncogenic properties in human hepatocarcinogenesis needs to be analyzed.

Do-022 Downregulation of the tumor suppressor gravin/AKAP12 in human hepatocellular carcinoma B. Goeppert1, G. Gdynia1, A. Warth1, M. Breinig1, M. Mittelbronn2, M. Vogel3, P. Schirmacher1, M. A. Kern1 1 Pathologisches Institut, Universitätsklinikum Heidelberg 2 Institut für Neuropathologie, UniversitätsSpital Zürich 3 Abteilung für Diagnostische Radiologie, Universitätsklinikum Tübingen Aims: SSeCKS, a Src-suppressed protein kinase C substrate, is the rodent orthologue of human gravin/AKAP12 that acts as a scaffold by binding protein kinase A and protein kinase C. Gravin is downregulated in several solid tumors and is implicated in tumorigenesis by inhibition of angiogenesis and cell cycle interference. However, so far no data concerning gravin expression in human hepatocellular carcinomas (HCCs) exist. Methods: Using tissue micro array (TMA) analysis, gravin expression was analysed by immunohistochemistry in 257 liver tissue samples including 15 “normal” livers, 37 dysplastic nodules (DNs), 161 HCCs (Grading: 34×G1, 96×G2, 24×G3, and 7×G4), 17 liver cirrhosis, and 27 peritumorous tissue samples. Additionally, the expression pattern was confirmed by western immunoblotting using protein lysates from human HCC tissues and HCC cell lines (HUH7, PLC, HepG2, Hep3B).

Results: The relative expression of gravin was strongly expressed in “normal” livers, DNs respectively and decreased significantly with dedifferentiation of HCC (p<0.001; p<0.01 respectively). Western immunoblotting of tissue lysates corroborated this finding. In HCC cell lines, gravin expression was still detectable in HuH7 and Hep3B. Conclusions: Our results are in line with various studies showing downregulation of the tumor suppressor gravin in different tumor entities. Accordingly, our findings suggest that gravin is a potential tumor suppressor also in human HCCs, and the loss may play a significant role in tumor progression. Further functional studies are needed to elucidate the molecular mechanism of tumor suppression by gravin in HCCs.

Do-023 The p53 Family Inhibitor deltaNp73 Induces Autonomous Growth and Hepatocarcinogenesis in Transgenic Mice J. Munding, K. John1, N. Miše1, A. Schmidt1, S. Buhlmann1, S.M. Ibrahim1, B. Pützer1, A. Tannapfel Institut für Pathologie, Ruhr-Universität Bochum 1 Department für Vektorologie und Gentherapie, Forschungsgruppe für Immungenetik, Universitätsklinikum Rostock Aims: p53 family proteins exert a wide spectrum of biological functions like differentiation, cell cycle arrest, apoptosis and chemosensitivity of tumors. In contrast N-terminally truncated p73 (deltaNp73) acts as a potent inhibitor of tumorsuppressor properties, implicating its participation in malignant transformation and oncogenesis. Several reports indicate upregulation of deltaNp73 in hepatocellular carcinoma (HCC) correlating with reduced survival, but little is known about the functional significance of deltaNp73 in tumorigenesis in vivo. Methods: Transgenic mice models in which deltaNp73 expression is directed to the liver by the albumin promoter were generated. Gene expression was tested by mRNA- and protein analyses. Results: Transgenic mice showed increased hepatocyte proliferation resulting in preneoplastic lesions (adenomas) at 3–4 months. 83% of 12 to 20 months old mice developed HCC. HCC-lesions displayed a significant increase of hyperphosphorylated inactive Rb, whereas p53-regulated inhibitors of cell cycle progression were downregulated in the tumors. Conclusions: Our data underlines the unique oncogenic capability of deltaNp73 to drive hepatocarcinogenesis in vivo, supporting its significance as a marker for disease severity in patients and as a target for cancer prevention. This model offers new opportunities to further delineate deltaNp73-mediated liver oncogenesis but may also enable the development of more effective cancer therapies.

Do-024 URI expression in hepatocellular carcinoma correlates with cell proliferation and impaired survival – a novel oncogene? J.P. Theurillat1,2*, M.O. Riener1*, S. Metzler2*, C. Hellerbrand3, N. Djouder2, P. Wild1, W. Jochum1, W. Krek2*, H. Moch1* *contributed equally 1 Institut für klinische Pathologie, UniversitätsSpital Zürich 2 Institut für Zellbiologie, ETH Zürich 3 Departement für Innere Medizin, Universitätsklinikum Regensburg Aims: URI is an unusually large member of prefoldins that functionally links nutrient availability with gene transcription. Recently, an anti-apoptotic role for URI at mitochondria has been described. Additionally, mild over-expression in vitro causes increased cell growth, further suggesting that URI could act as an oncogene in malignancies. Methods: In the present microarry study, primary hepatocellular carcinomas (n=140) were studied for URI expression using a monoclonal antibody. URI expression was compared with clinic-pathological parameters, proliferation, apoptosis and p53 protein expression. Results: Twenty-nine out of 140 primary carcinomas (20%) expressed URI. URI expression correlated directly with proliferation index (p<0.001) and tumor size (p=0.009). Eighty-six percent of URI positive carcinomas comDer Pathologe · Supplement 1 · 2008

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Abstracts pared to 31% of URI negative carcinomas showed a proliferation index higher than 10%. In line, survival analysis showed an association of URI expression with impaired patient outcome (p=0.049). Forty months after surgery, only 38% of the patients with URI positive tumors were alive compared to 59% in the group of URI negative patients. URI expression was paralleled with p53 expression (p=0.002). However, no correlation could be established between URI expression and apoptosis. Conclusions: The strong correlation of URI with cell proliferation, tumor size and impaired patient outcome adds further arguments in addition to in vitro findings that URI represents a novel oncogene.

Do-025 Cell-Cell Junctions in Normal Pancreas and Derived Tumors O. Cahyadi1,2, R. Zimbelmann1, P. Schirmacher2, W.W. Franke 1, B.K. Straub1,2 1 Abteilung Zellbiologie, Deutsches Krebsforschungszentrum Heidelberg 2 Pathologisches Institut, Universitätsklinikum Heidelberg Aims: Disintegration of cell-cell junctions is regarded as an important factor in carcinogenesis, just as in tumors of the pancreas. Methods: We have analysed the molecular structure of cell junctions in normal adult and fetal pancreas, derived tumors and cultured cells with immunofluorescence microscopy, protein biochemical and molecular biological methods including siRNA transfection strategies. Results: Regarding adherens junctions, E-cadherin was detected in acinar and ductal epithelial cells and in certain islet cells, there colocalizing with α-, and β-catenin, as well as protein p120ctn, whereas N-cadherin was positive in pancreatic islets and ducts as well as in the stroma. In pancreatic ducts, both E- and N-cadherin were found. Regarding desmosomes, desmoglein-2, desmocollin-2, plakoglobin, desmoplakins, plakophilin-2 and plakophilin-3 were localized in acinar and ductal epithelial cells as well as in the respective cultured cells. The tight junction proteins occludin, cingulin, JAM-A, and tricellulin were found in pancreatic acinar and ductal cells, together with certain claudins. Adenocarcinoma of the pancreas showed an overall reduced expression of E-cadherin and the other respective cell junction proteins, while Ncadherin was not significantly expressed. Additionally, in endocrine pancreas tumors E- and N-cadherin were both expressed but differentially localized. Conclusions: The pancreas is characterized by a distinct subset of cell junction proteins. Derived tumors may thereby be differentiated from other endodermal derivatives, thus improving pathological diagnosis. Experiments are underway to elucidate the functional role of these proteins during tumorigenesis.

Sitzung: AG Informatik in der Pathologie Do-026 Digital pathology – achievements and perspectives A. Roessner, R. Zwönitzer1, H. Hofmann2, J. Bernarding1, T. Kalinski Institut für Pathologie 1 Institut für Biometrie und Medizinische Informatik und 2 Medizinisches Rechenzentrum, Universitätsklinikum Magdeburg Aims: Virtual microscopy is well established for education in pathology. Virtual slide collections emerge in the internet. It is a small step to diagnostic virtual microscopy. Still, efforts are needed to integrate virtual microscopy in pathology information systems and to build up fully digital pathology. Methods: At the Department of Pathology, we established virtual microscopy using open standards, which provide a flexible basis for its integration in the fields of education, research and diagnostics. Results: The installations are continously enhanced and updated. The current status of the installations together with preliminary experiences and/or evaluations will be presented. Future perspectives will be denoted. Conclusions: The digital reorganization of pathology is an essential process, that will have an enourmous impact on the practice of pathology in the future.

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Do-027 Lossy image compression in digital pathology R. Zwönitzer, T. Kalinski1, H. Hofmann2, A. Roessner1, J. Bernarding Institut für Biometrie und Medizinische Informatik, 1 Institut für Pathologie und 2 Medizinisches Rechenzentrum, Universitätsklinikum Magdeburg Aims: Information systems, picture archiving, and image distribution systems are state of the art in hospitals today. Official standards like DICOM and HL7 assured success of those systems in radiology by defining formats and information models with regard to workflow aspects. However, integration of Digital Pathology into existing standards is essential for its practical success and acceptance by users. Those standards make use of royalty free compressions formats only. DICOM integrated JPEG2000 and its internet based distribution protocol JPIP. Methods: JPEG2000 was compared to its predecessor JPEG using digitized histological slides of up to 4 GB. Compression load was examined on different hardware. Results: JPEG2000 improved dramatically the performance serving medical needs of image compression and distribution (streaming), especially for Whole Slide Imaging. However, JPEG2000 needed large resources when encoding huge images. Also, Digital Patho-logy brings up new challenges such as managing the large data amounts. This requires new and powerful hardand software solutions for presenting and processing the data in real time as well as implementing new workflow concepts. Conclusions: Compression time might be reduced using more sophisticated algorithms. Using Digital Pathology in routine diagnostics, the acceptable information loss due to compression must be assessed by additional studies.

Do-028 3D virtual microscopy in diagnostic pathology T. Kalinski, R. Zwönitzer1, M. Evert, T. Günther, S. Sel, H. Hofmann2, J. Bernarding1, A. Roessner Institut für Pathologie 1 Institut für Biometrie und Medizinische Informatik 2 Medizinisches Rechenzentrum, Universitätsklinikum Magdeburg Aims: The limitation of ‘conventional’ 2D virtual microscopy to one focus plane may lead to a lack of information in virtual slides due to blurred areas outside the focus. Moreover, some structures, such as Helicobacter in gastric biopsies, require to be focused for exact diagnosis. 3D virtual microscopy would resolve such problems. Methods: Therefore, we composed 3D virtual slides from whole slide scans of gastric biopsies using 9 different focus planes in JPEG2000 format. The cases were diagnosed according to the modified Sydney classification by 3 pathologists in a blinded fashion using traditional light microscopy and 3D virtual microscopic slides with 1 to 9 focus planes. Results: Histological parameters, such as the grade and activity of gastritis and the occurrence of intestinal metaplasia, were diagnosed independent of the method used. However, the evaluation of Helicobacter colonization by 3D virtual microscopy matched the results of traditional light microscopy better than 2D virtual microscopy. Conclusions: 3D virtual microscopy enables virtual focusing, which is essential in diagnostic pathology. 3D virtual microscopy meets the requirements of diagnostic pathology comparable to traditional light microscopy.

Do-029 Open access virtual slide database http://pathorama.ch/ K. Glatz, D. Glatz1, M.J. Mihatsch, H. Moch2 Institut für Pathologie, Universitätsspital Basel 1 Universitätsrechenzentrum, Universität Basel 2 Institut für Klinische Pathologie, UniversitätsSpital Zürich Aims: Development of a publicly accessible online virtual slides atlas with premium quality histologic and cytologic reference slides. Provision of re-

sources for teaching and self-guided learning. Enabling contribution of virtual slides by multiple institutions. Methods: Histologic and cytologic preparations are scanned using commercially available slide scanners (Olympus .slide, Aperio ScanScope). Contributors feed ample categorized metadata including the slide URL into the central slide database while the slides are stored on servers at the contributing institutes. Thus the huge storage requirements for virtual slides can be distributed among multiple institutions. The metadata enables precise slide retrieval. Each slide including its metadata can either be used stand-alone (as an atlas: vSlides) or be re-used for several preexisting online components (e.g. histopathology course: HiPaKu), slide videos, or continuing medical education courses (vCollections). Technically and didactically vSlides and vCollections are a component of the e-learning platform PATHORAMA (http://pathorama.ch/). Results: Up to now, the slide atlas vSlides contains 112 virtual slides of two contributing institutions. Hundreds of slides covering a wide range of subspecialties of pathology and anatomy are soon to follow. Three slide seminars are accessible at vCollections. From 2008 onwards, the slides of all future slide seminars organized by the Swiss Society of Pathology and the Swiss section of the IAP will be available online in vCollections. Conclusions: An open access and modular concept turns virtual slide databases into versatile and efficient learning object repositories.

Do-030 Video podcasting: An eﬀective, inexpensive and attractive method for the teaching of pathology through Internet as complement to the virtual microscope A. Perez-Bouza, E. Breuer, F. Bärtling, A. Kneist, M. Merk, A. Donner, R. Knüchel Institut für Pathologie, Universitätsklinikum Aachen Aims: Digitalisation of histological samples represents a modern and attractive method for the teaching of histopathology. After a year experience with the so called virtual microscope (VM), we have now complemented the system by adding video files as podcast containing live macroscopy combined with digital histology in a simple and comprehensive manner. Methods: Video material with macroscopical preparation of organs was recorded with a conventional digital camera with 3,2 megapixel resolution. A so called screen-recorder software on a 20”” iMac (Apple inc.) was used to record videos with digital slides from the VM. Video files of 640×480pixel were processed and cut with iMovie. Here the spoken explanations were added. Files were exported as a high quality MPEG4-file of 5–10 minutes (10 MB/min). Videoclips were published on www.blip.tv and offered as RSSfeed through iTunes. For the reproduction of the videos video iPods can be used as portable devices. Results: First feedback from the students is very positive. Specially, the links between clinical, macroscopical and histlogical findings were found to be useful for understanding disease development and diagnosis. Conclusions: The combination of digital microscopy and video podcasting is a simple and attractive didactic offer. Founded by the medical faculty of the RWTH Aachen

Do-031 The use of telepathology in frozen section diagnosis J. Bertolini1, T. Gradistanac1, G. Heiland2, C. Wittekind1 1 Institut für Pathologie, Universitätsklinikum Leipzig 2 Chirurgische Klinik, Heinrich-Braun-Krankenhaus Zwickau Aims: We used the method of telepathology for intraoperative frozen-section consultation in a hospital in some distance to the university institute and will report on our experience. Methods: 508 unfixed specimens were examined in the usual procedure, which was done by residents, having experience of at least 150 frozen sections. From representative histological areas photos were taken and send to a consultant in the institute for confirmation of the diagnosis by telephone conference. The results were discussed with the operating team.

Results: The intervals between time-in and time-out varied between 7 and 30 minutes. Among 508 frozen-sections 71 (14%) revealed discrepancies between the frozen-section and the final diagnosis. Diagnostic difficulties were observed in the well known fields, namely frozen-sections of thyroid, pancreas, ovary, kidney tumours and renal cysts. In one case of thyroid and pancreatic lesions a tumour suspicion was raised and confirmed in the final histological diagnosis. There was no case of false positive diagnosis and one case with a false negative diagnosis (thyroid carcinoma). Conclusions: Telepathology of frozen sections in the described way is a safe and with regard to required time acceptable procedure. We experienced no unexpected diagnostic difficulties.

Do-032Virtual Microscopy in Clinical-Pathological Conference M. Andrulis1, E. Herpel1, P. Ganschow2, M. Kadmon2, P. Schirmacher1, P. Sinn1 1 Pathologisches Institut und 2 Chirurgische Klinik, Universitätsklinikum Heidelberg Aims: Virtual microscopy (vM) is becoming more and more established tool for medical education at universities. We wanted to set up a vM-based platform for interdisciplinary educational teaching – clinical pathological conference (CPC). Methods: An Aperio slide scanner was used to digitalize the histological tissue sections. An e-Learing System (Modular Object-Oriented Dynamic Learning Environment, “moodle”) was adapted to make digital slides accessible for the students. Results: The CPC gives the medical students an opportunity to study important clinical – pathological correlations of common human disease. A group of five to seven students have to prepare a short presentation based on a one single patient case. Typical morphological changes that are required for pathological diagnosis have to be detailed in the presentation. We integrated the vM and moodle to provide an access to the digitalized slides. The students are now using our web-based virtual microscope system for pathology teaching and for the preparation of the digital slides for the CPC presentation. Conclusions: vM is an excellent tool to communicate systemic pathology to medical students. Integration of vM in an interdisciplinary curriculum makes pathology more accessible, understandable, and attractive as a discipline.

Do-033 3D organotypic cultures to study cancer cells and tumor stroma interactions H. Dolznig1, C. Rupp1, C. Puri1, N. Schweifer2, C. Haslinger2, W. Sommergruber2, P. Garin-Chesa1,2 1 Klinisches Institut für Pathologie, Medizinische Universität Wien 2 Boehringer Ingelheim Austria, Wien Aims: Invasion and metastasis of tumor cells into normal tissues is accompanied by adaptive changes in the supporting stroma. Altered gene expression in non-transformed stromal cells provides potential targets for therapy, as shown with anti-angiogenic drugs. Recent evidence indicates that activated cancer associated fibroblasts are important promoters of tumor progression; however the molecular mechanisms behind these processes remain poorly understood. Methods: Therefore we have set up an immunohistochemically guided laser capture microdissection assay (iLCM) to dissect normal and activated tumor stromal fibroblasts from normal human tissues and carcinomas, to define the molecular signatures that characterize the “normal” vs the “activated” tumor stromal fibroblasts in vivo. In parallel, we have established a cell culture system in which human fibroblasts are co-cultured with human tumor cells in a 3D-system in the presence of ECM proteins. Results: This model allows live-imaging studies to monitor tumor cell growth, phenotypic characterization and functional experiments. Examples of molecules identified in the activated fibroblasts in vivo will be shown in the 3D co-cultures. Proteomic analysis was used to demonstrate the cross-talk between the various cellular components of the model. Moreover, the response to PI3 K inhibtion was monitored and quantified. In addition, induction of apoptosis, adhesion to ECM proteins, cell migration and morphogenesis can be studied. Der Pathologe · Supplement 1 · 2008

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Abstracts Conclusions: We have established a physiologically relevant assay system in which auto- and paracrine interactions between the tumor cells and the stroma can be examined in molecular and cellular terms. We believe that the effective utilization of this 3D model has an enormous potential in the validation of novel therapeutic targets.

Do-034 Bootstrap procedure veriﬁes the stability in hierarchical cluster analysis of immunohistochemical data – Performance of the algorithm was tested with tissue microarray data sets E. Korsching, K. Agelopoulos, H. Schmidt, W. Böcker, H. Bürger, E. Eltze, C. August Institut für Pathologie, Universitätsklinikum Münster Aims: Hierarchical cluster analysis is widely applied in the analysis of immunohistochemical data. The procedure is divided in three steps: a) the construction of a dissimilarity or similarity matrix, b) the application of a clustering algorithm and c) the graphical representation of the results. Depending on the clustering algorithm the procedure is not strictly deterministic if the distribution of the distances is dense. Furthermore there is not a really good quality measure concerning the stability of the presented groups. Here we step in and apply the well known bootstrap approach. Methods: The algorithm is based on S/R. The bootstrap is performed on the raw data per antibody staining preserving the inherent data characteristic. The raw data and the up to 10E3 bootstrap samples are all processed in our case with the ‘agnes’ (agglomerative nested) procedure. The raw cluster plot is annotated with the bootstrap weight per cluster node. Results: The procedure was applied to three different tumour entities: Invasive breast cancer, prostate cancer and oropharyngeal carcinomas. In all situations meta information was available to rate the outcome of the procedure. In all cases the weights of the first level nodes reflects observed behaviour. The higher nodes are of minor importance because the probability of fluctuations grows. These fluctuations result from differences in the distance matrix. Conclusions: The predictive quality of cluster analysis data can be improved and care in the interpretation of clustering results will be raised. The speed of the calculations is affordable and not a limiting factor, so that 10E3 bootstraps can be applied without problems in a standard situation with 500 cases with up to 20 immunohistochemical stainings.

Do-035 Quality Assurance in medical and scientiﬁc data management of Open European Nephrology Science Center T. Schrader, Y. Zou1, S. Hanß2, T. Schaaf2, C. Hahn, S. Niepage, T. Wetzel, D. Keune, T. Weckend3 Institut für Pathologie, Charité Universitätsmedizin Berlin 1 Fakultät III – Umwelt und Technik, Leuphana Universität Lüneburg 2 Institut für Medizinische Informatik, Charité Universitätsmedizin Berlin 3 IT-Zentrum, Charité Universitätsmedizin Berlin Aims: The Open European Nephrology Science Center (OpEN.SC) is supported by the German Research Foundation (DFG) as a Center of Research Information. The establishment of such a medical center is embedded in the international context of efforts to improve the availability of digitalized clinical data including Whole-Slide-Images (WSI). The OpEN.SC collects research relevant data from different national and international resources. A crucial point the assurance of data quality. Methods: The OpEN.SC system based on a Service Orientated Architecture to ensure a high flexibility and scalabilty of the application. Web services are distributed software modules and offer different functionalities; they are orchestrated by a Business Process Manager due to a modeled quality assurance process. Results: A systematic quality assurance process was modeled and descibed using the standard Business Process Execution Language. After a retrieval process cases can be selected for the QA process. A set of quality criteria are defined by the scientists and the cases are assigned to a consultant or an expert certified by an accepted organisation.

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Conclusions: The quality of medical and scientific data is a very complex problem field. Our system offers now a QA process for a specific purpose.

Do-036 From pathological report to information – concept dictionary for standardization of terms for export purposes from pathology management systems G. Haroske, T. Kramm, M. Mörz, M. Oberholzer1 Institut für Pathologie, Klinikum Dresden-Friedrichstadt 1 Institut für Pathologie, Universitätsspital Basel Aims: As to cope with the increasing demands for supplying information to a variety of documentation systems outside of pathology, the pathologists have to set the standards both for their genuine information content (e.g. by guidelines) and for the use of that information. The latter aspect has widely been neglected so far. Methods: Translations of pathology report guidelines for specified entities into an informatic concept dictionary were used for a framework of defined terms that can be used as a standard for information exchange, e.g. by XMLtechnology. Results: For the “ documentation requirements” of the guideline “Diagnostics of Colorectal Cancer” of German Pathologists a set of 53 features was extracted and translated in appropriate concepts. Each concept consists of 14 descriptors for names, definitions, codes, synonyma, data sources, data types, semantic interrelations, and administrative terms. All of them are compiled in an extendable concept dictionary. Attention was paid to have concepts as simple as possible for using them not only in one specific entity. The concepts have then been used for constructing an XML pathology report structure that enables an automatic export of relevant information from the PMS to external documentation systems, e.g. to hospital information systems or cancer registries. On the other hand, a structured reporting from a data set is possible, too. Conclusions: A pathology concept dictionary could be the basis of a specific namespace for a semantic web, enabling pathologists to provide a plenty of information from their routine reports to the still increasing demand for documentation in routine and research.

Do-037 Evaluation of a telepathology project in Tanzania H.-U. Völker1, M. Oberholzer2, G. Stauch3, H.-K. Müller-Hermelink1 1 Institut für Pathologie, Universitätsklinikum Würzburg 2 Institut für Pathologie, Universitätsspital Basel 3 Institut für Pathologie, Aurich Aims: Since April 2007, the St. Josef ’s Mission Hospital of Benedictians in Peramiho, Tanzania, is provided with histomorphological diagnoses via Telepathology (TP). For TP, iPath (Basel) is in use. The aim of this study was the evaluation of diagnostic safety. Methods: Telepathological diagnoses of 300 cases were revised by reinvestigation of the paraffin embedded material in Germany. The primary TP diagnosis based only on HE-stain. For definite diagnosis, all necessary diagnostic tools were used. Discrepancies of diagnoses were divided into acceptable and non-acceptable. The results were compared with existing analyses of TP in Cambodia and Honiara. Results: The level of diagnostic safety is high. In case of discrepancies, mostly acceptable differences occurred. Non-acceptable differences between diagnoses were rare (<2.5%). The main reason for non-acceptable misdiagnoses (e.g. malignant tumor as bengin) was an inadequat selection of digital pictures by the local staff (non-pathologists!). The results are comparable with the other projects. The most common problems happened in the diagnosis of mesenchyml tumors, malignant lymphomas and Kaposi sarcomas via TP. Conclusions: TP with a static system is suitable for the histomorphological diagnosing. However, the teaching of the local staff is one of the main important tasks in this field of medicine. Apart from diagnostic difficulties in some fields, the advantages of the fast diagnostic supply are prevailing.

Do-038 The evaluation of immunohistochemical results on salivary and oropharyngeal carcinomas by cluster and interrelation analyses reveals common and unique molecular principles E. Korsching, H. Schmidt, J. Alberty1, J. Wendker1, K. Agelopoulos, W. Böcker, C. August Institut für Pathologie und 1 HNO-Klinik, Universitätsklinikum Münster Aims: Oropharyngeal carcinomas are in nearly all cases tumours with epidermoid differentiation. Immunohistochemistry is helpful to better determine histologic heterogenity based on differentiation of stem cells. Such data might be important for illumination of pathways of tumour progression. Theoretical approaches could be useful to evaluate a large number of immunohistochemical data of oropharyngeal to decipher regulatory behaviour. To confirm the results a comparison of the data with that of salivary gland tumours which are known as tumors with both epidermoid and glandular differentiation would give clues to a generalized regulation scheme. Methods: Tissue microarrays of 157 oropharyngeal carcinomas (complete follow up) and of 57 parotideal carcinomas were investigated by semiquantitative scored immunohistochemistry (9 respective 28 markers). The data underwent a structural and interrelation analysis based on permutation approaches and hierarchical (bootstrap based) cluster analysis. Results: The comparative cluster analysis exhibited two main groups: The first one with a strong similarity between cytokeratin CK5/6 and CK 14 in a broader group with egfr and p53 (oroph.) respective p63 and p53 (parot.). The second group around CK 19 collected all the remaining factors. The comparative dependency analysis in both cases spanned a field of negative interaction between CK 5/6, CK 14 and androgen receptor and positive interaction to CK 19. The inverse pole was build by p63/egfr respective p53/p63. Conclusions: The presented analysis gave an excellent insight in the properties of tumor progression pathways of salivary and oropharyngeal carcinomas. In return the comparative situation itself gives also suggestions to improve the algorithms of the procedure.

Sitzung: AG Hämatopathologie Do-039 Hypermethylation and transcriptional inactivation of transcription factor genes in Myelodysplastic Syndrom (MDS) D. Römermann, K. Metzig, B. Hasemeier, F. Länger, B. Schlegelberger1, H. Kreipe, U. Lehmann Institut für Pathologie und 1 Institut für Zell- und Molekularpathologie, Medizinische Hochschule Hannover Aims: DNA methylation patterns of three transcription factor genes, for which aberrant methylation has been described in acute myeloid leukaemia (KLF-5, KLF-11, MAFB), was analyzed in a series of MDS patients. Methods: Genomic DNA was isolated from bone marrow biopsies from 127 patients with MDS or a secondary leukemia as well as from 25 control biopsies. For treatment with bisulfite published standard procedures were followed. The methylation level of MAFB, KLF-5, and KLF-11 was analyzed in all samples using “Combined Bisulfite Restriction Analysis (“COBRA”). All cases with clear methylation signals (n=41) as well as 16 controls were analysed using quantitative high resolution Pyrosequencing™. mRNA expression was analysed employing quantitative real-time PCR. Results: The different MDS subgroups display specific differences concerning aberrant methylation of these three genes. Hypermethylation of the KLF-5 is quite rare in MDS and secondary leukaemia, whereas KLF-11 and MAFB were hypermethylated in 30–40% of cases. However, KLF-11 displayed marked constitutive methylation in all controls. Only very extensive hypermethylation of this gene (>75%) correlates with reduced mRNA expression. Conclusions: The genes under study as well as the different MDS subgroups show characteristic differences in methylation patterns. Quite high constitu-

tive methylation can already be found in normal controls and only a quantitative detection method can identify disease-specific aberrations in DNA methylation.

Do-040 Increased of microRNA hsa-mir-223 in high risk MDS and secondary acute myeloid leukemia D. Römermann, K. Metzig, B. Hasemeier, F. Länger, B. Schlegelberger1, H. Kreipe, U. Lehmann Institut für Pathologie und 1 Institut für Zell- und Molekularpathologie, Medizinische Hochschule Hannover Aims: microRNA hsa-mir-223 is expressed specifically during granulocytic/ monocytic differentiation. Aim of this project is to figure out whether the expression of this particular microRNA is dysregulated during the development and progression of myeloid neoplasia. Methods: Total RNA was isolated from bone marrow trephines from patients with “low risk” MDS (n=20), “high risk” MDS (n=25) or a secondary acute leukaemia as well as from 25 control biopsies. Quantification of hsa-mir-223 expression was performed using quantitative real-time PCR after linker adaptation and microRNA specific cDNA synthesis. The expression level of two constitutively expressed small RNA molecules was used for normalization (RNU24, Z30). Results: Normalized to RNA input both small RNA genes showed a very similar expression level in all patients and controls. The microRNA hsa-mir223 is statistically significantly up-regulated in bone marrow biopsies from patients with “high risk” MDS or secondary acute leukaemia developing in MDS patients (p=0.005 bzw. 0.001). Conclusions: In bone marrow samples from patients with myeloid neoplasia the up-regulation of microRNA hsa-mir-223 correlates with disease progression, in contrasts to in vitro studies reporting the up-regulation during differentiation.Future studies have to elucidate the functional consequences of this up-regulation.

Do-041 MicroRNA proﬁling of formalin-ﬁxed and paraﬃn-embedded megakaryocytes in chronic myeloproliferative disorders K. Theophile, K. Hussein, H. Kreipe, O. Bock Institut für Pathologie, Medizinische Hochschule Hannover Aims: Primary myelofibrosis (PMF) and essential thrombocythemia (ET) belong to the group of Philadelphia chromosome negative chronic myeloproliferative disorders (CMPD). Both entities show a close early-onset phenotypic similarity, but are characterized by a significant difference in prognosis. As to date no molecular defect for a definite distinction between these disorders are available, we expanded the search for differently expressed markers by the rapidly growing field of microRNA expression profiling. Methods: Megakaryocytes of formalin-fixed and paraffin-embedded (FFPE) bone marrows of PMF (n=8), ET (n=2) and normal hematopoiesis (n=2) were isolated by laser-assisted microdissection. Following RNA extraction and microRNA-specific cDNA synthesis, the expression levels of 384 microRNAs were determined by TaqMan Low Density Arrays. Results: We identified several microRNA species that were differently expressed in megakaryocytes in CMPD compared to normal counterparts. Differences between PMF and ET also emerged but await confirmation by higher sample numbers. Conclusions: Since microRNAs have a relevant function in basic cellular processes such as proliferation and differentiation, our set of candidate microRNAs will allow important insights in the differences of megakaryocyte homeostasis in PMF and ET. This study provides first clues about the potential of microRNAs for subtyping of myeloproliferative disorders on the molecular level.

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Abstracts Do-042 Bone morphogenetic proteins (BMPs) are overexpressed in the bone marrow of primary myeloﬁbrosis and are apparently induced by ﬁbrogenic cytokines O. Bock, J. Höftmann, K. Theophile, K. Hussein, J. Schlué, H. Kreipe Institut für Pathologie, Medizinische Hochschule Hannover Aims: Primary myelofibrosis (PMF) is a chronic myeloproliferative disease characterized by progressive deposition of extracellular matrix components in the bone marrow. The process of myelofibrosis is thought to be reactive in nature and a broad range of fibrogenic growth factors have been demonstrated to be involved. Whether members of the bone morphogenetic protein (BMP) family are also involved in bone marrow matrix homeostasis in PMF has not yet been investigated. Methods: In this study, expression of BMP1, an activator of latent transforming growth factor beta-1 (TGFbeta-1) and processor of collagen precursors, and other BMP members by bone marrow cells in different stages of PMF (n=70) and controls (n=25) was analyzed by real-time RT-PCR and immunohistochemistry. Primary fibroblasts were cultured in vitro in the presence of TGFbeta-1 and basic fibroblast growth factor (bFGF). Results: In total bone marrow cells of PMF BMP1, -6, -7, and BMP receptor 2 (BMP-R2) were significantly increased in different stages of myelofibrosis compared to controls (p≤0.01). Bone marrow stromal cells and megakaryocytes were uncovered to be the major cellular source for BMP1 protein expression in advanced stages. Because TGFbeta-1 and bFGF have been shown to be important in the development of myelofibrosis, we studied the induction of BMPs by these cytokines in vitro. Fibroblasts treated with TGFbeta-1 showed an exaggerated up-regulation of BMP6, BMP1, and BMP-R2 suggesting that these stroma cells may be susceptible for BMP activation by cytokines with a proven role in the pathogenesis of PMF. Conclusions: We conclude that BMP family members may play an important role in the pathogenesis of myelofibrosis in PMF and are apparently induced by fibrogenic cytokines.

Do-043 KIT D816V-positive acute myeloid leukemia is usually associated with concomitant mastocytosis K. Sotlar, R. Fritsche-Polanz1, S. Colak2, K. Sonneck3, S. Florian3, W.R. Sperr3, H.P. Horny4, P. Valent3, M. Födinger5 Pathologisches Institut, Universitätsklinikum München (LMU) 1 Institut für medizinische und chemische Labordiagnostik, Hietzing Hospital Wien 2 Institut für Pathologie, Universitsklinikum Tübingen 3 Klinische Abteilung für Hämatologie und Hämostaseologie, Klinik für Innere Medizin I, Medizinische Universität Wien 4 Institut für Pathologie, Ansbach 5 Klinisches Institut für Medizinische und Chemische Labordiagnostik, Medizinische Universität Wien Aims: The KIT mutation D816 V is detectable in most patients with systemic mastocytosis (SM), including those with an associated hematologic non-MClineage disease. Recently, KIT D816 V has been reported in with acute myeloid leukemia (AML). However, it was not clarified whether these patients have co-existing SM. Methods: We investigated total DNA or mRNA from 174 patients with AML for the presence of KIT D816 V. In a subset of KIT D816V-positive patients, DNA form microdissected MC and leukemic blasts was also investigated for this mutation. Results: In 11/174 patients (6.3%), KIT D816 V was detectable. After thorough histologic analysis, all 11 cases were found to have associated SM. Microdissected tryptase+ MC in these patients displayed KIT D816 V in all cases tested, whereas CD34+ blasts exhibited KIT D816 V in only 3/9 patients investigated. In one patient with AML, SM with wt-KIT was detected. Conclusions: In summary, we could show that KIT D816 V in AML is highly associated with a co-existing SM (i.e., SM-AML). Moreover, AML blasts in

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these patients may also carry the transforming KIT mutation, which may be important when tyrosine-kinase inhibitors are considered for therapy.

Do-044 Immunohistochemical Proﬁling of Diﬀuse Large B-cell Lymphoma in a Prospective Randomized Trial H.-W. Bernd1, M. Pfreundschuh2, M. Ziepert3, A.C. Feller1 1 Institut für Pathologie, Universitätsklinikum S-H, Campus Lübeck, stellvertretend für die deutschen Referenzzentren für Lymphknotenpathologie 2 Klinik für Innere Medizin I, Universitätsklinikum Homburg/Saar 3 IMISE, Universität Leipzig Aims: The effords to develop a robust immunohistochemical classifier for prognostic class prediction in diffuse large B-cell lymphoma yielded in inconclusive and conflicting results so far. In this study, we performed IHC profiling in DLBCL cases from a large prospective randomized trial to possibly disclose prognostic subgroups and to test the published immunohistochemical classifiers in an independent prospective cohort. Methods: 414 cases of DLBCL, all of which are enrolled in the NHL-B trial of the DSHNHL, have been immunohistochemically studied with a broad panel of antibodies using tissue micro arrays. Results were correlated to clinical data and compared to published classifiers. Results: Amongst other results, the presence of CD23-positive FDC meshworks, a clonal light chain secretion by the tumour cells and expression of HLA-DR emerged as independent markers of improved OS in multivariate analyses. By applying to all cases previous published algorithms for classification into a GCB and a non-GCB group, we observed a strong trend for better OS in the GCB cases, but this was not significant (p=0,055). Thus, the reported significant prognostic value of class prediction according to suggested IHC classifiers could not be reproduced in our series. Conclusions: Results of several IHC studies are heterogeneous and disclose a limited reproducibility. Robust and reproducible immunohistochemical class prediction needs highly standarized work up and evaluation criteria that are universally accepted. None of the proposed IHC predictors is yet ready for clinical application.yet ready for clinical application.

Do-045 The Role of JunB in lymphoma development D. Laimer1, P. Vesely2, P. Pﬂegerl1, S. Lagger3, P. Staber2, C. Seiser3, G. Höﬂer2, L. Kenner1 1 Klinisches Institut für Pathologie, Medizinische Universität Wien 2 Institut für Pathologie, Medizinische Universität Graz 3 Universität Wien Aims: Anaplastic large-cell lymphoma (ALCL) is often associated with the generation of the fusion protein NPM-ALK. NPM-ALK positive ALCL is characterized by high expression of CD30 and of AP-1 transcription factor family members, mainly JunB. In this study we investigate mechanisms underlying tumor formation by the NPM-ALK fusion protein. JunB or one of its target genes could serve as diagnostic parameter for diagnosis or therapeutic target of ALCL lymphomas. Methods: In vitro models consist of different NPM-ALK positive or negative human ALCL cell lines. As in vivo model we breed transgenic mice expressing the human NPM-ALK fusion protein in T-cells and a T-cell specific JunB and/or cJun knockout (cre/lox system). Results: We showed that the Ap-1 complex in NPM-ALK positive cells largely consists of JunB and that NPM-ALK influences JunB via the Pi3 K/mTor signalling pathway. In NPM-ALK positive patient ALCL samples, we showed that mTor is active, while this is not the case in NPM-ALK negative ALCL samples. In the ongoing in vivo part of our project, we demonstrated that in contrast to JunB or cJun deficient mice, JunB/cJun deficient mice survive 70% longer than control mice. Tumors of JunB/cJun deficient mice have severely decreased mRNA/protein levels of PDGFRβ, which is essential for PI3 K/ mTor activation. Tumors of JunB/cJun deficient mice also show decreased proliferation and increased apoptosis rates, as well as decreased mRNA levels of several JunB target genes.

Conclusions: JunB is regulated by NPM-ALK via the PI3 K/mTor signalling pathway. Tumor formation is severely impaired in mice by a T-cell specific “knock-out” of JunB and cJun.

Do-046 Numeric and structural JAK2 aberrations and altered phosphorylated STAT (pSTAT) expression in lymphomas S. Höller, C. Meier, C. Bourgau, P. Hirschmann, A. Reiter1, P. Went, S. Dirnhofer, A. Tzankov Institut für Pathologie, Universitätsspital Basel 1 Abteilung für Hämatologie, Universitätsklinikum Mannheim Aims: The majority of Ph- chronic myeloproliferative diseases carry the oncogenically important activating point mutation V617F of the JAK2 gene. Gains and, occasionally, breaks in the JAK2 locus at 9p24 have been detected in primary mediastinal B-cell- (PMBCL), Hodgkin- (HL) and single peripheral T-cell lymphomas (PTCL). We aimed to asses the frequency of JAK2 aberrations and the expression of pSTAT in a large lymphoma collective. Methods: We analyzed 514 cases [128 diffuse large B-cell- (DLBCL), 50 PMBCL, 84 follicular- (FL), 39 small lymphocytic- (SLL), 18 mantle cell- (MCL), 26 angioimmunoblastic T-cell lymphomas (AILT), 18 PTCL and 152 HL], which were brought into a tissue microarray format, by means of fluorescent in situ hybridization with double-colored break-apart JAK2 probes and immunohistochemistry with anti-pSTAT and pJAK2 antibodies. Results: Gains of 9p24 were detectable in 35% of PMBCL, 33% of HL and in 14% of secondary DLBCL, while breaks were observed in one HL and one AILT case each. pJAK2 was prevalent in AILT followed by PTCL and PMBCL; pSTAT1 was more commonly expressed in HL, PMBCL and in a small number of primary DLBCL; pSTAT3 predominated in AILT, HL, PMBCL and PTCL; pSTAT5 was more common in HL, AILT, PTCL and PMBCL. Expression of pSTAT3 in AILT and pSTAT5 in PMBCL and in secondary DLBCL correlated with gains at 9p24. Only nuclear expression of pSTAT1 and pSTAT5 correlated with the JAK2 breaks in HL and AILT. pJAK2 and pSTAT5 correlated with better prognosis in FL. Conclusions: The JAK2/STAT pathway seems to be oncogenically relevant in HL, PMBCL, AILT and perhaps some PTCL and FL.

Do-047 Follicular Lymphomas With and Without Translocation t(14;18) Diﬀer in Gene Expression Proﬁles and Genetic Alterations E. Leich1, I. Salaverria2, S. Bea2, A. Zettl1, R.D. Gascoyne3, W.C. Chan4, R.M. Braziel5, L.M. Rimsza6, D.D. Weisenburger4, J. Delabie7, E.S. Jaﬀe8, T.A. Lister9, A.J. Norton9, L.M. Staudt10, E.M. Hartmann1, H.K. MuellerHermelink1, E. Campo2, G. Ott1,11, A. Rosenwald11 Institut für Pathologie, Universitätsklinikum Würzburg 2 Department of Pathology, Hospital Clinic, Barcelona, E 3 British Columbia Cancer Agency, Vancouver, CAN 4 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, USA 5 Southwest Oncology Group, Oregon Health and Science University, Portland, USA 6 Department of Pathology, University of Arizona, Tucson, USA 7 Norwegian Radium Hospital, Oslo, SWE 8 Laboratory of Pathology, National Cancer Institute, Bethesda, USA 9 Cancer Research UK, St. Bartholomews Hospital, London, UK 10 Metabolism Branch, National Cancer Institute, Bethesda, USA 11 Institut für klinische Pathologie, Robert-Bosch-Krankenhaus Stuttgart Aims: Identification of the frequency of t(14;18)-negative FL in a series of 164 cases of FL1–3a and comparison of genetic alterations and gene expression profiles between FL with and without t(14;18).Methods: Gene expression profiling (Dave et al., NEJM 351:2159–69, 2004), comparitive genomic hybridization, PCR, FISH, IHC. Results: Clinically, there was no difference between the t(14;18)-negative and -positive FL subgroups regarding median survival times. The t(14;18)-positive subgroup shows gains of 18q, not observed in the t(14;18)-negative cases. Cell

Cycle and NFKB target genes were found to be enriched in the t(14;18)-negative FL subset, whereas the t(14;18)-positive subgroup is defined by an enrichment of GCB markers. Conclusions: t(14;18)-negative FL differ from t(14;18)-positive FL regarding gene expression and genetic alterations.

Do-048 A comprehensive genetic and morphological analysis of MCL results in the delineation of distinct tumour proﬁles T. Katzenberger1, D. Kienle2, S. Stilgenbauer2, S. Höller1, C. Schilling1, U. Mäder3, B. Puppe1, C. Petzoldt1, S. Sander2, L. Bullinger2, H. Stöcklein1, J. Kalla1, E. Hartmann1, P. Adam1, M.M. Ott4, H.K. Müller-Hermelink1, A. Rosenwald1, G. Ott1,5 1 Institut für Pathologie, Universitätsklinikum Würzburg 2 Klinik für Innere Medizin III, Universitätsklinikum Ulm 3 Interdisziplinäres Zentrum für Klinische Forschung der Universität Würzburg 4 Institut für Pathologie, Bad Mergentheim 5 Institut für Klinische Pathologie, Robert-Bosch-Krankenhaus Stuttgart Aims: MCL is an aggressive lymphoid tumour characterized by the translocation t(11;14) and a poor clinical outcome. Recent studies revealed that an increased proliferation rate and certain chromosomal aberrations, such as deletions of TP53 and P16INK4a represent major adverse biological markers in this disease, although the molecular targets of chromosomal imbalances in MCL have not been identified for the large majority of loci affected. Methods: To correlate histomorphological and proliferation features of MCL with genetic findings we investigated 223 MCL by classical cytogenetic banding analyses (n=129) and/or FISH (n=157) covering the nineteen most common chromosomal alterations in MCL. Genetic findings were correlated to histomorphological features, proliferation data and clinical characteristics. Results and Conclusions: FISH analysis turned out to be more sensitive in the delineation of aberrations. Complex karyotypic alterations were associated with higher proliferation indices and inferior prognosis. A comprehensive analysis of biological features including genetic alterations in MCL by hierarchical clustering resulted in the delineation of four tumour subgroups differing with respect to their genetic constitution and suggesting different transformation or progression pathways. Moreover, in one of the groups identified, an unusually indolent clinical behaviour was associated with few secondary aberrations and the absence of known high-risk chromosomal aberrations pointing to the importance of karyotypic evolution in MCL.

Do-049 SNP Array Analysis Reveals Copy Number Alterations and Uniparental Disomy in Mantle Cell Lymphomas at High Resolution E. Hartmann1*, I. Salaverria2*, S. Beà2, A. Zettl1, P. Jares2, F. Bosch3, G. Ott1,4, H.-K. Müller-Hermelink1, E. Campo2#, A. Rosenwald1# 1 Institut für Pathologie, Universitätsklinikum Würzburg 2 Department of Pathology and 3 Department of Hematology, Hospital Clinic, University of Barcelona, E 4 Institut für Klinische Pathologie, Robert-Bosch Krankenhaus Stuttgart Aims: We investigated Mantle Cell Lymphoma (MCL) cell lines and primary samples with single nucleotide polymorphism (SNP) based arrays to determine the capability of this technique for high resolution copy number (CN) analysis and detection of loss of heterozygosity (LOH) in MCL and to refine altered regions of interest. Methods: We analyzed the 3 t(11;14)-positive MCL cell lines Granta-519, HBL-2 and JVM-2 and 5 primary tumor specimens from untreated MCL patients with both the 100 K and 500 K SNP array sets from Affymetrix and compared the results with data obtained by comparative genomic hybridization (CGH) approaches. Results: The results of both SNP array platforms were in excellent agreement, and confirmed and refined the alterations detected by published conventional and array CGH techniques. Moreover, we observed numerous additional small alterations that were not detectable by conventional CGH. Furthermore, Der Pathologe · Supplement 1 · 2008

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Abstracts we identified large regions of copy neutral LOH, also described as uniparental disomy, in the cell lines as well as in the primary tumor samples. Conclusions: The results demonstrate the capability of SNP array analysis for identifying CN alterations and copy neutral LOH at high resolution in MCL cell lines as well as in primary tumor samples.

Do-050 Quantitative immunohistochemistry reveals diﬀerences between follicular dendritic cells (FDC) in reactive germinal centers and follicular lymphoma M.K. Jin, W. Klapper Institut für Pathologie, Sektion Hämatopathologie und Lymphknotenregister, Universitätsklinikum S-H, Campus Kiel Aims: Follicular dendritic cells (FDC) are antigen presenting cells exclusively found in germinal centers (GC) under reactive conditions. Within the GC, FDC play a pivotal role in the regulation of B Cell selection and proliferation. In certain B-cell Non-Hodgkin-lymphomas like follicular lymphoma (FL) FDC can be detected as part of the reactive bystander cells of the tumor. Recently, gene expression profiling revealed expression of FDC related genes to be associated with the clinical course in follicular lymphoma (FL). The aim of our study was to characterize differences and commonalities of FDC in reactive GC and FL to gain insight into the possible role of FDC in the tumor development and maintenance. Methods: We studied quantitatively the expression of proteins known to be involved in FDC-B-cell interactions in reactive GC and FL. Fluorescent double staining using a pan-FDC marker (Ki-M4p) in combination with other antibodies was followed by image analysis (analySIS™) to assess quantitatively protein expression in FDC. Results: We were able to demonstrate, that compared to reactive GC, FDC in FL display quantitatively reduced expression of the adhesion molecule CD54, the complement receptor CD35 and the FC-receptor CD23. Conclusions: We have established a method to assess quantitatively protein expression in FDC. Using this technique we found that protein expression differs quantitatively between FDC in reactive GC and FL. The reduced expression of molecules involved in FDC-B-Cell interaction might suggest that FDC in FL are functionally impaired compared to their normal counterparts.

Do-051 The prognostic role of histopathology and T-cell subpopulations in follicular lymphoma – results from a prospective randomized trial of the German Low Grade Lymphoma Study Group (GLSG) K. Koch1, E. Hoster2, M. Unterhalt2, M. Dreyling2, W. Hiddemann2,W. Klapper1 1 Institut für Pathologie, Sektion Hämatopathologie und Lymphknotenregister, Universitätsklinikum S-H, Campus Kiel 2 Medizinische Klinik und Poliklinik 3, Universitätsklinikum München (LMU) Aims: In follicular lymphoma (FL) several studies have analyzed the prognostic significance of the T-cell content with discrepant results. Furthermore, the prognostic significance of histopathological heterogeneity e.g. reflected by growth pattern and tumor cell differentiation is unclear so far. Most studies published so far analyzed cohorts with heterogeneous treatment. Our aim was to assess systematically the prognostic significance of histopathology and T-cell sub-populations in FL, treated within a randomized, prospective trial. Methods: 155 FL were assessed for WHO-grade, diffuse growth, sclerosis and differentiation. Furthermore, a tissue-micro-array (TMA) was generated which contained FL treated within a clinical trial with progress after <17 month (n=11) and progress >82 month (n=11). Using this TMA as a feasibility-set, the T- and NK-cells were quantified by immunohistochemistry using antibodies against CD3, CD8, FoxP3, TIA, Granzyme B and CD56. Results: Tumor sclerosis but not WHO-grade, diffuse growth or differentiation was associated with poor prognosis. The extent and the distribution of T-cells in FL varied strikingly in the analyzed cohort. Clinico-pathological correlations will be presented. Conclusions: Due to its heterogeneous clinical course, prognostic biomarkers for follicular lymphoma are needed. We suggest that the analysis of samples

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from patients treated within prospective randomized trials is necessary to validate prognostic marker in FL.

Do-052 3’UTR deﬁcient Cyclin D1 mRNA is associated with high cyclin D1 mRNA levels and increased proliferation in mantle cell lymphoma, but not with blastoid morphology J. Slotta-Huspenina1, I. Koch1, L. de Leval2, K. Bink3, M. Kremer1, L. QuintanillaMartinez3, F. Fend1 1 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München 2 Institute of Pathology, Univ. Leuven, B 3 Institut für Pathologie, GSF Forschungszentrum Neuherberg Aims: The hallmark of MCL is overexpression of cyclin D1 (CyD1). Predominance of 3’UTR-deficient (Δ3’UTR) CyD1 mRNA was found to be associated with a “proliferation signature” and poor prognosis. We correlated the expression of CyD1 mRNA isoforms with morphological features and proliferation rate in MCL. Methods: Paraffin-embedded samples of 65 CyD1+ MCL, including 9 (14%) blastoid cases, were analysed by real-time RT-PCR for expression of total CyD1, Δ3’UTR isoform and CyD1α and β mRNA splice variants, relative to TBP as a reference gene. The findings were correlated with morphology, proliferation rate as assessed by MIB1 staining, and genomic deletions or mutations. Results: MCL cases expressed cyclin D1 mRNA with a mean ratio of 66.9. Predominance of Δ3’UTR mRNA was found in 15 (23%) samples, including 4 (6%) with complete loss of the standard transcript, accompanied by deletion of the UTR locus. Δ3’UTR predominance was significantly associated with high total CyD1 mRNA levels (p=0.004), and a tendency to increased proliferation. Proliferation rate was three times higher in blastoid than in classical cases (mean 43.3 versus 16.0) and especially high in a blastoid sample with complete 3’UTR loss (89.0). CyD1β transcript expression was low (mean: 7.7% of total mRNA) and did not correlate with morphology, proliferation rate, or the A870G splice site polymorphism. Conclusions: CyD1β transcript does not seem to play a role in MCL biology. In contrast, predominance of the Δ3’UTR CyD1 mRNA in MCL is associated with higher total CyD1 mRNA levels and may represent an alternative pathway for increased proliferative activity, unrelated and possibly additive to blastoid morphology.

Do-053 MicroRNA Expression Proﬁling Characterize Diﬀuse Large B-Cell Lymphomas and Follicular Lymphomas A. Röhle1, K.P. Höﬁg1, D. Repsilber2, C. Thorns1, K.O. Wesche1, M. Thiere1, W. Klapper3, M. Ziepert4, M. Löﬄer4, H. Merz1, A.C. Feller1 1 Institut für Pathologie, Universitätsklinikum S-H, Campus Lübeck 2 Forschungsinstitut für die Biologie landwirtschaftlicher Nutztiere (FBN), Dummerstorf 3 Institut für Pathologie, Universitätsklinikum S-H, Campus Kiel 4 Institut für Medizinische Informatik, Statistik und Epidemiologie (IMISE), Leipzig Aims: Recent studies indicate that miRNAs are mechanistically involved in the development of various human lymphomas and other malignancies and therefore represent a promising new class of biomarkers. We evaluated the miRNA expression profiles of 58 Diffuse Large B-Cell Lymphomas (DLBCL), 46 Follicular Lymphomas (FL) and 7 lymph nodes with chronic lymphadenitis. Methods: A quantitative PCR (qPCR)-based method was used to determine the expression levels of 157 miRNA species. The analysis was performed exclusively on FFPE tissues. Results: Comparison of the possible combinations of DLBCL-, FL- and lymph node profiles resulted in specific DLBCL- and FL-signatures, which include miRNAs with previously published function in hematopoiesis (such as miR-150 and miR-155). As compared to lymph nodes, some miRNAs are differentially regulated in both lymphoma types. Conversely, some miRNAs

show lymphoma-specific aberrant expression in DLBCL or FL. A classification tree was computed using four miRNAs to correctly identify 98 % of all cases that were analysed in this study. Conclusions: MicroRNAs are promising new biomarkers, involved in a multitude of human malignancies and – unlike mRNA – can be reliably detected and quantified in FFPE samples.

Do-054 Detailed deletion mapping of chromosome 17p reveals HIC1 as a novel tumor suppressor candidate in diﬀuse large B-cell lymphoma (DLBCL) H. Stöcklein1, J. Smardova2, J. Macak2, T. Katzenberger1, S. Höller1, S. Wessendorf3, G. Hutter4, M. Dreyling4, E. Haralambieva1, U. Mäder5, H.K. MüllerHermelink1, A. Rosenwald1, G. Ott1,6, J. Kalla1 1 Institut für Pathologie, Universitätsklinikum Würzburg 2 Department of Pathological Anatomy, Brno, CZ 3 Klinik für Innere Medizin III, Universitätsklinikum Ulm 4 Medizinische Klinik und Poliklinik III, Universitätsklinikum MünchenGrosshadern 5 Interdisziplinäres Tumorzentrum, Universitätsklinikum Würzburg 6 Institut für Klinische Pathologie, Robert-Bosch-Krankenhaus, Stuttgart Aims: Deletions in the short arm of chromosome 17 (del(17p)) involving TP53 occur in up to 20% of DLBCL. Although inactivation of both alleles of a tumor suppressor gene (TSG) is required for tumor development, the overlap between TP53 deletions and mutations is poorly understood in this entity, suggesting the existence of additional tumor suppressor candidates in 17p. Methods: Fluorescence in situ hybridization, chromosome banding, immunohistochemistry, direct sequencing, functional analysis of p53 (FASAY) and methylation-specific PCR. Results: A biallelic inactivation of TP53 was detected in 13/55 DLBCL, while 22/55 cases harbour a monoallelic TP53-alteration. A minimally deleted region, telomeric to the TP53 locus was defined in 11/55 cases, including the TSG HIC1. Furthermore, deletion of HIC1 was found in 28/55 TP53-deleted DLBCL. HIC1-hypermethylation was demonstrated in 30/55 DLBCL, and was associated with HIC1-deletion in 90% of the cases (27/30). Simultaneous inactivation of TP53 and HIC1 was correlated with worse clinical outcome. Conclusions: These findings strongly suggest HIC1 as a novel, clinical relevant TSG in DLBCL.

Do-055 PRDM 1/Blimp1 Expression is Associated with BCL6 and PAX5 Downregulation in a Subset of GCB-Type Diﬀuse Large B-Cell Lymphoma M. Andrulis1, A. Avramidou2, H.M. Jäck2, M. Hartmann3, S. Höller3, G. Ott3, A. Rosenwald3, H.-K. Müller-Hermelink3, A. Greiner1 1 Pathologisches Institut, Universitätsklinikum Heidelberg 2 Abt. für Molekulare Immunologie, Nikolaus-Fiebiger-Zentrum, Erlangen 3 Institut für Pathologie, Universitätsklinikum Würzburg Aims: Blimp1, BCL6, and PAX5 are key transcription factors that control terminal B-cell maturation. Impairment of their function may hamper B-cell differentiation and therefore drive lymphoma development. We wanted to dissect the transcriptional control of terminal differentiation in diffuse large B-cell lymphoma (DLBCL). Methods: 78 DLBCL cases were subgrouped into GCB and non-GCB subtypes and the co-expression of Blimp1 and its target genes BCL6 and PAX5 was analyzed. Additionally, the relationship between Blimp1 expression and plasmacytic differentiation was investigated. Results: Blimp1 was expressed in 12% (6/49) of GCB-type DLBCL and 28% (8/29) of non-GCB-type DLBCL. Interestingly, Blimp1 expression was associated with downregulation of BCL6 and PAX5, but not immunoblastic/plasmablastic differentiation in GCB-type DLBCL. Only two Blimp1+ GCB-type DLBCL cases aberrantly co-expressed Blimp1 and Bcl-6. Conclusions: In conclusion, Blimp1 expression characterizes a subset of GCBtype DLBCL which is defined by Blimp1+BCL-PAX5- phenotype, but does not exhibit features of plasmacytic differentiation.

Do-056 Small cell variant classical Hodgkin lymphoma: a histopathological study of 28 cases of classical Hodgkin lymphoma E. Shiozawa1,2, K. Jöhrens1, I. Anagnostopoulos1, H. Stein1 1 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin 2 Japan-Stipendium für deutschsprachige Pathologen, Deutsche Gesellschaft für Pathologie e.V. Aims: A histological diagnosis of classical Hodgkin lymphoma (CHL) has required demonstration of the presence of Hodgkin/Reed-Sternberg (HRS) cells, which have characteristic morphological features. In consulting on many diagnoses of CHL, we have noticed the presence of rare cases of CHL that have no cells with morphological features of HRS cells in the lesion, but the neoplastic cells of the lesion have an immunohistochemical phenotype consistent with HRS cells. We describe the epidemiology and clinicopathological features of these cases of CHL. Methods: The study included all cases of CHL which were newly diagnosed between 2003 and 2006 at the Consultation and Reference Center for Hematopathology at our institute. Results: A detailed review of 1158 cases of CHL showed that 57 (4.9%) were a new morphological variant of CHL without typical HRS cells. These cases were classified into histological subtypes: 24 of the nodular-sclerosis type (42%), 19 of the mixed-cellularity type (33%), and 8 of the lymphocyte-rich type (14%). 19 cases (33%) had a correlation with EBV infection. There were no specific immunohistochemical features in the variant of CHL. Conclusions: We conclude that a small number of CHL cases generate only non-specific neoplastic cells and we here propose that these cases represent a new morphological variant, which we refer to as small cell variant CHL.

Do-057 Autocrine NGFβ/TRKA signalling is an important survival factor for Hodgkin lymphoma derived cell lines C. Renné, S. Minner, R. Küppers1, M.-L. Hansmann, A. Bräuninger Institut für Pathologie, Universitätsklinikum Frankfurt am Main 1 Institut für Zellbiologie, Universitätsklinikum Essen Aims: Receptor tyrosine kinases (RTKs) are frequently involved in cellular transformation. The high expression of seven RTKs in primary HodgkinReed/Sternberg (HRS) cells in the majority of Hodgkin lymphoma cases has been observed. As gene dosage effects can contribute to the overexpression of genes we performed FISH analyses for the locus of the RTK TRKA with HRScell lines. Non neoplastic B cells, like HRS cells produce TRKA and its high affinity ligand NGFβ, which is a known autocrine survival factor for memory B cells. HRS cells are B-cell derived, NGFβ/TRKA signalling supports survival of B cells and TRKA is highly expressed in HRS cells, TRKA signalling could also be a survival factor in HRS cells. We thus analysed the role of TRKA on survival of four HL-derived cell lines as models for primary HRS cells. Methods: TRKA expression in primary HL cases was demonstrated by immunohistochemistry. TRKA, phospho-TRKA and NGFβ were detected by Western blotting or ELISA in cell lines. Survival of HL derived cell lines treated with K252a was measured by MTT assay. The number of the TRKA genomic loci in HL derived cell lines was accessed by FISH. Results: Immunohistochemically detectable TRKA expression is largely restricted to HRS cells of HL cases. TRKA expression in HL derived cell lines is much stronger than in non neoplastic B-cells. In KMH2 and L1236 minor gains of the TRKA locus were observed by FISH. HL derived cell lines carry functional and permanent activated TRKA. Addition of the TRKA inhibitor K252a to HL derived cell lines leads to diminished activation of TRKA and AKT which is accompanied by reduced cell survival. Conclusions: Activation of TRKA/NGFβ signalling is a survival factor in HRS cell lines and might be an possible therapeutical target.

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Abstracts Do-058 Lack Of PTPN1 (PTP1B) In U-HO1, A New Hodgkin-derived Cell Line, Protects Cells From Apoptosis S. Wegener, A. Mader, I. Melzner, S. Brüderlein, T. Barth, P. Möller Institut für Pathologie, Universitätsklinikum Ulm

Do-060 SOCS1 mutations and activation of the JAK/STAT-pathway in lymphocyte-predominant Hodgkin lymphoma A. Mottok, C. Renné, K. Willenbrock, M.-L. Hansmann, A. Bräuninger Institut für Pathologie, Universitätsklinikum Frankfurt am Main

PTPs regulate multiple cytokine and growth factor activated signaling pathways which are associated with malignancies like myelodysplastic syndromes and B-cell lymphomas. PTPN1 is a well studied non-receptor phophatase and involved in regulation of apoptosis. JAK2 was shown to be a substrate of PTPN1 which results in a negative regulation of the kinase activity of JAK2 and the subsequent activation of downstream targets like STAT5. The parental tumor of U-HO1, a nodular sclerosing classical Hodgkin lymphoma (cHL), and its resulting cell line proofed negative for PTPN1 in Western blot and by immunomorphology. We hence investigated the expression of PTPN1. PTPN1 cDNA of U-HO1 was markedly truncated: exon 2 to exon 8 were skipped translating into the following predicted short protein of 26 amino acids: MEMEKEFEQIDKSGSWAAIYQHESRH. To examine the role of lack in functional PTPN1 we transfected U-HO1 cells with wt-PTPN1. Stable wt-PTPN1 transfectants featured an increased dephosphorylation of phosphoSTAT5, a very slow proliferation, and underwent apoptosis to a much greater extent than mock transfectants. To see whether PTPN1 deficiency is occuring in the HRS-cells of cHL in vivo, we analyzed 61 samples from patients with cHL by immunohistochemistry. Only 15 of 61 cHL samples tested had PTPN1-positive neoplastic cells. Thus lack of PTPN1 is a common feature in HRS-cells in vivo. In summary our results show that PTPN1 plays a major role in deactivation of the JAK2-/STAT5 signaling pathway and its deficiency saves HRS-cells from apoptosis.

Aims: While the derivation of the lymphocytic and histiocytic (L&H) tumor cells of lpHL from germinal center (GC) B cells is well established, knowledge about their pathogenesis is limited. In a survey of JAK2 expression in lymphomas we observed high JAK2 expression in lpHL and analysed the causes and consequences of the high JAK2 expression in L&H cells. Methods: Immunohistochemistry (IHC) was performed for JAK2, p-JAK2, p-Stat 3, 5 and 6. For the analysis of SOCS1-mutations single CD20-positive L&H cells from lpHL cases were micromanipulated and then the complete coding region of SOCS1 was amplified in 3 overlapping fragments in a seminested two round PCR. All PCR products were directly sequenced. Results: IHC revealed high JAK2 expression in the vast majority of L&H cells in most lpHL cases (40/47 cases). While no activation of STAT3 and STAT5 was observed, STAT6 was phosphorylated in 49% of cases (21/43). Mutations of the SOCS1-gene were found in 6 of 12 cases. In 3 cases inactivating mutations were observed and presence of several replacement mutations in functionally important regions in the 3 other cases suggest that SOCS1 function was also impaired in these cases. Conclusions: These data suggest that in L&H cells SOCS1 function is impaired by mutations which are in most cases likely due to activity of somatic hypermutation (SHM). SOCS1 inactivation may cause constitutive activation of the JAK2/p-STAT6 pathway in most lpHL cases.

Do-059 Induction of a Hodgkin-like Phenotype by epigenetic Reprogramming of B cells M. Hummel, A. Ehlers, E. Oker, S. Bentink, D. Lenze, H. Stein 1 Institut für Pathologie, Charité – Berlin, Campus Benjamin Franklin 2 Institut für funktionelle Genomik, Computational Diagnostics Group, Regensburg Aims: A characteristic feature of the tumour cells (Hodgkin/Reed-Sternberg (HRS)) of classical Hodgkin lymphoma (cHL) is the loss of their B-cell phenotype despite their B-cell origin. It is suggested that epigenetic events such as DNA-methylation in the promoter region are involved in this silencing of B-cell associated genes. Furthermore it has been shown that the up-regulation of B-cell inappropriate genes may also contribute to the extinction of the B-cell program. Methods: We treated Hodgkin and B-cell lines with DNA-demethylation (5aza-dC) and histone-acetylation (TSA) reagents. Treated and untreated cell lines were analysed by Affymetrix GeneChips and numerous up- and down regulated genes were verified by quantitative RT-PCR. Chromatin-immunoprecipitation was carried out to determine the epigenetic modifications in the promoter region of the corresponding genes. Results: The treatment of Hodgkin cell lines with demethylating and acetylating reagents had no significant effect on the reactivation of the B-cell expression program. Instead, the treatment of B cells resulted in a complete loss of the B-cell phenotype and – in parallel – to an up-regulation of cHL characteristic genes. Conclusions: Our data show that DNA-demethylation and histone-acetylation induce in B cells a Hodgkin-like phenotype whereas in Hodgkin cells no re-activation of B-cell typical genes or suppression of B-cell inappropriate genes was observed. This finding suggests that the same genes which can be turned on in B cells by DNA-demethylation and histone-acetylation are constitutively active in Hodgkin cells.

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Sitzung: AG Oralpathologie Do-061 Expression of Platelet-Derived Growth Factor-AA and Platelet-Derived Growth Factor-alpha Receptor in Ameloblastomas I. Sulzbacher, N. Wick, B. Pichlhofer, P.R. Mazal Klinisches Institut für Pathologie, Medizinische Universität Wien Aims: Platelet-derived growth factor (PDGF)-AA isoform and its receptor, PDGF-alpha receptor (PDGFRA) regulate tooth development and growth. We investigated the expression of both proteins in ameloblastomas, to contribute to the understanding of the potential role of the PDGF/PDGFR system in this odontogenic neoplasm. Methods: Twenty–nine specimens of ameloblastoma were analyzed for PDGF-AA and PDGFRA expression using immunhistochemistry. The proliferation activity was investigated with the MIB-1 antibody. Additionally, capillary sequencing of genomic DNA was performed to search for mutations in therapeutically relevant exons 12 and 18 of the PDGFRA gene. Results: PDGF-AA and PDGFRA expression were detectable in all cases with the exception of one tumor. However, protein expression levels did neither correlate with each other nor with MIB-1 expression. Unicystic ameloblastomas did not differ from solid tumors in regard to PDGF-AA, PDGFRA and MIB-1 expression. One tumor revealed a somatic mutation of exon 12 of the PDGFRA gene. Conclusions: PDGF-AA and PDGFRA proteins are regularly expressed in variable levels in ameloblastomas, and somatic mutations of exon 12 and exon 18 of the PDGFRA gene are rare findings.

Do-062 Epithelial and mesenchymal marker expression and p16 occurrence in primary tumors and metastases of HPV infected and non infected oropharynx squamous cell carcinoma M. Stenner1, B. Yosef2, S. Preuss1, H.P. Dienes2, J.P. Klussmann1, M. Odenthal2 1 Klinik und Poliklinik für Hals-, Nasen-, Ohren-Heilkunde und 2 Institut für Pathologie, Universitätsklinikum Köln

Do-064 Aberrant promoter methylation in head neck squamous cell carcinomas I. Tischoﬀ, A. Weber1, B. Frerich2, A. Tannapfel Institut für Pathologie, Ruhr-Universität Bochum 1 HNO-Klinik, Klinikum Remscheid 2 MKG-Klinik, Universitätsklinikum Leipzig

Aims: The oropharynx squamous cell carcinoma (OSCC) is a high malignant and common type of human cancer. The mechanism of invasion and metastasis in tumor cells appear to be very complex and the understanding is incomplete. The present study aimed to analyse the phenotypical changes of epithelial tumor cells during tumor progression and metastasis of OSCC. Methods: A collective of 48 OSCC primary tumors classified according to TNM (grade 1 to 3) and the corresponding metastases were investigated by immunostaining of epithelial (E-cadherin, β-catenin) and mesenchymal marker proteins (a-SMA, vimentin). In addition, the cell cyclus protein p16 and the status of HPV infection were evaluated. Results: Vimentin-immunostaining was more prominent in primary OSCC of high grade than of low grade (p=0.02). The localisation of β-catenin, which is observed at the membrane of normal epithelium, differed in tumors. Thus, β-catenin was frequently observed in the nucleus of tumor cells indicating that the cells were activated. Primary OSCC, which were HPV infected showed more translocation of β-catenin into the nucleus than HPV-non-infected tumors (p=0.03). In addition the p16 expression strongly correlated with HPV-infection. Thus, p16 was strongly enhanced in tumors (p=0.04) and in metastases (p=0.01), which were HPV infected. Conclusion: The present study demonstrates that vimentin expression is a marker for tumor progression and that in addition to the p16 expression the translocation of β-catenin should be considered as a reporter for HPV infection.

Aims: In addition to genetic alterations, epigenetic inactivation of tumor suppressor genes by promoter methylation has been recognized as an important and alternative pathway in tumorigenesis. We analysed promoter methylation of nine tumor related genes in primary head neck squamous cell carcinomas. Methods: Promoter methylation of CDH1, SOCS1, SOCS3, DAPK, RASSF1A, BLU, SEMA3B, GSTP1 and NORE1A was determined by Methylation-specific PCR (MSP) in fresh frozen tissue of 30 head neck squamous cell carcinomas, corresponding normal mucosa and 18 corresponding lymph node metastases. The mRNA transcript was analysed by semiquantitative real-time RT-PCR. Results: DNA methylation in primary squamous cell carcinomas was detected for: SEMA3B 30/30 (100%), SOCS3 25/30 (83%), CDH1 25/30 (83%), DAPK 6/30 (20%), SOCS1 4/30 (13%), NORE1A 2/30 (6%), RASSF1A 1/30 (3%), GSTP1 and BLU 0%; in corresponding lymph node metastases for: SEMA3B 18/18 (100%), CDH1 18/18 (100%), SOCS3 12/18 (72%), SOCS1 4/18 (22%), RASSF1A 3/18 (17%), DAPK 2/18 (11%), NORE1A 1/18 (6%), GSTP1 and BLU 0%. No methylation occurred in corresponding normal mucosa. Conclusions: Our results show that promoter methylation leads to subsequent inactivation of several tumor related genes in primary head neck squamous cell carcinomas and corresponding lymph node metastasis.

Do-063 Immunohistochemical Snail expression in oral squamous cell carcinoma (OSCC) – correlation to metastasis formation and myoﬁbroblast development A. Berndt, K. Spiegel, M. Franz, P. Richter, A. Altendorf-Hofmann1, I. Virtanen2, H. Kosmehl3 Institut für Pathologie und 1 Tumorzentrum e.V. Universitätsklinikum Jena 2 Institute of Biomedicine/Anatomy, University of Helsinki, FIN 3 Institut für Pathologie, HELIOS-Klinikum, Erfurt Aims: Epithelial mesenchymal transition (EMT) is supposed as a prerequisite for metastasis formation in carcinomas and may be involved in the development of tumour associated fibroblasts. E-cadherin repressors like Snail play a crucial role in the process of phenotype transition. The study was aimed at elucidating the role of Snail mediated EMT for metastasis formation and myofibroblast development in OSCC. Methods: The immunohistochemical expression of Snail was semiquantitatively analysed in the invasive front of 134 OSCC and correlated to lymph node metastasis, stage, and grade. Furthermore, to elucidate the relation to myofibroblast development, the occurrence and spatial distribution of Snail positive cells were correlated to -smooth muscle actin (ASMA) positivity. Results in OSCC were compared to findings in the non metastatic basal cell carcinoma of the skin (BCC). Results: Snail was found in stromal, tumour, and endothelial cells. Although there is an increase of Snail positive stromal cells with raising grade and stage and the occurrence of nodal metastasis, a statistically significant correlation could not be evidenced. Snail showed a significant correlation and spatial colocalization to ASMA positive cells. Snail expression does not influence patient survival. In BCC, Snail could never be seen. Conclusions: The analysis of the immunohistochemical Snail expression cannot contribute to the prediction of metastatic potential and survival of OSCC patients. Nevertheless, snail induced EMT seems to be associated with myofibroblast transdifferentiation in OSCC with possible relevance to tumour biological behaviour.

Do-065 Growth factor induced phenotype changes in oral squamous cell carcinoma (OSCC) cells in vitro – a cell culture model for inducible epithelial to mesenchymal transition (EMT) in oral cancer? P. Richter, C. Umbreit, M. Franz, A. Berndt1, H. Kosmehl2, A. Berndt Institut für Pathologie, Universitätsklinikum Jena 1 Institut für Molekulare Pathogenese des Friedrich-Loeﬄer-Instituts, Bundesforschungsinstitut für Tiergesundheit, Standort Jena 2 Institut für Pathologie, HELIOS-Klinikum, Erfurt Aims: Invasion requires changes in carcinoma cells enabling scattering and migration. Epithelial mesenchymal transition (EMT) is a key step in this process and may be crucial for metastasis formation. Several growth factors are known to be involved in regulation of EMT in OSCC. The study was aimed at elucidating the role of TGFβ1 and EGF for phenotype transition of OSCC cells in vitro and if they are able to induce EMT. Methods: Characterization of OSCC cell lines (PE/CA-PJ15, 34, 41, 49) according morphology and immunohistochemical E-cadherin, vimentin, laminin-5 gamma2 chain (Ln-γ2) and snail expression. Stimulation with TGFβ1, EGF or both in combination over 5 h, 24 h and 4d. Analysis of phenotype changes and mRNA expression by immunohistochemistry and real time RT-PCR. Results: Cell lines differ in morphology: one with an epithelial like (PJ15) and one with a more mesenchymal appearance (PJ41) were used for stimulation experiments. In both cell lines, TGFβ1 and EGF induce snail mRNA-Expression. A down-regulation of E-cadherin and scattering could only be seen in PE15 stimulated with EGF or TGFβ1/EGF. Nevertheless, an up-regulation of vimentin mRNA and protein could be reached in both cell lines with TGFβ or co-stimulation. Up-regulation of vimentin is accompanied by an increase in Ln-γ2 synthesis. Conclusions: In OSCC cells in vitro (1) phenotype changes after growth factor stimulation depend on the differentiation status, (2) EGF/TGFβ1 co-stimulation is a potent inducer of EMT and may be useful as an in vitro model of phenotype transition, (3) EMT is accompanied by an up-regulation of the migration factor Ln-γ2.

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Abstracts Do-066 Detection of frequent genetic alterations on 16q in mucoepidermoid carcinomas H. Schmidt1, E. Korsching1, J. Alberty2, H.J. Holzhausen3, H. A. Baba4, W. Böcker1, C. August1 1 Institut für Pathologie und 2 HNO-Klinik, Universitätsklinikum Münster 3 Institut für Pathologie, Universitätsklinikum Halle 4 Institut für Pathologie und Neuropathologie, Universitätsklinikum Essen Aims: The classification of salivary gland tumours is based on its wide spectrum of differentiation. Genetic alterations responsible for tumour origin and progression are obscure. Previous CGH and microsatellite analyses showed several genetic hot spots especially on chromosome 6q. Our study focused on microsatellite markers frequently lost in invasive mamma carcinoma especially on 16q known as early events in progression of this tumour. Methods: Paraffin embedded tissues of 20 parotid mucoepidermoid carcinomas (MEC) were investigated by immunohistochemistry and by polymorphic microsatellite markers D7S522, D8S522, NEFL, D10S541 (PTEN), D13S153 (RB1), D16S400, D16S402, D16S422 and D17S855 (BRCA1) using a multiplex PCR-based analysis for the detection of allelic imbalances (AI). Results: Two different groups of tumours were detected by the panel of microsatellites. Confirming the results through mathematical cluster analyses one group was characterized by the frequent genetic imbalance for NEFL, PTEN, RB1 and for the marker on 16q. Tumours with this genetic background were frequently MEC of high grade malignancy showing a dominance of CK 5/6 and p63 compared to CK 8/18. Conclusions: The set of applied microsatellites is helpful to characterize MEC based on different ways of tumour progression in a better way. The two different groups also showed immunohistochemical differences with dominance of stem cell markers in MEC high grade and differences in follow up. AI on 16q seems to be involved in tumour progression similarly to the progression pathways known in mamma carcinoma.

Do-067 Cervical CUP – primary carcinomas of the tonsils and of the vallecula are small and can be missed G. Assmann1, C. Weiler1, P. Zengel2, T. Kirchner1, S. Ihrler1 1 Pathologisches Institut und 2 Klinik für Hals-, Nasen- und Ohrenkranke, Universitätsklinikum München (LMU) Aims: In cervical CUP (cancer of unknown primary) the primary tumours are often located in the nasopharynx, tonsils and vallecula. In our own casuistic observations primary tumours in the tonsils and in the vallecula were very small. Methods: 19 cases with 1) primary clinical manifestation with lymph node metastases and 2) subsequent histological detection of a carcinoma in the tonsils (n=8) or in the vallecula (n=11) were analyzed. Results: In 14/19 cases a primary tumour could not be localized in clinical and endoscopical examination. Large (and often cystic) lymph node metastases (average size 3,54 cm) are found dispite of mostly small primary tumours (average size 1,12 cm; 11 cases ≤0.8 cm). For the primary tumours, the histological differentiation was lymphoepithelial in 13 and transitional in 6 cases. Infiltration of the overlying mucosa was not present in 12/19 cases. 7/8 tonsillar tumours were located purely at the cryptal base. Conclusions: In analogy to lymphoepithelial carcinomas of the nasopharynx carcinomas of the tonsils and vallecula with lymphoepithelial or transitional histology represent a distinct tumour entity: It is characterized by 1) early and extensive nodal metastases and 2) often clinically occult primary tumour (CUP), which is, according to our findings, 3) due to small tumour size and 4) deep, submucosal growth. Our data demonstrate, that in case of cervical CUP both a standardized clinical biopsy including diagnostic tonsillectomy and a meticulous pathological workup is necessary to detect these clinically occult primary tumours.

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Do-068 Acinar enlargement in salivary sialadenosis: Consequence of functional insuﬃciency of myoepithelial cells? S. Ihrler1, C. Rath1,2, C. Weiler1, P. Zengel3, T. Kirchner1, J.D. Harrison4 1 Pathologisches Institut und 3 Klinik für Hals-, Nasen- und Ohrenkranke, Universitätsklinikum München (LMU) 2 Zentrum für Präventivzahnmedizin, Parodontologie und Kariologie, Universität Zürich 4 Dept of Oral Pathology, King‘s College London, UK Aims: The mechanism of acinar enlargement in sialadenosis is obscure, although peripheral polyneuropathy secondary to meta-bolic or hormonal systemic disorders is considered to be significant. Methods: Based on a fortuitous observation of diminished α-actin-positive myofilaments in myoepithelial cells in sialadenosis, 11 cases each of control and sialadenosis parotid glands were morphometrically analysed assisted by immunohistochemistry for α-actin, p63, and cytokeratin 14 (markers for myoepithelial cells), as well as for CK7, S100, and Ki67 including double staining. Results: In sialadenosis: the acini were much larger (1991,5 μm2 versus 1013,1 μm2); the number of myoepithelial cells as % per acinus was moderately reduced (77.2% versus 93.8%); the amount of α-actin-positive myofilaments as % per acinar circumference was greatly reduced (7.2% versus 47.2%; highly significant); and the proliferation rate of acinar cells (0.8% versus 2.0%) and of myoepithelial cells (0.0% versus 0.2%) was reduced. Conclusions: The great loss of myofilaments of myoepithelial cells is compatible with functional myoepithelial insufficiency that is secondary to peripheral polyneuropathy. Possibly the resultant loss of contractile support for the acini allows acinar cells to expand as secretory granules accumulate intracellularly to produce the massive enlargement of acinar cells characteristic of sialadenosis.

Do-069 Actinomyces in chronic palatinal tonsillitis – Is tonsillectomy the right choice to cure chronic tonsillitis due to bacterial infection? Histological study and self-experience U. Vogel Institut für Pathologie, Universitätsklinikum Tübingen Aims: For more than 100 years Helicobacter pylori was detected by pathologists in gastric specimens. However, as recently as 1984 Helicobacter pylori was finally accepted as a pathogen causing gastritis due to the publication of Marshall and Warren and is now treated by antibiotics. Certainly for more than 100 years pathologists describe Actinomyces spp. in the crypts of the palatine tonsil resection specimens. However, till now we receive palatine tonsil resection specimens of young and old patients in a constant high number providing a regular income for clinicians and pathologists and much pain for the patients immediately and also long years after tonsillectomy. Methods: In order to evaluate the incidence of Actinomyces spp. in palatine tonsillectomy specimens we screened 100 palatine tonsils of patients of all ages for Actinomyces spp. Results: In about 70% of palatine tonsillectomy specimens Actinomyces spp. could be detected. The dimensions of Actinomyces spp. within the crypts and the concomitant purulent exudate varied. Conclusions: The high incidence of Actinomyces spp. in palatine tonsillectomy specimens must not be neglected any longer as a cause for acute/chronic tonsillitis. As known by self-experience it is possible by simple and inexpensive methods to reduce the microbial growth in the mouth and stop the purulent exudate which causes the incriminating bad breath and the recurrent pain of swollen palatine tonsils. Thereby, it should be possible to prevent most of the tonsillectomies.

Do-070 Diﬀerential diagnosis of salivary tumours: prove of a basal cell component distincts oncytomas from acinic cell carcinomas S. Reu, C. Weiler, A. Sendelhofert, T. Kirchner, S. Ihrler Pathologisches Institut, Universitätsklinikum München (LMU) Aims: The differential diagnosis between acinic cell carcinomas (ACC) and oncocytomas of salivary glands can be difficult due to variation in histological appearance with overlapping morphological features. According to own casuistic observations, the presence of a basal cell component might assist in this differential diagnosis. Methods: On the basis of previous immunohistological investiga-tions of different salivary lesions, the semiquantitative expression of p63, CK5/6 and CK14 (all identifying basal cells), as well as of CK7, CK18, α-actin, PAS, αamylase and the proliferation index (Ki-67) are evaluated in 12 cases of salivary oncocytoma and 18 cases of ACC. The statistical differences are analysed using the U-test on the basis of a rank correlation. Results: Oncocytomas prove to contain a small basal cell compo-nent positive for p63, CK14, and CK5/6 (19,4% on average), whereas all ACC where devoid of basal cells. PAS-positivity and α-amylase-expression are significantly higher in ACC, and CK7-expression is significantly higher in oncocytomas, however, the values of these markers show a broad overlap between both tumour types. Conclusions: In those cases with a difficult differential diagnosis the immunohistochemical identification of a small basal cell component in salivary oncocytomas (especially with p63 and CK5/6) guarantees a clearcut distinction from ACC, which prove to be strictly negative for basal cells. This basal cell component in salivary oncocytomas is pathogenetically attributable to the development from striated ducts, harbouring basal cells. PAS, α-amylase, and CK7 may additionally be used as markers with, however, limited diagnostic value in the differential diagnosis between salivary oncocytomas and ACC.

Sitzung: AG Paidopathologie Do-071 Syndrome diagnosis in fetal pathology exempliﬁed by selected cases K. Schoner1, R. Bald2, K. Hinderhofer3, G. Bender4, C. Hofstaetter1, G. Schlüzer5, H. Rehder1,6 1 Institut für Pathologie, Universitätsklinikum Gießen und Marburg GmbH, Standort Marburg 2 Frauenklinik und Pränatalmedizin, Klinikum Leverkusen gGmbH 3 Institut für Humangenetik, Universitätsklinikum Heidelberg 4 Frauenklinik und Hebammenlehranstalt, Klinikum Oldenburg gGmbH 5 Pränatalmedizin und Genetik Nürnberg 6 Department für Medizingenetik, Medizinische Universität Wien Aims: Fetal pathology is concerned with the morphological diagnosis and analysis of disturbances in prenatal life leading to spontaneous abortion or termination of pregnancy. About 30% of aborted conceptuses present with malformations and the patho- logist may be the only one to ever see the malformed fetus. Especially in these cases statements about the etiology and recurrence risk of maldevelopment, the classification and syndromal pattern of malformations and the presence or absence of dysmor- phic signs are of major importance for goal directed investigations and genetic counselling and for appropriate prenatal measures in subsequent pregnancies. Methods: Fetal pathology comprises the recognition of inflammato-ry, toxic, hypoxic, circulatory and developmental disorders and the differentiation between genetic, teratogenic and sequential lesions. This certainly requires distinctive knowledge and experience in general pathology and embryology and in respective morphological techniques. On the other hand fetal pathology has to deal with genetics and syndromology including the application of molecular techniques. Results: The tight correlation between morphological and genetic investigations in fetal pathology will be exemplified by six selected cases, two presenting with hydrops, two with polydactyly and two with facial dysmorphism.

Conclusion: This underlines the need for a special genetic training and for an interdisciplinary status of the fetal pathologist.

Do-072 Severe respiratory insuﬃciency in a neonate: Diﬀerential diagnosis of lymphangiectasia T. Braunschweig, J. Sachweh1, B. Hermanns-Sachweh Institut für Pathologie und 1 Klinik für Kinderherzchirurgie, Universitätsklinikum Aachen Aims: Prolonged respiratory insufficiency is a significant source of morbidity and eventually may cause death. Early diagnosis of the underlying pathology may be crucial for optimal treatment, or at least may be a valuable tool to understand unfortunate outcome. We report on a male newborn presented with obstructive anomalous pulmonary venous return and severe lymphangiectasia. We present a diagnostic panel for differentiation of lymphangiectasia from other etiologies of severe respiratory failure. Methods: Thoracic organs were investigated macroscopically and histologically with H&E- and EvG- staining. In addition, electron microscopy and immunohistochemistry was carried out with antibodies against D2–40, CD34, pan-Cytokeratin, and CD68 to reveal the parenchymatous, vascular, and lymphatic changes. Results: Autopsy revealed obstructive anomalous pulmonary venous return and tubular hypoplasia of the aortic arch. Complete surgical correction of the cardiovascular malformations was done on the first day of life, however, the patient eventually died 6 weeks later because of intractable severe respiratory failure. Lung parenchyma showed marked clefts and dilated spaces surrounding the bronchovascular bundles. Immunohistochemistry and electron microscopy confirmed the origin of the dilated spaces to the lymphatic system. Conclusions: Immunohistochemic and electron microscopic means were effective to differentiate secondary severe lymphangiectasia from primary lymphangiomatosis, other blood vessel disorders and malformations and interstitial emphysema.

Do-073 Second report of a new congenital skeletal syndrome including microcephaly and rib hypoplasia M.S. Fahr, L. Seidmann, A.M. Müller, R. Schumacher, W. Coerdt Abt. Für Kinderpathologie, Institut für Pathologie, Universitätsklinikum Mainz Aims: One of the purposes of clinical syndrome classification is to improve perinatal genetic and sonographic counselling. Therefore it is crucial to interpret morphologic changes in accordance with syndrome patterns of malformation. Once there is suspicion of incongruence it might be necessary to discuss a new syndrome. Case report: We describe the first case of a male foetus with a congenital autosomal recessive syndrome which was first described by A. Duval et al. 1998. Clinically the foetus was suspicious for Seckel syndrome due to growth retardation, microcephaly and microretrognathia. Because of the abnormal sonographic findings the pregnancy of a non consanguineous couple was interrupted in the beginning of the 20th gestational week. The autopsy was significant for growth retardation, facial anomalies and microcephaly. The post-mortem x-ray studies reviled severe symmetrical hypoplasia of the cranial ribs, hypoplasia of the maxillary and the mandible. There was a normal male karyotype (XY) on chromosomal analysis by GTG-banded mitosis preparation. Conclusions: The similarity of our case to the two female foetuses described by A. Duval et al. is obvious. We think that there is enough evidence to conceder our autopsy findings more accordant to this new congenital skeletal syndrome than to the dysmorphology pattern of the Seckel syndrome.

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Abstracts Do-074 Vascular Lesions of Bone in Children, Adolescents and Young Adults E. Bruder1, A. Perez-Atayde2, G. Jundt1,3, A.I. Alomari4,8, J. Rischewski5, S.J. Fishman6,8, J.B. Mulliken7,8, H.P.W. Kozakewich2,8 1 Institut für Pathologie, 3 Knochentumor-Referenzzentrum und 5 Onkologie, Universitätsspital Basel 2 Department of Pathology, 4 Department of Radiology, 6 Department of Surgery, 7 Plastic Surgery and 8 Vascular Anomalies Center, Boston Children’s Hospital, Harvard Medical School, USA Background and Aims: Vascular lesions of bone are rare and their terminology is not standardized. In the binary classification approved by the International Society for the Study of Vascular Anomalies (ISSVA), vascular malformations as structural anomalies are distinguishable from proliferative vascular tumors. We compiled a large series of vascular lesions of bone in young individuals to characterize morphologic spectrum and ISSVA classification applicability. Methods: 77 vascular lesions, restricted to bone, with minimal contiguous soft tissue involvement and some multifocal lesions, from patients younger than 25 years were compiled from Boston Children’s Hospital and the Bone Tumor Reference Center Basel. Histology and radiographs were reviewed. Immunohistochemistry for D2–40, CD31, CD34, GLUT1 and MIB1 was performed on a subset of lesions. Results: The vascular lesions of bone were similar to their counterparts in skin and soft tissue. By using the ISSVA criteria, malformations (n=46) were more common than tumors (n=31); lymphatic and venous malformations were equally frequent. Hemangioendothelioma and epithelioid hemangioma were the most common tumors. We observed a previously unreported kaposiform hemangioendo-thelioma-like tumor in the context of lymphangiomatosis. Conclusion: Utilizing the ISSVA approach, the diverse terminology of vascular lesions of bone can be largely eliminated. Certain lesions of bone often regarded as tumors should be classified as malformations. We recommend the ISSVA classification be applied to vascular lesions of bone as standardized nomenclature

Do-075 Bioptical diagnosis of cherubism – actual immunohistochemical and molecular biological aspects T. Grob, R. Schmelzle1, H. Schäfer Institut für Pathologie und 1 ZMKG-Chirurgie, Universitätsklinikum Hamburg-Eppendorf Aims: Cherubism is a genetical disoder characterized by fibroblastic giant cell lesions in jaws causing characteristic facial deformation. While diagnosis is evident in case of knowledge of the family history and in completely developed states, diagnosis in uncharacteristic early stages and discrimination from other similar fibroblastic giant cell lesions is difficult. Case History: Excision material from an osteolytic lesion in the mandible of a 6 years old boy was investigated. Results and Discussion: Resection material from the mandible presented an osteolytic spindle cell lesion with several included osteoclastic multinucleated giant cells expressing histiocytic markers (CD 68, CD163), modest proliferative activity and lack of cellular atypia. The aspect was similar to central giant cell lesion of the jaw. Langerhans cell histiocytosis, nerve sheath and myogenic tumors were excluded by immunohistochemical negativity on CD1a, S100, desmin and myogenin. In contrast to the other considered differential diagnoses the present cherubism shows typical mutations of the SH3BP2 gene in 4p16.3 region encoding a c-Abl-binding protein. This mutation also excludes the Noonan-like/multiple -giant cell lesion syndrome which may present histologically similar jaw lesions but associated severe cardiovascular abnormalities and mutation of the PTPN11 gene in 12q.24.1. Whether patho-

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logical expression of the at the 4p16.3 site located FBGR3 gene is involved in pathogenesis of cherubism, is at present unknown. Conclusion: Certain early diagnosis of cherubism demands detection of typical mutation in the SH3BP2 gene in region 4p16.3.

Do-076 Genetic background of congenital ﬁbular aplasia or hypoplasia (FAH) A. Poeschl, T. Braunschweig, J. Senderek, B. Hermanns-Sachweh Institut für Pathologie, Universitätsklinikum Aachen Aims: Congenital a- or hypoplasia of the fibula (FAH) is the most common defect of congenital longitudinal skeletal disorders. The phenotypic aspect of this malformation varies from isolated affection of the fibula to involvement of the lateral foot skeleton as well as of the femur and/or the ulna. Macroscopic and histomorphologic findings were comparable to knock out mice deficient for hox c11. Thus we sought to proof if this link does also exist in patients and their relatives. Methods: Fourteen consecutive patients with FAH were encountered in this study. Histomorphologic investigations of tissue samples and molecular genetic means were applied to reveal pathologic changes and mutations in the homeobox genes hox c10 or c11. In 6 of those bone tissue and in 8 patients blood samples of the patients and their relatives were available. Results: Although all patients revealed histological findings comparable to the knock out mice, we were not able to identify the suspected mutation in any of these patients or their relatives. Conclusions: Patients with congenital a- or hypoplasia of the fibula revealed histomorphological changes comparable to knock-out mice deficient for hox c 11. However, this molecular genetic link was not found in our patients. In an upcoming investigation it will be tested if these patients have mutations of other candidate gene such as Hox d13, Shh, and Wnt7a.

Do-077 Perlman Syndrome: Report of a case emphasizing its similarity to and distinction from Beckwith-Wiedemann and Simpson-Golabi Syndromes C. Falkeis, B. Brunner1, G. Mikuz, E. Steichen2, C. Sergi Institut für Pathologie, 1 Universitäts-Klinik für Pädiatrie II und 2 Universitäts-Klinik für Pädiatrie IV, Medizinische Universität Innsbruck Background: Perlman syndrome (PS) is an autosomal recessively inherited overgrowth syndrome characterized by unusual face, fetal gigantism, visceromegaly, hydronephrosis, renal hamartomas with or without nephroblastomatosis or Wilms tumor. We report on a 5 weeks old female infant who exhibits typical features of PS. Case Report: Ultrasound during pregnancy showed myocardial hypertrophy, hepatomegaly and nephromegaly as well as placentomegaly. The girl was delivered at 32+4 weeks. Her birth weight was 3600 g (>97 centile). At birth, she was intubated because of respiratory distress syndrome. Subsequently, she was operated from stenotic isthmus aortae on the 2nd day. Postoperative longterm ventilation was necessary. Muscular hypotonia and the large abdominal organs as well as distinct facial features pointed to a genetic disorder. The EEG waves were suspicious for neurological involvement. Karyotype was 46, XX without structural anomalies. At autopsy, the infant showed generalized infant macrosomia, hepatomegaly, cardiomegaly, bronchopulmonary dysplasia, and hydronephrosis. Discussion: The presence of enlarged, echogenic kidneys in a newborn with generalized macrosomia points toward the diagnosis of an overgrowth syndrome. This phenotype includes Beckwith-Wiedemann, Perlman and Simpson-Golabi-Behmel syndromes. In this report we emphasize the importance of histological and genetic investigations to distinguish these overlapping syndromes.

Do-078 Persistent diagnostic relevance of electron microscopy in paediatric pathology – demonstrated in diﬀerent paediatric diseases including congenital brush border diseases H. Schäfer Institut für Pathologie, Universitätsklinikum Hamburg-Eppendorf

Do-080 Synovial sarcomas in children and young adolescents C. Vokuhl1, J. Stöfen1, S. Stegmaier2, E. Koscielniak2, I. Leuschner1 1 Institut für Pathologie, Sektion Kinderpathologie, Universitätsklinikum S-H, Campus Kiel 2 Olgahospital Stuttgart

Aims: Diagnostical electron microscopy has widely been substituted by immunohistochemistry and molecular biology. In some diseases, however, ultrastructural alterations are easier to identify or better defined than their molecular base. This presentation summarizes diseases with still diagnostically useful ultrastructural characteristics: storage diseases (i.e. Fabry’s disease), kidney diseases (i.e. minimal changes with nephrotic syndrome), congenital mitochondriopathies (i.e. a recently defined mitochondrial depletion syndrome) and some recently observed cases with congenital brush border defects. Methods: Tissue specimens were fixed in glutaraldehyde/OsO4 and embedded in epoxy resin according to routine methods. Results: In mitochondrial depletion syndrome mitochondria in hepatocytes showed mitochondrial irregularities (giant mitochondria, abnormalities in mitochondrial cristae) which can also be found in other types of primary mitochondriopathies. Bronchus biopsies in Zellweger’s syndrome presented the wellknown irregularities in dynein arms of ciliae, which are pathognomonic, but difficult to detect, and should, therefore, be correlated with in vitro study of ciliary motion in biopsies of respiratoy mucosa. Microvillous inclusion disease (=congenital microvillous atrophy) causes symptoms similar to celiac disease, but is characterized by diatary-resistance and high letality. Diagnosis can be made probable by conventional histology (PAS staining) and immunohistochemistry (i.e. CD10), but has to be confirmed by electron microspic detection of typical microvillar atrophy and intracytoplasmic inclusions. Conclusions: Electron microscopy is still an essential diagnostical tool in paediatric pathology.

Aims: Synovial sarcoma is a common soft tissue tumor which represents up to 10% of all soft tissue sarcomas. The tumors occur most often in adolescents and young adults, the median age of diagnosis is about 30 years. Synovial sarcomas are seen as high-grade sarcomas and in the last years several publications dealed with prognostic factors for patient survival. A poor clinical outcome could be shown, amongst others, for tumors larger than 5 cm, positive resection margins, high mitotic or high proliferative index, p53 mutation, histological subtype and the gene fusion type. In this study we investigated if the published prognostic factors in adults could also be useful in the pediatric age group. Methods: In a series of 63 primary synovial sarcomas in patients up to 18 years we investigated histological subtype, grade, mitotic activity, necrosis, vessel density and immunohistochemical expression of Ki-S5, Ki-S2, p53 with regard to the overall survival. Fusion transcripts were detected by RT-PCR. All data were analysed using SPSS 15.0 for Windows. Results: Based upon morphological parameters and immunohistological expression pattern we could not show a significant correlation between the investigated parameters and the overall survival. Conclusions: In contrast to recent studies, which stated that the parameters mentioned above could be used as prognostic factors in synovial sarcomas, we were not able to demonstrate this in the pediatric subgroup. These results could be an indication that pediatric and adult synovial sarcomas are two different tumor entities.

Do-079 Fetal malformations due to manganese over consumption M.S. Fahr, L. Seidmann, U. Zeigmeister, M. Locketz, W. Coerdt Abt. für Kinderpathologie, Institut für Pathologie, Universitätsklinikum Mainz Aims: We try to sensitise perinatal clinicians to screen for heavy metals in non specific syndromal malformations. In the following we present an exogenic induced foetus with limb malformations suspicious for a congenital arthrogryposis multiplex. Material and Methods: A foetus with multiple limb malformations showed on histology work-up calcifications mainly in the liver parenchyma and the meninges of the neural cord. The history of the mother was significant for a long time consumption of manganese polluted water from a private owned source. The amount of manganese in the water was more than 70 times above the threshold value of potable water which is 0.05 mg/l. We tried to detect manganese elements within the calcifications in the liver and the meninges. Therefore we used a REM (raster electron microscope) combined with an EDX (energy dispersive x-ray analysis) system which means that electron rays containing primary electrons are orientated on the calcifications. These primary electrons turn the elements in the target area in an activated state by striking electrons close to the core out of their orbital. Electrons further peripheral fall on the orbital closer to the core which results in emitting x-ray with energy corresponded to the energy difference. As each element has a significant own “finger print” of wave length material can be detected. Conclusion: Although we did repeated trials only calcium could be detected. We believe that the reason for a negative out come is the relatively small amount of suspected manganese in relation to the calcium in the examined area as well as the tissue processing

Do-081 Congenital salivary gland anlage tumor as a rare cause of neonatal airway obstruction A. Zimpfer, D. Kayser1, P. Schwabbauer, H.-J. Feickert2, I. Leuschner3, E.W. Herbst Institut für Pathologie, 1 Hals-Nasen-Ohrenheilkunde und 2 Kinder-und Jugendmedizin, Dietrich-Bonhoeﬀer-Klinikum Neubrandenburg 3 Institut für Pathologie, Universitätsklinikum S-H, Campus Kiel Aims: Upper respiratory tract obstruction in the neonatal period can result from a variety of conditions and may be difficult to differentiate especially when dysmorphic features are absent. We present a rare case of a salivary gland anlage tumor (SGAT) of the nasopharynx in a female baby. Methods: Case presentation and literature review. Results: A female neonate presented with signs of nasal airway obstruction after normal delivery. Capillary blood gases demonstra-ted respiratory distress. Symptoms of nasal obstruction aggravated in stress and position-dependent. The complex diagnostic workup resulted in the clinical diagnosis of a single polypoid mass lesion of the epipharynx. The lesion was excised. Macroscopically, a polypoid, firm, grey-white tumor of 20 mm in diameter with smooth surface was seen. On histology, a biphasic pattern of epithelial structures and solid spindle cell proliferates were seen. The epithelial structures were consistently immunoreactive for cytokeratin, whereas the spindle cells showed immunoreactivity for vimentin and muscle-specific actin. A SGAT of the nasopharynx was diagnosed. Conclusions: A wide range of inflammatory, congenital and neoplastic processes may cause nasal airway obstruction. Rarely, a SGAT of the nasopharynx, also referred to as congenital pleomorphic adenoma, a benign congenital mixed tumor is found. In the literature about 25 cases are reported, most of them male patients. Simple excision has been curative to date.

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Abstracts Do-081a Why are there so many appendices with chronic or missing signs of inﬂammation in daily paidopathological diagnostic? A.M. Müller1,M. Kaucevic1, W. Coerdt1, M. Heinz2, S. Turial3 1 Abt. für Kinderpathologie; 2 Institut für Medizinische Biometrie, Epidemiologie und Informatik und 3 Klinik für Kinderchirurgie; Universitätsklinikum Mainz Aims: The comparable high percentage of appendectomies with histopathological missing signs of acute inflammation in daily routine paidopathology raises the question of reliable (positive or negative) anamnestic and clinical data specifically associated with morphological chronic or fibrous appendicitis. Methods: 872 appendectomies (1/2000 -12/2006) from children and adolescents (5 mths – 15 yrs) were grouped histomorphologically into appendectomies with acute appendicitis (AA) resp. appendectomie without acute inflammation (chronic appendicitis (CA); no inflammatory signs (NI)). Presented medical history data, physical examination findings and basic laboratory data were compared with pathological diagnosis. Results: 444/872 appendices (52%) showed none or chronic signs of inflammation, 414/872 (48%) signs of acute inflammation. In CA/NI duration of symptoms was statistically longer (>2 weeks). Besides, in CA/NI percussion tenderness was found in 68% (AA: 87%), rebound tenderness in 47% (AA: 69 %), guarding in 22 % (AA: 47%), nausea in 55,2% (AA: 74,4%), vomiting in 30% (AA: 63%), free abdominal fluid in 14% (AA: 27%). There was no difference concerning psoas sign for the two groups. In CA/NI the median of leucocyte count was 8900/ml (AA: 15300/ml; n: 15500/ml), the median of CRP 2 mg/l (AA: 16,1 mg/l; n: -5 mg/l). Conclusions: Apart from duration of symptoms there are no signs specifically associated with histopathologically proven CN or NI. In fact, these patients clinically present in a relative high percentage with symptoms and clinical signs of acute appendicitis. As in case of doubt an appendectomy is performed, a comparable high percentage of appendices without signs of acute inflammation will remain in daily routine paidopathological diagnostic.

Sitzung: AG Dermatopathologie Do-082 Melanocytic lesions of the skin – overview and update E. Bierhoﬀ Institut für Dermatopathologie, Universitätsklinikum Bonn The histopathological diagnosis of melanocytic tumors has been the subject of countless studies. Although precise histopathological criteria for distinction of benign from malignant melanocytic proliferations have been established, some diagnostic criteria are still in debate and in some instances a precise diagnosis is not possible. Routine histological examination of the specimen is still the gold standard. There are no compelling data showing that any immunohistochemical marker provides informations beyond routine histology. Genetic studies may provide additional informations in the future, especially in controversial lesions. The lecture will give an overview of special settings e.g. proliferations of melanocytes within chronic sun-damaged skin, melanocytic proliferations at special skin sites, proliferation of melanocytes at sites of inflammatory skin disorders, combined nevi and recurrence (persistence) of melanocytic lesions. The lecture will further emphasize melanocytic proliferations with spitzoid morphology, which have been and still are one of the most commonly discussed problems in dermatopathology including conventional Spitz nevi and variants, atypical Spitz tumors and spitzoid malignant melanoma. The exact classification in some cases is still a matter of debate, e.g. it has been recently proposed that some Spitz nevi with deposits of melanocytes in lymph nodes may represent a subset and a type of „low-grade melanoma“ with much better prognosis than conventional melanoma with similar thickness.

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Do-083 Expression of acyl-CoA synthetase 5 in human epidermis J. Köster, N. Gaisa, J. Ehling, A. Reinartz, K. Raupach, U. Schneider, R. Knüchel, N. Gassler Institut für Pathologie, Universitätsklinikum Aachen Aims: In stratified epithelia like human epidermis, keratinocytes pass a differentiation process including generation of lipid-matrices. As recently shown for other epithelial cells, acyl-CoA synthetase 5 (ACSL5) is involved in lipidmetabolizing pathways. The aim of the present study was to investigate the expression of ACSL5 in human epidermis. Methods: Expression of ACSL5 in normal and diseased human epidermis was investigated with mRNA in situ-hybridization and immunohistochemial stainings. In situ data were supported with Western blot analysis and a qRTPCR approach. Functional studies were performed with HaCaT-cells. Results: ACSL5 was found in human epidermis including meshed and ridged skin. In all cases keratinocytes of the stratum spinosum were the main source for ACSL5 expression. Enhanced ACSL5 expression was seen in single keratinocytes, especially in chronic solar-exposed epidermis. In addition, co-staining for ACSL5 and TUNEL was occasionally found in superficial keratinocytes. Cell culture experiments revealed that ACSL5 expression was associated with differentiation of HaCaT cells to multilayered epidermal equivalents. Conclusions: ACSL5 could be a differentiation-associated enzyme in human epidermis with enhanced expression in stress response.

Do-084 Imiquimod downregulates Cyclin D1 and induces growth arrest in keratinocytes J. Pammer, M. Mildner1, N. Kozakowski, E. Tschachler1 Klinisches Institut für Pathologie und 1 Institut für Dermatologie, Medizinische Universität Wien Aims: Imiquimod is a topical immune response modifier with antiviral and antitumor effects which acts by stimulating dendritic cells and inducing apoptosis of tumor cells. In spite of its common use in dermatology, direct effects on keratinocytes (KC) have not yet been thoroughly investigated. Methods: Cultured human KC were studied by Western blotting and cell cycle analysis. Results: We show that in primary human KC imiquimod downregulates Cyclin D1 strongly and p21 and Id-1 weakly, whereas p27 is upregulated. By contrast, p53 is not regulated by imiquimod. Cell cycle analysis revealed a cell cycle arrest in G0/G1 and G2/M phase, matching the inverse regulation of cyclin D1 and p27. This regulation was not due to an inhibition of AKT and p42/44 ERK phosphorylation. Conclusions: The inhibition of mitosis and the regulation of Cyclin D1, p27 and Id-1 may be involved in the antiviral and antitumor effects of imiquimod. The exact mechanism of imiquimod’s antimitotic activity awaits further clarification.

Do-085 C-Jun is regulated by loss of E-Cadherin in malignant melanoma B. Spangler, S.Kuphal, L.Vardimon1, A.K. Bosserhoﬀ Institut für Pathologie, Universitätsklinikum Regensburg 1 Department of Biochemistry, Tel Aviv University, IL The transcription factor c-Jun is a key player in the process of cell proliferation and tumor progression. It forms homodimers or heterodimers with other members of the transcription factor superfamily AP-1, influencing the expression of a multitude of regulators of cell proliferation, migration and survival significantly involved in tumor development and metastasis. We could show by Western Blot analysis and immunhistochemical staining that c-Jun protein is upregulated in melanoma cells, whereas in melanocytes c-Jun protein is not expressed. Interestingly, there is a coincidence in the loss of E-Cadherin expression and upregulation in c-Jun protein in malignant melanoma. We, therefore, speculated whether E-Cadherin is a regulator of c-Jun. We examined three established model systems, which show a re-expression of

E-Cadherin in melanoma cells. Our data could show that loss of E-Cadherin expression during melanoma development induces c-Jun activity, whereas in melanocytes active cell-cell-contacts via E-cadherin have a negative impact on c-Jun, suggesting a direct link between E-Cadherin and c-Jun. Further experiments show that c-Jun regulation by E-cadherin is cell-contact dependent. Treatment of primary melanocytes with an anti E-Cadherin antibody destroying E-Cadherin dependent cell contacts between melanocytes, increased the c-Jun protein amounts in NHEM, which confirms the importance of cell-cell-contacts in regulating c-Jun. These data reveal that the active use of cell-cell contacts is important for ECadherin dependent downregulation of c-Jun. Loss of E-Cadherin during melanoma development induces c-Jun activity, suggesting a role of this regulation in melanoma development and progression.

Do-086 Molecular modulators of β-catenin activity in malignant melanoma S. Kuphal, A.K. Bosserhoﬀ Institut für Pathologie, Universitätsklinikum Regensburg The Wnt/β-catenin pathway is involved in various differentiation events during embryonic development and it participates in tumor formation when aberrantly activated. Recently, we have shown that β-catenin participates in an alternative pathway besides regulating LEF/TCF (T-cell factor/lymphoid enhancing factor)-mediated transcription by stimulating the transcriptional activity of NFkappaB. Molecular studies concentrating on colorectal cancer revealed activating mutations of apc (adenomatous polyposi coli), ctnnbi, btrc and tcf-4 genes which mimic Wnt stimulation. However, such mutations are rarely found during melanoma development. Therefore, we analyzed the Wnt/β-catenin signaling pathway and molecular modulators of β-catenin activity in this type of skin cancer. Interestingly, nuclear localization of βcatenin was rarely found in melanoma cell lines and melanomas in vivo which is in clear contrast to colon carcinoma cells. Consistently, the co-transcriptional activity of β-catenin via the transcription factors LEF/TCF regulating expression of β-catenin target genes can not be found in melanoma cell lines. Detailed analysis revealed that neither export/import mechanisms nor modifications through RelA, Chibby, PKC (protein kinase C), CK I (casein kinase I), CK II (casein kinase II), Wnt5a, Wnt agonists and GSK-3β are responsible for cytoplasmic β-catenin localization in malignant melanoma. Our data revealed that the melanoma-specific phosphorylation status of β-catenin is responsible for the cytoplasmic accumulation and signaling of β-catenin in melanoma cells. In summary, we present a new function of β-catenin in malignant melanoma cells resulting from a cytoplasmic localization in these cells. This study suggests a cell type specific regulation of β-catenin function modulated by phosphorylation.

Do-087 Dermal angiosarcoma show in a high percentage a lymphendothelial accentuated mixed phenotype A.M. Müller1, Y. Hunder1, A. Schneider2, H. Grieﬁngholt3,4 1 Abteilung für Kinderpathologie, Universitätsklinikum Mainz 2 Institut für Med. Biometrie, Epidemiologie u. Informatik, Universität Mainz 3 Institut für Pathologie, Bonn-Duisdorf 4 Institut für Pathologie am St. Franziskus-Hospital Münster Aims: During recent years it was proven that in angiosarcomas apart from hemangioendothelial tumor cells there are lymphangioendothelial tumor cells as well. We studied dermal angiosarcomas (DAS) – unrelated to lymphedema – concerning the percentage of lymphangioendothelial differentiation by conventional manual microscopy and computer assisted analysis. Methods: 32 DAS (diagnosis based on HE-staining) were immunohistochemically stained with the lymphendothelial antibodies D2–40 and Podoplanin. Analysis of the staining was based on 1) ACIS (Automated Cellular Imaging System), 2) manual microscopy. Results: According to ACIS 97% of the DAS (95%-Confidence interval:[82%, 100%]) showed a statistically definite (p<0,001) mixed lymph- and heman-

gioendothelial phenotype. In 1 case (3%) more than 85% of the tumor cells displayed a lymphendothelial differentiation. By manual microscopy 41% (D2–40) resp 32% (Podoplanin) of the tumors consisted of lymph- and hemangioendothelial cells; further 44% (D2–40) resp 55% (Podoplanin) of the DAS were diagnosed as pure lymphangiosarkomas. Conclusions: Nearly all of the studied DAS showed – apart from hemangioendothelial tumor cells – a unexpected high proportion of lymphendothelial tumor cells. Though ACIS is a reliable quantifying method, its technical limitations prevent diagnosis of true lymphangiosarcomas. It is an open question whether by application of lymphendothelial markers the percentage of diagnosed lymphangiosarcomas in other organs will increase as well.

Do-088 LEF-1 is a new candidate for transcriptional regulation of invasion in basal cell carcinomas L. Kriegl, D. Horst, T. Kirchner, A. Jung Pathologisches Institut, Universitätsklinikum München (LMU) Aims: Previous studies showed an involvement of the Hedgehog and Wnt/ β-catenin signalling pathway in the development of basal cell carcinomas (BCC). However the molecular mechanism regulating tumour invasion is still unknown. The present study aims to identify candidate factors for a transcriptional regulation of invasiveness in BCCs. Methods: 48 BCCs, including nodular (n=29), superficial (n=14), infiltrative (n=3), and sclerodermiform (n=2) types were analyzed by immunohistochemistry using β-Catenin, TCF-4, LEF-1 and TGF-β specific antibodies. Results: All examined BCCs showed a strong and distinct nuclear expression of LEF-1 in the palisading tumour cells at the invasive front of BCCs. 83% of the BCCs demonstrated diffusely distributed nuclear expression of TCF-4. Expression of β-Catenin was slightly increased, but no clear nuclear accumulation was seen. 31% of the BCCs displayed a distinct membranous expression of TGF-β especially in the palisading tumour cells at the front of invasion. Conclusions: As the expression pattern of LEF-1 strongly correlates with palisading tumour cells at the invasive front it seems to be a key factor for the regulation of invasiveness. No overlap between the expression of LEF-1 and of TCF-4 or β-Catenin was found. Therefore the Wnt pathway apparently plays no decisive role for upregulation and function of LEF-1 in BCCs. However a distinct membranous expression of TGF-β was found in 31% of the tumours particularly in the palisading cells expressing LEF-1. This indicates a relevant mechanism for induction of LEF-1 through the TGF-β/Smad pathway. Additional experiments employing cultivated cell lines are anticipated for the functional testing of this hypothesis.

Do-089 Analyses of a new antagonist of TGF-β/BMP signalling cascade which plays a role in skin sclerosis S. Arndt, S. Karrer1, A.K. Bosserhoﬀ Institut für Pathologie und 1 Institut für Dermatologie, Universitätsklinikum Regensburg Fibrotic diseases are characterized by excessive scaring due to an increased production, deposition and contraction of extracellular matrix. To understand the molecular basis of fibrotic disease, it is essential to appreciate how matrix deposition is normally controlled and how this process is deregulated in fibrogenesis. It is known that transforming growth factor-beta (TGF-β) is involved in sklerosis formation. Expression of TGF-β, TGF-β receptors or downstream mediators of TGF-β signalling cascade were shown to be deregulated in dermal fibrotic lesions of scleroderma patients. However, the underlying mechanisms are poorly understood. Recently, we identified a novel molecule with inhibitory function on bone morphogenic protein (BMP) signalling. Due to its function we named this molecule Fussel-15, for Functional smad suppressing element on chromosome 15. In this study, we used a collagen contraction model (wound healing model) to determine contraction capacity and gene expression of Fussel-15, BMP and TGF-β molecules in normal fibroblasts and fibroblast from skin sclerosis. InDer Pathologe · Supplement 1 · 2008

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Abstracts terestingly, we detected a specific expression pattern of Fussel-15 in fibroblasts of early wound healing processes and a deregulation of this molecule in fibroblasts of skin sclerosis. Additionally, we found differences in gene expression of several molecules of the TGF-β/BMP family in normal fibroblasts compared to those of scleroderma patients. To determine the function of Fussel-15 in vitro, knockdown and overexpression experiments were established. In summary, we could show that Fussel-15 is increased in sclerosis derived fibroblasts, Fussel-15 knockdown delayed and Fussel-15 over-expression enhanced collagen contraction in vitro.

Do-090 Autoimmune disease resembling SLE in mice lacking JunB P. Pﬂegerl1, E. Janig2, A. Soleiman1, P. Petzelbauer3, L. Kenner1,4 1 Klinisches Institut für Pathologie, Medizinische Universität Wien 2 Institut für Dermatologie, Medizinische Universität Graz 3 Institut für Dermatologie, Medizinische Universität Wien 4 Ludwig Boltzmann Institut für Krebsforschung, Wien Aims: Investigate the autoimmune phenotype of mice lacking epidermal JunB. Methods: To investigate the role of JunB in epidermal development, JunB was inactivated in keratinocytes of mice carrying a floxed JunB allele by crossing it to the K5-cre2 transgenic mouse(JunBep). We checked for UV-inducability of the JunBep phenotype. Serum was investigated for autoimmune parameters. FACS analysis was performed form lymphoid organs. JunBΔep mice, as well as skin of human SLE patients were investigated using IHC, Immunoflorescence as well as electron-microscopy. To gain insight in molecular patho-mechanism, JunB target gene analysis as well as ChIp analysis was performed. Results: 3–6 months old mice lacking JunB in epidermal cells develop skin lesions, Immuncomplex Glomerulonephritis and lymphocyte infiltrates in the liver, lung, kidneys and blood vessel walls. Lesioned skin displayed thickening of the epidermis with pronounced lymphocytes intermingled by granulocytes. UV radiation induced epidermal-Dermal as well as glomerula IgG deposits. CD4/CD8 ratio was dramatically raised in JunBep mice. IL-6 levels were selectively increased in sera from 3 and 6 months old JunBep mice. JunBep mice showed pathological levels of Histone H1,H2A, H2B, H3 and H4 protein. In skin samples of SLE patients JunB immunreactivity was almost absent. Crossing of JunBep with the Rag2-/- mice could rescue the kidney phenotype. Conclusions: This study describes a novel mechanism by which the absence of epidermal JunB protein causes a multiorgan disease similar to human SLE. The data demonstrate that the epidermis can act as an endocrine-like organ.

Do-091 Transcriptomal comparison of human dermal diabetic versus nondiabetic lymphatic endothelial cells ex vivo M. Hämmerle1, T.M. Keller1, B. Hantusch1, D. Stokic2, N. Wick1, E. Gurnhofer1, S. Thurner2, D. Kerjaschki1 1 Klinisches Institut für Pathologie und 2 Complex Systems Research Group, HNO, Medizinische Universität Wien Aims: Late stage diabetic type II disease is accompanied by severe microangiopathy. Here, we aimed to characterize the transcriptome of diabetic dermal lymphatic endothelial cells in comparison to healthy skin donors. Methods: Human microsvascular ex vivo lymphatic endothelial cells (LECs) were isolated from eight skin donors, four diabetic and four healthy patients. LECs were isolated and further separated from BECs by FACS using antiCD31, CD45 and podoplanin antibodies. The cells were immediately counted, lyzed and mRNA was extracted. The mRNA was amplified in two consecutive amplification rounds. Stringent quality controls by using the Agilent Bioanalyzer demonstrated purity of the material and enabled estimations of the probe amount. The derived cDNA species were hybridized to Affymetrix gene chips, and an extensive bioinformatic analysis was performed. Results: Basically, the presence of a characteristic set of lymphatic endothelial genes could be ascertained. Moreover, comparison of the gene expression

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profile of diabetic versus non-diabetic LEC samples identified genes differentiating these two populations. Conclusions: The direct ex vivo analysis of the LEC transcriptome may be helpful in understanding the pathogenesis and pathophysiology of diabetic disease

Do-092 Analysis of Various Monoclonal Antibodies to Tyrosinase Related Protein-1 (trp-1) as Diagnostic Reagents for Surgical Pathology A. Jungbluth1, D. Frosina1, B. Zaidi1, K.-J. Busam2 1 Ludwig Institute for Cancer Research and 2 Dept. of Pathology, Memorial Sloan-Kettering Cancer Center, New York, USA Aims: Various novel monoclonal antibodies (mAbs) mAbs to melanocyte differentiation antigens (MDAs) such as mAb A103 to Melan-A and mAb T311 to tyrosinase have broadened the spectrum of diagnostic tools in surgical pathology; trp-1 is another MDA, for which several mAbs are available. However, those have not been tested or have not found widespread use in surgical pathology. Methods: We analyzed anti-trp1 mAb TA99 generated in our lab and 2 commercial anti-trp-1 mAbs (mAb BD#23; Becton Dickinson and mAb G3E6, NovoCastra) as IHC reagents for surgical pathology: All three mAbs were tested in normal and tumorous frozen and paraffin-embedded tissues employing various antigen retrieval techniques. Results: TA99 worked well in frozen tissue. However, no reliable staining could be achieved in FFPE tissue; mAb BD#23 did not produce reliable staining in FFPE tissues compatible with trp-1 expression. Best results were seen with mAb G3E6 staining melanocytes in frozen and in FFPE tissue using standard AGR. However, in melanoma TMAs, G3E6 was positive in only approximately 50% of metastatic cases and negative in all desmoplastic melanomas while 6/6 angiomyolipomas were G3E6-positive. However, there was strong staining in non-melanocyte structures such serum precipitates in blood vessels and focally of endothelial cells as well as a subpopulation of inflammatory cells, obviously plasma cells. Conclusions: trp-1 has not yet been tested properly in regard of its usefulness as a melanocyte-associated antigen for surgical pathology. Our study indicates that the current anti-trp-1 mAbs are of limited use as diagnostic reagents in surgical pathology.

Symposium: Molekulare Infektionspathologie Fr-001 Pilze als Krankheitserreger und Heilsbringer in der Medizin A. Brakhage Jena Fr-002 Molekulare Mechanismen und Konsequenzen kardialer Virusinfektionen R. Kandolf Tübingen Fr-003 HPV bei nicht-gynäkologischen Tumoren I. Petersen Jena

Fr-004 Extra-gastrointestinal manifestations of Whipple’s disease and diagnostic algorithm based on modern methods C. Loddenkemper1, V. Moos2, A. Moter3, W. Geißdörfer4, H. Stein1, T. Schneider2 1 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin 2 Medizinische Klinik I und 3 Institut für Mikrobiologie, Charité – Universitätsmedizin Berlin 4 Institut für Klinische Mikrobiologie, Universitätsklinikum Erlangen Aims: Whipple’s disease (WD) is a rare chronic multisystemic infectious disease caused by Tropheryma whipplei with a potentially fatal oucome. Common symptoms are heterogeneous including arthropathy, weight loss, diarrhea and fever. Methods: Within the European research project on WD, we have evaluated biopsies from over 100 patients with Tropheryma whipplei infection. In addition, heart valves from patients with culture-negative endocarditis have been analyzed in two reference centers combining PAS staining, immunohistochemistry and PCR. Results: While pathologists are familiar with the typical PAS positive foamy macrophages in the duodenum, extra-intestinal manifestations e.g. in the CNS, lymph node, joint, bone, heart and skin may be easily overlooked or misinterpreted. In contrast to previous reports, focal lesions in WD confined to the CNS are extremely rare if a revised case definition based on PCR and sequencing as well as immunohistochemistry is applied (1/15 (7%) in our cases vs. 14/35 (40%) in the literature). Furthermore, Tropheryma whipplei was identified as the causative agent in a significant portion of heart valves (7%) after surgery due to culture-negative infective endocarditis in a large cohort of 224 patients. Conclusions: New diagnostic strategies detect non-gastrointestinal manifestations of Whipple’s disease more frequently. From our experience, the diagnostic algorithm in patients with suspected WD has to include modern methods like immunohistochemistry or PCR, especially in cases with extragastrointestinal involvement. To confirm the diagnosis of this rare disease and to initiate optimal treatment, referral to a specialized center should be considered.

Fr-005 Detection of granulomatous infections by PCR T. Goldmann, D.S. Lang, H. Schultz, E. Vollmer Klinische und Experimentelle Pathologie, Forschungszentrum Borstel Aims: The detection of infectious agents from paraffin-embedded tissues remains a central topic within molecular pathology, especially concerning granulomatous infections. The development of standardized procedures helps to overcome technical embarrasments given by the material to be analyzed. Methods: DNA-Extraction, Spectrophotometry, PCR, Electrophoresis, Prevention of contaminations. Results: The preservation of nucleic acids in formalin-fixed tissues impedes the application of molecular techniques to a large degree. Nevertheless, several key-points for a reliable as well as sensitive detection of infectious agents out of formalin-fixed, paraffin-embedded tissues have been elaborated during the last years in close cooperation of the panel laboratories for tuberculosis PCR in Germany. These include techniques for DNA-extraction which can be modified to “open” the bacterial cell wall. Special controls during DNA-extraction detect contaminations in the extraction-system. Spectrophotometry of the DNA delivers information about the amount and purity of template-DNA and PCR-inhibitors. The consiliation for standardized targets and primer-combinations to be used enhances the inter-laboratory comparability. Conclusions: Although the detection of granulomatous infections in formalin-fixed tissues remains a methdological challenge, we have shown that the application of appropriate techniques and standardization of the experimental procedures lead to sensitive, reliable and specific results. Nevertheless, due to the grade of preservation of DNA in formalin fixed materials, this can not

replace nor reach the quality of microbiological cultivation or the detection in fresh tissues.

Fr-006 Molecular Mimicry between Lysosomal Membrane Protein 2 (LAMP2) and the Bacterial Adhesin ﬁmH in Pauci-Immune Focal Necrotizing Glomerulonephritis (FNGN) R. Kain1, D. Cunningham2, O. Ashour1, A. Rees1,2, D. Kerjaschki1 1 Klinisches Institut für Pathologie, Medizinische Universität Wien 2 Immunology Programme, IMS, University of Aberdeen, UK Aims: To characterize the autoantibodies to lysosomal membrane protein-2 (LAMP-2) in pauci-immune FNGN; and to find out if they could be induced through molecular mimicry to bacterial proteins. Methods: LAMP-2 autoantibodies were detected by ELISA and pathogenic epitopes defined by peptide mapping. Bacterial homologies were identified from databases and relevant proteins tested for crossreactivity in Western blots and competitive binding assays. Anti-LAMP-2 antibody pathogenicity was assessed in vitro on endothelial cells and in vivo by injection into rats. Molecular mimicry was assessed by immunising rats with bacterial proteins. Results: Antibodies to LAMP-2 were detected in 78 of 84 (95%) of our patients with biopsy proven active FNGN. Antibodies to LAMP-2 cause tissue injury because passive administation induced FNGN with crescents in 14 WKY rats; and a monoclonal antibody to LAMP-2 induced apoptosis of blood microvascular endothelium in vitro whereas control monoclonals had no effect. Peptide mapping identified two common LAMP-2 epitopes and one (P41–49) was 100% homolous to the bacterial adhesin fimH. Binding studies showed patients’ anti-P41–49 autoantibodies cross-react with fimH. Consequently, 10 rats were immunized with fimH and all developed FNGN associated with anti-fimH antibodies that cross-reacted with LAMP-2. Finally, 9 of 13 prospectively studied patients had microbiologically proven infections with fimbriated pathogens shortly before presentating with with pauci-immune FNGN. Conclusions: FimH triggered autoimmunity to LAMP-2 provides a novel clinically relevant pathogenetic mechanism for the development of pauci-immune FNGN.

Symposium: Systempathologie des Gewebes (I) Fr-007 Virtuelles Gewebe M. Wagner Homburg Fr-008 Signaltheoretische Analyse histologischer Daten im Ortsfrequenzraum F. Weichert Dortmund Fr-009 Mathematische Modellierung in der Systembiologie am Beispiel der desmoplastischen Stromareaktion A. Groh Saarbrücken

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Abstracts Fr-010 Using Formal Ontology in Analyzing Complex Systems in Pathology – Data Integration and Standardization M. Brochhausen1, C. Brochhausen2, C.J. Kirkpatrick2 1 Institute for Formal Ontology and Medical Information Science (IFOMIS), Universität des Saarlandes, Saarbrücken 2 Institut für Pathologie, Universitätsklinikum Mainz

Symposium: Uro-/Nierenpathologie

Aims: Analyzing complex systems in pathology depends to a great extent on the identification of processes, functions and bearers in the pathological process. Storing and sharing relevant pathological and clinical data can be optimized by using ontology. An ontology may replace a data base schema with the advance of semantic interoperability. Both, research and therapy are in need of fast data integration. The Infectious Disease Ontology (IDO) provides a first step towards a complete disease ontology. Methods: Ontological representation and ontological engineering is a common method in biomedical informatics. The last years showed a huge increase in development of biomedical ontologies. It has been successfully used in anatomy, genetics and cancer management. The first step in ontological engineering is the analysis of the domain and the identification of entities. System pathology provides a new challenge for formal ontology, which focuses on the ontological status of functions and agents. Results: The IDO proves the importance of identifying multiple levels in complex processes in order to enable computerization. The methodology tested in its development can be used to represent any pathological processes. Conclusions: Ontology development provides a powerful methodology to analyze complex systems and break them down into processes and their bearers. The biomedical concept of function and its ontological status must be further clarified and evaluated by practical use in both, research and therapy. Ontology-based systems provide an optimal IT environment, since they facilitate data integration from different data bases and provide a basic standardization of possible values.

Fr-013 Angeborene Immunitätsreaktionen bei Glomerulonephritiden H.-J. Groene Heidelberg

Fr-011 Identiﬁcation of microRNAs regulated by p53 H. Hermeking1, V. Tarasov2, B. Verdoodt1, D. Lodygin2, G. Meister2, A. Menssen2, P. Jung1 1 Institut für Pathologie, Ruhr-Universität Bochum 2 Max-Planck-Institut für Biochemie, Martinsried

Aims: Mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are a common finding in bladder tumours of low stage and grade and are known to be less frequent in invasive tumours. Most of the macroscopically large papillary-invasive tumours, however, show a mixture of distinct grades of differentiation within the same tumour. The aim of this study was to investigate the FGFR3 mutation status within large papillary-invasive tumours and adjacent urothelium as well as connective tissue. Methods: Whole organ mapping formalin fixed tissue samples were microdissected and FGFR3 mutation analysis was performed by SNaPshot multiplex mutation detection assay. Tissue microarrays were stained with FGFR3 antibody and additionally p53 antibody. Results: The majority of papillary tumour parts showed FGFR3 mutations but no strong correlation with grading was found. Invasive tumour parts, investigated so far, harboured predominantly no FGFR3 mutations. Interestingly normal urothelium and connective tissue also showed FGFR3 mutations. Control immunhistochemical staining of p53 was linked to poorly differentiated invasive tumour parts, on the contrary positive staining for FGFR3 was associated with better differentiated tumour parts. Conclusions: Normal urothelium and connective tissue both harboured FGFR3 mutations. Differences in findings of these early progression protective mutations within large tumours could be interpreted as tumours caused by more than one clone of origin. N.T.G. was funded by a research travel grant of the DGP

Aims: MicroRNAs have recently been implicated in multiple aspects of cancer formation. In order to identify p53-regulated microRNAs we performed a genome screen. Methods: We induced p53 in a lung cancer cell line using a modified doxycycline-regulatable, episomal vector system. Libraries of small RNAs were generated and subjected to 454 sequencing. Results: We found that after p53-activation the abundance of thirty-four miRNAs was significantly increased, whereas sixteen miRNAs were suppressed. The induction of miR-34a was most pronounced among all differential regulations. Also expression of the primary miR-34a transcript was induced after p53 activation and by DNA damage in a p53-dependent manner. p53 occupied an evolutionarily conserved binding site proximal to the first non-coding exon of miR-34a. Ectopic miR-34a induced apoptosis and a cell cycle arrest in the G1-phase, thereby suppressing tumor cell proliferation. Other p53-induced miRNAs identified here may also have tumor suppressive potential as they are known to suppress the anti-apoptotic factor Bcl2 (miR-15a/16) and the oncogenes RAS and HMGA2 (let-7a). Conclusions: Our results for the first time directly integrate the regulation of miRNA expression into the transcriptional network regulated by p53. siRNAs corresponding to p53-induced miRNAs may have potential as cancer therapeutic agents as RNA interference based therapies are currently emerging.

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Fr-012 Endothelzellen und Nierentransplantatabstoßung H. Regele Wien

Fr-014 Lernt die Histopathologie von der Molekularpathologie? F. Hofstädter Regensburg Fr-015 Funktionen des VHL Proteins und Progression von Nierenkarzinomen H. Moch Zürich Fr-016 Distribution of FGFR3 mutations within extensive bladder carcinoma and whole urothelial linings N.T. Gaisa, R. Stöhr1, F.X. Real2, R. Knüchel Institut für Pathologie, Universitätsklinikum Aachen 1 Institut für Pathologie, Universitätsklinikum Erlangen 2 Molecular Pathology Unit, Spanish National Cancer Research Centre, Madrid, E

Fr-017 Characterization of T Cell Epitopes in Experimental Autoimmune Glomerulonephritis of DBA/1 Mice H. Hopfer, H.-J. Paust1, J.-E. Turner1, U. Hopfer1, M. Sachs2, A. Peters1, B. Kocaoglu2, U. Helmchen3, R.A.K. Stahl1, H.W. Mittrücker4, U. Panzer1 Institut für Pathologie, Universitätsspital Basel 1 III. Medizinische Klinik, 2 Institut für Pathologie, 3 Nierenregister und 4 Institut für Immunologie, Universitätsklinikum Hamburg-Eppendorf Aims: Experimental autoimmune glomerulonephritis (EAG), a model of human antiglomerular basement membrane disease, is induced in DBA/1 mice by repeated immunization with the Goodpasture antigen a3(IV)NC1. To study the contribution of T cell mediated immunity we mapped the T cell epitopes and studied if the peptides were nephritogenic. Methods: DBA/1 mice were immunized with recombinant a3(IV)NC1. Splenocytes were isolated on day 10 and restimulated with a set of overlapping 15mer peptides, spanning the whole a3(IV)NC1 sequence. DBA/1 mice were repeatedly immunized with the immunodominant peptides, a3(IV)NC1, or PBS, and followed for signs of disease. Results: In vitro restimulation with overlapping peptides revealed three immunodominant peptides generating an IFN-g response (AA10–24, AA91– 105, AA208–222). None of the peptides led to clinical signs of EAG in vivo. However, mice immunized with AA208–222 developed circulating antibodies against a3(IV)NC1 at 8 weeks in 6/6 mice and moderate to severe proteinuria at 18–24 weeks in 3/6 mice. Polyclonal stimulation of CD4+ renal cells induced proinflammatory IFN-g and IL-17 in these mice indicating renal inflammation. Conclusions: Our results suggest that only one of the three immunodominant peptides is nephritogenic inducing mild EAG. This is associated with an antibody response directed against the complete a3(IV)NC1 domain.

Symposium: Beste Forschungsbeiträge zur Gynäkopathologie Fr-018 Promoter methylation-associated loss of ID4 expression is a marker of tumour recurrence in human breast cancer E. Noetzel1, J. Veeck1, A. Hartmann2, R. Knüchel1, E. Dahl1 1 Institut für Pathologie, Arbeitsgruppe Molekulare Onkologie, Universitätsklinikum Aachen 2 Institut für Pathologie, Universitätsklinikum Erlangen Aims: The aim of this study was to unravel the role of the transcription factor Inhibitor of DNA binding 4 (ID4) in human breast carcinogenesis in more detail, especially the impact of ID4 promoter methylation on disease progression. Methods: The ID4 promoter methylation status of a large cohort of breast cancer specimens was determined via methylation-specific PCR and correlated with corresponding ID4 mRNA expression. In vitro demethylation analysis was performed in human breast cell lines. Using immunhistochemical staining, loss of ID4 protein expression was assessed and compared to ID4 promoter methylation and ID4 mRNA expression in matched samples of breast tumours and corresponding normal tissues. Statistical evaluations were performed with SPSS14.0. Results: Demethylating treatment of ID4 methylated breast cancer cell lines was associated with ID4 re-expression. ID4 promoter methylation was frequently observed in primary breast cancer samples (68.9%, 117/170). We found a very tight correlation (P<0.001) between ID4 promoter methylation and loss of ID4 mRNA expression in primary breast cancer specimens. Breast tumours with ID4 promoter methylation showed distinct loss of ID4 expression on both mRNA and protein level. Interestingly, ID4 promoter methylation was a factor for unfavourable recurrence-free survival (P=0.036) and increased the patients risk for lymph node metastases (P=0.030).

Conclusions: ID4 is a potential tumour suppressor gene in normal human breast tissue that undergoes epigenetic silencing during carcinogenesis, leading to an increased risk for tumour relapse. Thus, ID4 methylation status could serve as a prognostic biomarker in human breast cancer.

Fr-019 Early loss of heterozygosity on chromosome arm 16q in Flat Epithelial Atypia (FEA) of the breast detected by microsatellite analyses H. Schmidt, C. Dahrenmöller, K. Agelopoulos, D. Hungermann, W. Böcker Institut für Pathologie, Universitätsklinikum Münster Aims: With the improvement of breast carcinoma screening pre malignant cell lesions like Flat Epithelial Atypia (FEA) are detected more frequently. Several studies show that FEA have a risk of upgrade to invasive carcinoma, but is it a real precursor lesion? We have started a comparative genetic analysis of a panel of nine microsatellite markers on 6 different chromosomal regions to investigate whether FEA shows the same characteristic genetic alterations as DCIS and invasive carcinoma of the breast. Methods: FEA, DCIS and invasive carcinoma of the same patients were microdissected using PALM microlaser technology. DNA was isolated using the QIAamp DNA Mikro Kit (QIAGEN). We have investigated a set of the polymorphic microsatellite markers D7S522, D8S522, NEFL, D10S541 (PTEN), D13S153 (RB1), D16S400, D16S402, D16S422 and D17S855 (BRCA1) using multiplex PCR for the detection of allelic imbalances. Results: Most of the investigated FEA showed a lower frequency of loss of heterozygosity than DCIS or invasive carcinoma. We were able to detect the same alterations in FEA as in DCIS or invasive carcinoma in a lot of the investigated cases. Remarkable: the microsatellite marker on 16q showed more prevalent allelic imbalances in FEA than the other investigated markers. Conclusions: One of the hallmarks in the pathogenesis of a large subgroup of invasive breast cancer is the early loss of chromosome arm 16q. In this study we were able to detect frequent genetic alterations on chromosome 16q in FEA, DCIS and invasive carcinoma respectively in tissue samples from the same patient. This might lead to the assumption that FEA is a real precursor lesion in the low grade pathway.

Fr-020 The role of microRNAs (miRNAs) during postnatal mouse mammary gland development S. Sassen1,2, L. Goldstein2, C. Blenkiron3, E.A. Miska3, C. Caldas2,1 1 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München 2 Cancer Research UK Cambridge Research Institute and Department of Oncology and 3 The Wellcome Trust/ Cancer Research UK Gurdon Institute, University of Cambridge, UK Aims: This project tests the hypothesis that mammary gland development is regulated through dynamic changes in miRNA expression, and that deregulated expression of specific miRNAs may lead to malignant transformation of the breast. Methods: Expression data for 333 murine miRNAs was obtained by beadbased flow cytometric profiling (Luminex) on mouse mammary glands over a 16-point developmental time course including juvenile, puberty, mature virgin, gestation, lactation, and involution stages. In addition, whole genome mRNA expression (Illumina) data were obtained for all samples. Results: A total of 120 miRNAs were differentially regulated during mammary gland development. We identified groups of miRNAs that characterize key developmental stages. A subgroup of 18 conserved miRNAs significantly over- or under-expressed in human breast cancer versus normal breast tissue showed distinct patterns across breast development. Conclusions: Out of all known murine miRNAs (~360), about one third is present during mouse mammary gland development and expressed in a time specific pattern. A signature of 18 conserved miRNAs significantly differentially expressed in breast cancer vs. normal breast tissue showed distinct patterns according to key developmental stages. The comparison of miRNA and Der Pathologe · Supplement 1 · 2008

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Abstracts gene expression may provide candidate genes and pathways for the study of miRNA-target interactions.

Fr-021 Dynamic gene ampliﬁcation in the breast cancer cell line MDA-MB468 leads to diﬀerent coexisting genotypes K. Agelopoulos, H. Schmidt, E. Korsching, H. Buerger1, B. Brandt2 Institut für Pathologie, Universitätsklinikum Münster 1 Institut für Pathologie, Paderborn 2 Instiut für Tumorbiologie, Universitätsklinikum Hamburg-Eppendorf Aims: Intratumor genetic heterogeneity, a well-known characteristic of numerous cancers, often confounds a precise diagnosis and leads to therapeutical resistance. This study deals with such chromosomal variability which may be due to an inherent genetic instability affecting heterogeneity and clonal effects. Methods: Subpopulations of the breast cancer cell line MDA-MB-468 were isolated according to their EGFR-expression by FACS or by EGF incubation. Whole genome profiling (CGH; Mapping Arrays) and determination of egfr gene amplification (FISH; qPCR) was done directly after sorting or after several passages of cell culture. Results: Subpopulations differed in the amplification of the egfr-locus 7p11–14 showing egfr gene amplification rates of up to 60fold in high-level expressing populations and less than 2fold in low-level expressing populations. However, after several passages the original low-level cells showed a new amplification of the egfr gene. This new egfr amplification was as heterogeneous as the original amplification which can be detected in MDA-MB-468. Additional, spontaneously expressed fragile sites could be shown in FISH analyses which may affect cell culture heterogeneity. Conclusions: Dynamic gene amplification in the breast cancer cell line MDA-MB-468 leads to heterogeneous populations with different coexisting genotypes. Spontaneously expressed fragile sites potentially affect this heterogeneity. Understanding the precise chromosomal process would clarify mechanisms in vivo and improve both diagnosis and therapy of corresponding cancers.

Fr-022 The extracellular matrix protein ITIH5 is a novel prognostic marker in invasive breast cancer and its expression is abrogated by epigenetic gene silencing E. Breuer, J. Veeck, M. Chorovicer, A. Naami, R. Knüchel, E. Dahl Institut für Pathologie, Universitätsklinikum Aachen Aims: Recently, mRNA expression of ITIH5, encoding an ECM-stabilising protein, was found to be reduced in invasive breast cancer. Here, we aimed at analysing its protein expression in breast cancer, identifying the molecular cause of its expression loss and studying its biological function in breast tumorigenesis. Methods: Immunohistochemistry with a proprietary ITIH5-antibody was applied on a large tissue microarray including comprehensive clinical patient data. Epigenetic gene regulation was studied in cell lines and breast carcinomas by realtime PCR and methylation-specific PCR. Expression and epigenetic data were statistically correlated with patient characteristics and survival intervals. Stable ITIH5-transfectants were generated and assayed in cell biological experiments. Results: ITIH5 protein was abundantly expressed in normal breast tissues (n=10) and carcinomas in-situ (n=15), but lost in 42% (n=92/217) of invasive breast cancers. Loss of ITIH5 was associated with short RFS (p=0.037) and OS (p=0.044). Breast cell lines (n=6) and 40% (n=68/170) of primary breast carcinomas revealed strong ITIH5 promoter methylation in clear association with loss of mRNA expression (p<0.001; n=39). Methylation of ITIH5 was associated with lymph node (p=0.003) and distant metastases (p=0.047), short RFS (p=0.005), short OS (p=0.001), and remained an independent prognosticator in a multivariate Cox regression model (HR=9.0; p=0.033). Functionally, ITIH5-transfectants showed a less progressive behaviour than empty-vector control cells.

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Conclusions: ITIH5 is likely to be a novel metastasis-suppressor in breast cancer. Both, its protein expression and promoter methylation may serve as powerful prognostic markers in this disease, able to select patients with low risk of recurrence and disease progression.

Fr-023 Expression of Activated Leukocyte Cell Adhesion Molecule (ALCAM, CD166) in breast cancer correlates with presence of bone metastasis and is a predictive factor for response of taxane-free adjuvant chemotherapy E. Kilic, V. Müller1, K. Milde-Langosch1, M. Ihnen1 Institut für Pathologie, Universitätsspital Basel 1 Klinik und Poliklinik für Gynäkologie, Universitätsklinikum HamburgEppendorf Aims: ALCAM is a cell surface immunoglobulin expressed in normal and neoplastic tissues and is assumed to be implicated in tumorigenesis and tumour progression. We evaluated the expression of ALCAM in breast cancer (BC) in correlation to clinicopathological data in patients treated with Taxane-free adjuvant chemotherapy. Methods: Tissue specimens of 162 primary breast cancer patients were analyzed. Immunostaining (IHC) was performed using monoclonal ALCAM antibody. Western Blot (WB) analysis of ALCAM was performed and afterwards densitometrically evaluated. To validate our results ALCAM mRNA expression was evaluated by microarray analysis (Affimetrix). Results: At the invasive front of BCs ALCAM is strongly expressed in IHC. WB analysis shows a significant positive correlation of ALCAM protein levels with positive estrogen receptor status (p=0.040). In patients with relapse, bone metastasis correlates with high ALCAM levels, but not the presence of visceral metastasis. Histological type and grade of malignancy, tumour stage and age do not correlate with ALCAM expression. There was an improved disease free and overall survival in patients treated with adjuvant chemotherapy expressing high ALCAM compared to those with low ALCAM expression. Median mRNA expression of ALCAM was 4.6 fold higher in patients alive at the time of follow-up compared to those who died of breast cancer. Conclusions: High ALCAM expression might be a marker of more aggressive tumour behavior and an indicator for bone metastasis as well as a useful predictive factor of resistance to adjuvant Taxane-free chemotherapy.

Fr-024 Clonality of tubular breast carcinomas and associated low-grade DCIS, ﬂat epithelial atypia and lobular neoplasia S. Aulmann, Z. Elsawaf, R. Penzel, P. Schirmacher, H.P. Sinn Pathologisches Institut, Universitätsklinikum Heidelberg Aims: Invasive tubular carcinoma of the breast frequently is associated with non-invasive alterations of the terminal ductolobular unit, especially low grade DCIS, flat epithelial atypia (FEA), LCIS, or a combination of two or three of these lesions. However, it remains unclear if these changes are direct precursors of invasive tubular carcinoma or unrelated lesions. Methods: We have analyzed 21 cases of pure tubular carcinomas that were associated with low grade precursor lesions (18 FEA, 7 DCIS, 2 LCIS). DNA was isolated from microdissected invasive carcinomas and precursor lesions followed by analysis of the mitochondrial D-Loop sequence region using PCR, direct DNA-sequencing and phylogenetic tree clustering. Results: Identical patterns of mitochrondrial DNA heteroplasmy were found in 45% of FEA lesions associated with the tubular carcinomas (5/11 cases), 50% of DCIS (3/6 cases), and 0% of LCIS (0/2 cases). Conclusions: Flat epithelial atypia is clonally associated with coexisting invasive tubular carcinoma in a similar frequency as low grade ductal carcinoma in situ. Therefore, FEA appears to be a direct precursor of tubular carcinoma of the breast, at least in a significant proportion of cases.

Fr-025 Aquaporin 1 Overexpression is a Characteristic Feature of Basal-like Breast Carcinomas and is Associated with Poor Overall Survival F. Otterbach, R. Callies1, R. Kimmig1, K.W. Schmid, A. Bànkfalvi Institut für Pathologie und Neuropathologie und 1 Klinik für Frauenheilkunde und Geburtshilfe, Universitätsklinikum Essen Aims: Aquaporin 1 (AQP1) water channels facilitate water movement across cell membranes. In the female breast, AQP1 is expressed in endothelial and myoepithelial cells. AQP1 may play a role in tumour angiogenesis, cell migration and invasion. The aim of the present study was to investigate the expression and prognostic significance of AQP 1 in breast cancer. Methods: We immunohistochemically studied the expression of AQP1 in 203 invasive breast carcinomas and compared it with clinico-pathological parameters as well as biomarkers of basal-like breast carcinomas and overall survival. Results: AQP1 overexpression was found in 5.4% of all invasive carcinomas. Expression was significantly correlated with high tumour grade, absence of estrogen receptors, “triple-negativity”, expression of cytokeratin 14, expression of smooth muscle actin and medullary-like histology. In univariate analysis, AQP1 was significantly associated with poor prognosis, and proved to be an independent prognostic marker in multivariate analysis if stratified by age, size, lymph node status, histological grade and ER status. Conclusions: AQP1 expression is a novel characteristic feature of basal-like breast carcinomas with high prognostic impact. The invasive and metastatic potential of AQP1 expressing breast carcinomas may partly be explained by increased migration of tumour cells. A targeted tumour therapy for AQP1 expressing tumours can be assumed.

Fr-026 Basal and Myoepithelial Phenotype in Metastatic Mammary Carcinomas: A Prognostic Factor? P. Mainka1, D. Mayr1, S. Kahlert2, S. Marlow1, T. Kirchner1, J. Diebold3 1 Pathologisches Institut und 2 Frauenklinik Großhadern, Universitätsklinikum München (LMU) 3 Pathologisches Institut, Kantonsspital Luzern Aims: The aim of the present study was to evaluate the prognostic impact of basal and myoepithelial phenotype in breast carcinomas (BBC and MBC) after first detection of distant metastases. Methods: Paraffin embedded material of 244 primary breast carcinomas of patients with subsequent metastatic disease was stained immunohistochemically for CK 5/6, CK14, smooth-muscle actin, p63, estrogen receptor and progesterone receptor. BBC was defined as positive for CK5/6 and/or CK14 and MBC as positive for SMA and/or p63. Clinical and pathological data were available for all patients; follow up data for 96.3% (4 weeks to 150 months). Results: Until the end of follow up period 90.2% of patients died, 6.1% are still alive. 27.8% of tumours could be classified as BBC and 8.1% as MBC. Kaplan Meier analysis revealed a trend for reduced overall survival after first diagnosis of metastasis (OASM) for BBC and MBC. Differences in survival were significant for BBC (log-rank=5.0; p=0.025), but not for MBC. After stratification for hormone receptor status BBC lost its influence on OASM. CK5/6 and CK14 double positive tumours (n=19; 7.8%) were identified as a subgroup of BBC associated with reduced OASM (log-rank=10.3; p=0.001). In this group the association with reduced OASM was independent of hormone receptor status, lymph node status, grading and pattern of metastases or chemotherapeutic treatment. Conclusions: The association of BBC and MBC with reduced OASM in metastastic breast carcinomas is not independent from hormone receptor status. CK5/6+ CK14+ double positive tumours may be a subgroup of BBC with particularly unfavourable outcome

Symposium: Infektionspathologie Fr-027 The pathology and pathogenesis of avian inﬂuenza infection in humans J. Gu Peking Fr-028 Pathologie und Molekularpathologie der Human Papilloma VirusInfektion K. Sotlar München

Symposium: Systempathologie des Gewebes (II) Fr-029 Systembiologische Analyse zielgerichteter Therapien C. Sers Berlin Fr-030 Systems pathology of RAS oncogene-driven oncogenesis R. Schäfer, H.-P. Herzel1, C. Sers Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte 1 Institut für Theoretische Biologie, Humboldt Universität Berlin Aims: Systems analysis of oncogenesis requires the dissection of the entire circuitry of cancer cells and the interaction with the stroma and microenvironment. Systems analysis of cancer cells is hampered due to methodological constraints and the overall complexity of the multi-step process of tumorigenesis. We tried to integrate a reductionist approach based on oncogenedriven model tumor systems with state-of-the art analysis of transcriptomics, and the pertubation of signal transduction. Methods: We analysed RAS-oncogene transformed epithelial cells undergoing epithelial-mesenchymal transition in various steps. First we established a genome-wide profile of normal precursors versus transformed cells by microarray analysis and subtractive suppression hybridisation. Using pharmacological and genetic interference (RNAi) experiments targeting signalling kinases, we investigated both the regulation and function of critical target genes. Results: The conversion from the normal state to the transformed state triggered by RAS oncogenes is associated with the differential expression of hundreds of target genes involved in various aspects of malignant transformation comprising positive and negative growth regulation, motility, invasion, angiogenesis and metastasis. Pathway interference identified subsets of genes (modules), responding to the MAPK-pathway, the PI3K-pathway and yet undefined pathways. By comparing cell systems across mammalian species, we identified a limited number of target genes that directly triggered neoplastic properties. Conclusions: Elucidating the components of oncogenic signalling pathways helps to define the systemic wiring of target genes, their phenotypic effects and their potential impact as therapy targets.

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Abstracts Fr-031 Signal pathway proﬁling in formalin-ﬁxed cancer tissues: the emerging role of protein microarrays for diagnosis and therapy guidance K.-F. Becker1, H. Höﬂer1,2 1 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München 2 GSF-Forschungszentrum für Umwelt und Gesundheit Aims: Deregulated signal pathways can drive cancer growth, survival, invasion, and metastasis. Mapping these tumour cell networks in tissues will be critical for realizing the promise of patient-tailored molecular therapy. Until recently, however, it was not possible to objectively measure hyperactive or defective signalling proteins in routine clinical tissues. The aim of our research is to establish tools, standards and controls for translating proteomic pathway profiling to the bedside. Methods: We analysed 3 cell lines and more than 100 formalin-fixed primary cancer tissues, including biopsies. Proteins were extracted from formalinfixed histological tumour sections and printed on nitrocellulose-coated glass slides. The resultant protein lysate microarrays were probed with more than 30 rigorously validated (Western blot from formalin-fixed tissues) antibodies against phosphorylated or total proteins, respectively. Results: We found no differences between formalin-fixed and unfixed cells. We were able to quantify critical signalling endpoints in formalin-fixed clinical tissues. Clustered image analysis maps revealed biologically interpretable patterns of protein expression. As an example, we found that activation (phosphorylation) of ERK1/2 was independent of HER2 expression in breast cancer. Conclusions: Our vision is that monitoring phosphorylated proteins in formalin-fixed clinical tissues may allow us in the near future to infer the activity levels of proteins in a particular pathway as starting point for the design of individual therapy regimens without changing the routine clinical workflow for tissue analysis.

Symposium: Gynäkologische Pathologie Fr-032 EIN and WHO J. Baak Stavanger, N Fr-033 Expression of Focal Adhesion Kinase (pp125FAK) in endometrial cancer: clinicopathologic relevance B. Gabriel1, A. Hasenburg1, M. Waizenegger1, M. Orlowska-Volk2, M. Klar1, E. Stickeler1, A. zur Hausen2 Frauenklinik und 2 Institut für Pathologie, Universitätsklinikum Freiburg

(p=0.035), but not for LVSI. Multivariate Cox regression analysis identified histological tumor grade as significant independent predictor of survival. Conclusions: Our results suggest an important role of FAK in endometrial carcinogenesis. Furthermore, the histological tumor grade was an independent predictor of survival in our patient cohort.

Fr-034 Protein microarray analysis: EGFR signalling is associated with Snail stabilisation in endometrial cancers S. Hipp1, A. Walch2, H. Höﬂer1,2, K.-F Becker1 1 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München 2 Institut für Pathologie, GSF-Forschungszentrum Neuherberg Aims: We asked whether the p38-mitogen activated protein kinase (MAPK) and/or the AKT – glycogen synthase kinase-3 (GSK-3beta) pathways are involved in the regulation of the E-cadherin repressor Snail in human endometrial carcinoma. Methods: Proteins were extracted from 17 formalin-fixed and paraffin-embedded (FFPE) primary endometrioid endometrial carcinomas. 25 antibodies were used to precisely quantify signalling end points using protein lysate microarrays spotted onto nitrocellulose-coated glass slides. All antibody specificities were validated by Western blot. Results: Stimulation of the endometrial carcinoma cell line Ishikawa ERwith epidermal growth factor (EGF) leads to an increase of Snail protein expression and activation of p38-MAPK but does not increase phosphorylation of AKT and GSK-3beta. In the primary tumours analysed expression of activated EGFR (epidermal growth factor receptor, Tyr1086) and p38-MAPK (Thr180/Tyr182) correlated with increased levels of Snail protein as it was seen in the cell culture model. However, the inactive (phosphorylated) form of GSK-3beta (Ser9) was associated with expression of Snail. In addition, we observed a statistically significant inverse correlation between Snail and Ecadherin protein levels in these tumours. Conclusions: Our data suggest that EGFR and p38 MAPK activation but also GSK-3beta inactivation may be involved in the stabilisation of Snail protein in primary endometrial cancers, possibly resulting in down-regulation of Ecadherin.

State-of-the-art lecture: Systems Pathology – How to proceed with hyper-complex systems in pathology Fr-035 Systems Pathology – The new paradigm for multifactorial diseases H. Westerhoﬀ Amsterdam, NL

Symposium: Pathologie des Ovars Aims: pp125 FAK plays a pivotal role in tumor cell-signaling. FAK expression has been linked to tumor cell proliferation, invasion and metastasis. Here we investigate FAK expression in endometrial cancer to determine its impact on prognosis. Methods: Immunohistochemistry was performed on 134 resected endometrial cancer specimens. Kaplan-Meier survival analysis was used to assess the significance of FAK expression in the outcome of patients. Results: Specific FAK expression was found in the tumor cells, whereas normal endometrial epithelium showed barely any FAK expression. 120 (89%) revealed moderate and strong expression of FAK. Weak expression was found in 14 (11%) tumors. No statistical significant difference in patient survival depending on FAK expression was observed. However, a trend towards improved survival of patients with weak FAK expression was clearly observed. Accordingly, high expression of FAK correlated with higher histological grade (p=0.003), lymphovascular space invasion (LVSI) (p=0.003) and vascular space invasion (p=0.02). Furthermore, the survival analysis revealed prognostic significance for the variables tumor stage (p<0.01), histological type (p<0.01), histological grade (p=0.028), and pelvic lymph node status

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Fr-036 Histopathologische Zweitbegutachtung: Einschlusskriterium für klinische Studien bei Ovarialkarzinomen? F. Kommoss Mannheim Fr-037 Ovarialkarzinome: Spiegeln die Subtypen verschiedene Erkrankungen wieder? M. Köbel Vancouver

Fr-038 Wenn „Borderline“ aufhört borderline zu sein – klinische, pathologische und molekulare Parameter im Kontext von Langzeitverläufen seröser Ovarialtumoren A. Staebler Tübingen Fr-039 A molecular signature for identiﬁcation of platinum resistant ovarian cancer T. Fehm, M. Bonin1, J. Hoﬀmann1, M. Walter1, K. Sotlar2, D. Wallwiener, O. Riess1, E. Solomayer, H. Neubauer Frauenklinik und 1 Abteilung für Medizinische Genetik, Institut für Humangenetik, Microarray Facility, Universitätsklinikum Tübingen 2 Pathologisches Institut, Universitätsklinikum München (LMU) Aims: Ovarian cancer is one of the most common malignant tumors in women. Almost all patients are adjuvantly treated with platinum-based chemotherapy. Patients who suffer a relapse within 6 months are termed as platinum resistant. The goal of this study was to identify a geneset, which can predict resistance to platinum-based chemotherapy. Methods: Twelve platinum resistant and 12 platinum sensitive ovarian carcinomas were selected. RNA was isolated from fresh-frozen tissues and further analyzed on Illumina Human-6v2 BeadChips to determine differentially expressed genes. Data analysis with Genespring was followed by class prediction with Support Vector Machines (SVM) that was undertaken with a predictive set of 55 genes. It offered a correct classification into platinum resistant and platinum sensitive patients in all samples. Results: Analysis of our findings with Ingenuity Software showed a functional relevance to regulation of transcription, apoptosis and cell cycle. For validation of the predictive gene set, qRT-PCR was used to measure the mRNA expression level of 13 selected genes in 20 samples. By SVM analysis, 18 samples were found to be predicted correctly. The mean expression value in 10 of 13 genes was consistent with the trend observed in the microarray data. Conclusions: A predictive set of 55 genes was found that is able to classify ovarian carcinomas according to their sensitivity for platinum-based chemotherapy. The predictive power of the 55-gene set needs to be further validated in an independent set of ovarian cancer specimen.

Fr-040 Expression of the Endothelin axis in low-grade (type 1) surface epithelial neoplasms of the ovary – role in the adenoma-borderlinecarcinoma sequence of both serous and mucinous tumors A. Staebler, A. Jöcker, P. Kuhlmann, M. Schumacher, B. Karberg, R. Lelle1, L. Kiesel1, O. Buchweitz1 Institut für Pathologie und 1 Frauenklinik, Universitätsklinikum Münster Aims: Endothelin-1 (ET-1) and its receptors Endothelin-A and –B-Receptor (ETAR and ETBR) are implicated in ovarian carcinogenesis. Our aim is to evaluate the role of this signalling pathway in the development of borderline tumors (BLT) and low grade carcinomas (type 1 tumors) of different histological types. Methods: 216 cases of surface epithelial tumors of the ovary (23 Adenomas, 49 borderline, 30 low-grade and 114 high-grade invasive carcinomas) were analyzed by immunohistochemistry on tissue microarrays for expression of ETAR, ETBR and ET-1. Results: Increased expression ETAR and ETBR was observed significantly more frequently in invasive carcinomas (87%) than in borderline tumors (47%) and adenomas (0%), p<0,001. Analysis of individual subgroups showed a similar distribution: Serous tumors: ETAR: 92% of lowgrade carcinomas, and 85% of high-grade carcinomas, 36% of BLT, 0% of adenomas, p<0.001; ETBR: 79%, 63%, 50% and 0%, p<0,001. Mucinous tumors: ETAR: 90% of low-grade carcinomas, 77% of borderline tumors and 0% of adenomas, ETBR: 50%, 83%, 9%, p=0,002. Expression of ET-1 did no show any significant difference.

Conclusions: Over expression ETAR and ETBR is observed in the vast majority of invasive ovarian carcinomas, both low and high grade and in a subgroup of serous and mucinous borderline tumors but not in adenomas Therefore, endothelin signalling may play an essential role in the stepwise progression of both serous and mucinous borderline tumors to low-grade carcinomas.

Symposium: Aktuelle Habilitationen Fr-041 Clinico-pathologic and molecular characterisation of thymomas and thymic carcinomas R. J. Rieker Pathologisches Institut, Universitätsklinikum Heidelberg Aims: Evaluation of the morphology of thymomas and thymic carcinomas especially in regard to interobserver agreement and prognostic relevance; correlation with genetic aberrations to determine pathogenetic relations. Identification of novel therapeutic target structures. Methods: Retrospective Study, Comparative Genomic Hybridisation (CGH), Cluster analysis, Oncogenetic Tree Model, Western Blots. Results: The WHO classification showed good interobserver agreement (weighted kappa >0.87), significance for overall and disease free survival and a strong association with the stage according to Masaoka. Chromosomal imbalances detected by CGH were more frequent in thymic carcinomas compared to other subtypes and correlated with the stage according to Masaoka and WHO classification by cluster analyses. Oncogenetic tree models suggested that gains on chromosome 1q occured early in tumor development and were followed by losses on chromosome 6. Thymomas and thymic carcinomas showed significant and consistent COX-2 expression. Conclusions: The WHO classification shows a good prognostic power and correlates well with the stage according to Masaoka and with certain chromosomal imbalances detected by CGH. Some advanced thymomas and thymic carcinomas seem to arise through a multistage tumorigenic process. With COX-2, a possible new therapeutic target was identified.

Fr-042 Polo-like kinase 1 as a novel chemotherapeutic target in human solid cancers W. Weichert Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte Aims: The protein family of polo-like kinases (PLK) is known to play an outstanding role in the regulation of mitosis in normal and malignant cells. In several studies effects of PLK knockdown in cancer cell lines in vitro and PLK isoform expression patterns in vivo were investigated. Methods: PLK1 knockdown in cancer cell lines was done by either antisense or siRNA methods. Alamar Blue assays, confocal microscopy and western blots were performed to determine changes in cell number, morphology and expression of several key mitotic proteins. In addition, expression of PLK1 was assessed by immunohistochemistry in several large and well characterized cohorts of human solid cancers. Expression was correlated with clinicopathological data and patient survival. Results: PLK1 knockdown in cancer cell lines led to a decrease in cell number, to a mitotic arrest and an induction of apoptosis. In addition, an accumulation of mitosis associated proteins was observed. PLK1 protein expression was upregulated in 26% to 67% of cancer cases, depending on the tumor entity investigated. Usually expression was strongest in highly aggressive, locally advanced tumors. In some but not all tumor entities PLK1 expression had prognostic, partly independent significance for patient survival. Conclusions: The in vitro and in vivo data suggest that inhibition of PLK1 might represent an interesting new targeted chemotherapeutic approach for the treatment of a variety of human solid tumors.

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Abstracts Fr-043 Histological, Immunohistochemical and Molekularpathological Analyses of Ovarian Tumors: Correlation to Phenotype and Prognostic Outcome D. Mayr Pathologisches Institut, Universitätsklinikum München (LMU) Aims: Most tumors of the ovary are of epithelial origin. Little is known of the aetiology and until now no mechanism of malignant transformation has been identified. Most patients present in advanced stage because no early symptoms or diagnostic parameters exists. Variable prognosis points to different ways of tumorigenesis. Better understandings of tumor pathology and exacter sub-classifications with precise information for outcome are important for risk estimation and individually therapy. Methods: Paraffin embedded material of more than 400 ovarian tumors was analyzed histological, immunohistochemical and molecularpathological (CGH, FISH, PCR); correlation to phenotype and outcome was sought. Results: A universally accepted histopathological grading system (for e. Silverberg grading) for carcinomas, which correlates with prognosis, should be established. p53 accumulation is a prognostic factor in ovarian carcinomas. In Granulosa cell tumors most frequently numeric aberrations are seen. Endometriosis has no certain premalignant potential. In carcinomas the CCNE-1 immunoreactivity is of prognostic significance. Activation of the RAS-RAFpathway is often seen in epithelial borderline tumors. Conclusions: Cytogentic changes in sex cord stroma tumors, especially Granulosa cell tumors differ from those in epithelial cancer. The histological subtypes of epithelial tumors show differences in their results of RAS-RAFactivation; this points to various ways of development, both, of these different histological subtypes, and of invasive and non-invasive tumors. A conclusion for an adenoma-carcinoma-sequence can be drawn only for the small group of low grade carcinomas.

Fr-044 Tumor-stroma interactions: a crucial factor for the malignant potency of pancreatic cancer? I. Esposito Pathologisches Institut, Universitätsklinikum Heidelberg Aims: Interactions between stroma and epithelium play an important role in tumor biology, in particular in those neoplasms that arise in association with chronic inflammatory processes. Pancreatic ductal adenocarcinoma (PDAC) can be associated with long-standing chronic pancreatitis (CP) and is characterized by an intense desmoplastic stromal reaction. Aim of the study was the characterization of the desmoplastic stromal reaction in PDAC and the analysis of the contribution of different stromal cell types to the extremely aggressive behaviour of PDAC. Methods: Expression analysis of large numbers of PDAC-tissues and of PDAC precursors (PanIN) was performed using quantitative real-time PCR and/or immunohistochemistry. Functional analyses (growth and invasion assays) and drug effects were performed/tested on cultured pancreatic cancer cell lines and freshly isolated pancreatic stellate cells (PSC). Serum values were measured with the ELISA method. Correlations with relevant clinical parameters and survival were analyzed. Results: The composition of the stromal reaction in CP and PDAC concerning the number of infiltrating mast cells and macrophages and the expression of some extracellular matrix proteins (Tenascin C, Osteopontin) is comparable. Tumor cells can activate the surrounding stromal cells and induce the synthesis of molecules (VEGF, bFGF, Tenascin C, Periostin, Versican) that influence the growth and invasive ability of PDAC cells and can modify their response to chemotherapy. Activation of the stroma in a tumor-like fashion is already detectable around PanIN lesions. Conclusions: The extraordinary aggressiveness of PDAC resides -at least in part- in the relationship that the tumor cells establish with the surrounding tissue. The characterization of the desmoplastic stromal reaction in PDAC is essential in order to identify new therapeutic targets for the treatment of this deadly disease.

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Symposium: Mammapathologie in Screening und Prädiktion Fr-045 Radiologie im Mammographie-Screening: Fragen an den Pathologen S. Heywang-Köbrunner München Fr-046 Ergebnisse der Zweitbefundung im Mammographiescreening H. Kreipe Hannover Fr-047 Praktische Probleme im Mammographie-Screening: Kolumnarzellläsionen einschl. FEA und LIN J. Nährig München Fr-048 Prädiktion C. Petry Leverkusen

Symposium: Klinische Pathologie des Ovars Fr-049 Besonderheiten in der Behandlung des Ovarialkarzinoms, oder: Was der Pathologe vom Kliniker erfahren sollte J. Sehouli Berlin Fr-050 Molekulares Proﬁling und prädiktive Signaturen – Biomarkeranalysen beim Ovarialkarzinom C. Denkert Berlin Fr-051 Diﬀerential expression of secretoglobulins in normal ovary and in ovarian carcinoma – overexpression of Mammaglobin-1 (MBG-1) is linked to tumor progression A. Buckendahl2*, K. Fischer1*, D. Hornung3, C. Denkert2, S. Niesporek2, C. Ufer1, H. Schiebel1, H. Kuhn1, A. Borchert1 1 Institut für Biochemie und 2 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte 3 Klinik für Gynäkologie, Universitätsklinikum S-H, Campus Lübeck * Geteilte Erstautorenschaft Aims: Ovarian cancer has the highest mortality rate of gynecological malignancies, which is partly due to a lack of effective screening parameters. Members of the secretoglobulin family have recently been related to carcinogenesis in different organs and expression microarray studies suggested mammaglobin-2 as candidate marker. Methods: We characterized the distribution of the secretoglobulins lipophilin-B, mammaglobin-1 (MGB-1), and mammaglobin-2 (MGB-2) in human tissues and quantified their expression by real-time RT-PCR. In a second step we compared the expression levels of secretoglobulin mRNAs (real time RT-PCR) and MGB-1 protein (immunohistochemistry) in normal ovaries and ovarian carcinomas. Results: The three secretoglobulins are expressed at low quantities in most human tissues including ovary. We found significantly elevated concentrations of secretoglobulin mRNA in carcinoma compared to normal ovarian

tissue. For MGB-2 the most dramatic differences were observed whereas MGB-1 and lipophilin-B showed a moderate upregulation. For MGB-1 this upregulation was confirmed on the protein level and both nuclear and cytoplasmic localization was observed. Interestingly, overexpression of this protein correlated positively with FIGO stage, tumor grade and mitotic index. Conclusions: Secretoglobulins are upregulated in ovarian cancer on the mRNA and protein levels. This increased expression may be of clinical relevance since these gene products might be used as a diagnostic parameter. Furthermore, MGB-1 is linked to tumor progression.

Symposium: Meet the Expert (I) Fr-052 Gastrointestinale Tumoren T. Kirchner München Fr-053 Thymome und andere Mediastimaltumoren H. Marx Mannheim Fr-054 Hodentumoren G. Mikuz Innsbruck

Symposium: Freie Vorträge zur prädiktiven Pathologie Fr-055 Promoter-hypermethylation of Carbonic anhydrase IV is frequently found in colorectal cancer and correlates with tumor grade, CIN- and p53 status and may be useful as a prognostic and predictive marker in colon stage III cancer S. Schnall1, M. Bettstetter1, C. Vogel1, M. Klinkhammer-Schalke2, F. Hofstädter1, W. Dietmaier1 1 Institut für Pathologie und 2 Tumorzentrum, Universitätsklinikum Regensburg Aims: Carbonic anhydrases (CA) are a family of zinc-metalloenzymes. Several members of carbonic anhydrases have been shown to be associated with cancer and show prognostic features. Using Affymetrix chip arrays we found CA IV to be downregulated frequently in colorectal cancer. The aim of this study was to analyse whether downregulation of CA IV is caused by tumor specific promoter hypermethylation and to study the role of CA IV promoter methylation as a potential prognostic or predictive marker. Methods: A real-time PCR based quantitative promoter methylation analysis of CA IV was performed in 156 colon stage III cancer patients (lymph node positive). Sixty-three patients were treated by surgery only, 96 patients received additionally an adjuvant 5-fluorouracil (5-FU) based chemotherapy. We correlated CA IV methylation with molecular (microsatellity instability, chromosomal instability), clinical and pathological data. Results: We found a significant correlation of CA IV methylation with tumor grade (p=0.044), chromosomal instability (p=0.033) and p53 IHC (p<0.001) (positivity in G2-tu.: 40.0%, G3-tu.: 57.8%, CIN-neg-tu.: 53.8%, CIN-pos-tu.: 35.5%, p53-neg-tu.: 65.2%, p53-pos.-tu.: 33.3%). Patients with CA IV methylation and surgery treatment alone showed a significant shorter overall survival than patients without CA IV methylation (p=0.031, Kaplan-Meier analysis) but benefit from adjuvant 5-FU chemotherapy. Conclusions: CA IV methylation correlates with tumor grade, CIN- and p53 status and may be useful as a prognostic and predictive marker.

Fr-056 CD56 induces apoptosis and negative inotropy in ischemic cardiomyopathy S. Gattenlöhner1, C. Waller2, K. Schuh3, G. Ertl2, H.K. Müller-Hermelink1 1 Institut für Pathologie, Universitätsklinikum Würzburg 2 Medizinische Klinik I, Universitätsklinikum Mannheim 3 Physiologisches Institut, Universitätsklinikum Würzburg Aims: CD56 (NCAM) belongs to the family of Ca2+-independent cell adhesion molecules, CAMs, showing abundant expression mainly in fetal neural tissues. Recently, we found that CD56 is specificially overexpressed in ischemic cardiomyopathy (ICM) and regulated by the transcription factor RUNX1 (AML1). The aim of the study was to investigate the function of the CD56 expression in ICM in particular with respect to development of heart failure. Methods: Animal model for acute and chronic ischemia, RT-PCR and western Blot, stable cell transfection, micro cDNA array, FACS anaylsis and intracellular Ca+ measurements. Results and Conclusions: We first demonstrated in an animal model of acute heart ischemia a lacking CD56 expression in infarcted heart tissue and its upregulation in surviving cardiomyocytes, regulated by novel RUNX1 isoforms with activating and inhibiting function on the CD56 expression. Next we identified the highmolecular CD56140kDa isoform among the known structural and functional highly different CD56 variants as the exclusively expressed CD56 isoform in failing human hearts. In micro array cDNA chip analysis and subsequent quantitative RT-PCR using CD56 isoform specific stable transfectants of the murine cardiomyocyte cell line HL-1, the CD56140kDa stables as compared to controls and the other CD56 isoform showed an induction of apoptosis related genes as well as downregulation of pathways related to calcium channel signalling. Since the CD56140kD expressing cardiomyocytes showed an increased apoptosis (5-fold) in Annexin V FACS analysis and downregulation of the proliferation in MTT assay (3-fold) as well as a reduced calcium influx (8-fold) in Fluo-4AM intracellular measurement, we suggest that the overxpression of CD56140kD is functionally relevant for the development of ICM and blocking CD56140kD expression respectively its dependent signalling cascades might be a therapeutical target for the treatment of ischemic heart failure.

Fr-057 Global and gene-speciﬁc methylation patterns in patients treated with the demethylating agent 5-aza-2’-deoxycytidine (Decitabine) U. Lehmann, D. Römermann, K. Metzig, B. Hasemeier, F. Länger, C. Dobbelstein1, A. Ganser1, H. Kreipe Institut für Pathologie und 1 Abteilung für Hämatologie, Hämostaseologie, Onkologie, und Stammzelltransplantation, Medizinische Hochschule Hannover Aims: Treatment with demethylating agents shows promising response rates in patients with myeloid dysplastic syndrome (MDS) or myeloid leukaemia. Aim of this project is the analysis of this “epigenetic therapy” on a molecular level and to elucidate whether a quantitative methylation analysis can predict the individual response to this new therapy. Methods: Genomic DNA was isolated from sequential bone marrow biopsies from 15 patients with secondary acute myeloid leukaemia (2 to 6 biopsies per patient). Subsequently, DNA was treated with bisulfite following published standard procedures. The methylation level of the highly repetitive LINE-1 sequence and of the tumour suppressor gene p15INK4b gene was determined using quantitative high resolution Pyrosequencing™. Results: Patients showed marked differences concerning global methylation levels as well as gene-specific methylation at the beginning of therapy. During therapy with Decitabine LINE-1 as well as p15INK4b methylation was clearly reduced in a subgroup of patients. Reduction in methylation were accompanied by a good clinical response, whereas therapy refractory leukaemia did not show any significant alterations in methylation patterns. Conclusions: In a subgroup of patients, depending on the initial methylation level, Decitabine clearly induces demethylation. Thus, clinical response has a potentially predictive “molecular epigenetic correlate”.

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Abstracts Fr-058 Gene expression proﬁling reveals subtypes of ovarian cancers with clinical implications J. Budczies, C. Denkert, S. Niesporek, B. Györﬀy, J. Sehouli1,2, D. Könsgen1,2, H. Lage, M. Dietel Institut für Pathologie und 1 Klinik für Frauenheilkunde und Geburtshilfe, Charité – Universitätsmedizin Berlin, Campus Mitte 2 TOC-Network, Berlin Aims: Previous studies have already investigated the role of gene expression profiling in ovarian carcinoma and have shown that this method is able to identify gene expression profiles that predict survival in this type of tumors. However, unlike the situation in breast cancer, gene expression signatures in ovarian cancer have not been validated in independent data sets, so far. Methods: Expression analysis of an ovarian cancer cohort (Charité cohort, n=80) was carried out by hybridization to Affymetrix GeneChips. A semisupervised method developed by Bair and Tibshirani (supervised principle component analysis) was capable to detect two distinct tumor types that were associated with different prognosis. Results were validated on an independent cohort published by Dressman et al. (Duke cohort, n=117) that was profiled on the same plattform. Results: Kaplan-Meier analysis revealed significant differences in survival between the two tumors types both in a leave-one-out cross-validation on the Charité cohort and in a training-test protocol with the Duke cohort as test data. The hazard (univariate Cox model) was significantly different between the high risk and the low risk group with a hazard ratio arround 2. Conclusions: Gene expression profile based risk assessment has the potential to contribute to diagnosis of ovarian cancers and can help to stratify patients with the aim of a more individualized therapy.

Poster: Hämatopathologie Fr-059 Gelatinous Transformation of the Bone Marrow – A rare side eﬀect of body building? K. Engels, K.U. Chow1, M.-L. Hansmann, S. Kriener Institut für Pathologie, Universitätsklinikum Frankfurt am Main 1 Hematology-1Oncology Centre, Stresemannallee, Frankfurt am Main Aims: Gelatinous transformation (Syn.: serous fat atrophy) of the bone marrow has a wide spectrum of underlying disorders. We here present the case of an 42 year old male patient who submitted himself to a physician due to a subjective feeling of weakness and loss of weight (20 kg over the last months). Methods: Diagnostic procedures included an extensive serological analysis and a bone marrow trephine. The patient was extensively interviewed about his diet and his training activities. Results: The patient reported about a strict daily diet with two bread rolls with sausage, salad and protein, which he administered to himself over a period of at least 12 months. During this time the patient did sport for at least 21/2 hours per day including body building and athletics. On physical examination he was very muscular and seemed to have a complete loss of subcutaneous fat. Laboratory tests showed a leukopenia (2,9/nl) and an erythropenia (3,9 Mio/μl). Haemoglobin (13,7 g/dl) and haematocrit (0,39 l/l) were within the normal range while MCV (100 fl) and MCH (35 pg) were slightly elevated. A bone marrow trephine showed a hypocellular marrow with a regular maturing haematopoiesis with a dominant erythropoeisis and an extreme gelatinous transformation. Infections (e.g. HIV, tuberculosis), a malignant disease, and metabolic disorders were excluded. The patient was submitted to a specialist for eating disorders. Conclusions: Not only consuming diseases or metabolic disorders may cause a gelatinous transformation of the bone marrow. It may also be caused by a prolonged diet with an inadequate calorie and fat intake in combination with a physically demanding sport. Haematologic changes and a loss of power may result.

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Fr-060 Transcriptional down-regulation of VEGF receptors (FLT1, KDR) and inhibitor of endothelial cell migration endostatin in primary myeloﬁbrosis (PMF) are early events and independent of the stage of disease J. Höftmann, I. Preußner, K. Theophile, J. Schlué, H. Kreipe, O. Bock Institut für Pathologie, Medizinische Hochschule Hannover Aims: Primary myelofibrosis (PMF) is a chronic myeloproliferative disorder showing reactive proliferation of bone marrow stroma cells. Another hallmark in PMF bone marrow architecture is increased angiogenesis probably contributing to the abnormal progenitor cell trafficking into peripheral blood and distant organs. In search for new molecular markers reflecting the bone marrow pathology in PMF we investigated FLT1 and KDR, both of them receptors for vascular-endothelial growth factor (VEGF), and the designated inhibitor of endothelial cell migration endostatin, a fragment of collagen-type –18 (COL18). Methods: mRNA targets in bone marrow cells were quantified by real-time RT-PCR and in part by immunohistochemistry. Primary human fibroblasts were treated in vitro with basic fibroblast growth factor (bFGF) in order to investigate a potential effect on COL18 transcription. Results: We found a significant down-regulation of FLT1 and KDR transcripts (up to 25-fold and 50-fold, respectively) in PMF bone marrow cells (p≤0.001). Interestingly, this notable decrease was independent of the stage of disease, i.e. prefibrotic and advanced phases. Endostatin was likewise down-regulated up to 30-fold in both phases. Conclusions: We conclude that transcriptional down-regulation of FLT-1 and KDR reflects the already increased angiogenesis even in early stages of PMF. The notable decrease of endostatin expression obviously contributes to vessel formation through unrestricted promotion of endothelial cell migration. In summary, the bone marrow environment in PMF reflects a pro-angiogenic state independent of the degree of myelofibrosis.

Fr-061 No aberrant expression of miRNA 10a, 126, 20a and 17–5-p by megakaryocytes in Philadelphia chromosome-negative myeloproliferative disordes K. Hussein, W. Dralle, K. Theophile, H. Kreipe, O. Bock Institut für Pathologie, Medizinische Hochschule Hannover Aims: Micro RNA (miRNA) are a new class of short regulatory RNA molecules (~20 bp) which are able to inhibit mRNA expression by complimentary miRNA/mRNA binding. Micro-array analysis of human megakaryocytic (MK) cell lines revealed a subset of miRNA which are associated with MK differentiation in vitro (Garzon et al., 2006). Our aim was to evaluate aberrant miRNA expression in Philadelphia chromosome-negative myeloproliferative disorders (Ph- CMPD) derived MK in vivo. Typically, in these diseases MK cells show aberrant differentiation (e.g. enlarged cytoplasm, polyploidy). Therefore, pathological MK differentiation in Ph- CMPD might be based on deregulation of miRNA homeostasis. Methods: MK from different Ph- CMPD entities (n=46) and non-malignant controls (n=16) were collected from individual archived bone marrow samples by laser microdissection. Subsequently, total RNA was extracted and was analysed for miRNA expression with a real-time PCR assay. RNA amounts allowed the analysis of 4 candidates (miRNA 10a, 126, 20a, 17–5-p; selected from previously published data). Results: i) in vitro data on MK-differentiation-associated miRNA were reproducible in vivo ii) In the subset of analysed miRNA none was significantly altered in Ph- CMPD. Conclusions: i) Sorted MK cells from archived bone marrow are suitable for miRNA expression analysis ii) 10a, 126, 20a and 17–5-p, known to be involved in MK differentiation, are not aberrantly expressed in Ph- CMPD iii) other miRNA species appeared, however, to be differentially expressed in PhCMPD derived MK (see Theophile et al.)

Fr-062 Ocular marginal zone lymphoma in a young patient with SLE – a case report with a 10 year survey of extranodal MALT-lymphoma in the Austrian state Salzburg R. Kemmerling, O. Dietze, D. Neureiter Institut für Pathologie, Paracelsus Medizinische Privatuniversität Salzburg

Fr-064 Array-based expression analysis of single megakaryocytes in primary myeloﬁbrosis (PMF) for determination of aberrantly expressed genes involved in apoptosis K. Theophile, H. Kreipe, O. Bock Institut für Pathologie, Medizinische Hochschule Hannover

Aims: We present a young female patient with an ocular marginal zone lymphoma in circumstances of autoimmunity. Furthermore we give a complete 10 year survey of the status of MALT-Lymphoma in the Austrian state Salzburg. Methods: A 36 year old female patient was admitted to the clinic with a prominent lesion of the ocular adnexa. To compare this case with the overall incidence rate, aetiology, remission status and progression of extranodal marginal zone lymphoma a retrospective digital data query covering the years 1997 to 2006 was carried out. Results: Histological, immunohistochemical and molecular analysis revealed an infiltration of a conjunctive marginal zone lymphoma. Microbiological analysis were negative. The further clinical examinations showed a systemic lupus erythematodes with detection of auto-antibodies. Retrospective investigation revealed an incidence rate of 56 new cases with extranodal MALT lymphoma (1997–2006): stomach (53.6%), ocular (12.5%), skin (10.7%), thyroid gland (7.1%), parotid gland (5.3%), intestine (7.2%) and mamma (3.6%). In about 31.5% of the cases infectious agents or an autoimmunity disease could be evaluated. Progression and lethality rate was low. Patients with extranodal MALT-lymphoma had a mean age of 62.8±13.7 years (ocular MALT-lymphoma: 59.1±21.8 years) and showed female predominance (male/female:25/31). Conclusions: Comparable to the literature extranodal marginal lymphoma are a rare and relative stable disease with lower age onset in cases of primary ocular location.

Aims: One of the predominant morphologic features of PMF is exaggerated proliferation of atypical megakaryocytes which form large and dense cellular clusters. Whether accumulation of megakaryocytes is due to impaired apoptosis is not fully understood. To extend the limited understanding of the underlying pathologic processes, we used TaqMan Low Density Arrays to examine potentially involved genes. Methods: We determined the expression levels of 48 genes involved in apoptosis and cellular differentiation in formalin-fixed and paraffin-embedded megakaryocytes and total bone marrow of PMF (n=22) and megakaryocyte hyperplasia (n=10). To this end, megakaryocytes were isolated from bone marrow by laser-assisted microdissection. After RNA extraction, the cDNA of total bone marrow was directly quantified, whereas the cDNA of megakaryocytes was pre-amplified prior to quantification. Results: We could identify several genes showing up- or down-regulation in both megakaryocytes and total bone marrow. Some of these genes exposed equal tendencies in single cells as well as total cellularity, but others showed different or even opposite trends. Conclusions: A clear-cut up-regulation of anti-apoptotic genes and downregulation of pro-apoptotic genes could not be established in this study. Nevertheless, the aberrant expression of several apoptosis-relevant genes in PMF pathogenesis could be clearly demonstrated.

Fr-063 Detection of the systemic Mastocytosis speciﬁc ckit D816 V mutation in paraﬃn-embedded tissue by tetra-primed ARMSR R. Penzel, S. Aulmann, G. Mechtersheimer, P. Schirmacher Pathologisches Institut, Universitätsklinikum Heidelberg Aims: Mastocytosis is a heterogeneous disease of bone marrow origin and characterized by abnormal growth and/or accumulation of clonal mast cells (MC) in one or more organs. In systemic mastocytosis (SM), at least one extracutaneous organ is involved by definition. The somatic ckit D816 V activating mutation is most frequent (90%) in systemic mastocytosis and one of the minor diagnostic criteria. Furthermore, D816 V represents a potential drug target as reported for the treatment of D816V-mutant c-kit with Dasatinib, a dual SRC/ABL kinase inhibitor. We established an allele-specific amplification-refractory mutation system to detect this clinacally relevant mutation in an inexpensive and sensitive manner. Methods: We designed a tetra-primed allel-specific amplification-refractory mutation system to detect the ckit D816 V in a single PCR reaction. DNA of 15 mastocytosis patients was extracted from paraffin-embedded tissues and analyzed by ARMS. Results: The results were compared to conventional ckit exon 17 DNA-sequencing. Conclusions: Examination of a series of 15 SM-cases demonstrates the feasibility and sensitivity of the ckit D816V-ARMS and revealed the poor sensitivety of DNA-sequencing to detect this mutation due to the low abundance of neoplastic MC caracterstic for this disease.

Fr-065 Expression of Hedgehog Ligands in Myelodysplastic Syndrome – a preliminary Study R. Kemmerling, B. Allinger, D. Neureiter Institut für Pathologie, Paracelsus Medizinische Privatuniversität Salzburg Aims: Apoptosis, proliferation and morphogenesis are hallmarks of haematopoietic differentiation thus being associated with hedgehog signalling. Methods: 10 cases with refractory cytopenia with multilineage dysplasia (according WHO criteria) as well as 5 cases with normal haematopoiesis were analysed. Routine staining (Hematoxylin-Eosin staining and Naphthol-ASD-Chloracetatesterase-reaction) were used to estimate the dysplastic changes of granulopoiesis, erythropoiesis and megakaryocytopoiesis. Immunohistochemical studies were carried out with monoclonal antibodies (dessert hedgehog (DHH) M/N, sonic hedgehog (SHH) and indian hedgehog (IHH)). Expression levels were semi-quantitative evaluated. Results: Overall, the expression of hedgehog ligands was significant higher in granulopoiesis than erythro- or megakaryopoiesis (p<0.05). The expression of IHH was significant lower in dysplastic granulopoiesis of cases than controls (p=0.019). Higher expression of IHH could be found in the erythropoiesis of cases than controls (p=0.042). Additionally, the expression of SHH was slightly higher in erythro- and megakaryocytopoiesis of case than controls. The correlation analysis revealed a significant positive association of IHH and SHH with the degree of the dysplasia inside erythro- and megakaryocytopoiesis (p<0.05). Conclusions: Preliminary immunohistochemical investigations revealed a heterogeneous and haematopetic cell lineage associated expression of hedgehog ligands in cases of myelodysplastic syndrome possibly being involved in the pathogenesis.

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Abstracts Fr-066 MALDI imaging in the diagnosis of classical Hodgkin lymphoma K. Schwamborn, R.C. Krieg, R. Knüchel, G. Ott1, A. Rosenwald2, A. Wellmann Institut für Pathologie, Universitätsklinikum Aachen 1 Institut für Pathologie, Robert-Bosch-Krankenhaus Stuttgart 2 Institut für Pathologie, Universitätsklinikum Würzburg Aims: Classical Hodgkin lymphoma (cHL) is a distinctive lymphoma subtype characterized by the rareness of tumor cells. Although it represents one of the most frequent lymphomas in the Western world the biology of this highly variable disease is not completely understood. Matrix assisted laser desorption ionization (MALDI) imaging is a novel technology resulting in proteomic information with spatial distribution by utilizing tissue sections. Methods: Frozen sections of lymphatic tissue (32 cHL and 22 lymphadenitis) were mounted onto conductive glass slides (Bruker Daltonics, Bremen) and evenly spray coated with matrix solution. Each sample was overlaid with a virtual grid of measuring points. Mass spectra were obtained using a Reflex IV MALDI-TOF-MS (Bruker). Removing matrix after MS analysis allows for H&E staining and pathological survey. Spectra were analyzed using ClinProTools software 2.0 (Bruker). Results: Utilizing different statistical evaluation strategies lymphoma clusters within lymph nodes could be distinguished from lymphadenitis with an over all cross validation, sensitivity and specificity up to 86.65%, 83.92% and 89.37%, respectively. Conclusions: MALDI imaging correlates protein expression values with immediate histologic data. In contrast to immunohistochemistry unlimited proteins are scanned in parallel. Additionally, the identification of a disease specific protein pattern might facilitate the understanding of cHL and could lead to uncover new biomarkers.

Fr-067 Changes of immunophenotype in B-NHL after Rituximab treat-ment: Impact for diagnostic evaluation and follow-up C. Aumueller1, C. Meyer, Z. Bueschenfelde2, I. Koch1, M. Perker3, L. Quintanilla-Martinez4, F. Fend1, M. Kremer1 1 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München 4 Institut für Pathologie, GSF-Forschungszentrum Neuherberg 2 III Med. Klinik, Klinikum rechts der Isar der TU München 3 Hämatologische Praxis, Weilheim Aims: Rituximab, a chimeric anti-CD20 antibody has shown signifycant efficacy in patients with B-NHL. Loss or downregulation of CD20 expression during rituximab therapy has been observed and made responsible for secondary resistance. Methods: A total of 82 formalin-fixed, paraffin-embedded lymph node and bone marrow biopsies of 48 patients with well characterized B-NHL (28 FL, 37 MCL, 3 MZL, and 14 DLBCL) obtained before and after therapy were analyzed for expression of CD20 and other B-cell markers (CD79a, CD22), using immunohistochemistry, flow cytometry, gene rearrangement (IgH PCR), as well as mRNA (QRT-PCR) studies. The post-therapy samples were taken between 0–16 months after initation of rituximab therapy. Results: Twenty-three of 82 (28%) studied cases achieved complete remissions without immunophenotypical or molecular evidence of lymphoma in the post-therapy samples. Fifty-six cases showed persistent disease or developed relapse (54% FL, 84% MCL, 67% MZL, 79% DLBCL). Loss or significant decrease of CD20 staining was noticed in 25/56 (44%) cases at relapse (46% FL, 41% MCL, 50% MZL, 50% DLBCL), and in non-neoplastic B-cells in18/23 (78%) cases with remission (84% FL, 83% MCL, 100% MZL, 33% DLBCL). CD20 downregulation was clearly time-dependent, with the highest frequency of CD20 loss within the first three months of rituxi-mab therapy. CD79a and CD22 expression was not altered by Rituximab treatment. There was a high concordance between the results of immunohistochemistry, flow cytometry and molecular studies. Conclusions: Loss or decrease of CD20 expression in a time-dependent fashion is a common phenomenon in a significant number of B-NHL, possibly

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as “tumor escape mechanism”. Therefore, a panel of antibodies is required for the appropriate evaluation of rituximab-treated B-NHL.

Fr-068 Follicular lymphoma of the duodenum (FL-D): a distinct mucosal variant of FL A. Chott, M. Raderer, B. Streubel, G. Seitz3, M. Stolte4 Klinisches Institut für Pathologie und Innere Medizin I, Medizinische Universität Wien 3 Institut für Pathologie am Klinikum Bamberg 4 Institut für Pathologie, Klinikum Bayreuth Aims: Patients with FL usually present with widespread nodal disease. Stimulated by accumulating primary FLs diagnosed on duodenal biopsies, an Austrian-German collaboration was started and the findings on a large series are presented here. Methods: Endoscopic biopsies were studied histologically, immunohistologically, by PCR and FISH for clonality and BCL2 rearrangement; in 4 cases karyotyping was performed. The patients’ records were reviewed with special emphasis on endoscopic findings and staging. Results: 50 patients (M:F=1:1.5, med. age, 64y; range, 32–82y) with stage EI disease were studied. The vast majority presented with warty polyps around the papilla of Vater, had FL grade 1 involving mucosa ±submucosa, were bcl2+, CD10+, and carried the t(14;18). Karyotyping revealed t(14;18) as the sole aberration in 4/4 cases. The median follow-up was 43 months, ranging from 6–148 months. The 23 patients who were followed by watchful waiting showed stable local disease, radiotherapy in 13 patients resulted in complete remission. Except one patient who developed mesenteric lymph node involvement after 5 years, none of them disseminated or progressed to DLBCL. Calculation on the frequency of FL-D reavealed that it can be expected in roughly 1 of 4000 duodenal biopsies. Conclusions: Presumably at least in part because of a t(14;18) only karyotype, FL-D is a remarkably indolent disease restricted to mucosa/submucosa which usually does not transform or disseminate and can be managed by watchful waiting or radiation.

Fr-069 CD52 expression in peripheral T-cell lymphoma E. Geissinger1, I. Bonzheim1, S. Roth1, A. Rosenwald1, H.K. Müller-Hermelink1, T. Rüdiger2 1 Institut für Pathologie, Universitätsklinikum Würzburg 2 Institut für Pathologie, Karlsruhe Aims: Peripheral T-cell lymphomas (PTCL) are rare neoplasms in which the tumor cells can hardly be distinguished from reactive by-standers on morphological grounds. To improve the poor prognosis of these tumors, new therapeutic strategies are under investigation. One new agent is the anti-CD52 antibody (alemtuzumab). However, a detailed analysis of CD52 expression in the various PTCL, with an accurate discrimination between the expression in the tumor cells versus reactive bystander cells, does not exist. Methods: We investigated the expression of CD52 in tumor cells and in reactive T- and B-cells in 9 angioimmunoblastic T-cell lymphomas (AILT), 8 PTCL-not otherwise specified (NOS) and in 18 anaplastic large cell lymphomas (ALCL, 9 ALK-, 9 ALK+) using fluo-rescence double and triple stains (CD52 in combination with a spe-cific antibody directed against the T-cell receptor Vβ-segment re-arranged in the tumor cells as determined by PCR (AILT and PTCL-NOS) or in combination with CD30 (ALCL); CD52+CD79a+CD3). Results: The tumor cells in all AILT and PTCL-NOS showed con-sistent medium to strong expression of CD52, while the tumor cells in all ALCL, irrespective of the ALK status, did not reveal a specific membranous positivity. However, the background infiltrate in all PTCL showed a strong positivity for CD52 in up to 90% of both, reactive T- and B-cells. Conclusions: These results offer two potential mechanisms of action of the anti-CD52 antibody in PTCL, namely a direct effect against CD52-positive tumor cells or, alternatively, a disturbance of the microenvironment by depleting

CD52-positive, reactive B- and T-cells. Major prospective clinical trials will have to show whether this antibody has similar or divergent clinical efficacy between various PTCL entities, especially within CD52-negative ALK- ALCL.

Fr-070 Homozygous Deletion of p15 (CDKN2B) in classical Hodgkin lymphoma S. Hartmann1, J. Hüsken1, I. Martin-Subero2, R. Siebert2, R. Küppers3, A. Bräuninger1, M.-L. Hansmann1 1 Institut für Pathologie, Universitätsklinikum Frankfurt am Main 2 Institut für Humangenetik, Universitätsklinikum S-H, Campus Kiel 3 Institut für Zellbiologie, Universitätsklinikum Essen Aims: Homozygous deletions of p15 (CDKN2B), an inhibitor of CDK4 and CDK6 and therefore regulator of cell growth, have been widely described in Non-Hodgkin lymphomas. So far deletions of p15 have neither been reported in Hodgkin lymphoma cell lines nor in primary Hodgkin lymphomas. Methods: Seminested quantitative two round PCRs using the ΔΔCt method were performed on DNA isolated from microdissected Hodgkin-and-ReedSternberg cells and reactive lymphocytes from the same lymph node. The first round PCR was conducted as multiplex reaction with a control gene. Results: A strongly reduced copy number for p15 DNA in the Hodgkin and Reed-Sternberg cells compared to reactive lymphocytes in one case indicated a homozygous deletion. Conclusions: Therefore, deletions of p15 and consequent deregulation of CDK might be of importance for tumor growth of Hodgkin lymphoma.

Fr-071 Search for mutations in aberrantly expressed receptor tyrosine kinases in Hodgkin-Reed/Sternberg cells of Hodgkin lymphoma F. Lang, M.-L. Hansmann, A. Bräuninger, A. Mottok Institut für Pathologie, Universitätsklinikum Frankfurt am Main Aims: In Hodgkin-Reed/Sternberg (HRS) cells of classical Hodgkin lymphoma (HL) seven aberrantly expressed receptor tyrosine kinases (RTKs) (TIE1, TRKB, TRKA, EphB1, DDR2, RON and PDGFRα) can be detected by immunohistochemistry (IHC). IHC with antibodies specific for the activated forms of the RTKs demonstrated activation in several cases. RTKs play important roles in tumor pathogenesis and are often constitutively activated by mutations. We therefore analyzed if mutations could contribute to the activation of the aberrantly expressed RTKs in HRS cells. Methods: HRS cells were micromanipulated from frozen tissue sections using an ultraviolet laser beam (PALM microdissection system). Pools of 60– 100 HRS cells were digested by Proteinase K and aliquots representing about 25 cells were used for amplification of the exons encoding the transmembrane and intracellular parts of the RTKs. PCR products were directly sequenced by using an automated sequencing system. Sequences were compared with published germline sequences to identify mutations. Results: For each of the RTKs TRKB, EphB1 and TIE1 the exons encoding the transmembrane and intracellular parts of the RTKs (TRKB: 9 exons, EphB1:9 exons, TIE1:8 exons) of ten HL cases and four HRS cell lines (L1236, L428, HDLM2, KMH2) were sequenced. The sequences will be compared to the RTK germline serquences. Conclusions: The analysis of exons encoding the transmembrane and intracellular parts of ten cases aberrantly expressing TRKB, EphB1 and TIE1 will reveal if mutations contribute in a significant fraction of cases to the activation of the RTKs in HRS cells.

Fr-072 Gene Expression Proﬁling of Thymomas and Thymic Carcinomas B. Huang1, P.S. Tröbel2, S. Kneitz3, L. Li4, A. Marx2, H.K. Müller-Hermelink1 1 Pathologisches Institut, Universitätsklinikum Würzburg 3 Interdisziplinäres Zentrum für Klinische Forschung der Universität Würzburg 2 Pathologisches Institut und 4 Zentrum für Medizinische Forschung, Universitätsklinikum Mannheim Aims: The oncogenesis of thymomas and thymic carcinomas is partially understood and whether thymomas exhibit a medullary or cortical differentiation is controversial. The molecular classification based on gene expression profiles contribute to diagnosis and understand the pathomechanism. Methods: Using customer designed cDNA microarrays and the JMP ® Genomics statistical platform, we investigated 30 cases of the epithelial-rich WHO type A and B3 thymomas and thymic squamous cell carcinomas and compared the gene expression finding with chromosome aberrations and clinical stages. Results: Distinct gene expression profiles of type A, B3 and C thymoma subtypes based on a subset of 37 differentially expressed genes enables an accurate classification. There were different top genes with distinct functions and possible related pathways. Genes belonging to adhesion molecule pathway : ITGA6, ITM2B, NEO1 and NET1 were important for type A, while oncogenes such as MAF and DIAPH2 were overexpressed in B3 thymomas. Gene related with transcription: RFX5, MEOX1, ARNT2 and EVI1 played a major role in C. Another molecular feature is the substantial promiscuity among a medullary versus cortical histogenetic signature in various thymoma subtypes. The overexpression of genes located in 16q (NFATC3) in B3 thymomas than in thymic carcinoma correlated also with corresponding chromosomal aberrations. Conclusions: Gene expression profiles support the WHO histological classification of thymomas and thymic carcinomas. The histogenesis of thymomas may be explained in term of a new stem cell model of thymus epithelial cells.

Fr-073 Hemophagocytosis and Macrophage Activation as Modulators of Inﬂammation and Mediators of Septic Spleen M. Kurrer, C. Schaer1, G. Schoedon1, A. Imhof1, D. Schaer1 Pathologisches Institut, Kantonsspital Aarau 1 Medizinische Klinik, UniversitätsSpital Zürich Aims: The septic spleen is a pathognomonic sign of septicemia at autopsy. Others have shown that neutrophils are not increased in septic spleens. We aimed to analyse whether patients with sepsis would develop hemophagocytosis and macrophage activation that could represent an anti-inflammatory feed back loop in sepsis and could represent a mediator of septic spleen. Hemophagocytosis is a hallmark of macrophage activation syndromes but has not consistently been implicated in pathophysiolocical events in sepsis. Methods: Immunohistochemistry and immunofluorescence for CD68, CD163, heme oxigenase 1 (HO-1) and ferritin in macrophages and CD8, granzyme B and perforin in T cells in autopsy bone marrow, liver and spleen from 28 patients with fatal sepsis in comparison to 8 control patients. Further, EBV was detected by EBER in situ hybridization. Results: Bone marrows in fatal sepsis showed infiltration of CD163+/HO-1+/ Ferritin+ macrophages, increased CD8+/granzyme B+/perforin+ T cells and morphological change of macrophages from dendritic to polygonal. Within these macrophages mature and immature hematopoietic cells were detected, representing evidence of hemophagocytosis. There was no evidence of EBV reactivation. Spleen showed hemophagocytically active macrophages in sinusoidal distribution. Conclusions: Hemophagocytosis accompanies fatal sepsis, represents a likely cause of HO-1 upregulation in macrophages and subsequent production of anti-inflammatory metabolites of heme degradation, and may represent a significant negative immunomodulator in inflammation. Activated macrophages detected in the spleen of patients with fatal sepsis should represent a likely mediator of septic spleen.

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Abstracts Fr-074 Epidemiology of Malignant Lymphoma and Leukaemia: Implications for Classiﬁcations and Biological Entities M. Kurrer, D. Poncini1, V. Stucki1, S. Giger1, D. Korol2, R. Maurer3, N. ProbstHensch2, H. Moch1 Pathologisches Institut, Kantonsspital Aarau 1 Institut für Klinische Pathologie, UniversitätsSpital Zürich 2 Kantonalzürcher Krebsregister, Universität Zürich 3 Institut für Pathologie, Stadtspital Triemli, Zürich Aims: Epidemiological data (ED) on lymphoma and leukaemia (LL) have been valuable for clinical use. We intended to analyse and use ED to confirm and develop hypotheses on the biology of LL. Methods: Retrospective analysis of 11000 LL registered in the Cancer Registry of the Canton of Zurich from 1980 until 2004) – complemented for Hodgkin lymphoma (HL) and mediastinal large B cell lymphoma (MBCL) by a review of histological and clinical data of patients from the University Hospital Zurich and Stadtspital Triemli. Results: LL show an exponential increase with age (EIA), that is characteristic for B and T cell lymphoma (BCL and TCL) and hematopoietic stem cell diseases (HPSCD). LL show a male:female ratio (MF) of about 1.75. Distinct lymphomas differ: hairy cell leukaemia (HCL) (flat age curve (FAC), MF 4), HL (nodular sclerosis (NS) and nodular lymphocyte predominant (NLP) age peak at 25, mixed cellularity (MC) EIA, classical lymphocyte rich (cLR) FAC, overall MF1.2), MBCL (age peak at 33, MF 0.5), ALL/AML (decrease after birth until adolescence and later EIA), marginal zone lymphoma (MZL) (MF 0.8) and follicular lymphoma (FL) (EIA until age 65 followed by FAC, MF 0.8). Conclusions: Cancerogenic factors add up differently in B cells, T cell and HPSC. In HPSCD epidemiological curves confirm two clinical forms. NS/ MBCL/NLP, cLR and MC/most BCL appear epidemiologically homogeneous. Within BCL FL/MZL and HCL appear distinct. Outlook: ED confirm known entities and may guide hypothesis generation for biological relatedness of LL.

Fr-075 Chronic lymphocytic leucemia (B-CLL) is the most frequent haematologic malignancy simultaneously occurring with epithelial neoplasms (carcinomas) A. Metzger, P. Adam, H.K. Müller-Hermelink Institut für Pathologie, Universitätsklinikum Würzburg Aims: Incidentally, in patients presenting with a solid tumor disease (e.g. carcinoma) a simultaneous haematological neoplasm is found. This study aimed at the identification and systematical analysis of such patients with co-incidence of an epithelial and a haematological tumour. The latter were characterised regarding frequency, type of carcinoma and gender predominance. Methods: A database of all carcinomas and haematological diseases diagnosed at the Institute of Pathology at the University of Würzburg in a time period of 10 years (1996–2006) was created and analysed to find patients with both an epithelial tumour and a haematological disease. In the cases with chronic lymphocytic leukaemia (B-CLL) an additional immuno-histochemical staining was performed using an antibody against ZAP70 in order to elucidate the mutational status in these cases. Results: From 1996 to 2006 altogether 52099 cases of carcinoma were diagnosed. Of these, 239 patients suffered additionally from a haematological disease. The 3 most frequent hematological diseases associated with a carcinoma were chronic lymphocytic leukemia/B-CLL (45 cases), plasmozytoma and MPS (each 41 cases). In the immuno-staining for ZAP70, 26 cases were negative, and 18 positive. Conclusions: Chronic lymphocytic leucemia (B-CLL) is the most frequent haematologic malignancy simultaneously occurring with epithelial neoplasms (carcinomas). 60% of the cases showed an expression of ZAP70, suggesting a non-mutated sequence of the B-cell receptor genes. Only in 40% evidence for an antigene influence on the malignant lymphocytic clone was detected.

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Fr-076 Trend reversal in the frequency of invasive mycoses in hematological neoplasias: Autopsy results 1976–2005 K. Donhuijsen1, P. Petersen1, K.W. Schmid2 1 Institut für Pathologie, Städtisches Klinikum Braunschweig 2 Institut für Pathologie und Neuropathologie, Universitätsklinikum Essen Aims: Invasive mycoses are a leading complication for patients with hematological neoplasias; over two decades they have increased with the increasing aggressiveness of the therapeutic procedure. Is there a continuous increase despite intensified antimycotic prophylaxis? Methods: Retrospective in autopsy results of invasive mycoses over thirty years (1976–2005) from a single institution were analysed according to basic disease, frequency and kind of mycoses, involved organs, hematopoietic transplantation and cause of death. Results: 340 of 1591 postmortem examined patients (21.4%) revealed an invasive mycosis. Percentages increased from about 10% before 1980 to 30% in the nineties but decreased to 21% until 2005. The frequency of mycoses decreased significantly for transplanted (47.5% to 30.3%) as well as for nontransplanted patients (29.8% to 16.4%). The mycosis-related cause of death showed an overall decrease from 30% to 21%. Aspergilloses predominated increasingly, whereas candidoses diminished. Mycotic complications mostly concerned the lungs. Conclusions: The autopsy results signal a trend reversal in the leading complication of an aggressive therapy in hematological neoplasias. There is some reason to assume positive effects of antimycotic strategies.

Poster: Kardiopathologie Fr-077 Antioxidative treatment with Tempol alters plaque composition, but not plaque size in Apoliprotein E-deﬁcient mice with impaired renal function S. Huebner, V. Campean, N. Cordasic1, B. Klanke1, J. Jacobi1, K.F. Hilgers1, K. Amann Pathologisches Institut und 1 Klinik für Nephrologie und Hypertensiologie, Universitätsklinikum Erlangen Aims: Oxidative stress may contribute to the accelerated atherosclerosis in chronic renal failure (CRF). The aim of our study was to investigate the effect of antioxidant treatment with Tempol on inflammation and athero-sclerotic plaque formation. Methods: ApoE-deficient mice (apoE-/-, n=73, both sexes, age 3 months) were either subjected to sham operation (sham), uninephrectomy (UNX) to induce mild renal dysfunction, or subtotal nephrectomy (SNX) to induce CRF. Each group was further divided into 2 subgroups: one half was trea-ted with Tempol (1 g/L drinking water, n=10–13 ) over a period of 4 months, the other half was left untreated (n=10–14). Blood pressure was recorded via carotid catheters. Atherosclerotic plaques at the proximal aorta were analysed using morphometry and immunohistochemistry. Protein expres-sion was evaluated using a semiquantitative score. Results: Blood pressure and serum parameters were not significantly affected by Tempol treatment. Markers of inflammation (ICAM, VCAM, CD40, CD154, CRP) and oxidative stress (nitrotyrosine) were higher after UNX and particularly after SNX and markedly lowered by Tempol treat-ment. Morphometric analyses showed increased aortic plaque size UNX- and SNX-ApoE-/compared to sham. Tempol markedly altered plaque composition and tended to decrease plaque size and volume in all groups. Of note, in all experimental groups female mice had significant higher plaque areas than males. Conclusions: Tempol treatment showed marked anti-inflammatory effect in the Apo E-/- mouse with mild renal dysfunction and CRF. Moreover, it changed the composition of the aortic plaques whereas it had at most a mild effect on aortic plaque size. To treat the aggravated atherosclerosis of chronic

renal failure, antioxidative strategies may exert beneficial effects on plaque vulnerability, but not on plaque size.

Fr-078 Analysis of B- and T-cell inﬁltrates in kidney biopsies from pediatric patients with idiopathic nephrotic syndrome K. Benz1, K. Dittrich, V. Campean1, J. Dötsch, K. Amann1 Kinderklinik und 1 Pathologisches Institut, Universitätsklinikum Erlangen Aims: There is increasing evidence that in addition to T-cells also B-cells may play an important role in the pathogenesis of idiopathic nephrotic syndrome (NS). This is confirmed by the successful use of the anti-CD20 antibody Rituximab as a rescue therapy in NS. There is, however, no quantitative data on the number and distribution of infiltrating B- and T-cells in kidney biopsies from patients with NS. Methods: Kidney biopsies from 25 paediatric patients (11f, 14m) were analysed using immunohistochemistry with antibodies against CD3, CD4, CD8 and CD20 and quantitative morphology. Kidney biopsies with normal renal morphology and thin basement disease as underlying diagnosis were used as controls. Results: Overall, in the kidney biopsies the number of infiltrating T-cells (CD3+, CD4+, CD8+) was 5–10 fold higher than the number of infiltrating B-cells. In patients with FSGS more CD3+ interstitial and glomerular cells were seen than in patients with minimal change GN. There were, however, no differences with respect to CD4+, CD8+ or CD20+ interstitial or glomerular cells. When patients were divided according to their clinical response in 3 subgroups, i.e. 1. frequent relapsing NS, 2. steroid dependent NS and 3. steroid resistant NS, the number of interstitial CD3+ and CD8+ cells was significantly higher in frequent relapsing NS than in steroid dependent NS. Conclusions: In kidney biopsies infiltrating B-cells are less frequent than Tcells and patients with frequent relapsing NS had the highest cell number. In contrast to differences in the number of CD3+ and CD8+ infiltrating cells, no differences could be found with respect to CD4+ and CD20+ cells. In view of the latter finding it is very unlikely that the number of infiltrating B-cells in kidney biopsies may be used to predict the clinical response to the anti-CD20 antibody Rituximab.

Fr-079 Genetically determined low nephron number is not associated with alterations in postglomerular structure K. Benz, V. Campean1, N. Cordasic2, B. Klanke2, A. Hartner, K.F. Hilgers, K. Amann1 Kinderklinik, 1 Pathologisches Institut und 2 Medizinische Klinik IV, Universitätsklinikum Erlangen Aims: An association between low nephron number and subse-quent development of hypertension in later life has been demon-strated. The underlying pathomechanisms, however, are yet un-known. Apart from altered salt handling and activation of the RAAS structural changes of postglomerular structures are discussed. GDNF (Glial cell line derived neurotrophic growth factor) heterozy-gous mice (GDNF+/–) show low nephron number at birth and are mildly hypertensive at 14 months of age (+18 mmHg). We used this animal model for a detailed investigation of postglomerular struc-tural and functional changes. Methods: In 22 mices (10 GDNF+/–, 12 C57B6 wildtype controls) at the age of 26 weeks blood pressure (bp) measurements were per-formed and blood samples taken. After perfusion fixation, kidneys were investigated using immunohistochemistry (for markers of nephron segments and tubular transporter systems) and quantita-tive morphology. Results: There was no sig. difference in bp, serum and urinary parameters between GDNF+/– and controls. GDNF+/– mice had 30% lower nephron number per kidney (12400 vs. 8900, p<0.05) and increased glomerular volume (+48%). Renal damage scores, glomerular and tubular proliferation, number and length

of intrarenal arteries and capillaries as well as expression and localisation of tubular transporters were not sig. different between GDNF+/– and controls. Conclusions: Lower nephron number is not associated with post-glomerular changes of renal structure or tubular function which may predispose to hypertension. Whether other pathomechanisms like altered salt handling are involved is currently under investigation.

Fr-080 Eﬀect of an Erythropoesis-stimulating agent on renal inﬂammation in Apolipoprotein E-deﬁcient mice with impaired renal function B. Karpe, V. Câmpean, N. Cordasic1, B. Klanke1, K. Hilgers1, K. Amann Pathologisches Institut und 1 Klinik für Nephrologie und Hypertensiologie, Universitätsklinikum Erlangen Aims: The effect of anemia on progression of renal disease is controversial. Previous studies suggest that Erythropoesis-stimulating agents (ESA) may ameliorate renal dysfunction and injury in animal models. We evaluated markers of oxidative stress and inflammation in the kidney of the ApoE-/mouse model with impaired renal function. Methods: Sham operated (SHAM) uninephrectomized (UNX) and 5/6 nephrectomized (SNX) 3 months old male ApoE-/- mice were randomly assigned to saline (n=29) or treatment (n=23) with the ESA Darbepoetin alpha 0,33 μg (DPO) once per week for 4 months. In addition, 10fold dose was given to six further SNX mice. Morphometric analysis was performed on PAS, Sirius and HE stained sections, semiquantitativ scoring on immunohistochemical slides and QPCR on isolated cDNA. Results: Hematocrit was lower in UNX (51.1%±3.6) and SNX (44.8%±2.1) than in SHAM (56.6%±5.5), and was significantly increased in SNX by treatment (ESA: 59.6%±7.8, high-dose ESA: 64%±8,74). ESA tended to increase glomerulosclerosis in SHAM and UNX as well as tubular damage in SNX. Formation of Nitrotyrosine, expression of CD 40 and activation of Caspase-3 in SHAM and UNX, but not SNX was ameliorated by ESA. Conclusions: UNX and SNX led to increased renal expression of markers of inflammation and oxidative stress. ESA treatment showed a trend towards anti-inflammatory and anti-oxidative effects in early stages of chronic renal failure. In advanced stages of renal dysfunction high-dose ESA even worsened renal injury.

Fr-081 Vascular thickening, calciﬁcation and inﬂammation in patients with early and late stages of renal failure I. Varga, D. Neureiter1, V. Campean, A. Reimann2, M. Weyand2, K.F. Hilgers3, K. Amann Pathologisches Institut, Universitätsklinikum Erlangen 1 Pathologisches Institut, Salzburger Landeskliniken 2 Klinik für Herz-Chirurgie und 3 Med. Klinik 4, Universitätsklinikum Erlangen Aims: Death from cardiovascular causes is the major cause of death in patients with endstage renal failure. While patients with renal failure have a high prevalence of classical coronary risk factors, there is increasing evidence that atherosclerosis, i.e. the evolution and composition of plaques is different in renal compared to non-renal patients. Currently, there is a lack of data concerning the morphological characteristics of vascular structural changes and atherosclerosis in patients with different stages of renal failure. Therefore, the present study compares changes in different vessels obtained at cardiac surgery between patients with early and late stages of renal dysfunction and non-renal control patients. Methods: 50 patients undergoing cardiac bypass surgery were devided into 3 groups:1. patients with S-creatinine <1.3 mg/dl (control group, n=24), 2. patients with moderately elevated S-creatinine (1.3–2.0 mg/dl, early CRF, n=14), 3. patients with established renal failure (S-creatinine >2.0 mg/dl or dialysis treatment, CRF, n=12). A. mammaria int., aorta, small subcutaneous arterioles and V. saphena were analysed using quan-titative morphometry and Der Pathologe · Supplement 1 · 2008

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Abstracts immunohistochemistry for markers of inflamma-tion (CRP, CD40, CD154, iNOS, eNOS, ICAM, VCAM). Then, univariate analysis and correlation analyses were performed. Results: Media thickness and inflammation score of the A. mammaria int. and aorta was sig. higher in CRF patients than in controls. In contrast, significant inflammation of all vessels and calcification of the aortic media was already present in early CRF. Calcification of the aortic intima and of V. saphena was significantly more pronounced in CRF patients than in and controls. Of note, CaxP product correlated well with markers of inflammation, but not with calcification itself. Conclusions: Early stages of CRF are associated with local upregulation of proinflammatory molecules in the vascular wall and calcification of the aortic media. These findings point to the importance of systemic and local microinflammation in CRF and may shed new light on the possibly overestimated role of the CaxP product for vessel calcification.

Fr-082 Roles of Survivin and Cyclin D1 in cardiac hypertrophy and “reverse remodelling” after ventricular unloading J. Wohlschlaeger, J. Schmitz K, K.W. Schmid, C. Vahlhaus1, C. Schmid2, H.A. Baba Institut für Pathologie und Neuropathologie, Universitätsklinikum Essen 1 Klinik und Poliklinik für Kardiologie und 2 Klinik und Poliklinik für Thorax- und Herzchirurgie, Universitätsklinikum Münster Aims: In chronic heart failure (CHF), D-type cyclins are associated with cardiac hypertrophy. Survivin is highly expressed in proliferating tissues and was recently demonstrated to initiate cell cycle progression by enhancing the formation of CyclinD1/cdk4 complexes by inhibiting p16INK4a. Aim of the study was to investigate the roles of Survivin and Cyclin D1 in cardiac hypertrophy and “reverse remodelling” after mechanical support by LVAD. Methods: In 20 paired myocardial samples (before and after LVAD), the cardiomyocyte diameters and the protein expression of Survivin, Cyclin D1 and PCNA were immunohistochemically investigated and morphometrically quantified. Results: Both Survivin and Cyclin D1 expression correlate significantly with cardiomyocyte diameters in CHF, whereas PCNA expression does not. Hemodynamic unloading is associated with a significant decrease of Survivin, CyclinD1 and PCNA. A significant positive correlation between Survivin and Cyclin D1 expression was noted prior to, but not after unloading. Conclusions: Both Survivin and Cyclin D1 expression are associated with cardiac hypertrophy in human CHF. Given the positive correlation between the two molecules, a Survivin-enhanced cardiomyocyte progression to the S phase of the cell cycle via increased CyclinD1/cdk4 complexes associated with aggravated hypertrophic growth during CHF is suggested.

Fr-083 Blood pressure-independent eﬀects of salt on vascular structure – fetal programming or adult remodeling? G. Piecha1,2, N. Koleganova1,2, A. Geldyyev1, L.E. Becker1, A. Müller1, E. Ritz2, M.-L. Gross1 1 Pathologisches Institut und 2 Medizinische Klinik, Universitätsklinikum Heidelberg High salt intake may lead to hypertension and adverse cardiovascular outcomes. Some salt mediated forms of target organ damage are blood pressure-independent.. There are studies suggesting that high salt intakes during pregnancy influence blood pressure in the offspring. It was the pupose of the present study to clarify whether high salt intake in pregnant rats will also alter vascular morphology in the offspring. Sprague-Dawley rats were fed low (0.15%), normal (1.3%), or high (8.0%) salt diet during pregnancy and weaning. The offspring were separated from mothers at 4 weeks of age and maintained on the same or switched to low or high salt diet. Systolic blood pressure was controlled by tail plethysmogra-

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phy and intraaortal measurement. Vascular geometry was assessed at 7 and 12 weeks postnatally. No significant differences in blood pressure were observed between the groups. At 12 weeks the aortic wall thickness was significantly higher in female offspring continuously on high salt (99.0±12.3 μm), switched form high to low salt (102.7±14.2), and switched from low to high salt (103.3±16.9) compared to those on low (89.4±16.3) and medium (90.3±11.3) salt diet. The same relationship was observed in male offspring and in the carotid arteries and in the wall to lumen ratio in carotid arteries and aorta of both males and females. There was no significant difference, however, in vascular geometry at 7 weeks postnatally. High salt intake in pregnant rats increases aortic wall thickness in the offspring without elevating blood pressure and this persisted even if the offspring was maintained on low salt diet after delivery.

Fr-084 Histologic and Histomorphometric Examinations of the Wall of the Ascending Aorta in Patients with Bicuspid Aortic Valves U. Wolf1, J. Knörig2, R. Hetzer1, R. Meyer1 1 Deutsches Herzzentrum Berlin 2 SANA-Herzzentrum Cottbus Aims: In patients with bicuspid aortic valves there is an accumulation of aortic valve disease (stenosis, insufficiency) beside dilatation of the ascending aorta. Based on histologic and histomorphometric examinations the aim of this study was to analyze whether the localized aortic dilatation is an expression of a structural aberration or consequence of a haemodynamically adaptation. Methods: With an image analysis system we messured the content of fibrosis, the thickness of the elastic lamelllae and the the distances between the elastic lamellae as well in 34 patients with bicuspid aotic valves and existing or lack of dilatation. 100 surgical patients with tricuspid aortic valves served as a control. Results: Patients with bicuspid aortic valves had a lower content of fibrosis (13,51+/–6,32%) as patients with tricuspid aortic valves (16,62+/–7,53%). The elastic lamellae are faint thinner in patients with bicuspid aortic valves (2,85+/–0,26 μm) as in such cases with tricuspid aortic valves (2,94+/– 0,24 μm) and the distances between the elastic lamellae is similar in both groups (BAV 22,57+/–6,87 μm vs. TAV 22,02+/–6,40 μm). Conclusions: The wall of the ascending aorta in patients with bicuspid aortic valves has in comparison with patients with tricuspid aortic valves a lower content of fibsosis and minor thinner elastic lamellae. The distances of the elastic lamellae are not importantly different. Serious structurelly varieties between the both examination groups could not be detected.

Fr-085 Ultrastructure of subendocardial Purkinje ﬁbres (sPF) with contraction bands in ischemia-reperfusion injury depends on the kind of myocardial protection and on the temperature P. Schnabel1, A. Schmiedl2, J. Richter3 1 Pathologisches Institut, Universitätsklinikum Heidelberg 2 Institut für funktionale und angewandte Anatomie, Medizinische Hochschule Hannover 3 Abt. Elektronenmikroskopie, Zentrum Anatomie, Universitätsklinikum Göttingen Aims: Experimentally, contraction banding (CB) in sPF due to global ischemia/ reperfusion appeared to depend on the temperature and on the kind of cardiac arrest. Therefore we compared the fine structure of sPF under different conditions. Methods: 82 canine hearts were arrested by coronary perfusion with St. Thomas’ hospital (STH) or HTK solution (Custodiol®) or by topic cooling with Tutofusin® (4°C) and aortic cross clamping (pure ischemia: PI). Perfusion or immersion fixation was performed with cacodylate-buffered aldehyde immediately following cardiac arrest, after ischemia at 5 or 25°C, and after

21 min reperfusion with a modified Tyrode’s solution (35°C). Transmission electron microscopy, and electron spectroscopic imaging were applied. Results: CB occurred in sPF during ischemia/ reperfusion during PI and after STH, but not after HTK cardioplegia. At 5°C, CB was associated with dark unswollen mitochondria revealing normal matrix granules, additionally annular granular or fine granular inclusions. At 25°C severe mitochondrial swelling, loss of normal matrix granules and of matrix structure, fragmentation and partial loss of cristae, additionally amorphous matrix densities were seen. Conclusions: CB occurs in presence of oxygen and calcium in PI or after STH, not after HTK cardioplegia (intracellular calcium concentration). CB during ischemia at 5°C is associated with high cellular ATP concentrations and calcium overload, at 25°C with low ATP concentrations a residual mitochondrial energy production.

Fr-086 Eﬀects of chronic and acute ischemia on the ultrastructure of subendocardial Purkinje ﬁbres (sPF) and immediately adjacent working myocardium (sWM) in explanted human hearts with ischemic (ICM) or dilative cardiomyopathy (DCM) P. Schnabel 1, A. Haag1, E. Herpel1, A. Warth1, A. Koch2, F.-U. Sack2, T.J. Dengler3, H.A. Katus3, M. Karck2, P. Schirmacher1 1 Pathologisches Institut, 2 Abt. Herzchirurgie und 3 Abt. Kardiologie und Pulmonologie, Universitätsklinikum Heidelberg Aims: Clinically, life-threatening arrhythmias occur in ICM as well as in DCM. Experimentally, a significantly (sig.) higher susceptibility of sPF to global ischemia compared to sWM has been shown by ultrastructural stereology. Therefore we compared the fine structure of sPF and sWM in explanted hearts with ICM and DCM. Patients and Methods: Patient age (mean±SD; n=5) was 49.4±9.5 years in ICM, and 40.5±18.5 in DCM. Immediately after explantation hearts were incubated in HTK solution (Custodiol®, 4°C) for 30 min at 20°C. Perfusion fixation with buffered glutaraldehyde was performed after perfusion with Rheo-Makrodex plus Procain-Hydro-chloride. Stereologically, the point and intersection counting method (Weibel) was used for mitochondrial swelling (surface-to-volume ratio), and for cellular swelling (quotient of volume densities). Results: The amount of sPF was sig. reduced in ICM compared to DCM, whereas the degree of subendocardial fibrosis did not differ. After 30 min of acute global ischemia mitochondrial swelling in sPF and sWM was sig. higher in DCM than in ICM. Cellular swelling of sPF and sWM showed a tendency to higher values in DCM. Conclusions: In explanted ICM hearts chronic ischemia causes a sig. loss of sPF compared to DCM. However, acute global ischemia leads to sig. reduced mitochondrial swelling and to a trend of less cell edema in the remaining sPF in ICM compared to DCM. This can be explained by preconditioning to ischemia in ICM.

Fr-087 Lymphatic precollectors contain a novel, specialized subpopulation of podoplaninlow, CCL27 secreting lymphatic endothelial cells D. Haluza, N. Wick, D. Kerjaschki Klinisches Institut für Pathologie, Medizinische Universität Wien Aims: The precise localization of lymphatic vessels in tissues and their potential mechanistic role in inflammatory processes was made possible by the availability of novel molecular markers, such as the membrane mucoprotein podoplanin. We have discovered two distinct subpopulations of lymphatic endothelial cells (LEC) in human skin that were defined by their distinct cell surface density of podoplanin, LECpodo-low and LECpodo-high. Methods: LECs were isolated from human skin and purified by FACS on the basis of their expression of podoplanin. DNA chip screens of ex vivo preparations revealed selectively expressed marker proteins that provided a basis for the analysis of LECpodo-low characteristic phenotype and functions in vitro and

in vivo using immunofluorescence staining, Western Blotting and quantitative PCR. Results: LECpodo-low in tissues and in cell culture selectively express pro-inflammatory factors, including CCL27, and are restricted to lymphatic precollectors that originate from initial LECpodo-high lymphatic capillaries. In human inflammatory skin diseases the pathogenic CCR10+ T lymphocytes accumulate preferentially around and within CCL27+ LECpodo-low vessels. In transmigration assays, isolated CCR10+ T cells are chemotactically attracted by LECpodo-low, in a CCL27 dependent fashion, but not by LECpodo-high. Conclusions: These observations raise the possibility that LECpodo-low containing precollectors constitute a specialized segment of the initial lymphatic microvasculature that is involved in trafficking of CCR10+ T lymphocytes in skin inflammation.

Fr-088 Nestin in mature endothelial cells: not an exclusive marker of proliferation C. Brochhausen, C.B. Wiedenroth, S. Halstenberg, C.J. Kirkpatrick Institut für Pathologie, Universitätsklinikum Mainz Aims: The intermediate filament nestin is expressed by endothelial cells (ECs) of regenerative tissues and malignant tumors. It is widely believed that nestin is a marker for proliferative endothelium only. In the present study we analyzed the expression of nestin in the cardiovascular tree in situ and in cultured human ECs of different sources. Methods: Human aorta (n=10), A. and V. renalis (n=10), liver (n=10), V. cava (n=10), lung (n=10), endocardium (n=10), hemangiomata (n=10), and lymphangiomata (n=10) were analyzed immunohistochemically using the peroxidase method. Monoclonal antibodies against nestin, CD31, D2–40, and Ki67 were used. For in vitro analyses, we cultured human umbilical vein ECs (HUVEC), human pulmonary microvascular ECs (HPMECs), an immortalized HPMECs cell line (HPMECs-ST1), and a hemangiosarcoma cell line (ISO-HAS). The cells were immunostained for the expression of CD31, actin and nestin in subconfluent and confluent cultures. Results: In all analyzed blood vessels, the majority of the endothelial cells reacted positively with antibodies against nestin. positivity for CD31 and negativity for D2–40 served as controles. Ki67 was only found in a few endothelial cells. The endothelium of lymphatic vessels reacted negative for nestin and positive for D2–40 and CD31. Immunoreactivity for nestin was found in all types of cultured ECs, both in confluent and in subconfluent populations, with marked differences in the intensity between the various sources. Actin was found in all cultured ECs. Conclusions: Nestin is not an exclusive marker for proliferative endothelium. However, nestin discriminates blood vessels from lymphatic endothelium and can therefore serve to discriminate endothelium from lymphatic and blood vessel origin.

Fr-089 Microvasculopathy does not aﬀect the favorable outcome in female heart transplant recipients N.E. Hiemann, C. Knosalla, E. Wellnhofer1, H.B. Lehmkuhl, R. Hetzer, R. Meyer Klinik für Herz, Thorax- und Gefäßchirurgie und 1 Klinik für Innere Medizin – Kardiologie, Deutsches Herzzentrum Berlin Aims: Women show a more favorable long-term outcome after heart transplantation (HTx) than men, and we tested whether stenotic microvasculopathy (MVP) affects the more beneficial course in female recipients. Methods: We studied 873 consecutive patients (35/151 pre-menopausal women [aged <40years]; median follow-up 12.4 (95% CI 11.4–13.5) years) who received a primary heart transplant between 1986 and 2002. In 7,750 biopsies harvested within the first post-transplant year endothelial disease and stenotic MVP were evaluated by light microscopy (H&E). Kaplan-Meier and Cox regression analyses were performed for major cardiac events (lethal myocardial infarction, sudden cardiac death, graft failure, cardiac re-transplantation). Results: Endothelial disease was more frequent in pre-menopausal women (72% vs. men 56%; p=0.025), while stenotic MVP was found equally in men Der Pathologe · Supplement 1 · 2008

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Abstracts (38%) and women (pre-menopausal 41%, post-menopausal 32%). Allografts from pre-menopausal female-to-male transplants more frequently developed endothelial disease (78% vs. 65%; p=0.021) and stenotic MVP (46% vs. 28%, p=0.024). Beyond the first 5 post-transplant years women presented less often major cardiac events as compared to men, independently of donor gender and stenotic microvasculopathy (p=0.0001). Multivariate regression analysis found women at lower risk for major cardiac events (RR 0.38; 95%CI 0.17– 0.81), while stenotic MVP (RR 2.15; 95%CI 1.42–3.26) and treated diabetes (RR 1.65; 95%CI 1.08–2.52) represented a higher risk for major cardiac events. Conclusions: Stenotic MVP has prognostic impact on the survival of male and female cardiac recipients; however, it does not affect the more beneficial course of women in the long-term follow-up.

Fr-090 A protective role of Cxcr4 unveils the importance of neutrophils in atherosclerosis A. Zernecke1,3, Y.D. Talab1, I. Bot2, J. Bornemann3, E. Shagdarsuren1, E.A. Biessen2, C.Weber1, R. Knüchel3, B. Hermanns-Sachweh3 1 Institut für Kardiovaskuläre Molekularbiologie, Universitätsklinikum Aachen 2 Division of Biopharmaceutics, Gorlaeus Laboratories, Leiden University, NL 3 Institut für Pathologie, Universitätsklinikum Aachen Aims: The CXCL12/CXCR4 chemokine-receptor axis controls haematopoiesis, organ development, and angiogenesis but its role in the inflammatory pathogenesis of atherosclerosis is unknown. Methods: Apolipoprotein E-deficient (Apoe-/-) mice received osmotic pumps for continuous treatment with AMD3465 (AMD) or PBS or were reconstituted with Cxcr4-/- or WT BM. The extent of atherosclerosis was assessed in aortic roots and aortas by staining with oil-red-O. Immunohistochemistry, -fluoresence/ transmission electron microscopy was performed on aortic roots. Results: Interference with Cxcl12/Cxcr4 by the small-molecule antagonist AMD or by genetic Cxcr4 deficiency aggravated diet-induced atherosclerosis in Apoe-/- mice. Chronic blockade of Cxcr4 caused leukocytosis and an expansion of neutrophils, and increased neutrophil content in plaques, associated with apoptosis and a pro-inflammatory phenotype. Whereas circulating neutrophils were recruited to atherosclerotic lesions, depletion of neutrophils prevented its exacerbation after blocking Cxcr4. Conclusions: Disrupting Cxcl12/Cxcr4 thus promotes lesion formation through deranged neutrophil homeostasis, indicating that Cxcl12/Cxcr4 controls the important contribution of neutrophils to atherogenesis in mice.

Fr-091 C1-esterase inhibitor protects against neointima formation after arterial injury in atherosclerosis prone mice A. Zernecke1,3, E. Shagdarsuren1, K. Bidzhekov1, E.A. Liehn1, W.A. Buurman2, C.Weber1, R. Knüchel-Clarke3 1 Institut für Kardiovaskuläre Molekularbiologie, Universitätsklinikum Aachen 2 Department of General Surgery, Maastricht University, NL 3 Institut für Pathologie, Universitätsklinikum Aachen Aims: While an activation of the complement system has been implicated in the progression of human atherosclerosis, its function during arterial remodeling after injury has not been investigated. Here we examined the contribution of the complement cascade to neointima formation in apolipoprotein E-deficient (Apoe-/-) mice. Methods: Apoe-/- mice fed an atherogenic diet were subjected to wire-induced endothelial denudation of the carotid artery and treated with a C1esterase inhibitor (C1-Inh) or vehicle perioperatively and subsequently every 2 days. Mice were sacrificed after 3 weeks. Results: A significant reduction in serum triglyceride levels was observed in C1-Inh-treated mice, while cholesterol levels did not differ. Neointimal area was significantly reduced in C1-Inh-treated mice versus controls. This was associated with a significant decrease in neointimal macrophage and CD3+

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T cell content. The peak in serum C3 levels after injury was markedly downregulated by C1-Inh, as evident by ELISA. Immunohistochemistry revealed strong expression of C3 and C3c, which colocalized to plaque macrophages and was reduced in C1-Inh-treated mice. C1-Inh impaired leukocyte recruitment to carotid arteries after injury in vivo. Conclusions: C1-Inh limits neointimal plaque formation and inflammation. This may involve the blockade of complement activation, the inhibition of leukocyte recruitment and reduced triglyceride levels, thus providing a multimodal approach to treat arterial disease.

Fr-092 Analysis of cardiovascular disease as cause of death in autopsy reports of the Charité, 1900–1999 C. Proch1, R. Meyer1, T. Schnalke2, M. Dietel3, R. Hetzer1 1 Klinik für Herz-, Thorax- und Gefäßchirurgie, Deutsches Herzzentrum Berlin 2 Berliner Medizinhistorisches Museum der Charité – Universitätsmedizin Berlin 3 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte Aims: We investigated demographic changes in the nature of cardiovascular disease as the cause of death in the whole of the 20th century, using the autopsy reports of the Institute of Pathology, Rudolf Virchow House, Charité. Methods: 60,446 autopsy reports prepared between 1 January 1900 and 31 December 1999 (approx. 96% of all autopsies) were evaluated. The causes of death were classified in accordance with the ninth revision of the ICD and the encoded data were transferred to an SPSS file (Version 14) for statistical evaluation. Results: Analysis showed that cardiovascular diseases were among the main causes of death (alongside neoplasia) during the whole century (21.6%). At the beginning of the century infections and parasitic diseases were the main cause of death (25%) but after 1950 these were less important. The percentage of deaths caused by neoplasia did not change during the course of the century, whereas the proportion of cardiovascular diseases increased from 14.8% to 40.3%. At the beginning of the century inflammatory heart disease (31.8%) and arterial disease (38.8%) were predominant; at the end, the ischemic heart diseases (41.1%) were in first place. Conclusions: Cardiovascular diseases head the list of the main causes of death, with the percentage increasing throughout the past century. However, ischemic heart disease increased while the inflammatory heart diseases decreased.

Poster: Endokrine Pathologie Fr-093 Metachronous appearance of Oncocytoma and oncocytic adenomatous (nodular) hyperplasia in the submandibular glands of a 77 year old female patient P. Knöß1, M. Knöß1, V. Krenn1, P. Schwerdtfeger2, M. Otto1 1 Zentrum für Histologie, Zytologie und Molekulare Diagnostik, Trier 2 Abteilung HNO, Mutterhaus der Borromäerinnen, Trier Aims: We report the case of a female patient presenting with metachronous appearance of oncocytoma and adenomatous (nodular) oncocytic hyperplasia in the submandibular glands. Methods: Case report and review of literature. Results: The patient presented with a painless swelling of the left submandibular gland. She had a history of goiter with multifocal autonomy, parotideal cystadenoma and oncocytoma of the right submandibular gland. There were echoless poorly circumscribed nodes in sonography. Histologically the specimen showed multiple macroscopically brownish coloured nodes of oncocytic cells measuring up to 1 cm without evidence of necrosis or perineural invasion indicating a benign process. Salivary gland tissue was present within the nodes. The oncocytic cells were positive for Pancytokeratin and negative for S100, p53 and Actin.

Conclusions: Oncocytic lesions rarely affect the submandibular gland, accounting for less than 1% of all salivary lesions. The WHO classification describes diffuse oncocytosis, focal adenomatous (nodular) oncocytic hyperplasia and oncocytoma. As to our concern meta-chronous appearance of oncocytoma and multifocal adenomatous (nodular) oncocytosis has not been described in literature yet. A definite differential diagnosis of oncocytic lesions of the salivary glands requires both clinical and histopathological findings.

Fr-094 Desmoplasia in medullary thyroid carcinoma: a reliable indicator of metastatic potential O. Koperek, C. Scheuba1, M. Cherenko2, N. Neuhold3, C. De Micco2, K.W. Schmid4, B. Niederle1 (BN), K. Kaserer Klinisches Institut für Pathologie und 1 Klinik für Chirurgie, Medizinische Universität Wien 2 Laboratory of Pathology, Marseille, F 3 Institut für Pathologie und Bakteriologie, Kaiserin Elisabeth Spital, Wien 4 Institut für Pathologie und Neuropathologie, Universitätsklinikum Essen Aims: To evaluate the reliability of desmoplasia as a reproducible morphologic parameter indicating the metastatic potential of medullary thyroid carcinoma (MTC). Methods: 120 cases of MTC of the Medical University of Vienna, Austria, and 76 cases of the School of Medicine of Marseille, France, were analyzed for the presence of desmoplastic stroma reaction by 4 endocrine pathologists. Intra- and interobserver concordance were assessed The Austrian cases were additionally analyzed for various morphologic parameters. Results: Intra- and interobserver concordance were highly significant with a kappa value of 0,883 for intraobserver reliability and 0,837; 0,79; 0,758 respectively when pathologists NN, CDM, and KWS reviewed the Austrian cases. The cases from France were reviewed by CDM and KK with a kappa value of 0,759. None of the cases that were categorized as desmoplasia negative by any of the investigators showed lymph node metastasis. No other distinct morphologic characteristics could be assigned to the MTCs without desmoplasia. Conclusions: Our data indicate desmoplasia as a reliable and highly reproducible parameter with regard to lymph node metastatic potential.

Fr-095 Diﬀerent regulation of microRNA in hyalinizing trabecular tumour and papillary thyroid carcinoma S.Y. Sheu, E. Vogel, K. Worm, S. Schwertheim, F. Grabellus, K.W Schmid Institut für Pathologie und Neuropathologie, Universitätsklinikum Essen Aims: There is much controversy about hyalinizing trabecular tumours (HTT) regarding their biological behaviour. Because of morphological (nuclear features) and pathogenetic (RET/PTC rearrangements) similarities with papillary thyroid carcinomas (PTC) they are considered to be a variant of PTC. Using a set of microRNAs typically upregulated in PTC we compared the microRNA expression profile of PTC with those in HTT. Methods: Paraffin embedded tissue specimen of 20 patients with HTT were investigated for the expression of a set of microRNAs (miR-146b, -181b, -21, -222 and 221) using real-time quantitative stem-loop RT-PCR. Tissues of 10 normal thyroids and 10 PTC served as control. Results: miRNA expression profiles of PTC and HTT are completely different. PTC show an upregulation of all investigated types of microRNAs whereas HTT and normal tissues disclose a similar regulation profile. Conclusions: Although there are similarities between HTT and PTC in histopathology and some molecular pathologic features the different regulation of the investigated microRNAs is against the assumption of HTT being a variant of PTC probably favouring its classification as a benign tumour.

Fr-096 Primary lymph node gastrinoma or occult duodenal microgastrinoma with lymph node metastases in a MEN1 patient: the need for a systematic search for the primary tumor T. Henopp1, M. Anlauf1, T. Enosawa1,2, A. Schmitt3, O. Gimm4,5, M. Brauckhoﬀ4, H. Dralle4, A. Musil6, S. Hauptmann6, A. Perren3,7, G. Klöppel1 1 Institut für Pathologie, Universitätklinikum S-H, Campus Kiel 2 First Department of Pathology, Showa University, J 3 Institut für Pathologie, UniversitätsSpital Zürich 4 Klinik für Allgemein-, Viszeral- und Gefäßchirurgie, Universitätsklinikum Halle 5 Department of Surgery, University of Linköping, S 6 Institut für Pathologie, Universitätsklinikum Halle 7 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München Aims: Gastrinoma tissue has been found frequently in lymph nodes located near the duodenum without a known primary tumor. Therefore It has been suggested that a primary lymph node gastrinoma exists. We report on a 38year-old woman suffering from multiple endocrine neoplasia type 1 (MEN1) with a proven menin gene mutation and a history of primary hyperparathyroidism followed by Cushing’s disease, the detection of tumors of the pituitary, the pancreas and an elevated serum gastrin level. An octreotide scan revealed three foci in the upper abdomen. Methods / Results: Total pancreatoduodenectomy was performed and conventional histopathological examination revealed a neuroendocrine tumor of the pancreas expressing glucagon accompanied by several microadenomas. In addition, four gastrin positive regional lymph nodes were found, but no gastrinoma in either the duodenum or the pancreas. Only after the entire duodenal and pancreatic tissue had been screened, including an evaluation of 65 tissue specimens, two microgastrinomas (450 and 800 μm in diameter) were identified in the duodenum. Conclusions: Duodenal gastrinomas that give rise to lymph node metastases may be so tiny that they are easily overlooked in a routine examination and that systematic tissue monitoring is required to identify them.

Fr-097 QPRT, a potential new diagnostic marker for follicular thyroid carcinoma based on gene expression N. Hinsch, M. Frank, C. Vorländer1, C. Döring, M.-L. Hansmann Institut für Pathologie, Universitätsklinikum Frankfurt am Main 1 Abteilung für Chirurgie, Bürgerhospital Frankfurt am Main Aims: Aim of this study is the identification of new diagnostic markers for follicular thyroid carcinoma, which can be applied in routine diagnostics. Methods: We performed global gene expression analysis of 4 follicular adenomas, 4 microinvasive follicular carcinomas and 4 invasive follicular carcinomas. Three genes, which showed the highest differences in gene expression between adenoma and carcinoma, were further evaluated using immunohistochemistry of 24 samples and qRT-PCR. One antibody was tested further using a set of 126 samples and validated by western blot analysis. Results: 23 genes were underexpressed in follicular carcinoma with a fold change lower than –5. One gene (QPRT) was overexpressed in follicular carcinoma with a fold change higher than 5. Of the genes used for evaluation (dermatopontin, S100A8/9 and QPRT), only one gene, QPRT, turned out to be discriminating between follicular thyroid adenoma and carcinoma. 65% of follicular carcinomas were immunohistochemical QPRT-positive while 76% of follicular adenomas were QPRT-negative. The correlation between the diagnosis based on HE-staining and the immunohistochemical diagnosis was 71%. Conclusions: QPRT is a new immunohistochemical marker which may help to discriminate between follicular thyroid adenoma and carcinoma.

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Abstracts Fr-098 Expression of glucose transporter GLUT1 in adrenocortical carcinomas is associated with signiﬁcant shorter survival H.-U. Völker1, P. Adam1, S. Hahner2, H.K. Müller-Hermelink1, B. Allolio2, M. Fassnacht2 1 Institut für Pathologie und 2 Medizinische Klinik, Universitätsklinikum Würzburg Aims: Adrenocortical carcinomas (ACC) are tumors with poor prognosis and limited therapeutic options. Valid prognostic parameters have not yet been established. As the clinical course is very heterogeneous, we investigated key proteins in glucose metabolism to screen for differences in tumor energy supply and prognosis. Methods: Tissue microarrays (TMAs) of 167 tumor samples from patients with ACC collected by the German ACC registry (www.nebennierenkarzinom.de) were investigated immunohistochemically with antibodies against GLUT1, GLUT3, p-Akt, M2PK, TKTL1, and HIF1α. The staining intensity was assessed semiquantitatively (negative, weak, moderate, and strong). Survival analysis was performed using the log rank test. Results: GLUT1 was expressed in 41/125 evaluable ACC samples. In univariate analysis, expression of GLUT1 was associated with a significant shorter survival (median overall survival 1.8y vs. 5y, P<0.0008). Median disease free survival was 0.8y vs. 3.5y (P<0.0003). Increasing intenstiy of expression correlated with more aggressive clinical behaviour. The other markers did not correlate significantly. Conclusions: Expression of GLUT1 is inversely correlated with survival in ACC. Possible therapeutic options (e.g. glucose withdrawal, target specific therapy) based on this data should be subject to further clinical investigation.

Fr-099 Pancreatic somatostatin-producing neuroendocrine tumors (SOMNETs): Frequency, types, biological behavior, functional activity and association with hereditary syndromes N. Garbrecht1, M. Anlauf1, A. Schmitt2, P.U. Heitz2, H. Moch2, P. Komminoth3, A. Perren4, G. Klöppel1 1 Institut für Pathologie, Universitätsklinikum S-H, Campus Kiel 2 Institut für Pathologie, UniversitätsSpital Zürich 3 Institut für Pathologie, Kantonsspital Baden 4 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München Aims: Pancreatic SOM-NETs have been repeatedly described, but our knowledge of these tumors is largely based on case reports. Methods: In a series of 541 pancreatic NETs we investigated: the (1) frequency, (2) histological types, (3) classification according to WHO and TNM, (4) biological behavior and follow-up, (5) functional activity and (6) association with hereditary syndromes. Results: Pancreatic SOM-NETs accounted for 4% (n=21 cases) of all pancreatic NETs. Somatostatin was expressed in NETs (n=19), a gangliocytic paraganglioma (GCPG) and a poorly differentiated neuroendocrine carcinoma (pdNEC). SOM-NETs are usally malignant (41%) or of unknown biological behavior (53%). 37,5% of the patients had a relapse of the disase. The GCPG showed benign behavior after enucleation. SOM-NETs occurred sporadically (90%) or in association with MEN1 (10%). All MEN1-associated SOM-NETs were benign. In all patients the symptoms were unspecific and the existence of a somatostatinoma syndrome could not be verified. Conclusions: SOM-NETs are the fourth most common pancreatic NETs. Sporadic SOM-NETs and pdNECs require complete resection. The GCPG was benign by behaving and enucleation was sufficient. In addition to sporadic SOM-NETs there are MEN1-associated ones.

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Poster: Orthopädische Pathologie Fr-100 Isolation and enrichment of side populations as putative stem cells in osteosarcoma cell lines K. Agelopoulos, W. Kleinekemper, H. Schmidt, E. Korsching, H. Buerger1 Institut für Pathologie, Universitätsklinikum Münster – EuroBoNeT 1 Institut für Pathologie, Paderborn Aims: Side populations are getting more and more important as putative stem cells in many tissues as well as in cancers. As characterization of side populations is often hindered by their low proportion enrichment of these cells before isolation would be helpful for subsequent analyses. Methods: Isolation of side populations was done in ostesarcoma cell lines by means of FACS (FACSVantage SE, Becton Dickinson). Isolated cells were used for cell culture using different coatings of cell culture flasks and varying FCS contents of media. After several passages of cell culture the side population fraction was again determined. Results: Out of the tested osteosarcoma cell lines only ZK58 and MNNGHOS showed reproducible fractions of side population cells. After culture of side population cells under normal growth conditions the primary detected side population proportions of up to 0.43% could be reproduced in the reanalyses. But, when cell culture was started using increased FCS contents and coated flasks the side population proportion was clearly raised and reached more than 2%. Conclusions: Using an adjusted cell culture design can conserve side population characteristics whereas standard culture conditions lead to loss of these characteristics. By this, enrichment of side populations can be performed quite easily what ensures continuative analyses e.g. determination of stem cell properties.

Fr-101 Expression of MMPs and TIMPs in human bone lining cells indicating a possible catabolic function in bone remodelling? C. Dierkes, M. Kreisel, A. Schulz, J.-C. Wolﬀ, L. Fink Institut für Pathologie, Universitätsklinikum Gießen-Marburg Aims: We aimed to establish laser-assisted microdissection for isolation of endosteal lining cells from native human bone tissue in order to analyse mRNA expression of enzymes related to bone matrix degeneration and bone remodelling. Methods: Fresh human bone from femoral heads was cut into small cubes. Consecutively kryosectioning was performed followed by staining with Nuclear Fast Red for 2 s. Laser-assisted microdissection was performed using PALM and Leica systems. 4 to 8 pooled single cells c-DNA was synthesized and introduced to a qualitative single-cell PCR. Furthermore, immunofluorescence studies were performed on iliac crest biopsies. Results: We established a protocol for laser-assisted microdissection to isolate single cells from native human bone tissue with special focus on cells of the endosteal lineage. Both, PALM LPC and Leica LMD turned out to allow a fast and precise isolation of bone lining cells. Cell specific m-RNA expression was proved by identification of Cbfa1 (Run-X2) mRNA expression in the isolated cells. Furthermore, we investigated mRNA of MMP-13 and TIMP-1 in endosteal lineage cells. Conclusions: Laser-assisted microdissection in combination with molecular methods proved to be a suitable approach to examine catabolic (and anabolic) functions of bone lining cells directly taken out of their complex microenvironment. The anabolic functions of osteoblasts have been studied intensively during the last decades but little is known about the “quiescent” form, commonly called lining cell. It is assumed that these cells are responsible for long time preservation of bone. Our study clearly shows that bone lining cells produce proteases for bone matrix degradation and are probably an active player in the process of slow bone loss with increasing age.

Fr-102 Precise tumor mapping following neoadjuvant chemotherapy in osteosarcoma – an easy approach using conventional image processing software D. Baumhoer1, G. Jundt1,2 1 Institut für Pathologie und 2 Knochentumor-Referenzzentrum am Institut für Pathologie, Universitätsspital Basel Aims: Histologic response to neoadjuvant chemotherapy is an established prognostic factor in osteosarcoma patients. Generally, tumor necrosis of more than 90% is classified as a “good” response and is associated with a favourable prognosis. The ratio of viable and necrotic tumor is estimated in an entire slab of the largest tumor diameter and can be impeded by non-coherent or irregularly defined tumor areas. Methods: We used conventional image processing software to calculate the ratio of viable and necrotic tumor precisely. Briefly, we performed tumor mapping according to routine protocols, identified and marked the viable and necrotic tumor areas with a pen and subsequently scanned all sections batch-wise with an image scanner. Then, the image processing software was used to select the areas of interest, total tumor area and viable tumor area, and to measure the corresponding number of pixels with the histogram option followed by calculating the percentage viable tumor area to total tumor area. Results and Conclusions: We herewith suggest an easy, accurate and reproducible way to calculate the ratio of total and viable tumor following neoadjuvant chemotherapy in osteosarcoma. This approach is especially of interest in cases that present with viable tumor areas close to 10% at initial estimation.

Fr-103 The role of osteoprotegerin and ist ligand in bone terms containing osteoclastic giant cells K. Sturm, A. Schulz Institut für Pathologie, Universitätsklinikum Gießen-Marburg Aims: The interaction of osteoprotegerin (OPG) and its ligand (OPG-L) is accepted as the probably most important regulation process for the development of osteoclasts. The question raises whether this receptor-ligand mechanism applies to osteoclastic giant cells in bone tumors as well. Methods: 92 giant cell containing tumors (63 osteosarcomas, 19 giant cell tumors of bone, 9 tenosynovial giantcell tumors, 1 giantcell pancreatic carcinoma) were studied by immunhistochemistry of OPG and OPG-L in “Multi-tissue-Arrays”. Each of them contained 15 tumorous specimens and various control tissues. Conventional immunohistochemistry (APAAP) was performed using monoclonal antibodies against OPG and its ligand (R&D Systems, MAB 8051, MAB 10541). Results: Immunoreactivity for OPG was found in the cytoplasm and the nuclei of osteoclastic giant cells and in a certain part of mononuclear cells within the tumor tissues. OPG-L occurred as cytoplasmic and membranous labelling of giant cells but in some mononuclear cell types as well. Conclusions: The OPG and OPG-L interaction is present in tumors developing osteoclastic giant cells. The types of mononuclear cells delivering the signal (OPG-L) and bearing the receptor(OPG) remain to be identified by a next step of investigation.

Fr-104 Histological characterization and classiﬁcation of tissue samples removed during orthopaedic surgery for degenerative disc disease C. Weiler, V. Weiler, M. Lopez-Ramos, A. Nerlich1 Pathologisches Institut, Universitätsklinikum München (LMU) 1 Institut für Pathologie, Akademisches Krankenhaus Bogenhausen, München Aims: I. To characterize an orthopaedic spine center patient population in terms of underlying diseases, gender, age and body mass index. II. To apply our recently developed histologic degeneration score (HDS) to the surgicaly obtained disc material. III. To correlate the amount of histologic disc

alterations with the clinical data and – if available – with the body mass index (BMI). Methods: Excised disc material from 1134 patients (699 men/435 women) with a age-range 12–105 years (mean 57 y.) was graded based on our classification system (HDS). The degree of histological changes was correlated with gender, distribution, age and BMI. Results: The HDS (0–18 points) showed significantly higher values in the Nucleus pulposus (NP) than in the Anulus fibrosus (AF) (mean: NP 11,5/AF 8) with a significantly higher frequency of histomorphological alterations in men in comparison to women. Furthermore, the HDS revealed a positive significant correlation between the BMI and the extent of histological changes. No statistical age relation of the degenerative lesions was seen. Conclusions: I. Histologic disc alterations in surgical specimen can reliably be graded based on our histologic disc degeneration score. II. Histomorphological changes are more pronounced in the NP than in the AF. III. Men showed higher levels of degenerative changes than women. IV. The BMI showed a positive correlation with the HDI scores, indicating that overweight increases the risk of disc degeneration.

Fr-105 Is p63 a helpful marker in the diﬀerential diagnosis of giant cell tumour of the rib? A case report and review of the literature S. Scheil-Bertram, J. Schirren1, K. Hauptmann2, A. Fisseler-Eckhoﬀ Institut für Pathologie und Zytologie und 1 Klinik für Thoraxchirurgie, Dr. Horst-Schmidt-Kliniken (HSK) Wiesbaden 2 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte Giant cell tumours (GCT) of the rib are very rare. So far, less than 20 cases have been reported in the world literature. Recently, p63 was suggested to be helpful in differential diagnosis of giant cell lesions (Dickson et al, 2007). Aims: We report on a case of GCT of a 9th rib and discuss its differential diagnosis. Methods: In total 52 giant cell lesions of bone and soft tissue (20 bone, 32 soft tissue) were stained routinely and immunohistochemically (p63, CD68; both DAKO) stained. In the present case biopsy and surgical specimens, serological parameters, radiographs and computed tomography (CT) were available. Results: The postoperative histopathological examination of the resected specimen confirmed the preoperative diagnosis of giant cell tumour. Interestingly, 43.6% of mononuclear cells of the GCT demonstrated p63 immunoreactivity while none of CD68 positive mononuclear and the CD68 giant cells demonstrated p63 immunoreactivity. So far, only two cases of the giant cell lesions examined were negative for p63. Conclusions: GCT of the rib is a rare entity and p63 is not a helpful marker in its difficult differential diagnosis.

Fr-106 Inactivation of p53 promotes genomic instability in Fos-induced osteosarcomas W. Jochum, R. Schuler, F. Nötzli, M. Bawohl, E.F. Wagner1 Institut für Klinische Pathologie, UniversitätsSpital Zürich 1 Forschungsinstitut für Molekulare Pathologie (IMP) Aims: Mutations of the TP53 tumor suppressor gene are frequent in sporadic osteosarcomas and patients with germ line TP53 mutations (Li-Fraumeni syndrome) show an increased incidence of osteosarcomas. Our aim was to investigate the mechanistic role of TP53 inactivation in the pathogenesis of osteosarcoma using Fos transgenic mice as a disease model. Methods: Fos transgenic mice lacking functional p53 were generated. Mice were analysed using radiography. Tumor tissues were studied using conventional histology, immunohistochemistry, static DNA cytometry and interphase FISH with centromeric probes for chromosomes 11, 16, 17 and 18. Results: Radiographic analysis revealed that p53–/– Fos transgenic mice developed more bone tumors with an earlier onset than p53+/+ and p53+/– Fos transgenic littermates. Histologically bone tumors showed morphological features of low-grade chondroblastic osteosarcoma with comparable areas of chondroblastic differentiation in p53+/+, p53+/– and p53–/– mice. About Der Pathologe · Supplement 1 · 2008

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Abstracts 30% of p53–/– tumors contained areas of anaplastic tumor morphology. Static DNA cytometry revealed that p53–/– osteosarcomas contained more tumor cells with an increased DNA content (>4c) than controls. For all four chromosomes analysed, the proportion of tumor cells with 3 and more signals was significantly higher in p53–/– osteosarcomas indicating numerical chromosomal instability. Tumor cell proliferation was higher in p53–/– osteosarcomas. Conclusions: These results indicate that loss of p53 promotes genomic instability in osteosarcomas and suggests that p53 inactivation is a critical step during osteosarcoma pathogenesis.

Fr-107 Catabolic properties of endosteal bone cells studied by immunoﬂuorescence of matrixmetalloproteinases M. Kreisel, C. Dierkes, A. Schulz Institut für Pathologie, Universitätsklinikum Gießen-Marburg Aims: The loss of bone with increasing age is due to a negative balance of active “bone-remodelling” by osteoblasts and osteoclasts. Since more than 80% of endosteal surfaces of the adult skeleton are covered by flat “bone lining cells” the maintenance of the skeleton may depend on this cell type as well. We postulate, that lining cells may possess catabolic abilities. To prove this hypothesis, the expression of MMP`s was investigated by immunofluorescence staining of iliac crest bone biopsies. Methods: We studied 61 iliac crest bone biopsies in age cohorts <45 years, 45–70 years and >70 years, to examine the immunoreactivity of MMP 2, 13, 14 and their antagonists TIMP 1, 2, 3, 4 by immunofluorescence. The remodelling of bone was measured by histomorphometry. Results: Our histomorphometric measurements confirmed that 90% of endosteal surfaces were covered by lining cells. Particularly MMP 13 and 14, were present in 86% and 96% of the examined “lining cells”. 33% of the “lining cells” contained MMP 2. TIMP 1 and 2 could be detected in 96% and 76%, whereas TIMP 3 and 4 occurred in 6% and 20% of the “lining cells” only. Conclusions: Endosteal “bone lining cells”, covering by far the largest extent of endosteal bone surfaces, do have catabolic properties enabling them to play an important role in bone matrix degradation. The biological control of this cell type remains to be elucidated as a novel key process for age-related bone loss.

Fr-108 SOX Gene Expression in Human Osteoarthritic Cartilage – Indication of Phenotypic Instability J. Haag, P.M. Gebhard, T. Aigner Institut für Pathologie, Universitätsklinikum Leipzig Aims: While the developmental role of the SOX transcription factors in fetal chondrocyte differentiation is well documented, but much less is known about the expression of SOX family members in normal and osteoarthritic adult cartilage. The aim of the present study was therefore to present a thorough analysis of SOX gene expression in normal and osteoarthritic human adult cartilage. Methods: Total RNA was isolated directly from human adult normal and osteoarthritic knee cartilage and transcribed into cDNA. The expression of the SOX transcription factor family members was analyzed by gene expression profiling using the Affymetrix gene chip technology. Exact transcript levels of SOX genes 5, 6, 8, 9 and 10 were determined by quantitative real-time PCR. Results: Most members of the SOX transcription factor family showed no or very low expression levels in adult normal and osteoarthritic cartilage. In contrast, SOX9 expression was fairly high in normal cartilage, amounting to approximately 20 percent of GAPDH levels. SOX9 transcript levels were substantially reduced in OA. SOX6 levels were reduced albeit starting from a low basis expression in normal tissue. Conclusions: The presented data indicate that the role of SOX transcription factor family in adult human cartilage is most probably restricted to a few members with SOX9 being the most prominent. Furthermore, the reduction

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of SOX9 and 6 transcript levels in osteoarthritic chondrocytes might be responsible for the loss of phenotypic stability of OA chondrocytes.

Fr-109 Comparative analysis of the expression of bone degrading proteins from the small integrin-binding ligand N-linked glycoprotein family and RANKL/OPG ratio in aseptic versus septic interface membranes N. Koleganova1, G. Krohmer2, P. Hagjicostas3, B. Fink4, I. Berger1 1 Pathologisches Institut und 2 Chirurgische Klinik, Universitätsklinikum Heidelberg 3 Schwarzwald-Baar Orthopädische Klinik 4 Orthopädische Klinik Markgroningen Aims: The present study aims to evaluate the expression profile of proteins involved in bone degradation in septic versus aseptic interface membranes. Methods: 102 interface membranes resected at revision arthroplasty in patients with septic (30 patients) and aseptic (72 patients) prosthesis loosening were examined. Comparative analysis of the expression of CD68, CD45, bone sialoprotein (BSP), osteopontin (OPN) and the RANKL/Osteoprotegerin (OPG) ratio was performed using histology, immunohistochemistry, flow cytometry. Results: All investigated interface membranes showed an elevated expression of RANKL, BSP and OPN. RANKL/OPG ratio was significantly higher in aseptic compared to septic interface membranes. Expression of OPN was similar in wear particle type of aseptic interface membranes and in septic interface membranes. Aseptic interface membranes with degenerative changes showed significantly higher expression of OPN compared to other types of interface membranes. Conclusions: An elevated expression of bone degrading proteins such as RANKL, BSP and OPN is common in interface membranes. This can induce periprosthetic osteolysis independently from the histological type of interface membrane, prosthesis survival and age of patients. The evidence suggesting a role of the bone degrading proteins in the periprosthetic osteolysis suggests that the interface membranes might respond to the treatment with specific inhibitors of these proteins

Fr-110 Retransplantation of autologous chondrocytes – morphological outcome of autologous chondrocyte transplantation and analysis of the used scaﬀold material – A case report C. Brochhausen1, S. Biesterfeld1, A. Hofmann2, J. Decking3, C.J. Kirkpatrick1 1 Institut für Pathologie 2 Unfallchirurgische Klinik und Poliklinik und 3 Orthopädische Klinik, Universitätsklinikum Mainz Aims: Trauma, inflammation and degeneration are the main reasons for articular cartilage defects. Autologous chondrocyte transplantation (ACT) is a widely used method to treat focal defects based on tissue engineering strategies. However, it remains unclear whether the regenerated tissue will develop properties characteristic of the hyaline cartilage phenotype. Methods: We describe a 43-year-old female patient, who presented with knee pain and functional limitations after ACT. Due to severe disorders a revision and chondrocyte re-transplantation were performed. The resected tissue as well as the tissue engineered construct were analysed histomorphologically and histochemically. Results: Macroscopically multiple fragments of a mainly fibrous, partly chondroid tissue as well as a flat white sponge without visible tissue could be found. Histologically, the tissue fragments consisted of a mainly fibrous chondroid tissue with areas of a membraneous, fibrin-rich connective tissue. Small areas of hyaline cartilage could also be found. No acute inflammatory reaction could be detected. The scaffold material consisted of a collagen-rich sponge with longitudinally and partially perpendicularly oriented fibres. Round-shaped, matrix-producing cells were embedded in the scaffold as revealed by alcian blue staining. Conclusions: The present case demonstrates that commercially available tissue engineered cartilage may result in a mainly fibrous-type tissue with low

cell numbers without any signs of chondroid clusters or cartilage formation. Further evaluation of ACT constructs before and after implantation are necessary to understand the underlying mechanisms.

Fr-111 Innovative Models for the in vitro analysis of chondrocytes – scaffolds, drug release and new characterization methods C. Brochhausen1, S. Halstenberg1, R. Zehbe2, N. Sanchez1, B. Watzer3, R.E. Unger1, H. Schubert2, C.J. Kirkpatrick1 1 Institut für Pathologie, Universitätsklinikum Mainz 2 Institut für Materialwissenschaften und -technologien, Berlin 3 Eicosanoidlabor, Mutter Kind Zentrum, Universitätsklinikum GießenMarburg Aims: Degenerative and rheumatic diseases often result in severe cartilage damage. To understand the underlying pathomechanisms several in vitro systems are used. In our approach we adopt methodologies from tissue engineering to analyze the behaviour of cultured chondrocytes. Methods: Cell culture: Human chondrocytes were seeded on a collagen- derived, oriented scaffold with and without prostaglandin supplementation. Drug release system: PGE2 was immobilized in polylactide-based microspheres, the release was measured by tandem gas chromatography /mass spectrometry over a period of 154 hours. Three-dimensional characterization: Microspheres and cells were integrated in the scaffold. The cell and microsphere distribution was analyzed by synchrotron-μCT. Results: After 14 days in vitro chondrocytes without PGE2 showed a fibroblast-like phenotype, whereas with PGE2 the chondrocytes had a rounded chondroid phenotype. The delivery of PGE2 from polylactide-based microspheres resulted in a prolonged release over 154 hours. SR-μCT revealed the three dimensional distribution of microspheres and cells throughout the scaffold. Conclusions: Three dimensional scaffolds offer interesting opportunities to analyze chondrocytes in a complex microenvironment and SR-μCT allows the analysis of three-dimensional cellular distribution. Polylactide-based microspheres can be used to incorporate further cytokines in a drug-delivery system to simulate various pathophysiologic conditions and control differentiation. Thus, tissue engineering strategies provide an innovative input in the investigation of cartilage damage and its repair.

Fr-112 Relevance of Endoglin (CD105) and FDG-PET for diagnosis of septic / aseptic prosthesis loosening B. Hermanns-Sachweh, K. Dautzenberg, M. Gebhard1, T. Mumme2 Institut für Pathologie, 1 Klinik für Nuklearmedizin und 2 Orthopädische Klinik, Universitätsklinikum Aachen Aims: Septic and aseptic prosthesis loosening are typical long-term complications and may require different treatment strategies. The aim of this study was to provide a diagnostic tool for differentiation of these causalities. Methods: Patients with clincial signs of prosthesis loosening were investigated by FDG-Positron Emission Tomography (PET). Periprosthetic tissue, obtained during the subsequent surgery was analyzed in 37 patients by conventional histology and immunohistochemically (antibodies against VEGF, CD105, CD 34, CD31, factor VIII). Results: Periprosthetic tissue from patiens suffering from septic loosening revealed an increased number of granulocytes as well as a marked neovascularization positive for Endoglin (CD105). In patients suffering from aseptic loosening focally increased vascularization was seen as well, however, these areas were never positive for CD105. Conclusions: FDG-PET is effective in differentiation of septic from aseptic prosthesis loosening and thus, is helpful to initiate the respective therapy. The correlative of an accumulation in FDG-PET is an increased tissue turnover with CD105-positive angioneogenesis. Based on these data, a screening for increased Endoglin serum levels in patients with prosthesis loosening is planned.

Poster: Gynäkopathologie Fr-113 A RNAi-induced knockdown of the putative tumour suppressor gene SFRP1 causes changes in cell-adhesion in human MCF10 breast cells F. Wiesmann, R. Knüchel, E. Dahl Institut für Pathologie, Universitätsklinikum Aachen Aims: Silencing of the WNT antagonist “Secreted Frizzled Related Protein 1” (SFRP1) due to promoter methylation has previously been associated with progression in many human tumour entities such as breast cancer. In this study we wanted to analyze the molecular and cellular consequences that result from a forced loss of SFRP1 in an in vitro model. Methods: Artificial silencing of SFRP1 was induced in cell culture by applying SFRP1 gene specific siRNAs to MCF10A mammary cells by lipofection. Transfection efficiency and gene-specific knockdown were assessed after 48 h, 96 h, 144 h and 192 h after transfection using realtime PCR analysis for mRNA levels and Western blot and immuno-cytochemistry for protein levels. Additionally, RNAi induced changes in expression were analyzed by Affymetrix profiling experiments using the U133 Plus 2.0 micro array. Results: A significant reduction of SFRP1 mRNA expression down to 5.8% (fold change=16.4) of the original expression level could be achieved after 96 hours and was verified in triplicate analysis. Effects on SFRP1 protein expression could be observed 144 h after initial transfection. At this time point of incubation, there was a clear induction of known WNT target genes including genes from the matrix-metalloproteinase family. Furthermore, we observed a significant reduction of genes which are thought to modulate cell adhesion. Conclusions: SFRP1 is a putative tumour suppressor gene lost in a high percentage of human breast cancer. However, little is known about the resulting molecular consequences. This is the first study which identifies and validates target molecules from loss of SFRP1-signalling in human breast cancer in an in vitro model. Our preliminary data describe a close correlation between loss of SFRP1 and changes in the molecular composition of cell adhesion molecules. Functional assay are underway. The array study may decipher potential novel cancer drug targets in the WNT pathway. Supported by a grant from the Fritz Thyssen Stiftung

Fr-114 The ubiquitin-like molecule interferon-stimulated gene 15 (ISG15) is a novel prognostic marker in human breast cancer N. Bektas1, A. Hartmann2, R. Knüchel1, E. Dahl1 1 Institut für Pathologie, Universitätsklinikum Aachen 2 Institut für Pathologie, Universitätsklinikum Regensburg Aims: ISG15 is a ubiquitin-like molecule (UbLs) that is strongly upregulated by type I interferon as a primary response to diverse microbial and cellular stress stimuli. However, alterations in the ISG15 signalling pathway have also been found in several human tumour entities. In the current study, we present for the first time a systematic characterization of ISG15 expression in human breast cancer and normal breast tissue both at the mRNA and the protein level. Methods: ISG15 immunohistochemistry was performed on a tissue microarray containing invasive breast carcinoma (n=179) and normal breast tissue (n=51). In addition, ISG15 expression in breast cancer was analysed by quantitative real-time PCR, cancer profiling array and Western blot. SPSS software was used for statistical analysis. Results: ISG15 is overexpressed in breast carcinoma relative to normal breast tissue both on the RNA and protein level. We found a significant correlation between ISG15 expression and the oestrogen receptor status (p=0.028). Recurrence-free survival analysis showed a significant correlation (p=0.012) between ISG15 overexpression and unfavourable prognosis. Conclusions: ISG15 may represent a novel breast tumour marker with prognostic significance that is closely linked to oestrogen receptor expression. Recently, micorarray experiments determined strong dysregulation of ISG15 expression in response to diverse cancer chemotherapeutic agents. Thus, ISG15 could represent a new drug target in breast cancer treatment. Der Pathologe · Supplement 1 · 2008

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Abstracts Fr-115 Dizygotic twin gestation consisting of a complete mole and a normal conceptus A.M. Müller1, M.S. Fahr1, W. Coerdt1, U. Zechner2, T. Maltaris3 1 Abteilung für Kinderpathologie, 2 Institut für Humangenetik und 3 Frauenklinik, Universitätsklinikum Mainz Aims: The simultaneous occurrence of complete hydatidiforme mole and a fetus as twin pregnancy is rare (incidence: 1:22.000–1:100.000). We report a twin pregnancy of a morphologically normal fetus (XY) with macro – and microscopically normal placenta and a complete hydatiform mole (XX). Case report: A 33-year old gravida I presented herself in the obstetric department in the 22nd gestational week being in labour. In ultrasound, a partial mole and a living fetus was diagnosed. Unquenchable active uterine contractions led to spontaneous abortion. Postmortem examination of the fetus showed a morphological regular male fetus. Histology of the placenta demonstrated in part a normally developed placenta side to side with a complete hydatidiform mole. Genetically, the fetus and the morphological normal placenta showed a 46, XY karyotype while the complete mole displayed a 46, XX karyotype which was confirmed to be of androgenetic origin by microsatellite DNA analysis. Conclusions: Combination of (histo)morphological and cytogenetical diagnostic led to the diagnosis of a dizygotic twin gestation with a complete mole and exclusion of the clinical diagnosis of a partial mole. As in some studies the rate of subsequent development of persistent trophoblastic tumour (PTT) is regarded to be higher in patients with dizygotic twin gestation (consisting of a complete mole and a normal conceptus) than in patients with single complete mole, this entity should be accurately diagnosed. Combination of histomorphological and genetical analysis allows a correct diagnosis and exclusion of a partial mole.

Fr-116 A simple and inexpensive method for the construction of paraﬃn tissue microarrays from needle biopsy specimens Example with breast needle biopsy specimens U. Vogel, B. Bültmann Institut für Pathologie, Universitätsklinikum Tübingen Aims: Customarily, resection material is used for paraffin tissue microarrays (PTMAs). However, sometimes needle biopsy specimens (NBSs) are the only available material of a patient. Therefore, we looked for a simple and inexpensive technique to construct PTMAs using NBSs. Methods: H&E stained slides of 94 NBSs of breast cancer were screened for those areas where the carcinoma was equally distributed within a 4 mm long section. These parts of the NBSs were punched of the paraffin block using a 4 mm in diameter biopsy punch, melted in a routine steel mold, picked up with a 25-gauge needle and manually transferred to the holes of a predrilled recipient block. Then the PTMA was cut with a rotary microtome to ensure that the paraffin tissue core biopsies (PTCBs) reached the surface of the PTMA to get contact to a double-sided adhesive tape. Then the PTMA was melted on a standard hot plate at 65°C to ensure a strong contact between the PTCBs and the surrounding paraffin. Results: A PTMA with 84 PTCBs of NBSs could be successfully constructed. There was no difference in cutting, staining and evaluation of the sections in comparison to PTMAs made of resection material. About 11% of the originally screened 94 NBSs were not punched due to a patchy distribution of the carcinoma. On the average 15% of the PTCBs used in the PTMA were non-informative due to missing tumor cells when evaluating the different immunohistochemical stainings (e.g. estrogen receptor). Conclusions: With this simple and inexpensive method it is possible to use NBSs for the construction of PTMAs and to make NBSs accessible for high throughput examinations.

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Fr-117 Expression and functional analysis of PGRMC1 in breast cancer H. Neubauer, G. Adam, K. Sotlar1, U. Vogel2, M.A. Cahill3, D. Wallwiener, H. Seeger, E. Solomayer, T. Fehm Frauenklinik, Universitätsklinikum Tübingen 1 Pathologisches Institut, Universitätsklinikum München (LMU) 2 Institut für Pathologie, Universitätsklinikum Tübingen 3 ProteoSys AG, Mainz Aims: In a proteomic screening approach we identified progesterone receptor membrane component 1 (PGRMC1) as a protein upregulated in frozen ERnegative breast cancer tissue samples making PGRMC1 a potential therapeutic target. The aim of this project was to characterize the potential biological function of PGRMC1 by structure analysis and generation of different mutant forms. Methods: Several PGRMC1-specific hybridoma were generated and tested by western blot analysis. Multiple immune fluorescence for PGRMC1, estrogen receptor alpha and the hypoxia marker glucose transporter 1 (Glut1) was applied on tissue microarrays containing different breast cancer specimens. Additionally, posttranslational modification of PGRMC1 was investigated by phosphatase treatment of tissue lysates. Functional analysis was performed by stimulating transfected MCF-7 cells with a membrane-impermeable progesterone:BSA:fluoresceinisothio-cyanate conjugate. Results: PGRMC1 is posttranslationally modified. Stimulation of PGRMC1 expressing MCF-7 cells resulted in an increased proliferation compared to control cells grown in growth factor / hormone reduced medium. Western blot analysis of hybridoma produced a PGRMC1-specific signal at the predicted molecular weight of 28 kDa which is not detected after preincubation of the hybridoma with recombinant PGRMC1 protein. In immune fluorescence analysis of breast cancer tissue sections presumably myoepithelial cells were highly positive for PGRMC1. Besides that PGRMC1 expression was upregulated in Glut1 positive, hypoxic areas in ductal carcinoma in situ of comedo type. Conclusion: These data indicate that PGRMC1 is expressed in breast cancer and might functionally play a role in cell proliferation.

Fr-117a A concept for pathologic evaluation of sentinel lymph node biopsy C. Jayasinghe, H. Rott1, H.-J. Terpe Institut für Pathologie, Klinikum Leverkusen 1 Frauenklinik, Klinikum Leverkusen Aims: The sentinel lymph node biopsy (SNLB) is by now accepted as the gold standard to determine the lymph node status in patients with breast carcinoma. There are several techniques for examining SNLs intra- and postoperative. In the present study we demonstrate a method for careful evaluation of SNLs in daily routine with a very low error rate. Methods: 228 SNLs of 105 patients were processed. Intraoperative examination was performed macroscopically laminating the SNL in 3 mm slices and investigating the cut surface carefully by loupe. A suspicious cut surface was aditionally evaluated by frozen section. All specimens of negative macroscopic findings were completely examined histologically in paraffin sections with 200 μm intervals (postoperative exploration). Results: 75.2 % (79 patients) had negative intraoperative SLNs. 18.1 % (19 patients) had macroscopically detected positive SLNs. 6.7 % (7 patients) were in gross examination negative and in postoperative performed histological evaluation positiv (3 cases with micrometastasis ≤ 2 mm and 4 cases with 3 mm metastasis). In 14 of the 79 patients with negative SNLs the cut surface was macroscopically sorted suspicious. Conclusions: Our results confirm the concept of intraoperative gross evaluation of SLNs in combination with frozen sections depending on macroscopic findings. With this method the risk of losing tumor tissue, especially in case of micrometastasis, is minimised.

Fr-118 Case report to diﬀerential diagnosis of borderline tumors of the ovary A. Dellmann1, H. Franz2, I. Schmitz3, A. Tannapfel3, K. Donhuijsen1 1 Institut für Pathologie und 2 Frauenklinik, Städtisches Klinikum Braunschweig Institut für Pathologie 3 Institut für Pathologie, Universitätsklinikum Bochum Aims: The WHO tumor classification defines several types of borderline tumors of the ovary (BOT), mostly serous or mucinous BOT. We observed a case with BOT criteria related to the very rare tumors of the rete ovarii. Methods: A cystic lesion of the right ovary accidentally detected in sectio cesarea of a 39-year-old woman was partly resected. At 1.5 cm biopsy of the tumor we diagnosed a special type of an adenomatous tumor with indefinite malignant potential. Therefore some weeks later the right side ovary with a 4×3×2 cm tumor was resected totally and further investigations inclusive electron microscope and K-ras PCR were done. Results: Histological criterias and immunohistochemical results fullfilled the criterias of adenomatous tumor of the rete ovarii especially with Aktin SMpositive surrounding tissue of the small and large cystic structures. However the leiomyomatous differentiation is only expressed in part more myofibroblastic than well-differentiated leiomyogenic. Remarkably the proliferation activities were discrepant between the first and the second biopsy and diminished some weeks after the first surgery, probably as a sign of normalizing hormone status after birth. Conclusions: The diagnosis of cystic adenomatous tumors has to reflect the possibility of rete ovarii tumors and the diagnostic tool of immunohistochemical detection of smooth muscle differentiation. The possibility of borderline tumors of ovary has to be respected even in the very small group of adenomatous tumors of the rete ovarii.

toid DCs.These data suggest that the character of inhaled Ag may influence APC phenotype, function, and the subsequent immune response.

Fr-120 Egr1 (Early growth response 1) is downregulated during alveo-larization and exerts antiproliferative eﬀects on lung cells J.-C. Wolﬀ, R. Voswinckel, J. Wilhelm, U. Seay, A. Zakrzewicz, W. Seeger, L. Fink Institut für Pathologie und Med. Klinik II / UGLC, Universitätsklinikum Gießen-Marburg Aims: Besides others, we found the Egr1 gene to be regulated in different types of lung growth. We now aim to identify its specific effects. Methods: In microarray studies of C57BL/6 mouse lungs, we found several genes to be significantly regulated after birth (induction of alveolarization) as well as when undergoing compensatory lung growth following unilateral lung resection. Real-time PCR validations were followed by expression and localization studies using western blot and immunohistochemistry. We then evaluated the effects of some candidate genes in cell culture using overexpression and knock-down strategies. Results: Only about 25 genes were found to be regulated in both types of lung growth, and half of them even in different directions. Egr1, a transcription factor involved in multiple cellular responses, was one of the most interesting candidate genes as it is involved in many processes necessary for lung growth. We were able to show an anti-proliferative effect of Egr1. As the gene was found to be down-regulated in newborn mice, the overall influence was proproliferative. Ongoing studies focus on changes in adhesion and / or migration activity of cells as well as on effects regarding Egr1-dependent pathways. Conclusions: Egr1 may be a potential target gene for the development of new therapeutic strategies for destructive lung diseases. It was found to be regulated in postnatal as well as compensatory lung growth and negatively regulates proliferation of pulmonary cells.

Poster: Lunge Fr-119 Characterization of lung APCs taking up soluble and particulate aeroantigens D. Reiner1, R. Lee1, P. Singh1, O. Hoﬀmann 3, G. Stingl1, M.M. Epstein1, G. Dekan2 1 Klinische Abteilung für Immundermatologie und Infektiöse Hautkrankheiten, Universitätsklinik für Dermatologie und 2 Klinisches Institut für Pathologie, Medizinische Universität Wien 3 Institut für Pharmakologie and Toxikologie, Universität Wien Aims: Antigen (Ag) presenting cells (APC) in lungs are involved in immune responses to inhaled Ags. Many soluble, particulate, and viral Ags are routinely inhaled, captured by lung APCs, and induce either immune responses or immune tolerance. The exact subpopulations of lung APCs involved remains unclear. Our aim is to compare and contrast the phenotype of lung APCs taking up inhaled soluble, particulate, and viral Ags. Methods: Mice were instilled intranasally with either 10mcg of soluble ovalbumin (OVA) Alexa-Fluor594 conjugates (OVA594), 5% FITC-labelled latex beads, 109 PFU of modified green fluorescent protein expressing-vaccinia virus, or 10% India ink alone or combined with OVA594. Airway cells and frozen lung sections were stained with mAbs directed against cell surface Ags and evaluated by immunofluorescence microscopy. Results: Sixty minutes after intranasal instillation, 50% of airway cells were OVA594+ CD11c+CD11b-MHC class II-. Gr-1 and B220 were negative but they expressed Mac and DC markers, MOMA-2, F4/80, CD68, CD206, and CD205. Remarkably, India ink particles and latex beads were taken up by APCs with a similar phenotype. In contrast vaccinia virus was taken up by CD11b+ CD11c+ MHCII+ CD86+. Conclusions: The same APCs take up soluble, particulate and viruses during inhalation, express a mixed DC and Mac phenotype, and are not plasmacy-

Fr-121 Fatal pneumococcal infection, dysgammaglobulinaemia and lymphytic hypoplasia in a 38 year old previously healthy patient A. Zimpfer, G. Edelhoﬀ1, B. Nebel1 , H.K. Koch Gemeinschaftspraxis für Pathologie Prof. Koch, Dr. Hellerich, Dr. Venzke, Freiburg 1 Anaesthesie und Intensivmedizin, Kreiskrankenhaus, Emmendingen Aims: Common variable immunodeficiency (CVID) is the most common primary immunodefiency characterized by low levels of immunoglobulins. Recurrent infections of the respiratory and/or gastrointestinal tract and autoimmune phenomena are typical signs of CVID in the adult. We report a case of a 38-year-old male patient with overwhelming pneumococcal pneumonia and sepsis. Methods / Results: Following autopsy and histology, severe inflammatory lung damage was responsible for respiratory failure. The enlarged spleen unusually displayed a reduced white pulp without well-formed secon-dary follicles. Further investigations showed neither in lymphatic node nor in the tonsils secondary lymph follicles. Hemophagocytic histiocytes intermingled with bland looking lymphocyted were quite frequent in different organs, e.g. bone marrow and spleen illustrating the clinical diagnosis of hemophagocytic lymphohistiocytosis. Retrospectively, the patient had had repeatedly episodes of respiratory tract infections as a child and adult. Immunoglobulin levels were analysed and IgM and IgG levels found significantly lowered. So far, characterisation of specific genetic defects failed due to technical problems. Conclusions: Clinical, laboratory and autoptical findings were consistent with a primary immunodeficiency syndrome, especially as a form of CVID manifested in adulthood. The presentation of this case with lymphatic hypoplasia is exceptional insofar as this is only seen in a small subgroup of CVID patients. The presentation of this case with lymphatic hypoplasia is exceptional insofar as this is only seen in a small subgroup of CVID patients.

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Abstracts Fr-122 Immunhistochemical detection of extracellular matrix proteins, matrix metallo-proteinases and their tissue inhibitors in human nonsmall cell lung cancer (NSCLC): Diﬀerences between intrapulmonal, lymphatic and cerebral metastases C. Schopf1, E. Herpel1, D. Kieslich de Hol1, A. Warth1, H. Hoﬀmann2, P. Schirmacher1, P. Schnabel1 1 Pathologisches Institut, Universitätsklinikum Heidelberg 2 Chirurg. Abt., Thoraxklinik am Universitätsklinikum Heidelberg

Fr-124 TTF1 ampliﬁcation deﬁnes TTF1 overexpression in lung adenocarcinomas and has prognostic signiﬁcance S. Perner1,3, F. Demichelis1, L. Johnson1, B. Weir2, J. Barletta1, M. Meyerson2, L. Chirieac1, M.A. Rubin1 1 Pathology, Brigham and Women’s Hospital, Boston, MA, USA 2 Medical Oncology, Dana-Farber Cancer Institute, Boston and Cancer Program, Broad Institute, Cambridge, MA, USA 3 Institut für Pathologie, Universitätsklinikum Ulm

Aims: We studied whether and to which extent there might be differences in the distribution of extracellular matrix proteins (MPs) matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs (TIMPs) within different pulmonal (same lobe or different lobe), cerebral or mediastinal lymph node metastases of NSCLC. Methods: From 87 patients (suffering from NSCLC stage III, IV) showing metastases in intrapulmonal (same lobe: pT4 (n=36) or different lobe: pM1 pulmonal (n=17)), cerebral (n=16) or mediastinal lymph node (pN2; n=45) sites, paraffin embedded samples were investigated. Immunohistochemistry for Collagen I and IV, Fibronectin, MMP-1, MMP-9, TIMP-1 and TIMP-2 was performed. Analyses were done using a semi-quantitative scoring system or the point counting method (stereology). Results: Collagen I was expressed significantly higher (p=0.0001) in cerebral than in intrapulmonal (pT4 and pM1) metastases. Intrapulmonal significantly more Collagen I (p=0.018) and Collagen IV (p=0.025) were expressed in the pM1 than in the pT4 metastatic lesions. Collagen IV was expressed significantly higher (p=0.027) in intrapulmonal (pM1) than in mediastinal lymph node metastases. Cerebral metastases expressed significantly more (p=0.0001) MMP-9 and TIMP-1 than mediastinal lymph node metastases. Conclusions: Metastatic lesions of NSCLC show significant differences in the composition of MPs, MMPs and TIMPs. Different ways of metastatic spreading and local environmental conditions might also contribute to different distributions of MPs, MMPs and TIMPs.

Aims: Lung cancer is the leading cause of cancer death worldwide. We reported a high-level amplification of 14q13.3 as the most common recurrent event (~12% of tumors) in lung adenocarcinomas. Within this interval, we identified TTF1 (thyroid transcription factor 1) as lineage-specific transcription factor and novel proto-oncogene. Although TTF1 is already known to be expressed in virtually all lung adenocarcinomas and thus used as a diagnostic marker, we asked whether TTF1 amplification was associated with the degree of protein expression and outcome. Methods: We interrogated 198 lung adenocarcinomas for TTF1 protein expression by immunohistochemistry and for TTF1 amplification status by FISH. Kaplan-Meier survival analysis was performed on 70 patients to assess if TTF1 amplification status is associated with outcome. Results: There is a significantly higher TTF1 expression in adenocarcinomas with TTF1 amplification as compared to adenocarcinomas without amplification (p=0.02; Wilcoxon test ). Also, there is a trend that patients with TTF1 amplification have worse outcome than patients without (p=0.15). Conclusions: This study demonstrates for the first time that the routinely used diagnostic lung cancer marker TTF1 may also be a prognostic biomarker for lung adenocarcinomas. Future work attempts to confirm the prognostic significance of TTF1 amplification/over expression.

Fr-123 Inactivation of IGFBP-rP1 in human lung cancer is related to DNA methylation Y. Chen1, P. Lund2, M. Pacyna-Gengelbach2, N. Deutschmann2, I. Petersen1 1 Institut für Pathologie, Universitätsklinikum Jena 2 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte

Fr-125 Comparison of pulmonary adenocarcinomas and squamous cell carcinomas: Immunohistochemical detection and quantiﬁcation of extracellular matrix proteins (MPs), metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) P. Schnabel1, E. Herpel1, C. Schopf1, D. Kieslich de Hol1, A. Warth1, H. Hoﬀmann2, P. Schirmacher1 1 Pathologisches Institut, Universitätsklinikum Heidelberg 2 Chirurg. Abt., Thoraxklinik am Universitätsklinikum Heidelberg

Aims: The aim of this study was to investigate the mechanism responsible for the downregulation of the IGFBP-rP1 gene in lung cancer. Methods: Real-time RT-PCR was applied to analyze the mRNA expression of IGFBP-rP1 in lung cancer. The methylation status of IGFBP-rP1 was evaluated by demethylation test and bisulphate sequencing. Results: We found that 11 out of 16 lung cancer cell lines showed markedly decreased expression compared to normal bronchial epithelial cells (HBEC) and small airway epithelial cells (SAEC) by real-time RT-PCR. To explore the mechanism responsible for the gene silencing, we treated the 4 lung cancer cell lines H2170, H226, SHP77 and H2030 lacking the expression of IGFBPrP1 with the demethylation agent 5-aza-2’-deoxycytidine. The restoration of IGFBP-rP1 protein was detected in the 3 out of 4 cell lines by Western blot analysis. Moreover, methylation status of the IGFBP-rP1 5’ CpG islands was analyzed by bisulphate sequencing in 6 patients with primary lung tumor. A heterogeneous methylation pattern was found in exon/intron 1 of IGFBP-rP1 in all 6 samples, while in 5 normal lung tissues, only a spontaneous methylation was detected. Conclusions: Our study revealed the first description of the methylation status of the IGFBP-rP1 of exon/intron1 region in primary lung tumors. This result suggests that the inactivation of IGFBP-rP1 in lung cancer may be explained by promoter hypermethylation.

Aims: Clinically, pulmonary adenocarcinomas (AD) and squamous cell carcinomas (SQ) together are regarded as “non small cell lung cancer” (NSCLC). We investigated whether and to which extent there might be differences in the distribution of important components of the extracellular matrix between AD and SQ. Methods: From 102 patients 64 suffered from AD, and 38 from SQ (stage III, IV, control group stage I). Paraffin embedded tumour samples were investigated by immunohistochemistry using antibodies against Collagen I and IV, Fibronectin, MMP-1, MMP-2, MMP-9, TIMP-1 and TIMP-2. Qualitative, semiquantitative and morphometric results were obtained especially volume fractions of the respective MPs, MMPs and TIMPs were analyzed using the point counting method (according to E.R. WEIBEL). Results: Collagen IV (p=0.011), MMP-1 (p=0.007), TIMP-1 (p=0.026), and TIMP-2 (p=0.005) were expressed significantly higher in the respective positively stained structures or cells of AD than of SQ. No statistical significant differences were found for Fibronectin, Collagen I, MMP-2 and MMP-9. Conclusions: Pulmonary adenocarcinomas and squamous cell carcinomas show statistically significant differences in the composition of important components of the extracellular matrix. Thus, with regards to diagnostics and specific therapy pulmonary adenocarcinomas and squamous cell carcinomas should be regarded as two distinctly different entities and not as “NSCLCs”.

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Fr-126 Fhl-1 as a mediator of pulmonary arterial hypertension G. Kwapiszewska, M. Wygrecka, L. Marsh, S. Schmitt, R. Trösser, W. Seeger, O. Eickelberg, L. Fink, N. Weissmann Lung Center, Universitätsklinikum Giessen-Marburg

Fr-128 The HOPE-technique permits enhanced detection of EGF-R in Non Small Cell Lung Cancer D. Kähler, T. Goldmann, H. Schultz, E. Vollmer Klinische und Experimentelle Pathologie, Forschungszentrum Borstel

Aims: Exposure to hypoxia causes vasoconstriction of the pulmonary arteries, eventually resulting in vascular remodelling. To investigate the processes underlying hypoxic remodelling in the lung, we utilised the hypoxic mouse model of pulmonary hypertension in combination with 2D-PAGE. Methods: One of most significantly up regulated proteins was Fhl-1 (6.6 fold). Immunohistochemical staining localised Fhl-1 expression to pulmonary arteries. Laser-microdissection of this compartment revealed significantly higher expression of Fhl-1 in mice exposed to prolonged hypoxia. Similarly, consistent upregulation of Fhl-1 was observed in laser-microdissected pulmonary arteries from 5 IPAH patients compared to healthy donors (>4 fold increase, p=0.002). Additionally, in PASMC isolated from IPAH patients expression of Fhl-1 was elevated as compared to donors. In vitro exposure of PASMC to hypoxia further enhanced Fhl-1 expression. Hypoxic induction of Fhl-1, was controlled by the transcription factors (HIF and EGR) as shown by siRNA and EMSA studies. Overexpression of Fhl-1 led to enhance proliferation and migration of PASMC, while siRNA knock-down attenuated these effects. Results: To further elucidate the molecular function of Fhl-1, we applied coimmunoprecipitation and MALDI-TOF to identify potential Fhl-1 interacting partners. We identified Talin1, which has a role in cytoskeletal organisation and cell migration. Immunofluorescence staining of human PASMC, as well immunohistochemistry in human lungs demonstrated colocalization of Fhl-1 and Talin1 in vivo. Conclusions: Fhl-1 has previously been documented to be induced during development of heart and skeletal muscle hypertrophy. However, we demonstrate for the first time the involvement of Fhl-1 in hypoxic vascular remodelling in the lung, and its association with human IPAH disease.

Aims: Due to large ongoing clinical studies on targeted therapy facing EGF-R, determination of the receptor-status of EGF-R has become an issue in pathology. Like in the detection of Her2, immunohistochemistry needs hardly to standardize target retrieval techniques and has to be backed up by FISH. Here we analyzed the effects of formalin-fixation upon the immunohistochemical detection of EGF-R and compared the results with corresponding Hope-fixed samples of Non Small Cell Lung Cancer. Methods: HOPE-technique, Immunohistochemistry, RNA-extraction, Reverse Transcription, RT-PCR; FISH Results: Compared to the formalin-fixed specimens we observed a higher proportion of EGF-R score 3 positive in the tissues fixed by the HOPE-technique. Negative controls without primary antibodies remained negative, while the signals were clearly located on the cell membranes showing up as rings. To verify this, we additionally performed RT-PCR targeting EGF-R mRNA and FISH for detetion of gene amplification from the HOPE-fixed material, the results of which were in accordance to immunohistochemistry. Conclusions: The application of immunohistochemical techniques for the semiquantitative evaluation of biomarkers is common practice and serves for decisions on therapeutic interventions. Due to the effect of formalin onto antigenic structures, target retrieval techniques are necessary, which are difficult to standardize. We have shown here in one example that tissues treated by the HOPE-technique can serve for more realistic results. We found a higher portion of score 3 positive tumors than described in the recent literature. Further studies are ongoing.

Fr-127 Frequency of EGFR drug sensitivity and resistance mutations in lung adenocarcinoma V. Tischler, K. Struckmann, D. Zimmermann, B. Bode, W. Weder2, R. Stahel1, H. Moch, A. Soltermann Institut für Klinische Pathologie, 1 Klinik für Onkologie und 2 Klinik für Herz- und Gefässchirurgie, UniversitätsSpital Zürich

Fr-129 The cutting (w)edge – Comparative evaluation of renal baseline biopsies obtained by two diﬀerent methods Z. Bagó-Horváth, A. Soleiman, M. Bodingbauer1, F. Mühlbacher1, H. Regele Klinisches Institut für Pathologie und 1 Universitätsklinik für Chirurgie, Medizinische Universität Wien

Aims: Mostly patients with somatic sensitivity mutations of the epidermal growth factor receceptor (EGFR) kinase domain will show a clinical response to tyrosine kinase inhibitors. Since resistance mutations have recently been described, we aimed for EGFR genotyping prior to targeted therapy. Methods: Sixty patients with surgically resected lung adenocarcinoma and without prior therapy were analysed. The EGFR status included genotyping of exons 18–21, fluorescence in situ hybridization and immunohistochemistry. Samples containing <50% tumour cells were micro-dissected before analysis. Results: Of the 60 patients, 27 (45%) were males, 33 (55%) females. 45 (75%) were (former) smokers. We found gene mutations in 13 patients (22%), gene amplifications in 12 (20%) and both in 4 patients (7%). 8 of the 13 patients with gene mutation (62%) never smoked, 5 of these 8 non-smokers (62%) were females. Of the 13 gene mutations, 11 (85%) were of drug sensitivity and 2 (15%) of drug resistance type (exon 20 insertions, p.D770_N771insG and p.N771_P772insT). 12 of 13 mutated tumours (92%) showed increased protein expression. Conclusions: In 60 patients with surgically resected lung adenocarcinoma, EGFR mutations and amplifications were found in 22% and 20%, respectively. Mutations referring to both drug-sensitivity and resistance were detected and were more frequent in non-smokers and females, the rate of resistance mutations being 15%. Hence, assessment of EGFR status prior to targeted therapy is suggested.

Poster: Varia

Aims: Here we report about a new, standardized method for obtaining baseline kidney biopsy specimens at the time of transplantation. Instead of taking wedge biopsies, a skin punch biopsy tool was utilized for this purpose, to obtain a biopsy sample also representative of the deeper cortical zones. Besides providing a more adequate sampling of small arterial vessels, this method allows a prompt sutural closure of the biopsy defect. Methods: We compared 80 biopsy specimens taken by either method (35 wedge biopsy specimens vs. 45 punch biopsies) with respect to the number of glomerula and arterial vessels they contained, as well as their predictive value regarding chronic donor-related damage by comparing baseline biopsies with early allograft biopsies of the same patient. Results: Although wedge biopsy samples contained a significantly higher number of glomerula, there was no difference regarding the number of small arteries. However, “punch” biopsy samples, when compared to traditional wedge biopsies, displayed a significantly lower standard deviation regarding the number of glomerula contained, indicating lower inter-individual variation of biopsy sample size. Both methods appeared to be reliable in detecting chronic donor-related damage. Conclusions: We conclude that the use of skin punch biopsy tools for obtaining baseline biopsies from transplanted kidneys is a safe and effective method for assessment of donor-related damage of the organ.

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Abstracts Fr-130 Quantitative melting curve analysis to identify gene copy number alterations T. Böhm, L. Fink, J. Wilhelm Institut für Pathologie, Universitätsklinikum Giessen und Marburg Aims: The loss of tumour suppressor genes and the gain of (proto-) oncogenes is a common event in tumourigenesis. The involved genes contribute to the phenotype of the tumour. Thus, knowledge about genes with altered copy numbers is important for tumour classification and choosing the best treatment strategy. Alterations of gene copy numbers should be detected using a simple, fast, and reliable technique. Methods: Multiplex PCR with subsequent melting curve analysis using SYBR Green I. The amplification of the target gene and a reference gene is multiplexed. The primers are designed to yield PCR products with different melting temperatures. The melting curves of the reaction measured after PCR gives two distinct melting peaks. The curves are corrected for temperature quench and the ratio of the peak areas is used as a measure of the relative target gene abundance. The comparison to the ratio obtained from non-neoplastic specimens is used to identify a copy number alteration in the tumour sample. Results: The proof-of-principle for the applicability of the method is given by the analysis of plasmid titrations simulating different degrees of copy number alterations. The method allowed the reliable detection of single allele losses or gains in as little as 20% of the cells. The method proofed to be robust against the number of PCR cycles performed and against the amount of material initially used. Although the results are not quantitative, the method performed considerably better in identifying copy number alterations than the quantification by real-time quantitative PCR. Conclusions: Quantitative melting curve analysis can reliably identify alterations in gene copy numbers and thus may be useful to detect losses of tumour suppressor genes or the amplification of (proto-)oncogenes in tumour samples.

Fr-131 Expression of new specialized keratins in squamous cell carcinomas of diﬀerent origin M. Divo, L. Langbein1, A. Schmidt, H. Grieﬁngholt, R. Moll Institut für Pathologie, Universitätsklinikum Gießen-Marburg, Standort Marburg 1 Deutsches Krebsforschungszentrum Heidelberg Aims: Keratins are a complex group of epithelial differentiation markers which may aid in identifying the origin of metastases or in stratifying certain tumor types. The family of human keratins finally has extended to 54 functional genes, with pronounced complexity in stratified epithelia, particularly in hair follicle structures. We have asked whether some newly identified, specialized (hair follicle-associated) keratins are expressed in squamous cell carcinomas (SCCs) and whether there may be correlations to their site of origin. Methods: Using specific antibodies against hair follicle-associated K75 (companion layer), K71 (inner root sheath) and K85 (hair keratin), we have immunohistochemically investigated 66 cases of primary SCCs (resection specimens) of various sites of origin (skin, oral cavity and pharynx, larynx, lung, cervix). Results: K71 and K85 were completely negative. Focal immunostaining (up to 5% of tumor cells) for K75 was found in 25 cases (38%). While the majority of larynx carcinomas (11/15 cases, 73%) was K75-positive, SCCs of other sites revealed positive cells in totally 14/51 cases (27%), showing statistical significance (p=0.002; Fisher’s exact test). There were no significant correlations to the grading and the degree of keratinization. Conclusions: In view of the pronounced restriction of K75 among normal tissues, its expression (albeit focal) in SCCs of various sites was surprising. This may be interpreted as a sort of aberrant diffe-rentiation in malignantly transformed cells. Future studies should clarify the mechanisms of gene regulation of K75 and the biological significance of the high incidence of K75 in larynx carcinomas. The study of larger series and of metastases is required to find out whe-ther K75 may contribute to predict the origin of metastatic SCCs.

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Fr-132 Two cases of retroperitoneal xanthoﬁbrogranuloma suggesting Erdheim-Chester-syndrom M. Gajda1, H.J. Holzhausen2, S. Oswald3, T. Seidel3, G. Neumann4, S. Schuenemann3, C. Bielecki3, A. Stallmach3, S. Hauptmann2, I. Petersen1 1 Institut für Pathologie, Universitätsklinikum Jena 2 Institut für Pathologie, Universitätsklinikum Halle-Wittenberg 3 Klinik für Innere Medizin II und 4 Institut für Diagnostische und Interventionelle Radiologie, Universitätsklinikum Jena Aims: To describe two cases suggestive for the diagnosis of Erdheim-ChesterSyndrom (ECS). Methods: One autopsy case was accessible to comprehensive macroscopic and microscopic evaluation supplemented by Immunohistiochemistry (IHC). In a second case multiple biopsies were available for routine stainings and IHC analysis. Results: Around one month after hysterectomy and adnexectomy a 54 year old female patient died from interstitial pneumonia and massive tumor manifestations in the mediastinum, heart, kidney and the femoral bones. The autopsy revealed a generalized retroperitoneal xanthofibrogranuloma. IHC showed a tumour infiltrate that was partially positive for S100 and CD68. The second case consisted of a 51 year old man with unilateral pain of the knee. Examinations by X-ray and CT showed an enlargement of the mediastinum with deviation of the trachea. Further tumour manifestation were suggested in the retroperitoneum, cerebrum, orbita and long bones (osteosclerotic foci). Biopsy of the retroperitoneum revealed a xanthgranulomatous, inflammation like lesion that by IHC was positive for MAC387, KP1 and negative for CD1a. Similar findings were detectable in a bone marrow biopsy. Both cases were interpreted a Erdheim-Chester-disease. Conclusions: ECS is a malignant histiocytosis with unfavorable prognosis. Originally though to be rare, our case reports add new findings to the meanwhile reported 250 cases.

Fr-133 A case report: accidently detected generalized amyloidosis in autopsy T. Gradistanac1, M. Mochalski2, C. Röcken3, C. Wittekind1 1 Institut für Pathologie und 2 Herzzentrum, Universitätsklinikum Leipzig 3 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte Aims: Amyloidosis can mimicry a giant spectrum of clinical features. This possibly results in therapeutic attempts which display only therapy of symptoms without really knowing the underlying disease. Additionally, genesis of amyloidosis is manifold. Therefore, comparing clinical features and histomorphological results as we do in autopsy can be helpful. Methods: Autopsy of a 68 year old woman was performed. Clinically was the patient for admission because of an insufficiency of the mitral valve. In the preoperative course she showed suddenly hypotony and bradycardy and died in succession because of an electromechanic decoupling. No further anamnestic readings were known. Results: Autopsy showed a generalized amyloidosis including the whole arterial vascular system with partial lumen stenosis especially in lung, myocardium, whole gastrointestinal tract, liver, kidneys and others. In lung and myocardium also interstitial amyloidosis with changes of tissue up to 75% in lung and 80% in myocardium with involvement of the heart valves and consequent heart hypertrophy and dilatation could be verified. Consequently an acute left- and right sided heart insufficiency as cause of death was detected. Conclusions: This case report emphasizes the importance of autopsy as a tool of revealing rare diseases and thus adding to quality management in medicine. This is particularly true for diseases as amyloidosis with a broad spectrum of symptoms.

Fr-134 Immunhistochemical detection of extracelullar matrix proteins, metalloproteinases and their tissue inhibitors in human non-small cell lung cancer (NSCLC): Diﬀerences between zone of growing edge and tumour centre E. Herpel1, C. Schopf1, D. Kieslich de Hol1, A. Warth1, H. Hoﬀmann2, P. Schirmacher1, P. Schnabel1 1 Pathologisches Institut, Universitätsklinikum Heidelberg 2 Chirurgische Abt., Thoraxklinik am Universitätsklinikum Heidelberg Aims: We investigated whether and to which extent there might be differences in the distribution of extracellular matrix proteins (MPs), matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs (TIMPs) within different areas of a primary tumour. Methods: Paraffin embedded tumour samples from 107 patients suffering from NSCLC (stage III, IV, control group stage I) were taken from the zone of growing edge and the tumour centre. Then immunohistochemistry was performed using antibodies against Collagen I and IV, Fibronetin, MMP-1, MMP-2, MMP-9, TIMP-1 and TIMP-2. Afterwards the volume fractions of the respective MPs, MMPs and TIMPs were analysed using the point counting method. Results: Collagen I and Fibronectin were expressed significantly higher (p=0.0001) in the tumour centre than in the invasion front, whereas collagen IV was expressed significantly higher in the invasion front. All MMPs were found significantly higher (p=0.0001) in the tumour centre than in the invasion front. No statistical significant differences were found for TIMP1 and TIMP-2. Conclusions: Zone of growing edge and central tumour parts differ in the composition of extracellular matrix proteins and the distribution of MMPs and TIMPs which suggests different processes in remodelling and may provide implications for specific therapy

Fr-135 Immunohistochemical Detection of Intrapulmonary / Mediastinal Lymph Nodes Metastases including Micrometastases in Non- Small Cell Lung Cancer: Relevance for Prognosis and Survival E. Herpel1, E. Palm1, D. Kieslich de Hol1, A. Warth1, H. Hoﬀmann2, P. Schirmacher1, P. Schnabel1 1 Institut für Pathologie und 2 Chirurgische Abt., Thoraxklinik, Universitätsklinikum Heidelberg Aims: (Micro-/)Metastasis to intrapulmonary and mediastinal lymph nodes is an important prognostic factor. Micrometastasis is defined as the presence of minimal amount of cancer cells that pathological examination cannot detect with hematoxilin/eosin-staining (H&E). We examined firstly, whether and how many micrometastases/ metastases are detectable by immunohistochemistry (IH) compared to H&E staining, secondly, whether this might influence the N-stage or the total disease free survival (DFS). Methods: We investigated all intrapulmonal, peribronchial and me-diastinal lymph nodes available from 172 patients suffering from non-small cell lung cancer (NSCLC). From each of the 5421 lymph nodes serial sections were performed and investigated by IH using cytoceratin markers KL-1, CK7, CK5/6, and the epithelial marker BerEP4. Then results were compared to the routine H&E staining. Results: H&E staining revealed 126 metastases/ micrometastases (M/ M), IH with KL-1 233 M/ M, CK 7 133 M/ M, CK5/6 15 M/ M, and BerEP4 52 M/ M. The N-stage was upgraded in 32 patients from pN0 to pN1 which was not associated with a reduction of DFS. In 9 patients it was upgraded from pN0 or pN1 to pN2 which was associated with a significant (p=0.007) reduction of DFS. Conclusions: IH may be a valuable tool in detection of metastases/ micrometastases in addition to H&E staining leading to a prognostic relevant upgrading of the N-stage. The upgrading from pN0 or pN1 to pN2 is associated with a reduction of the disease free survival.

Fr-136 The role of HIF hydroxylases in tumor angiogenesis and progression A. Klotzsche, J. Kalucka, B. Wielockx, G. Breier Institut für Pathologie, Universitätsklinikum Dresden Aims: Hypoxia plays a major role in tumor vascularization and progression. The cellular hypoxia response is mediated by hypoxia-inducible factors (HIFs) which in turn are regulated HIF prolyl hydroxylases (PHDs). The goal of our project is to elucidate the function of PHDs in tumor progression and metastasis. Methods: We use the following experimental approaches: (i) over-expression or inhibition of PHDs in tumor cells, (ii) endothelial selective over-expression of PHDs in tumor endothelial cells, and (iii) conditional ablation of PHDs in mice. Results: Mouse LM-8 osteosarcoma cells were stably transfected with expression vectors for PHD2 and PHD4. PHD-2 overexpression led to a reduction of HIF-1α and HIF-2α levels in hypoxic tumor cells, whereas PHD-4 affected HIF-2α only. Following transplantation into the hind limb of mice, LM8 cells transfected with hPHD-2 or hPHD-4 showed a significantly reduced tumor growth compared to control tumors. Analysis of tumor sections revealed a significant reduction of the vessel area in LM8 tumors overexpressing PHD-2 but not in tumors overexpressing PHD-4. Conclusions: Our data indicate that PHDs play an important role in tumor progression and that modulation of their expression might be a promising strategy for the treatment of cancer.

Fr-137 Paraﬃn tissue microarrays constructed with a cutting board, a board of steel spacers and a soldering iron U. Vogel, B. Bültmann Institut für Pathologie, Universitätsklinikum Tübingen Aims: The construction of paraffin tissue microarrays (PTMAs) consists of putting paraffin tissue core biopsies (PTCBs) from donor blocks (e.g. paraffin tissue blocks of daily pathological work) into preformed holes of recipient blocks (formerly blank paraffin blocks). Because of the different thickness of the tissue in the donor blocks the PTCBs are of different length. In consequence, the sections of the deeper portions of the PTMA don´t contain all of the PTCBs thereby diminishing the efficacy of the PTMA technique. Methods: To overcome this drawback and to cut the PTCBs to a certain length we manufactured a cutting board out of plexiglass with a thickness of 4 mm. 144 holes were drilled into this board which were filled completely by at least one PTCB. The holes of the PTMAs were poured in advance using a board equipped with 144 steel pins as spacers. When the holes of the cutting board were filled the PTCBs were injected from the cutting board into the holes of a PTMA simultaneously using the board of steel pins. Then, the paraffins of the PTMA and the PTCBs were melted using a soldering iron to ensure a solid contact of the paraffins. Results: Several PTMAs were constructed and cut with a standard rotary microtome. Per PTMA up to 1200 sections could be obtained which comprised nearly all of the 144 PTCBs. Using the steel spacers, the transfer of the PTCBs from the cutting board to the holes of the PTMAs was very comfortable and fast. Moreover, using the soldering iron for melting, the PTCBs did not topple over. Conclusions: Using the cutting board, the board of steel spacers and the soldering iron it was possible to construct efficiently PTMAs which did not show the thinning out of PTCBs in deeper sections.

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Abstracts Fr-138 Internet based telecytology using iPath H. Adams1, H. Spieler2, K. Kunze3, H. Neumann4, P. Dalquen1, K. Brauchli1, M. Oberholzer1, L. Bubendorf1 1 Institut für Pathologie, Universitätsspital Basel 2 Institut für Pathologie, Kantonsspital St. Gallen 3 Institut für Pathologie, Universitätsklinikum Dresden 4 Institut für Pathologie Nordhorn Aims: To test if the telemedical system iPath (http://ipath.ch) can be used for cytological diagnoses via the internet. Material and Methods: Representative microfotographs of 141 consecutive cytological cases together with a short clinical history were placed into iPath and independently evaluated by four external experts. Internal diagnosis was used as reference. The 141 cases contained 15 different categories including fine needle aspirations (37,6%), effusions (24,1%) and bladder washings (13,4%). The diagnosis was confirmed by histology, immuncytochemistry, FISH-analysis, or by unequivocal clinical data. Results: The reference cytologist (LB) diagnosed 60 (42.6%) cases als malignant, 10 (7.1%) as suspicious for malignancy, 69 (48.9%) as benign and 2 (1.4%) as most likely benign. In the telecytological consultations 69.7%–82.5% of all 564 diagnoses were correct (true positive or true negative; mean: 77.6%). When suspicious cases were considered as positive, benign and malignant diagnoses were concordant with the reference in 84,5% and 90.2%, respectively (mean values). 11,1% of the diagnoses were false negative, and 10.1% were false positive. False negatives were seen in non small cell lung cancer, breast cancer, and adenocarcinomas from various sites. False positive diagnoses were most common in the urinary tract (20,6%) and in pleural effusions (18,8%). Conclusions: iPath allows reliable cytological diagnoses but also highlight fields of discordance. The system offers the a wide range of possibilities for education in diagnostic cytology.

Fr-139 Comparison of miRNA expression proﬁles in fresh-frozen and FFPE samples A. Ferlinz1, S. Günther1, Y. Wang1, J. Li2, J.J. O‘Leary2, O. Sheils2 1 Applied Biosystems, Darmstadt 2 Department of Histopathology, University of Dublin, IRL Aims: MicroRNAs (miRNAs), non-protein coding RNAs, negatively regulate the expression of genes by translational repression of their targeted mRNAs. Archival formalin-fixed paraffin-embedded (FFPE) specimens represent excellent resources for biomarker discovery and the study of human diseases in general. These tissues have not been widely used due to the poor quality of RNA extracted from FFPE blocks. In our studies we examined the reliability of miRNA detection in FFPE samples compared to fresh-frozen samples. Methods: Nthy-ori 3–1, a normal thyroid follicular epithelial cell line that has been transfected with a plasmid encoding for the SV40 large T gene was grown to confluence. Suspended cells were aliquot and pelleted (a) snap frozen and (b) formalin fixed and paraffin embedded. RNA was extracted with either the mirVana miRNA Isolation Kit for the cell line or the RecoverAll Total Nucleic Acid Isolation Kit (Ambion) for the FFPE cells. MicroRNA expression levels were detected through quantitative PCR using the Applied Biosystems TaqMan microRNA assays and the Applied Biosystems 7900HT Fast Real-time PCR system. Results: RNA isolation from approx. 2×106 of FFPE cells and 1.7×105 snap frozen cells yielded in approx. 10 μg of total RNA each. There was a good correlation of miRNA expression pattern in between FFPE and snap frozen cells with R2>0.95. Conclusions: Degradation and modification of mRNAs makes a reliable gene expression analysis from FFPE samples challenging. However, small RNA molecules appear to be less affected by these processes and are recovered more easily. We confirmed that miRNA species may be successfully analysed from archival sources and be used as biomarkers for cancer diagnosis and prognosis.

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Fr-140 CAMPATH-1H: A potential therapy for skeletal tumors? R. Guenther1, A. Noske1, A. Gruetzkau2, P. Schlag3, P. Tunn3, M. Dietel1, I. Melcher4, K. Schaser4, H.U. Kasper5, V. Krenn6, C. Sers1 1 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte 2 Deutsches Rheumaforschungszentrum Berlin 3 Robert-Rössle-Klinik, Charité – Universitätsmedizin Berlin, Campus BerlinBuch 4 Zentrum für muskuloskeletale Chirurgie, Charité – Universitätsmedizin Berlin, Campus Mitte 5 Institut für Pathologie, Universitätsklinikum Köln 6 Gemeinschaftspraxis für Pathologie Trier Aims: Bone tumors are rare neoplasms of the skeletal system. Earlier investigations suggested that CD52, a GPI-anchored protein expressed on leucocytes and cells of the male genital tract, is overexpressed in mesenchymal neoplasms of the bone. The current study aimed to further analyze the expression of CD52 on several skeletal tumors and to evaluate the potential for treatment of CD52 expressing skeletal tumors with CAMPATH-1H, an antibody directed against the CD52 antigen. Methods: RT-PCR, immunohisto-chemical staining and flow cytometry were used to analyze the expression of CD52. MNNG/HOS osteosarcoma cells were treated with CAMPATH-1H and proliferation of the cells was measured using MTT-assays. Results: We confirmed the expression of CD52 mRNA and protein both in vivo and in vitro in benign and malign skeletal tumors and their non-neoplastic counterparts. In general, the malign tumors showed a higher CD52expresion compared to benign entities. Treatment of MNNG/HOS cells with CAMPATH-1H led to a complement- and antibody-dependent reduction of viable osteosarcoma cells. Conclusions: In this study we describe for the first time the extra- and intracellular expression of CD52 in mesenchymal tumors. Higher expression of CD52 in malignant tumors suggests a functional involvement of CD52 in tumor progression. CAMPATH-1H-induced reduction of tumor cell growth might indicate a potential use of this antibody in tumor therapy.

Fr-141 Overexpression of EphA2 and EFNA1 in human osteosarcoma and a potential role in oncogenic signal transduction R. Guenther1, A. Noske1, P. Schlag2, P. Tunn2, L. Herbst1, N. Schmidt1, P. Wachs1, M. Dietel1, V. Krenn3, C. Sers1 1 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte 2 Robert-Rössle-Klinik, Charité – Universitätsmedizin Berlin, Campus BerlinBuch 3 Gemeinschaftspraxis für Pathologie, Trier Aims: The interaction between the EphA2 receptor and its ligand EFNA1 plays a role in tumor growth and metastasis. Osteosarcomas are a common tumor type in children with a high metastatic potential. We used genomewide microarray analysis to identify genes differentially expressed between normal bone and osteosarcomas and found up-regulation of EFNA1 and EphA2 in osteosarcoma tissue. Methods: Expression of Ephrins in normal and malignant human osteogenic tissues was analyzed by immunohistochemistry. Signal transduction following EphA2 activation by the ligand was studied in a human osteoblast and several osteosarcoma cell lines using EFNA1/Fc soluble ligand. Results: The study verified an increased expression of EFNA1 and EphA2 in human osteosarcoma tissue as compared to normal bone. We observed timeand dose-dependent suppression of EphA2 expression in osteosarcoma cell lines through EFNA1/Fc. EphA2 and EFNA1 are targets of the Mek/Erk pathway and stimulation of EphA2 by EFNA1/Fc leads to an activation of Raf, Mek and Erk. This stimulation resulted in an activation of the transcription factors Elk1 and cJun. MTT-assays showed significantly higher proliferation of high EphA2 expressing MNNG/HOS cells after ligand-treatment. Conclusions: Our results show that EphA2 and EFNA1 are overexpressed in osteosarcomas suggesting a role of EhpA2 dependent signalling during osteo-

genic tumorigenesis. We suggest that a possible positive feedback loop regulates EphA2-expression through the activation of Raf/Mek/Erk activity.

Fr-142 microRNAome analysis in human germ cell tumors (GCTs) S. Günther1, L.H.J. Looijenga2, A.J.M. Gillis2, A. Ferlinz1 1 Applied Biosystems, Darmstadt 2 Department of Pathology, Josephine Nefkens Institute, Erasmus MCUniversity Medical Center Rotterdam, NL Aims: MicroRNAs (miRNAs), non-protein coding RNAs, negatively regulate the expression of genes by translational repression of their targeted mRNAs. miRNAs can play a role in cancer development both as oncogenes and as tumor suppressor genes. Our aim was the confirmation of previous results that miRNAs 371–373 are involved in overruling cellular senescence induced by oncogenic stress in GCTs. Methods: Samples were obtained after gonadectomy, before treatment by either chemotherapy or irradiation. Representative samples were snap-frozen in liquid nitrogen. RNA enriched for small RNA species was isolated using the mirVana miRNA Isolation Kit from Ambion. RNA quantity and quality were determined by a high-resolution electrophoresis bioanalyser (Agilent) and by UV-absorbance. Expression profiling of 156 mature miRNAs was done through quantitative PCR using the TaqMan MicroRNA Assays Human Panel from Applied Biosystems and a fully automated Applied Biosystems 7900HT Real-time PCR system. Results: After normalization to allow inter-sample analysis, the previous miRNA 371–373 cluster finding was confirmed. Hierarchical cluster analysis of the miRNA expression levels resulted in a clustering of the in vivo samples based on their maturation status. Conclusions: The clustering of the in vivo samples based on their maturation status parallels normal embryogenesis and supports the model that miRNAs are involved in regulating differentiation of stem cells, retained in GCTs. This is novel information on the role of miRNAs in the development of malignant GCTs.

Fr-143 Establishment of human thymoma and thymic carcinoma cell lines R.J. Rieker, V. Ehemann, M. Breinig, B. Gunawan1, C. Steger2, H. Dienemann3, P. Schnabel, H.J. Schulten1, C. Schlaeger4, B. Radlwimmer4, P. Lichter4, P. Schirmacher, M.A. Kern Pathologisches Institut, Universitätsklinikum Heidelberg 1 Zentrum Pathologie, Universitätsklinikum Göttingen 2 Institut für Pathologie, Medizinische Universität Innsbruck 3 Thoraxklinik Heidelberg- Rohrbach am Universitätsklinikum Heidelberg 4 Deutsches Krebsforschungszentrum Heidelberg Aims: Several new therapeutic approaches in advanced thymomas and thymic carcinomas emerged during the last years. However, no sufficient in vitro model has been available to analyze the molecular mechanisms responsible for thymic malignancies and to evaluate novel therapeutic targets. Therefore we established and characterized a stable thymoma and a thymic carcinoma cell line. Methods: Cell lines were characterised by immunohistochemistry, flow cytometric, cell cycle analysis, cytogenetic analysis and cell viability measurement. Results: FACS analyses revealed an aneuploidy of the poorly differentiated, CD5 positive thymic carcinoma and the type B1 thymoma. The aneuploid cell fraction of the thymic carcinoma cell line was characterized by a high proliferation index of 55.9%, in contrast to a lower proliferation rate of the aneuploid cell fraction of the thymoma (19.7%). Array-based comparative genomic hybridization (aCGH) and conventional cytogenetic analysis of the thymoma revealed only minor imbalances whereas the thymic carcinoma was characterized by a complex karyotype in the hyperdiploid range that was readily defined with multicolor FISH (mFISH). Application of a selective COX-2 inhibitor dose-dependently reduced cell viability in both cell lines.

Conclusions: In summary, we established the first stable human thymomaand thymic carcinoma cell line, offering the possibility to perform extensive in vitro studies that are a basis for the analysis of novel therapeutic targets.

Fr-144 Fibroblasts derived from diﬀerent origins induce distinct eﬀects in a 3D co-culture carcinoma model C. Rupp1, H. Dolznig1, C. Puri1, W. Sommergruber2, P. Garin-Chesa1,2 1 Klinisches Institut für Pathologie, Medizinische Universität Wien 2 Boehringer Ingelheim Austria, Wien Aims: Recent evidence indicates that activated cancer associated fibroblasts play an important role in tumour progression. Expression profiling studies using cultured fibroblasts from different anatomical locations have shown to display distinct gene expression signatures. This heterogeneity may also affect tumour cell migration, proliferation and survival during invasion and metastasis. To test this hypothesis we study the effects of different fibroblasts on tumour cells in a recently developed 3D co-culture system. Methods: We have established an in vitro model in which human fibroblasts derived from skin, embryonic, and adult normal colon or freshly isolated from colon carcinoma samples, are co-cultured with human tumour cell spheroids (LS174T) in a 3D-system in the presence of extracellular matrix (collagen I). Realtime RT-PCR and Western blot analysis were used in combination with live-imaging microscopy to monitor tumour cell growth and invasion. Morphological analysis and IHC on FFPE- and frozen samples and in gel IF studies were performed, followed by confocal microscopy. Results: Invasion of tumor cells into the matrix was significantly increased when tumor cell (LS174T) spheroids were cultured in the presence of cancerassociated or embryonic fibroblasts as compared to normal skin fibroblasts. In addition, data on tumor cell apoptosis, proliferation, and morphological changes will be presented. Conclusions: We could demonstrate that our model system is feasible to study the influence of fibroblasts of diverse origin on tumor cell growth behaviour. This system allows monitoring the tumor-stroma interaction in time course experiments at the morphological and molecular level.

Fr-145 Fluorescence in situ hybridization in the diagnosis of malignant mesothelioma in pleural eﬀusions S. Savic1, B. Bode2, H. Loosli3, P. Spieler4, R. Schönegg4, H. Moch2, L. Bubendorﬀ1 1 Institut für Pathologie, Universitätsspital Basel 2 Institut für klinische Pathologie, UniversitätsSpital Zürich 3 Institut für Pathologie, Universität Bern 4 Institut für Pathologie, Kantonsspital St.Gallen Aims: Cytological distinction of malignant mesothelioma from reactive mesothelial cells in effusions is notoriously difficult. Deletion of 9p21 and polysomy of chromosome 7 are common chromosomal alterations in malignant mesothelioma, and can be detected by FISH using the UroVysion™™ multitarget FISH assay (Abbott). We evaluated this assay in the diagnosis of malignant mesothelioma in pleural effusions. Methods: 47 Papanicolaou-stained smears from pleural effusions of histologically confirmed malignant mesotheliomas were retrospectively hybridized with the mixture of fluorescent probes to the centromeric regions of chromosome 3, 7 and 17 and to the 9p21 locus. 13 Papanicolaou-stained smears from benign pleural effusions with reactive mesothelial cells were tested as negative controls. Results: 38/47 (80.9%) pleural effusions with biopsy proven malignant mesothelioma had chromosomal aberrations with loss of 9p21 as the most common finding. All 13 benign pleural effusions with reactive mesothelial cells were FISH negative. Sensitivity, specificity, positive and negative predictive values for detection of malignant mesotheliomas by FISH were 80.9%, 100%, 100%, and 59%, respectively. Conclusions: UroVysion multi-target FISH is a helpful tool in the diagnosis of malignant mesothelioma in effusions. Der Pathologe · Supplement 1 · 2008

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Abstracts Fr-146 Analyses of microRNA in tissues after formalin-ﬁxation U. Siebolts*, H. Varnholt*, U. Drebber, F. Schulze, I. Wedemeyer, H.P. Dienes, C. Wickenhauser, M. Odenthal Institut für Pathologie, Universitätsklinikum Köln *equally contributed

Fr-148 mRNA analysis of aged blood specimens S. Wiegert1,2, G. Mall1, I. Petersen2, M. Michael1,3 1 Institut für Rechtsmedizin, 2 Institut für Pathologie und 3 Institut für Transfusionsmedizin, Universitätsklinikum Jena

Aims: MicroRNAs (miRNAs) are short, noncoding RNAs, that interfere with and down-regulate cellular transcripts. They play a crucial role in carcinogenesis and miRNA expression profiling of human tumors has already identified signatures associated with tumor staging, progression and prognosis. However, formalin fixation causes RNA fragmentation of routinely processed pathological materials. Therefore, we studied the effects of fixation and prolonged storage in paraffin blocks on miRNA accessibility for diagnostic purpose. Methods: Mouse livers were fixed for 12, 24 and 72 hours in buffered or unbuffered formalin. In addition, routinely processed tissues of different human organs were taken. Total RNA from snap-frozen and deparaffinized and proteinase K lysed FFPE tissues were used for phenol based extraction. MiRNA122 and miRNA-16 were reverse-transcribed and amplified by Real Time PCR using reagents from Applied Biosystems. Results: Quantification of the liver specific miRNA-122 demonstrates equal miRNA accessibility after cryoconservation and different times of fixation. No reduction in miRNA accessibility was observed after prolonged fixation times (72 h). MiRNA could be quantitatively analysed in biopsies from different organs, such as liver, colon and mamma, even when stored for more than 20 years. Conclusions: In conclusion, PCR validation revealed an adequate quality of miRNA obtained from FFPE tissues regardless of length of storage, which is suitable for quantitative analyses of miRNA pattern. Thus we have shown that the routinely processed FFPE tissues are suitable materials for comprehensive miRNA expression analyses using real-time PCR.

Aims: Investigations of gene expression in autopsy cases are only rarely performed due to the assumed poor quality of the mRNA. However, it harbours potential important application, e.g. for the determination of the post-mortem interval. There is a particular lack of information regarding the early and late post-mortem phase. Methods: Whole blood samples from 6 living individuals were taken and stored for 178 days under room temperature. Total RNA was extracted at different time periods and mRNA expression of three genes (GAPDH, beta-actin, Interleukin-1 beta) was quantified by Real-Time-PCR (beta-actin) and by laser-induced fluorescence capillary electrophoresis after RT-PCR (all three genes). Results: mRNA was detectable over the entire time period for the two housekeeping genes while IL-1b expression was lost after 14 days. GAPDH was the most reliably gene accessible by fluorescence capillary electrophoresis. A decrease of beta-actin expression was clearly quantifiable by RT-PCR and an association between storage time and RNA-level/Ct-value could be determined. Conclusions: Our study could show that RNA from blood specimens stored at ambient temperatures is much more stable than generally assumed. Degradation is dependent on the gene type and occurs at different velocities. The information may thus be used for assessing the RNA quality of specimens used in diagnostics and research. After the investigation of the influence of environmental conditions it may be specifically adapted for the determination of post-mortem intervals.

Fr-147 Lymphatic Neoangiogenesis in Rheumatoid Arthritis Contributes to Traﬃcking of Immunologically Active Cells A. Soleiman, R. Kalt, I. Raab, K. Nagy-Bojarszki, S. Kandutsch, P. Birner, G. Schett1,2, J. Smolen2, D. Kerjaschki, J. Grisar 2 Klinisches Institut für Pathologie, Medizinische Universität Wien 1 Abteilung für Innere Medizin 3, Universitätsklinikum Erlangen 2 Abteilung für Rheumatologie, Medizinische Universität Wien

Fr-149 Targeting heterodimeric EGFR/HER2 receptor complexes with a novel bispeciﬁc small molecule tyrosine kinase inhibitor in EGFR dependent human malignant glioma cells proves a highly eﬃcient treatment approach S. Berezowska, A.-L. Grosu1, J. Schlegel2 Pathologisches Institut, Universitätsklinikum München (LMU) 1 Klinik für Strahlentherapie und Radiologische Onkologie, MRI, und 2 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München

Rheumatoid arthritis is an autoimmune disease that leads to joint destruction due to synovial autoimmune inflammation and systemic generalisation. Local organisation and dynamics of inflammatory cells are dependent on extensive synovial neovascularisation that provides transport conduits for immunologically active cells. However, exit routes for cell trafficking out of inflamed synovium leading to systemic propagation of autoimmunity are not well characterized. Utilizing a lymphatic endothelial marker panel, we demonstrate a massive TNF and IL-1b–triggered increase of synovial lymphatic capillaries in rheumatoid arthritis compared to osteoarthritis or normal. Lymphatic capillaries proliferate in areas of organized nodular infiltrates that contain B-, T-lymphocytes, macrophages and dendritic cells. Excess of proinflammatory cytokines TNF and IL-1b triggers lymphangioproliferation via synoviocytes thereby initiating NF-kB-dependent production of active lymphangiotrophic growth factor VEGF-C in vitro and in vivo Chemokine receptor CCR7+ cells are attracted by lymphatic endothelial-derived secondary lymphoid chemokine SLC/CCL21, accumulate around newly formed lymphatic vessels, enter the lumen and migrate with the lymph. These results suggest that lymphangiogenesis triggered by TNF/ IL-1b-driven production of VEGF-C in synoviocytes is involved in organisation of RA inflammatory infiltrates thereby providing exit conduits for immunologically active cells that bear the potential to propagate autoimmune disease.

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Aims: Primary Glioblastoma multiforme (GBM) belongs to the EGFR driven neoplasms. It concomitantly expresses high levels of HER2 receptors, and therefore we hypothesize, that targeted blockade of EGFR/HER2 heterodimeric receptor complexes will prove to be a most efficient and radiosensitizing therapeutical approach. Methods: Two primary human GBM cell lines with different amounts of EGFR and HER2 receptors (LN229: EGFR +, HER2+++; LN18: EGFR+++, HER2+) were treated with novel bispecific small molecule TKIs (NOVARTIS) and Tyrphostin AG1478, and irradiated with 0–6 Gy. Protein lysates were drawn 12 h after 48 h following treatment cells were assayed for proliferation or plated for clonogenic survival, which was assessed 10 days later. Results: The TKIs showed different effects in the cell lines. Proliferation of LN229 cells was efficiently and uniformly blocked at low concentrations, whereas growth inhibition of LN18 cells was only achieved at substantially higher concentrations. Bispecific TKI proved significantly more efficient than AG1478, especially in LN18 cell lines. The radiosensitizing effect of bispecific treatment was confirmed by colony forming assays. Western Blot analysis revealed the strong impact of bispecific TKI on both MAPK and especially AKT signaling pathways. Conclusions: Taken together our results underline the importance of EGFR/ HER2 heterodimeric receptor complexes for GBM cell proliferation and survival. Furthermore and clinically highly important we suggest that therapeu-

tic success in targeting the EGFR may not be proportional to the EGFR-levels expressed by the tumor cells.

Fr-150 2D-PAGE from formalin-ﬁxed and paraﬃn-embedded tissues P. Porschewski1, A. Weiss1, K.-F. Becker2 1 Qiagen GMBH Hilden 2 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München Aims: Formalin-fixed and paraffin-embedded (FFPE) tissues are a particular challenge for proteomic analysis. Consequently, any successful analysis using FFPE tissues has great impact on translational clinical research as more than a billion FFPE tissue blocks exists world-wide. In a first feasibility study, we wanted to demonstrate that it is possible to use proteins isolated from FFPE tissues in 2-D polyacrylamid gel electrophoresis (PAGE) applications. Methods: Proteins were extracted from different FFPE tissues and quantified using the method of Lowry. Three 10 μm sections were used for each protein extraction. Western blot analysis for beta-actin was used to demonstrate reproducibility of protein extraction. 2D-PAGE was performed using 250 μg total protein per gel (24 cm, pH3–10). Results: Our data clearly show that it is possible to use proteins isolated from FFPE tissue in 2-D gel electrophoresis applications. We have used 2-D PAGE followed by visualization of the proteins using different staining methods and 2-D difference gel electrophoresis (DIGE) to reveal tyrosine kinase-dependent signalling pathways and to quantify single components of those pathways. Differentially expressed proteins could be analyzed by mass spectrometry. Conclusions: Based on our recently developed technology for the extraction of intact proteins from FFPE tissue (1) we have improved the current method which will now allow the analysis of millions of clinical samples by 2-D gel electrophoresis in a multiplex manner. Becker, K.F. et al. () J Pathol :–

Fr-151 Frequency of Mutations in Patients with Tansthyretin Amyloidosis – Sequencing of DNA Extracted from Formalin Fixed Paraﬃn Embedded Tissue M. Eriksson, T. Todorov1, J. Büttner1, M. Fändrich2, S.Yumlu, H. Schmidt1,3, C. Röcken Institut für Pathologie und 1 Medizinische Klinik mit Schwerpunkt Gastroenterologie, Hepatologie and Endokrinologie, Charité – Universitätsmedizin Berlin, Campus Mitte 2 Leibniz-Institut für Altersforschung, Jena 3 Transplantationshepatologie, Universitätsklinikum Münster Aims: Transthyretin (TTR) constitutes the amyloidic fibrillar protein found in hereditary TTR amyloidosis (ATTR) and senile systemic amyloidosis, earlier called systemic cardiac amyloidosis. Hereditary ATTR is due to mutations in the TTR gene, whereas only the wildtype of TTR is involved in the age-associated senile systemic amyloidosis. ATTR is routinely diagnosed immunohistochemically, but this does not reveal whether the disease is of the hereditary type or not. This study aims to examine the frequency of hereditary TTR amyloidosis in patients diagnosed with TTR amyloidosis. Methods: In total, 31 patients from 2006 and 2007 in Germany were diagnosed with ATTR. Total genomic DNA was extracted from formalin fixed paraffin embedded (FFPE) tissue biopsies from 21 of these patients and the four exons of the TTR gene were sequenced. DNA isolated from blood from two patients was previously analysed. For some patients, tissue was not available. Results: Of the 31 patients, 8 patients with mutation were found. Five mutations, including the most common mutation (p.30Met>Val) and one not previously described mutation were found. Patients with mutation were all under 80 years old. Conclusions: More than 20% of the ATTR patients studied carry a mutation in the TTR gene. The frequency of mutations in this gene in ATTR patients seems to be higher than previously believed.

Fr-152 Diagnosis of Systemic Amyloidosis by Luminescent Conjugated Polymers K. Ikenberg*#, K.P.R. Nilsson2*, A. Aslund3, S. Fransson2, P. Konradsson3, A. Aguzzi2, H. Moch Institut für Klinische Pathologie und 1 Institut für Neuropathologie, UniversitätsSpital Zürich 2 Department of Chemistry, Linköping University, S # Present address: Hautklinik, Universitätsklinikum Tübingen *Equally contributing authors Aims: For the treatment of amyloid associated diseases specific and sensitive diagnostics are essential due to the fact of a promising but in some cases aggressive treatment, depending on the subtype of amyloid. We investigated the diagnostic potential of Luminescent Conjugated Polymers (LCPs) which contain swiveling thiophene backbones the geometry of which modulates their fluorescence (in contrast to e.g. thioflavin T (ThT) and Congo red). Methods: Over 80 samples from more than 40 patients received at the University Hospital Zurich were analysed by conventional histology, immunohistochemistry and LCP-fluorescence-signaling. Results: We could demonstrate with different LCPs a strong colocalization of Congo red stained amyloid and LCP-fluorescent-signals, demonstrating a high level of specifity. Due to an intensive fluorescent signal and strong binding of the LCPs, the sensitivity is highly increased compared to Congo red staining. In addition, by using an individual anionic LCP (PTAA), we could define classes of stained deposites on a subset of tissue samples which were analysed on a tissue microarray. Correlation of histology (including immunohistochemistry), clinical data and LCPs-binding-profiles was additionally analysed and strongly supports these findings. Conclusions: Congo red staining will stay the gold standard for the detection of amyloid. Here we show that the additional use of LCPs as a cheap, reliable and fast staining-probe could provide the opportunity to diagnose amyloid associated diseases earlier (sensitivity) and more accurate (specifity) in routine diagnostics.

Fr-153 Cellular heparanase expression: a potential prognostic marker in squamous cell carcinomas of human head and neck tumours? C. Mogler1, G. Dyckhoﬀ2, C. Herold-Mende2,3, E. Jenetzky4, P. Beckhove5, B.M. Helmke1 1 Pathologisches Institut, 2 Hals-Nasen-Ohrenklinik, 3 Neurochirurgische Klinik und 4 Medizinische Biometrie, Universitätsklinikum Heidelberg 5 Deutsches Krebsforschungszentrum, Heidelberg Aims: Head and neck squamous cell carcinomas (HNCC) are characterized by a poor prognosis due to aggressive tumor growth. Expression of the extracellular matrix degrading enzyme heparanase was associated with poorer prognosis in several cancers. As recently described, cellular heparanase expression in HNCC also might have influence on prognosis and overall survival. Methods: We analyzed the cellular expression of the active form of heparanase in 71 human HNSCC using immunohistochemistry and compared the expression with the clinicopathologic data of the corresponding patients in order to validate potential prognostic significance. Results: Heparanase expression was detected in 40 of 71 (56.34%) tumor cases. Heparanase was localized in disseminated tumor cells, in tumor cell clusters and in tumor cells at the tumor invasion front. Results were compared with clinical data. In statistical analysis our comparison showed significant benefit on overall survival and could not confirm preexisting data. With involvement of TNM classification as a prognostic significant value impact of heparanase lost significance. Conclusions: According to our data, cellular heparanase expression detected by immunohistochemistry seems to have life prolonging influence on pa-

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Abstracts tients with squamous cell carcinomas of human head and neck tumors but does not have equal explanatory power compared to TNM classification.

Fr-154 Case-report: Mutation of Fibrinogen Aα Chain Gene in a Patient with Hereditary Renal Amyloidosis M. Knoess1, P. Knoess1, M. Otto1, T. Ackermann2, F. Gohlke2, C. Röcken3, J. Kriegsmann1 1 Zentrum für Histologie, Zytologie und Molekulare Diagnostik, Trier 2 Nephrologische Schwerpunktpraxis Dialysezentrum, Mechernich 3 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte Aims: We report on a 63 year old patient presenting with a rare mutation in the fibrinogen Aα chain gene and resulting in renal amyloidosis. Methods: Case report and review of literature. Results: The patient presented with nephrotic syndrome with deficiency of immunoglobulin G and M and renal insufficiency, hyperphosphatemia, hyperuricemia and anemia. The renal biopsy was examined by light and electron microscopy as well as immunohistochemistry and revealed fibrillary glomerulopathy. The diagnosis of renal amyloidosis was established. Further xaminations first failed to determine the type of amyloidosis. Blood analysis then detected a mutation in the fibrinogen Aα chain and therefore, the diagnosis of renal amyloidosis associated with this condition was proved. Conclusions: This is one of the very rare cases of renal amyloidosis caused by mutation of fibrinogen Aα gene.

Fr-155 Microarray analysis of Ewing’s sarcoma family of tumors reveals characteristic gene expression signatures associated with metastasis and resistance to chemotherapy K.-L. Schäfer, U. Dirksen1, M. Eisenacher2, D.H. Wai3, R. Diallo-Danebrock, S. Heikaus, H.E. Gabbert, C. Poremba Institut für Pathologie, Universitätsklinikum Düsseldorf 1 Klinik für Pädiatrische Hämatologie und Onkologie Universitätsklinikum Münster 2 Integrierte Funktionelle Genomik (IFG), Universitätsklinikum Münster 3 Childrens Hospital of Los Angeles, CA, USA Aims: The aim of this study was to identify genes and signalling pathways, which are involved in metastasis and response of tumor cells to chemotherapy, since these parameters represent the clinically most important prognostic parameters of this tumor entity. Methods: Expression profiles of 27 ESFT specimens were analyzed using Affymetrix U133A microarrays. Results: Functional annotation of differentially regulated genes in metastatic vs. localized tumors revealed 29 over-represented pathways including PDGF, TP53, NOTCH, and WNT1-signaling. Regression of primary tumors induced by polychemotherapy was found to be correlated with the expression of genes involved in angiogenesis, apoptosis, ubiquitin proteasome pathway, and PI3 kinase and p53 pathways. These findings could be confirmed by in vitro cytotoxicity assays. A set of 46 marker genes correctly classifies these tumors as responding vs. non-responding. Conclusions: Expression signatures of initial tumor biopsies can help to identify ESFT patients at high risk to develop tumor metastasis or to suffer from a therapy refractory cancer.

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Fr-156 Quality control procedures at the tissue bank of the National Center of Tumor Diseases (NCT) Heidelberg: An overview on receipt, processing and delivery of tissue specimens E. Herpel1, J. Lehmann-Koch2, B. Goeppert2, C.V. Kalle2, P. Schirmacher1, F. Autschbach1 1 Pathologische Institut und 2 Nationales Centrum für Tumorerkrankungen (NCT), Universitätsklinikum Heidelberg Aims: To harmonize and to uniform the process of tissue banking at the NCT Heidelberg we developed uniform standard operating procedures (SOPs) and quality assurance measures (QA) for our participating institutions. Methods: SOPs and QAs regarding patient information and declaration of consent as well as the collection, freezing, storage, retrieval and tracking of tissue samples were generated. Operating theatre workflows for the handling of specimens were developed. We also generated management tools for the central database to govern associated clinical data, to coordinate requests and to provide project assistance when required. Regulations concerning the acquisition of tissues by researchers and sample delivery on request were formulated. Results: Not all SOPs and QAs were applicable at every institution. Some operational sequences, e.g. concerning the process of specimen handling at the operation theatre or the organization of patient information have to be adjusted to local conditions in the participating departments. Conclusions: Development of compulsory measures (SOPs) and process recommendations which have to be re-checked constantly are important issues to ensure high sample quality and useability. Total conformity of workflows within different participating institutions is not practicable

Fr-157 Comparative analysis of EGFR expression, gene copy number and mutations in early stage NSCLC S. Bode, J. Schirren1, S. Scheil-Bertram, A. Kruger, A. Fisseler-Eckhoﬀ Institut für Pathologie und Zytologie und 1 Klinik für Thoraxchirurgie, Dr. Horst Schmidt Kliniken (HSK) Wiesbaden Aims: Recent studies on EGFR aim at EGFR as prognostic and predictive factor in advanced NSCLC to identify patients who especially benefit from therapy with tyrosine kinase inhibitors. Methods: We analyzed expression, genomic gains and mutations of EGFR in 80 surgically treated NSCLC in early tumour stage (pT1pN0, pT1pN1, pT2pN0) having regard to tumour histology. EGFR locus was investigated by immunohistochemistry, FISH analysis and analysis of most common EGFR mutations in exon 19 and L858R. Results: EGFR expression was positive in the majority of enrolled cases (68%), preferentially in squamous cell carcinoma (14/15, 93%) and adenocarcinoma without bronchioloalveolar features (34/49, 69%). Genomic gain (amplification/high polysomy) was observed in 14 cases (18%) independently of histology, whereas 8 of 10 detected mutations concerned adenocarcinoma. There was no correlation between genomic gain and mutation status. All tumours with mutation or amplification also showed EGFR expression. No significant correlations between EGFR status and gender, median age at presentation, median tumour size, lymph node metastasis, median recurrence free survival and differentiation were observed, though there was a trend towards more frequently EGFR expression in G2 (38/49, 78%) than in G1 (16/31, 52%) carcinoma. Conclusions: Further studies on EGFR status in different entities of NSCLC could improve understanding of tumourigenesis and selection of patients for targeted therapies.

Fr-158 Multifactorial anti-cancer eﬀects of di-galloyl resveratrol encompass apoptosis, cell cycle arrest, and inhibition of lymphendothelial gap formation in vitro S. Madlener1, P. Saiko2, N. Stark1, R. Popescu1, M. Gridling1, N.T.-P. Vo1, I. Herbacek3, A. Davidovits1, S. Venkateswarlu4, S. Geleﬀ1, M. Grusch3, G. Trimurtulu4, D. Kerjaschki, M. Fritzer-Szekeres2, T. Szekeres2, G. Krupitza1 1 Klinisches Institut für Pathologie, 2 Klinisches Institut für Medizinische und Chemische Labordiagnostik und 3 Klinik für Innere Medizin I und Krebszentrum, Medizinische Universität Wien 4 Laila Impex Research Center, Unit I, Phase III, Jawahar Autonagar, Vijayawada 520 007, IND Aims: Di-galloyl-resveratrol (di-GA) is a synthetic compound aimed to combine the biological effects of the plant polyhydroxyphenols gallic acid and resveratrol, which are both radical scavengers and COX-inhibitors exhibiting anticancer activity probably be due to the adjacent free hydroxyl groups. Hence this compound was tested in HL-60 leukemia cells. Methods: Western blotting, HOPI-stain, FACS, microscopy, proliferation assay, HPLC, pulse labeling, cell co-cultivation. Results: di-GA activated caspase 3 and dose-dependently induced apoptosis. It further inhibited cell division in the G1 phase by three different mechanisms: downregulation of cyclin D1, induction of the Cdk-inhibitor p21Cip, and inhibition of ribonucleotide reductase activity, which resulted in reduced dCTP and dTTP levels. di-GA inhibited the retraction and subsequent gap formation of human lymphendothelial cells which was triggered by MCF-7 cancer cell spheroids. Conclusions: Gap formation in the lymphendothelium adjacent to a bulk of cancer cells can be considered as a port for metastatic spread. Therefore, diGA exhibited 3 distinct anti-cancer activities: anti-metastatic-, pro-apoptotic, and growth-inhibitory properties in vitro.

Fr-159 Evidence for a tumor suppressive function of PDGFRL in human cancer S. Seitz1, J. Strissel1, I. Petersen1,2, A. Meindl3 1 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte 2 Institut für Pathologie, Universitätsklinikum Jena 3 Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe, Universitätsklinikum München (LMU) Aims: Several lines of evidence suggest that PDGFRL (platelet-derived growth factor-beta-like tumor suppressor) is a putative tumor suppressor in human carcinogenesis. No biological function has yet been assigned to the gene. However, its homology to the ligand-binding domain of PDGFRβ may suggest its involvement in growth-control pathways. The aim of the study was to elucidate the role of PDGFRL in carcinogenesis. Methods: In this study, we carry out an extensive expression and genetic analysis of PDGFRL in human tumors of diverse tissue origin and transfection studies in human breast cancer cell lines. Results: We find that PDGFRL is significantly downregulated in breast, pancreas and thyroid tumors compared to corresponding normal tissues. Neither mutations in the coding region of the gene nor epigenetic mechanisms are responsible for loss of protein expression. Furthermore, we show that stable transfection of the gene into PDGFRL deficient breast cancer cell lines results in an increase of gene expression and a strongly reduced anchorage-dependent and -independent growth and absence of tumorigenicity in nude mice. Conclusions: Our data strongly suggest that human PDGFRL represents a (breast) tumor suppressor gene which complements a molecular defect in tumor cells resulting in a normalized cellular phenotype in vitro and tumor suppression in vivo.

Fr-160 Synchrotron μCT – an innovative tool for three dimensional tissue analysis C. Brochhausen1, R. Zehbe2, A. Heibel3, U. Gross4, H. Schubert2, C.J. Kirkpatrick1 1 Institut für Pathologie, Universitätsklinikum Mainz 2 Institut für Materialwissenschaften und Technologien, TU, Berlin 3 Deutsches Elektronen-Synchrotron -DESY, Petra III, Hamburg 4 Labor für Biomaterialforschung, Charité – Universitätsmedizin Berlin Aims: Three dimensional tissue analyses are an ongoing challenge for the morphological understanding of the dynamic processes in health and disease. Especially morphogenesis, cell-matrix-interactions, invasion and the structural organization of the interface between different tissue types are important targets. However, the feasibilities of such analyses are limited. In our study we report on the complete three dimensional characterization of different tissues (articular and epiphyseal cartilage) using synchrotron μCT (SR-CT). Methods: Knees from 4 week old Sprague Dawley rats including articular cartilage and the growth plate were embedded in paraffin for histological analysis. After that a representative part of the tissue was deparaffinised for SR-μCT analysis, which was performed at four levels. A Gd2O2S-scintillator was used with a CCD-camera to record projected images. The sample was rotated in steps of 180°/ 1200 and was exposed to the beam at 14 keV for 2.0 s. 3D reconstruction was performed using filtered backprojection. Results: In articular and growth plate cartilage the morphology of single cells was detected. Furthermore, the three dimensional cell distribution and organization of the interface between bone and cartilage was visualized. Conclusions: Our study shows for the first time the detection of single cells in non metal-labelled biological samples using SR-μCT in a quality equal to conventional histology. In current studies the 3D structure of cartilage damage and tumour invasion is being analysed. SR-μCT offers new and interesting perspectives for our morphological understanding of the steric dimensions of tissues and mechanisms involved in disease and repair.

Fr-161 Are tissue banks of paraﬃn blocks compromised by a mycotic contamination? H. Hannig, A. Dellmann, K. Donhuijsen Institut für Pathologie, Städtisches Klinikum Braunschweig Aims: Nowadays tissue banks of paraffin blocks are established institutions, suitable even for PCR methods. When conducting a PCR-detection of mycotic infections on paraffinized tissue the question arose whether a mycotic contamination of the paraffin blocks, which are often stored for several years, can lead to wrong positive PCR results. Methods: From the tissue bank of paraffin blocks of our institute we selected twenty blocks with histological proven aspergillosis and twenty blocks without mycotic infection, each material stored longer than five years. Fungal DNA was extracted using a standard protocol. Aspergillus DNA was detected with a Two-step-PCR, amplifying 747 base pairs of the Asp. fumigatus and 690 base pairs of the Asp. flavus. The PCR products were separated by the GeneScan-Analyzer. Results: The paraffin embedded tissue with histologically proved aspergillosis also revealed positive results after using the PCR method. Paraffin material from the aspergillosis-positive blocks showed just as negative results in the PCR as tissue blocks completely without aspergillosis. Conclusion: Our results indicate that there is no important risk of a mycotic contamination of paraffin-embedded tissue, especially with respect to aspergillosis, even after a storage of several years. Therefore, paraffin blocks can be used for PCR investigations.

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Abstracts Fr-162 Central Research Infrastructure for molecular Pathology (CRIP) C. Schröder1, K. Heidtke1, K. Zatloukal2, M. Dietel3, H. Stein4, H. Höﬂer5, A. Stege3, M. Hummel4, F. Bier1 1 Fraunhofer Institut für Biomedizinische Technik, Potsdam 2 Institut für Pathologie, Medizinische Universität Graz 3 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte 4 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin 5 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München Aims: Drug and biomarker development requires the analysis of large numbers of human tissue specimens in order to identify the most promising molecular structures. Pathology departments at university hospitals collected human tissue specimens for decades and processed them within the course of diagnostic routine. Access to these resources for research projects should overcome their fragmentation, enhance their annotation with clinical data, establish common SOPs and secure the donor’s privacy. Methods: CRIP represents a gateway to the tissue repositories located at different pathology institutes. The available tissue samples remain in the cooperating hospitals and are made accessible for research projects by an intelligent search tool and a semiautomated workflow. CRIP is in full compliance with ethical and legal standards and has been approved as a model concept by the “Berliner Beauftragter für Datenschutz und Informationsfreiheit”. Results / Conclusions: CRIP provides access to the anonymized data of annotated tissue specimens available in the cooperating institutes of pathology. It advances and facilitates the design and accomplishment of tissue-based research projects and allows the combination of specimens and data from different repositories in one research project. CRIP is open to further partner institutes. It supports harmonization of standards and may provide a German-Austrian node in an evolving European network of tissue banks.

Fr-163 Recruitment and activation of (myo)ﬁbroblasts in lung allografts undergoing chronic injury D. Jonigk, K. Theophile, K. Hussein, U. Lehmann, F. Länger, H. Kreipe Institut für Pathologie, Medizinische Hochschule Hannover Aims: After recent years have witnessed a breakthrough in the management of acute allograft rejection by new regimes of immunosuppression, chronic allograft dysfunction has emerged as the remaining major and unsolved problem of modern transplantation medicine. Long term loss of transplant function probably has diverse causes and uniformly leads to fibrotic remodeling of transplanted organs. Bronchiolitis obliterans (BOS) has to be considered the main cause for 5-year-survival after lung transplantation not exceeding 50 % on a worldwide scale. This project aims to gain a deeper insight into the mechanisms which govern the genesis of bronchiolitis obliterans (BOS). Methods: By combining laser microdissection with pre-amplification and consecutive quantitative real-time PCR on the one hand and double labelling with fluorescence microscopy on the other, the cellular sources of pro-fibrotic cytokines like TGF-β, SMAD 1/3/5, FGF(R), PDGF-α, etc in BO-lesions are analyzed. BO-lesions in lung allografts (n=15) are being compared to BOlesions, which have arisen due to other causes in non-transplanted lungs (n = 10). Results: So far the results of RT-PCR assays and immunohistochemistry show a distinctive pattern of gene expression in chronically injured lung allografts developing bronchiolitis obliterans, which differs from non-transplanted cases. Conclusions: BO-lesions in lung allografts appear to represent a unique entity of fibrotic remodelling in chronically injured transplants.

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Symposium: Pathologie und zielgerichtete Therapie (I) Sa-001 Targeting cancer stem cells? C. Klein Regensburg Sa-002 Tyrosinkinasen beim Nierenkarzinom H. Moch Basel Sa-003 YB-1 als neues Target H. Lage Berlin Sa-004 Breast cancer resistance protein expression in high-risk breast cancer subgroup treated with high-dose or conventional dose-dense chemotherapy E. Ting1, S. Mohrmann2, O. Gluz2, H.E. Gabbert1, U. Nitz2, C. Poremba1, R. Diallo-Danebrock1 1 Institut für Pathologie und 2 Brustzentrum, Universitätsklinikum Düsseldorf Aims: Breast cancer resistance protein (BCRP) is a half-molecule ATP-binding-cassette transporter that causes multi-drug resistance to various anticancer agents. In this study, we retrospectively evaluated the predictive impact of BCRP in a high risk subgroup of breast cancer patients (>9 axillary node metastases) who received high-dose (HDC) or dose-dense (DDC) conventional chemotherapy (WSG AM01 phase III trial, median follow-up of 48.6 months) and correlated these findings with the event-free survival (EFS) and the overall survival (OS). Methods: 403 patients were randomly assigned to DDC or to two courses of EC followed by two courses of HDC. Treatment arms were well balanced for age, menopausal status, tumor size, grade, number of node metastases and ER/PR status. BCRP expression was evaluated immunohistochemically using tissue microarrays containing breast cancer samples from 236 patients (with available tumor blocks). Results: There was a significant OS benefit for patients receiving HDC (p=0.00047). Those patients with BCRP-positive breast cancers who received HDC had significantly better OS (p=0,02) and EFS (p=0,01) compared to patients who received DDC. Conclusions: These results indicate that tandem HDC may reduce drug resistance since even high-risk breast cancer patients whose tumors expressed the drug-resistance protein BCRP had significant survival benefit from HDC compared to those patients who received DDC.

Sa-005 Analysis of EGFR status in NSCLC treated with Geﬁtinib S. Bode, N. Frickhofen1, J. Schirren2, C. Müller-Tidow3, A. Fisseler-Eckhoﬀ Institut für Pathologie und Zytologie, 1 Klinik Innere Medizin III, und 2 Klinik für Thoraxchirurgie, Dr. Horst Schmidt Kliniken (HSK) Wiesbaden 3 Labor für Hämatologie und Onkologie, Universitätsklinikum Münster Aims: Tyrosine kinase inhibitors are used in second line therapy of NSCLC in advanced tumour stages. Recent studies suggest that especially patients with somatic EGFR mutations benefit from this therapeutic strategy. Therefore 68 patients (m=44, f=24, median age 60y) with advanced NSCLC (stageIIIB/ IV) were treated with Gefitinib from 05/02 to 07/05. 75% of enrolled patients (51/68) had received chemotherapy before. Methods: Expression and genomic gain of EGFR locus was analyzed in 19 representive patients by immunohisto-

chemistry and FISH analysis. Most common somatic mutations (exon 19 deletion, L858R point mutation in exon 21) were investigated in 26 cases. Results were correlated to gender, smoking history and histologic tumour type. Results: EGFR mutations were detected in 15% (4/26), whereas EGFR expression and genomic gain was observed in 26% (5/19) and 16% (3/19) respectively. There was a correlation between subjective (23/68) or objective (22/68) response and EGFR status. Objective response was seen in all patients with genomic gain or mutation and in 4 of 5 cases with positive EGFR expression. Significant correlations between response and gender, smoking history and tumour type could not be detected. Conclusions: EGFR status, especially if measured by FISH analysis and mutation analysis, is an important predictive factor for clinical outcome of NSCLC patients, treated with Gefitinib.

Symposium: Beste Forschungsbeiträge Sa-006 Microbiological diversity analysis of extranodal marginal zone B-cell lymphoma of MALT-type in the lung (BALT lymphoma) by 16S rDNA heterogeneity analysis using RFLP and phylogenetics (SHARPScreening) P. Adam1, U. Hentschel2, C. Gernert2, S. Schmitt2, E. Haralambieva1, G. Ott1, H.K. Müller-Hermelink1 1 Institut für Pathologie, und 2 Zentrum für Infektionsforschung, Universitätsklinikum Würzburg Aims: For several anatomical localisations of extranodal marginal zone B-cell lymphoma of MALT type (eMZBCL) an association with chronic inflammation caused by microbiological agents (e.g. Helicobacter pylori in the stomach) has been described. In the lung a link between lymphomagenesis and a defined causative organism is missing. Methods: A comprehensive diversity survey using 16S-rDNA library construction followed by restriction-fragment-length-polymorphism (RFLP) analysis, sequencing and phylogenetic tree construction was employed on nine cases each of BALT lymphoma and control lung tissues (normal fetal lung, pneumonitis, cancer). Results: This highly sensitive method, hereafter termed “SHARP-Screening”, allowed for the identification of the entire bacterial population in the tissue in a cultivation-independent manner. Noteworthy, in eight of the nine cases of BALT lymphoma, bacteria of the Alcaligenaceae family (Alcaligenes, Achromobacter, AKIW733), were detected, whereas none of the control cases showed the presence of these clades. Conclusions: 16S-rDNA library construction in combination with RFLPscreening and phylogenetic analyses, hereafter described as “SHARP-Screening” is a novel, molecular and cultivation-independent tool for the analysis of the microbial environment in chronic inflammation processes, giving rise to extranodal marginal zone B-cell lymphomas of MALT-type. Betaproteobacteria of the Alcaligenaceae family may be affiliated with and possibly involved in the lymphomagenesis of BALT lymphomas.

Sa-007 In vivo roles of the glycosphingolipids Gb3 and iGb3 in hemolyticuremic syndrome and iNKT cell function S. Porubsky, B. Luckow1, M. Bonrouhi, A. Speak2, V. Cerundolo3, F. Platt2, H.-J. Gröne Zelluläre und Molekulare Pathologie, Deutsches Krebsforschungszentrum Heidelberg 1 Medizinische Poliklinik, Universitätsklinikum München (LMU) 2 Department of Pharmacology and 3 Weatherall Institute of Molecular Medicine, University of Oxford, UK The glycosphingolipids globotrihexosylceramide (Gb3, CD77) and isoglobotrihexosylceramide (iGb3) are isomers differing only in one glycosidic linkage and have been implicated in several processes of the innate and adaptive immune system.

Aims: 1) To verify the function of Gb3 in the pathogenesis of hemolytic-uremic syndrome as the cellular receptor responsible for cytotoxicity caused by verotoxin (VT) elaborated by Shigella and certain strains of E.coli. 2) To investigate in vivo the previously implicated function of iGb3 as the endogenous lipid ligand responsible for positive selection of invariant natural killer T-cells (iNKT; Zhou et al., Science 2004), which have an essential regulatory function in infection, tumor rejection and tolerance. Methods: Generation of mice deficient in Gb3 and iGb3 synthesizing enzymes. VT injection into Gb3-deficient mice. Analysis of iNKT cell development and function by flow cytometry and by administration of the exogenous agonist alpha-galactosylceramide in iGb3 deficient mice. Results: Ad 1) Gb3 deficient mice were insensitive to otherwise lethal doses of VT. Ad 2) iGb3 deficient mice showed normal numbers of iNKT cells. Furthermore the thymic development, T-cell receptor composition as well as the function of iNKT cells evolving in iGb3-deficient mice were unaffected. Conclusions: 1) Gb3 is the cellular receptor mediating verotoxin cytotoxicity in hemolytic uremic syndrome. 2) In contrast to previous indirect implications, iGb3 cannot be regarded as the endogenous iNKT selecting lipid ligand.

Sa-008 IHE: Modeling anatomic pathology workﬂow concerning the progress in the standards HL7 & DICOM T. Schrader, B. Beckwith1, M.G. Rojo2, J. Gilbertson3, C. Daniel4 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte 1 Department of Pathology, Havard Medical School and Beth Israel Deconess Medical Center, Boston, USA 2 Department of Pathology, Hospital General de Ciudad, Ciudad Real, E 3 Pathology Informatics Case Comprehensive Cancer Center, Case Western Reservere University, USA 4 ADICAP, INSERM UMR_S 729 Paris, Univ René Descartes, Paris, F Aims: For medical decision making, correct consistent and complete information is essential. The anatomical pathology as a diagnostic discipline has a central information exchange function and uses data from the clinical departments for the diagnostic process. The IHE (Integrating the Healthcare Enterprise) created an integration profile based on HL7 and DICOM standards. Methods: The integration profile “Pathology Workflow” (PWF) establishes the continuity and integrity of basic pathology data acquired by examination order for an inpatient as well as outpatient. Seven actors and six transactions are involved to maintain the consistency of information and speciment management. Results: The workflow of anatomical pathology was modeled concerning the aspects of ordering examinations, image and report management. The core of this model is the definition of a specimen identification and description model based on the standards DICOM & HL7. Conclusions: The Pathology Workflow is a cornerstone for a systematic quality assessment based on comparable models and workflow. The IHE supports the integration of different information systems.

Sa-009 Linking Gene Expression Signatures with von Hippel-Lindau Gene Mutation Types in Clear Cell Renal Cell Carcinoma V.D. Luu, P. Zimmermann1, K. Mertz2, G. Boysen, H. Moch, P. Schraml Institut für Klinische Pathologie, UniversitätsSpital Zürich 1 Institut für Pﬂanzenwissenschaften, ETH Zürich 2 Uniklinik für Dermatologie, Medizinische Universität Wien Aims: The von Hippel-Lindau (VHL) tumor suppressor is a multifunctional protein. VHL mutations are common in sporadic clear cell renal cell carcinoma (ccRCC). Different mutation types may alter specifically gene expression profiles, which consequently may have significant impacts on the course of the disease. We aim to identify gene expression signatures that correlate with specific VHL gene mutation types in RCC. Methods: We have selected 120 samples of clear cell and papillary RCC from the tumor biobank of the Zurich University Hospital for transcriptome analysis using Affymetrix gene chips. The sampling was chosen such that it allows the

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Abstracts study of signalling pathways in combination with the VHL gene mutation type in renal cell tumorigenesis using a sophisticated database (Genevestigator). Results: In a preliminary approach we compared the gene expression profiles of 22 ccRCC and 12 pRCC samples. By applying Significance Analysis of Microarrays (SAM) we identified genes which were differentially regulated among these two groups. Many of these genes are known biomarkers for ccRCC and pRCC, respectively. Hierarchical clustering based on the expression profile of the most differentially regulated genes resulted in a significant stratification between the two RCC populations. Conclusions: Our results clearly argue for the high quality of the tissue samples and the suitability of the chosen microarray high throughput platform. Correlations between gene expression signatures and specific VHL mutation types are currently being analyzed.

Sa-010 Immunophenotyping without antibodies – new perspectives in the characterisation of lymphomas M. Tinguely, A. Hofmann1, D. Bausch-Fluck1, H. Moch, B. Wollscheid1 Institut für klinische Pathologie, UniversitätsSpital, Zürich 1 Institut für Molekulare System Biologie, ETH, Zürich Aims: Cell surface proteins are key mediators for signal transduction and promising targets for the distinction of different lymphoma subtypes and subsequent therapeutic intervention. Here, we used gel-free proteomic technologies to identify differentially expressed cell surface proteins on human Hodgkin and B-NHL cell lines and compared them to the cell surface subproteome of normal lymphocytes and primary patient tissue. Methods: Cell Surface Capturing (CSC) technology was used combined with subsequent quantitative mass-spectrometric (MS) analysis to globally identify cell surface expressed proteins as classification markers. Differentially identified proteins were validated by IHC on a multi-lymphoma tissue microarray to obtain statistical significance. All proteins identified were annotated and integrated into the SISYPHUS Lymphoma Cell Surface Protein Atlas, a new open source repository for MS identified cell surface proteins. Results: We identified and relatively quantified more than 300 bona fide cell surface proteins from RAMOS B cells and patient derived lymphoma tissues. From the 80 CD annotated proteins so far identified, 17 were verified by IHC as potential classifcation markers. Conclusions: The CSC technology allows to globally identify cell surface proteins which can’t be identified and quantified otherwise. We hypothesize that quantitative cell surface protein expression patterns represent a molecular fingerprint for the classification of different lymphoma subtypes. Cell surface protein fingerprints allow for the new design of experiments towards the understanding of signal transduction networks within malignantly transformed cells.

Sa-011 Protumorigenic dysregulation of stathmin family members in nonsmall cell lung cancer (NSCLC) S. Singer1, E. Herpel1, M. Keith1, V. Ehemann1, P.A. Schnabel1, T. Muley2, M. Meister2, P. Huber3, H. Hoﬀmann2, P. Schirmacher1, K. Breuhahn1 1 Pathologisches Institut, Universitätsklinikum Heidelberg 2 Chirurgische Abt., Thoraxklinik am Universitätsklinikum Heidelberg 3 Abteilung Strahlentherapie, Deutsches Krebsforschungszentrum Heidelberg Aims: Stathmin is the founding member of a protein family of microtubuledestabilizers including the stathmin-like proteins SCG-10, SCLIP and RB3. It has been reported to be upregulated in different tumor entities but less is known about the functional relevance and redundancies of stathmin family members in NSCLC. Methods: Taqman-PCR and Westernblot was used for Stathmin, SCG-10, SCLIP and RB3 expression-profiling in 37 NSCLC patients comparing tumor/ non-tumor sample pairs. After siRNA mediated knock down of stathmin in NSCLC cell lines (Calu1 and Calu6) we measured cell viability (MTT-Assay), proliferation, apoptosis, cell cycle distribution (FACS), migration (Scratch-As-

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say), and invasion (3D-Invasion-Assay). Furthermore, the sensitivity to chemotherapeutic agents (Paclitaxel, Vinblastine, Cisplatin, Gemcitabin) and irradiation (2–10 Gy) was determined (MTT-Assay) after stathmin inhibition. Results: Stathmin and SCLIP were overexpressed in approximately 80% and 40% of NSCLCs, respectively. No significant induction of SCG-10 and RB3 was detectable. Inhibition of stathmin lead to reduced cell viability, proliferation, migration and invasion and sensitzed tumor cells to radio/chemotherapy. Although stathmin was fully depleted in both cell lines all effects were more prominent in Calu1 than in Calu6. Conclusions: Overexpression of stathmin exerts protumorigenic effects in NSCLC cells and may represent a favourable therapeutic target. Since Calu6 also showed overexpression of SCLIP our findings indicate that a functional redundancy of stathmin-like proteins may partially compensate the loss of stathmin expression.

Sa-012 U-HO1, A Novel Cell Line Derived From A Primary Refractory Case Of Classical Hodgkin Lymphoma P. Möller, A. Mader, T. Barth, S. Brüderlein Institut für Pathologie, Klinikum Ulm The Hodgkin cell line U-HO1 was established from a malignant pleural effusion of the 23-yr-old patient Andreas Mader during the end stage of refractory nodular sclerosing classical Hodgkin lymphoma. Since its establishment, U-HO1 has maintained stable characteristics during the last 2 years in vitro and has a doubling time of about 4 days under standard culture conditions. U-HO1 lacks HLA-ABC but expresses MHC class II antigens and surface expresses CD15 and CD30 in the absence of CD19 and CD20. Karyotype analysis of U-HO1 revealed an hyperdiploid karyotype: 50,XY,d el(1)(p13.2p31.1),der(2)t(2;10)(q35;q16.1)add(2)(p11.2).rev ish amp(2)(p13p23), t(4;6)(p12;p11.1),t(5;22)(q35.1;q13.2), +der(6)t(4;6)(p12;p11.1)del(6)(q22.3q26 ),der(10)t(2;10)(q35;q26.1),+t(12;18)(p11.1;q11.2),del(15)(q11.2q15),+der(18)t(1 2;18)(p11.1;q11.2),+del(20)(q13.1),ins(21;15)(p11.2;q11.2q15),der(22)t(5;22)(q35. 1;q13.2).ish t(9;19)(p24;?) [43]/ 50,sl,del(8)(q24.1)[6]/50,sl,del(7)(q36.3)[2]/ 50,sl,del(7)(q11.23)[2]. CGH analysis revealed the following imbalances: ish cgh dim(1)(p13p31)(p12q21), enh(2)(p13p23),dim(4)(q31.3qter), enh(6)(q22q27),enh(12), enh(18),enh(20)(q13.1pter). Fish analysis showed an amplification of REL and BCL-11A of about six fold on chromosome 2(p13p23). Thus, U-HO1 is prototypical for classical Hodgkin lymphoma in every aspect tested so far. However, compared to the conventional Hodgkin lymphoma cell lines, which have highly complex karyotypes, U-HO1 proved far less genetically aberrant suggesting that the few imbalances suffice to develop the full-blown phenotype of refractory Hodgkin’s disease.

Symposium: Orthopädische Pathologie Sa-013 Der neue Facharzt für Orthopädie: Neue Erwartungen des Orthopäden an den Pathologen W. Rüther Hamburg Sa-014 Diﬀerentialdiagnostik der Synovialitis L. Morawietz Berlin Sa-015 Chondrogene Tumoren des Skelettes G. Jundt Basel

Sa-016 Klassiﬁkation bei Protheseninsuﬃzienz und Partikelbestimmung M. Otto Trier Sa-017 Osteogene Tumoren K. Hauptmann Berlin Sa-018 Riesenzellreiche Tumoren des Knochens – Diﬀerentialdiagnose S. Lang Wien

Symposium: Pathologie und zielgerichtete Therapie (II) Sa-019 Inhibitoren von Histon-Demethylasen: Neue Möglichkeiten einer molekular gerichteten Therapie geting cancer stem cells? R. Büttner Bonn Sa-020 Biomarker als notwendige Prädiktoren molekular gerichteter Therapien T. Henkel Kassel Sa-021 Novel RUNX1 isoforms determine the fate of Acute Myeloid Leukemia cells by controlling CD56 expression S. Gattenlöhner1, E. Serﬂing1, A. Marx2, H.K. Müller-Hermelink1 1 Institut für Pathologie, Universitätsklinikum Würzburg 2 Institut für Pathologie, Universitätsklinikum Mannheim Aims: CD56 (NCAM) belongs to the family of Ca2+-independent cell adhesion molecules, CAMs showing abundant expression mainly in fetal neural tissues and several other organs postnatally. Strong CD56 expression occurs in ~15–20% of acute myeloid leukemias (AMLs) and is an independent negative prognostic marker. But what controls CD56 expression and whether it determines the aggressive AML phenotype is unknown. Recently, we found that the transcription factor RUNX1 (AML1) regulates CD56 expression in cardiac ischemia. Since RUNX1 is among the most frequently mutated genes in AMLs, we asked whether RUNX1 controls CD56 expression in AML cells. Methods: cDNA library synthesis and screening, RT-PCR and western blot, promotor studies and cell transfection, siRNA assay. Results and Conclusions: We show here that CD56 expression on AML cells correlates with an abnormal expression pattern of RUNX1 isoforms. Whereas full-length p48 RUNX1 (p48) upregulated CD56 in AML cells, three previously unknown shorter RUNX1 isoforms, p38a, p30 and p24, suppressed CD56 expression. Both p48 and CD56 induced nuclear translocation of NFkB and increased bcl2L12 expression, and inhibition of this pathway by siRNA-mediated p48 knock-down or NF-kB blockade, substantially increased apoptosis in CD56(+) AML cell lines. These findings indicate the potential for new therapy of CD56high AML by suppression of the “overactive” RUNX1/ CD56/NF-kB signalling pathway(s).

Symposium: Virtuelle Mikroskopie Sa-022 Virtuelle Mikroskopie und Routinediagnostik P. Hufnagl Berlin Sa-023 Virtuelle Mikroskopie und Bildanalyse G. Kayser Freiburg Sa-024 Virtual microscopy as a novel method for high-throughput evaluation of immunohistochemical stainings on breast cancer core biopsies from neoadjuvant clinical trials B. Müller, K. Schlüns1, S. Niesporek1, S. Loibl2, M. Komor2, G. von Minckwitz2, C. Denkert1 1 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte 2 German Breast Group, Neu-Isenburg Aims: Virtual microscopy is still an advanced method for histopathological evaluation of tissue sections. We developed a system for the high-throughput evaluation of immunohistochemical stainings on breast cancer core biopsies. Methods: Immunohistochemical stainings of various biomarkers were evaluated in a cohort of 219 patients who had been included in the GeparDuo trial, a multicenter study on neoadjuvant chemotherapy for breast cancer, performed by the German Breast Group [von Minckwitz G, J Clin Oncol 23, 2676 (2005)]. For immunohistochemistry, tissue microarrays (TMAs) were constructed from core biopsies. Stained slides were digitized by a Zeiss-Mirax slide scanner. Subsequently, the digital slides were evaluated using a custom made software for whole slide imaging. Results: A total of 121 slides with 2772 tissue samples were scanned and evaluated as digital slides. 119 out of 121 slides were scanned successfully. Navigation on the TMA and identification of small tissue samples was more convenient by the virtual microscope compared to conventional analysis. Expression of biomarkers such as ER and PR was comparable between TMAs and large sections from the core biopsies. Conclusions: For the first time, we used digital microscopy for the evaluation of a series of immunohistochemical stainings of a large cohort of breast cancer patients. TMA production from core biopsies and virtual microscopy are highly promising technologies for rapid and tissue-saving large-scale evaluation of immunohistochemical data.

Symposium: Freie Vorträge Gynäkologische Pathologie Sa-025 Loss of SFRP1 expression in ovarian cancer and its association with DNA promoter methylation J. Veeck, S. Alkaya, D. Koensgen1, R. Knüchel, J. Sehouli1, E. Dahl Institut für Pathologie, Universitätsklinikum Aachen 1 Klinik für Frauenheilkunde und Geburtshilfe, Charité – Universitätsmedizin Berlin Aims: In ovarian cancer, low-frequent methylation of the Wnt antagonist SFRP1 has recently been reported in a small cohort. Yet, SFRP1 expression has never been analysed in this disease. Our aim was to determine the level of SFRP1 expression and its correlation with epigenetic regulation in primary ovarian cancer and metastatic tissues. Methods: SFRP1 expression in normal/cancerous ovarian tissues and in metastatic tissues was studied by Northern blot, immunohistochemistry and

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Abstracts realtime PCR. Methylation-specific PCR was applied on malignant ovarian cell lines, primary ovarian carcinomas and distant metastases. Results: SFRP1 was strongly expressed in normal ovary and fallopian tubes, but reduced in 12/14 tumors when compared to its adjacent normal tissue. Additionally, 85% of tumors (n=46) showed significant expression loss. Realtime PCR of laser-microdissected normal/cancerous tissue pairs (n=10) revealed a median 20-fold downregulation in the tumour. Of 21 metastases, 16 (76%) had reduced SFRP1 expression. The SFRP1 promoter was methylated in 2 of 3 cell lines, 25% of 48 primary ovarian tumors and 33% of 21 metastases in clear association to mRNA expression loss (n=67; p=0.0016). Moreover, the methylation status was identical in 5 matched pairs of primary tumor and associated metastase. However, 58% of tumors exhibited loss of SFRP1 expression in the absence of SFRP1 promoter methylation. Conclusions: SFRP1 promoter methylation causes loss of SFRP1 expression only in a fraction of ovarian tumors, indicating that other inactivating mechanisms are active in this disease. Since expression and methylation patterns were similar in primary ovarian cancers and matched metastases, we suggest that loss of SFRP1 is associated with initiation, but not progression of ovarian cancer.

FIGO III, were constructed (two samples per case). Immunohistochemical expression of p53, EpCAM, hsp27, PTEN and CyclinE was examined. Immunostaining results were scored with regard to percentage of positive tumor cells (<10%, 10–50%, 51–80%, >80%) and relative immunostaining intensity (weak, moderate, strong). Correlation to survival was sought. Results: 236 cases of p53 (97.1%), 237 cases of EpCAM (97.5%), 238 cases of hsp27 (97.9%), 228 cases of PTEN (93.8%) and until now 97cases of CyclinE (39.9%) could be analysed successfully. Negative immunohistochemistry was observed in 39.1% for p53, 37.9% for EpCAM, 37.0% for hsp27, 50.6% for PTEN and 18.5% for CyclinE. 25.9% of the cases were strong positive for p53, 23.5% for EpCAM, 23.5% for hsp27, 9.1% for PTEN and 2.9% for CyclinE. None of the investigated proteins showed a correlation between expression (percentage and/or intensity) and shortened survival. Conclusions: Our results suggested that overexpression of p53, EpCAM, hsp27, PTEN and CyclinE may be generally a prognostic factor in ovarian carcinomas, but no specific protein was analyzed that could play a particular role in reduced survival of patients with advanced ovarian carcinoma (FIGO III).

Sa-026 A SNP in the 3’-UTR of the human MDMX gene is strongly associated with decreased disease-free and overall survival of ovarian carcinoma patients A. Böhnke1, J. Jung1, N. Sbezny1, A. Fiedler1, D. Kubitza1, E. Gradhand1, K. Zeng1, S. Hauptmann1, F. Bartel1 1 Institut für Pathologie, Universitätsklinikum Halle

Sa-028 Alpha-methylacyl CoA racemase in primary ovarian adenocarcinoma: expression and survival analysis S. Scheil-Bertram, A. du Bois1, P. Harter1, P. Tylus, A. Fisseler-Eckhoﬀ Institut für Pathologie und Zytologie und 1 Klinik für Gynäkologie und Gynäkologische Onkologie, Dr. Horst-SchmidtKliniken (HSK) Wiesbaden

Aims: The MDM 2 homologue, MDMX, is another critical negative regulator of p53 tumor suppressor protein. Numerous studies confirming a clinical significance of MMD2 have been published. However, only a few data exist so far, on the importance of MDMX alteration sin ovarian carcinomas. Methods: The aim of our study was therefore to determine the status of MDMX gene amplification, its mRNA and protein expression in a series of 107 ovarian carcinomas. We performed sequencing analyses to identify mutations and/or SNPs in the MDMX gene. Results: The MDMX gene was amplified in 15% of the cases. This was associated with a shorter average survival time of 35 months compared with 58 months for patients with a single-copy MDMX gene (p=0.048; log-Ranktest), and a 2-fold increased risk of tumor-related death (p=0.05). A SNP (SNP34091) was identified in the 3’-UTR, 31 bp downstream of the stop codon. The wild-type state (A/A) of SNP34091 was found more frequently in patients with high-grade tumors compared to low-grade carcinomas (62.5% vs. 45.5%) with the remaining cases being heterozygous (A/C). The A-allele was associated with increased expression of the full-length MDMX-mRNA and the MDMX protein. Importantly, in a multivariate Cox-regression we found that patients with ER-negative tumors and homozygous for the A-allele had an 8-fold increased risk of recurrence (p=0,009) and 7-fold increased risk of tumor-related death (p=0,014). Conclusions: Our data suggest that HDMX alterations, especially the SNP34091, play an important role in ovarian carcinoma progression.

Alpha-methylacyl CoA racemase (AMACR) is a mitochondrial and peroxisomal enzyme involved in the metabolism of isomeric transformation of fatty acids entering the beta-oxidation pathway. AMACR serves as a useful marker to establish a diagnosis of prostatic malignancy; however, limited information is available in regard to its presence in primary ovarian adenocarcinoma (OC). In previous studies AMACR was expressed weakly in up to 14% of ovarian neoplasms. Aims: We investigated AMACR expression within a spectrum of OCs and its correlation with patients’ survival. Methods: A retrospective study of primary ovarian serous adenocarcinoma treated by surgery and chemotherapy [2000–2004, n=71; median age 67 years; FIGO 3 IA-C, 6 IIA-C, 46 IIIB-C, 16 IV; Grading: 2 G1, 28 G2, 43 G3]. Multi tissue arrays of all cases were stained with the monoclonal anti-AMACR antibody p504S (clone 13H4). Results: The overall median survival was 43 months. 15% of ovarian carcinoma demonstrated AMACR immunoreactivity (IR). The Kaplan-Meier analysis revealed that the survival of patients with AMACR-positive OC was not significantly better (median survival 68 month; confidence interval 0.000 to 149.7). Conclusions: AMACR is expressed in a small subpopulation of OC, but this is not positively correlated with the outcome of advanced OC.

Sa-027 Protein over-expression of tumorgenesis relevant genes in advanced ovarian cancer: A comprehensive immunohisto-chemical analysis on tissue microarrays C. Völklein, J. Boda, A. Sendelhofert, A. Heier, T. Kirchner, D. Mayr Pathologisches Institut, Universitätsklinikum München (LMU)

Sa-029 Early lesions of the Breast F.A. Tavassoli New Haven

Aims: Clinical outcome in patients with advanced ovarian cancer (FIGO III) is worse. However, these patients show distinct differences in their time of survival, probably justified on different responsiveness to chemotherapy. The aim of this study was to assess relevant cancer genes by their protein overexpression (p53, EpCAM, hsp27, PTEN and CyclinE), which may have an impact on overall survival in this group of patients with poor prognosis. Methods: Paraffin embedded material and survival data of 243 patients were available. Tissue microarrays (TMAs) containing 243 invasive carcinomas,

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State-of-the-art lecture: Early lesions of the Breast

Symposium: Zielgerichtete Therapie beim Kolonkarzinom Sa-030 Aktuelle Therapie des metastasierten kolorektalen Karzinoms – was wünscht sich der Onkologe vom Pathologen? A. Reinacher-Schick Bochum Sa-031 Die Bedeutung der molekularen Pathogenese für die zielgerichtete Therapie beim kolorektalen Karzinom M. Kloor Heidelberg Sa-032 Molekulare Targets beim Kolonkarzinom: VEGF, EGFR – was noch? C. Röcken Berlin Sa-033 Tailored therapy in patients with malignant mediastinal tumors P. Ströbel1, P. Hohenberger2, A. Marx1 1 Institut für Pathologie und 2 Sektion Thoraxchirurgie und spez. Chirurgische Onkologie, Universitätsklinikum Mannheim Aims: Approximately 50% of patients with advanced thymomas and thymic carcinomas are candidates for second line treatments. Due to the rarity of the disease, no prospective data on such treatments are currently available. Using sophisticated molecular methods, we aimed at the prospective evaluation of critical signalling pathways in individual tumors to predict response to novel targeted treatments. Methods: Based on extensive previous work using a large collection of archival tumor samples analyzed by gene sequencing, microarray techniques and phospho-proteomics, new cases are routinely screened for activation of receptor tyrosine kinases and activation of intracellular downstream signalling cascades such as MAP kinases both at the gene expression and protein level by multiplex PCR and phospho-protein arrays. In selected cases, pre-clinical studies using ex vivo tumor cell cultures treated with potential drugs are performed. The results are prospectively transferred into therapeutic decisions by a committed interdisciplinary team. Results: 5 patients have so far been prospectively analyzed and treated according to the evidence of the most promising pre-clinical studies. However, the therapeutic response and further clinical course were variable, although our studies could also be used to re-design the chosen treatments. Conclusions: Preliminary experience with several patients shows the validity of our approach, but also highlights salient problems in translational research and the need for further and more extensive studies.

Symposium: Biobanking in Pathology Sa-034 Erfahrungen bei Einrichtung und Betrieb von BB A. Stege, M. Hummel Berlin Sa-035 Gewebebanking im NCT Heidelberg – Innovative Plattform für die Translationale Tumorforschung P. Schirmacher Heidelberg

Sa-036 Biobanking and Biomolecular Resources K. Zatloukal Graz Sa-037 Reproducible gene expression proﬁling of archived tissues using the QuantiGene Assay J. Davies, K. Wilkens, P. Turner, W. Yang, B. Maqsodi, G. McMaster, F. Wang1, J. Flanagan1, Y. Luo1 Panomics Inc., Fremont CA 94555 USA 1 Advanced Cell Diagnostics Inc., Fremont CA 94555 USA Formalin fixation of human tissue specimens for long-term storage modifies RNA, making it difficult to extract and recalcitrant to enzymatic manipulations such as reverse transcription. Therefore this sample type has not been amenable to PCR-based approaches to gene expression profiling. Panomics’ QuantiGene assay, however, overcomes the challenges associated with this sample type, enabling sensitive and reliable measurements of RNA in FFPE samples. The QuantiGene assay is a hybridization-based method for RNA quantitation based on branched DNA (bDNA) technology, obviating the need for RNA isolation, reverse transcription and amplification. Target RNA is captured directly from tissue homogenates and immobilized on a solid surface. Signal from captured RNA is amplified via sequential hybridization of DNA amplification molecules. Both singleplex and multiplex QuantiGene assays (up to 30-plex) are available. Here we demonstrate that QuantiGene assay signals are not affectted by formalin fixation, and are reduced only 2–4 fold even on highly degraded RNA typically found in FFPE tissues. We show that measurements of RNA from FFPE samples made by the QuantiGene assay were more sensitive and more reproducible than data from qRT-PCR assays. Finally, we demonstate the applicability of the QuantiGene assay signal amplification strategy to RNA in situ hybridization. Images from multiplexed in situ hybridization assays demonstrating single copy RNA detection will be shown. Therefore, the QuantiGene method can deliver sensitive, quantitative, and contextual information on RNA expression and localization in clinically-relevant samples.

Sa-038 Standardization and quality control procedures in establishing a tumor biobank for cancer research P. Schraml, S. Dettwiler, S. Steu, M. Baucamp, G. von Dach, M. Bawohl, M. Storz, H. Moch Institut für Klinische Pathologie, UniversitätsSpital Zürich Aims: In order to translate the knowledge of the complex nature of tumor-associated pathways into clinical applications it is necessary to analyze numerous, well-documented human tumor tissues at the DNA, RNA and protein levels. For this purpose we are establishing a tumor biobank by introducing standardization and quality control procedures. Methods: Standardization for collection, storage, processing, annotating, and tracking of frozen tumor samples and matched normal tissues. Results: A standardized procedure for tissue freezing and processing applicable to both intra-operative frozen section diagnosis and tissue banking was introduced 2006. Plastic molds that fit exactly in cryocassettes are used in which tissue pieces (up to 20×15×5 mm) can be placed and embedded in OCT. Tissues are snap frozen by immersing the closed cryocassette in pre-cooled isopentane at –80°C using the mechanic freezer SnapFrost®. To prevent desiccation the surface of cut tissue is re-sealed transparently with diluted OCT using a brush. About 800 frozen tumor samples are collected routinely per year. HE sections from all frozen tissue specimens are reviewed and documented. DNA, RNA and protein integrity is analyzed randomly. Conclusions: This procedure simplifies significantly tissue handling and processing, and preserves excellently the integrity of cellular and molecular structures in tumor tissues. By continuing the standardized upgrading of the Der Pathologe · Supplement 1 · 2008

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Abstracts tumor biobank in consideration of legal and ethical aspects, a significant potential for cancer research projects will be available.

Conclusions: We have developed tools and methods that allow for the effective easy integration of series of virtual microscopy educational slides into Moodle, an open-source e-learning system.

Symposium: Meet the Expert II Symposium: Freie Vorträge zu Varia Sa-039 Interstitielle Lungenerkrankungen K. Junker, Brasch Bremen Sa-040 Intraepitheliale Neoplasie/Dysplasie in chronisch entzündlichen Darmerkrankungen G. Baretton Dresden Sa-041 Entzündliche Lebererkrankungen H.P. Dienes Köln

Symposium: Virtuelle Mikroskopie Sa-042 Virtuelle Mikroskopie in Lehre und Ausbildung H.P. Sinn Heidelberg Sa-043 Virtuelle Mikroskopie als Werkzeug der Systempathologie N. Grabe Heidelberg Sa-044 Integration of Virtual Microscopy into an Open-Source E-Learning System M. Andrulis Pathologisches Institut, Universitätsklinikum Heidelberg Aims: Running a pathology course for general or systemic pathology that integrates virtual microscopy requires a suitable e-learning platform that allows for an effective linkage to the virtual microscopy (VM) server. This task is not trivial, because the interface should be intuitive, easy to be maintained and expandable since many different virtual images have to be made accessible per course. Methods: A standard installation of the e-learning software Moodle was configured for general pathology, neuropathology and systemic pathology courses. Moodle has a fixed and an user contributed set of modules that can be used for specific purposes, such as interactive training, testing, evaluation, and others. We have expanded this set of tools with an adapted version of the database module and scripts that perform the addition and linking of sets of virtual slides. Results: By the use of these methods within the Moodle framework we can manage e-learning courses that are easy to navigate for the student as well as easy to maintain for the administrator. The database module is used to display the text information for the virtual slides and provides thumbnail images and links to the image server. Linking and displaying preview images can also be accomplished with an external PHP script that is invoked using an iframe tag in the HTML module of Moodle. In addition to virtual microscopy, Moodle provides much other functionality to pathology courses, such as authentication, communication, monitoring, and more that are needed for a modern e-Learning system.

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Sa-045 MicroRNAs as regulators of Ras-dependent gene expression A. Gross, O. Tchernitsa, R. Schäfer Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte Aims: Transcriptome alterations underlie the oncogenic potential of the signal-transducing RAS proteins. In previous studies we performed genomewide surveys of mRNA expression patterns of RAS onocgene-transformed cells and identified several hundred mRNAs that were up-regulated as well as down-regulated on transition from the normal to the transformed state. Recently it was found that micro-RNAs (miRNAs), small, non coding RNAs, regulate about 30–40% of all genes by translational repression and mRNA degradation. Therefore, we compared miRNA expression profiles in normal epithelial cells and RAS-transformed derivatives. Methods: We interrogated microarrays representing the currently known and a number of predicted miRNAs. Cells were obtained from two different model systems for epithelial cancer. Transformation of human embryonic kidney (HEK) cells was achieved by transfection with mutated human H-RAS gene and transformation of rat ovarian epithelial cells (ROSE) with K-RAS gene, respectively. Oncogenic signalling downstream of RAS was blocked by the MEK inhibitor U0126. Microarray results were validated by QuantiMir RT-PCR. Results: We found 44 up- and 65 down-regulated miRNAs in transformed HEK and 29 up- and 51 down-regulated miRNAs in transformed ROSE cells. The contribution of the MAPK pathway to these changes was about 50% in HEK and about 80% in ROSE cells. Fifteen miRNAs were commonly up- and 33 down-regulated in the two cell systems. Conclusions: The integration of mRNA and miRNA expression profiles will allow a better understanding of gene regulation in cancer cells, elucidate the cellular circuitry at the systems level and provide novel tools for blocking oncogenic activity.

Sa-046 EGFR protein overexpression and EGFR gene copy number gains in prostate cancers A. Erbersdobler, T. Schlomm1, P. Kirstein, J. Köllermann, T. Steuber2, H.K. Chun2, A. Haese2, M. Graefen1, R. Simon, H. Huland1,2, G. Sauter Institut für Pathologie, Universitätsklinikum Hamburg-Eppendorf 1 Martiniklinik Hamburg 2 Klinik und Poliklinik für Urologie, Universitätsklinikum HamburgEppendorf Aims: Epidermal Growth Factor Receptor (EGFR) is a protein involved in progression of many cancer types and an important therapeutic target. To determine its role in prostate cancer we analyzed 2497 prostate cancers with clinical follow up data on the protein and DNA level. Methods: Tissue samples from each tumor were brought into a tissue microarray format and analyzed by immunohistochemistry and fluorescence in situ hybridization (FISH). A subset of cancers was also sequenced for EGFR exon 18–21 mutations. Results: Detectable EGFR expression was found in 18% of cancers and was significantly associated with high grade, advanced stage, and high risk for PSA recurrence (P<0.0001, each). FISH analysis with a dual labeling probe for centromere 7 and EGFR showed increased EGFR copy numbers in 3.3% of cases. EGFR copy number gains were mostly due to an overrepresentation of the entire chromosome. EGFR copy number increase was associated with EGFR protein expression (P<0.0001), high grade (P<0.0001), and advanced stage (P=0.0056). Only one cancer had a high level amplification with more than 20 EGFR gene

copies per cell. This amplification was heterogeneous involving only about 30% of tumor cells. EGFR mutations were not found in 35 analyzed cases. Conclusions: Increased EGFR expression is often seen in prostate cancer and associated with a high risk of tumor recurrence. The significant association of EGFR copy number gains with immunohistochemical positivity argues for a role of minimal gene copy number changes for protein expression.

Sa-047 Elucidating transcriptional networks triggering oncogenesis by systems analysis I. Stelniec, O. Tchernitsa, S. Legewie1, H.-P. Herzel1, C. Sers, R. Schäfer Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte 1 Institut für Theoretische Biologie, Humboldt Universität Berlin Aims: Genetic alterations in receptor tyrosine kinases (RTKs) and downstream effector proteins KRAS and BRAF have been recognized as a major driving force in many types of cancer. While the contribution of the RTK/ RAS/MAPK signalling pathway the genetic program, malignant transformation and clinical properties of cancer cells have already been investigated in some detail, the specific roles of transcription factors are still poorly understood. We are studying the function and hierarchical organisation of transcription factors deregulated in an ovarian cancer model system. Methods: Based on a genome-wide survey of transformation-associated target genes in KRAS oncogene-expressing rat ovarian surface epithelial cells (ROSE), we identified eight up-regulated transcription factors. Their function was analysed by transient RNA interference followed by gene expression profiling using a customized RAS pathway target microarray. Results: Transient silencing of transcription factor expression resulted in a significant reduction of anchorage-independent pro-liferation and reversed epithelial–mesenchymal transition. Micro-array analysis showed that these factors regulate 85% of target genes differentially expressed on transition from normaly to the KRAS-transformed state. Overlapping and distinct transcription factor-related target profiles helped to establish hierarchical relationships between them. Conclusions: Integrated approaches combining transcription factor knockdown and transcriptional profiling will help to better under-stand and interfere with intrinsic cancer progression.

Sa-048 Characterization of a novel human breast cancer cell line derived from a lobular carcinoma M. Christgen1, H. Bruchhardt1, D. Steinemann2, D. Gadzicki2, T. Focken2, T. Krech1, C. Hadamitzky3, M. Ballmaier4, B. Schlegelberger2, H. Kreipe1, U. Lehmann1 1 Institut für Pathologie, 2 Institut für Zell- und Molekularpathologie, 3 Institut für Anatomie und 4 Institut für Pädiatrische Hämatologie, Medizinische Hochschule Hannover Aims: Investigation of lobular breast cancer (LBC) at a cell biological level is limited by the challenge of maintaining primary LBC cells in vitro and the lack of well-characterized LBC cell lines. Therefore, we started to establish new cell lines from patients with lobular breast cancer. Methods: Carcinoma cells from the malignant effusions of a 72 years-old female with peritoneal metastases from lobular breast cancer were cultivated in vitro for now more than two years. STR-PCR, array-CGH, and FISH were employed for genetic characterization. Quantitative real-time PCR, immunohistochemistry, FACS and Western-Blotting were used for expression studies. Results: STR-PCR-based DNA fingerprinting of the new cell line, named IPH-926, demonstrated genetic identity with primary tissue of the patient. IPH-926 cells strongly expressed TACSTD1, encoding for ESA/EpCam, were Ber–EP4–positive, but lacked expression of CDH1 mRNA and E–cadherin protein. IPH-926 cells also showed a distinct genomic gain at 8p12-p11.2, spanning approximately from 35 to 43 Mb. Additionally to inactivation of E–cadherin, amplification of this genomic region encompassing the FGFR1 gene is a hallmark of LBC.

Conclusions: IPH-926 represents a novel human breast cancer cell line with characteristic features of lobular breast cancer and will serve as a valuable tool to further investigate this tumor entity.

Sa-049 Defects of the TASK1 potassium channel in adrenal glands cause disturbed adrenocortical zonation and hyper-aldosteronism A. Weber, S. Bandulik1, C. Sterner1, W. Wisden2, J. Barhanin3, R. Warth1 Institut für Klinische Pathologie, UniversitätsSpital Zürich 1 Institut für Physiologie, Universitätsklinikum Regensburg 2 Institute of Medical Sciences, Aberdeen, UK 3 IPMC, CNRS, Nice, F Aims: TASK1 (KCNK3) is a potassium channel highly expressed in adrenal glands. To characterize TASK1 function, adrenal glands of task1 knock-out (task1-/-) mice were morphologically and functionally characterized. To evaluate the significance of theses studies for human diseases, normal and diseased human adrenal glands were analysed for TASK1 expression. Methods: In situ hybridization, immunofluorescence, RT-PCR and Western blotting were performed on mouse and human adrenals. Blood pressure, heart rates, electrolytes, and aldosterone levels were determined in mice. Results: Task1-/-mice before puberty and adult female task1-/- mice showed a impaired mineralocorticoid homeostasis with severe hyperaldosteronism and arterial hypertension. This was paralleled by severe adrenocortical zonation defects in the outer adrenal cortex. Disturbed patterns of TASK1 expression were also seen in human adrenal adenomas. Conclusions: TASK1 is essential for adrenocortical zonation. The zonation defects in task1-/- mice shown in this study demonstrate a morphological basis for hyperaldosteronism even in the absence of adrenocortical hyperplasia or neoplasia. The disturbed patterns of TASK1 expression in human diseased adrenal glands suggest that TASK1 also plays a role in human adrenal gland diseases.

Poster: Gastrointestinale Pathologie Sa-050 Frequency of epidermal growth factor (EGFR) ampliﬁcation, mutation and overexpression in esophageal cancer and correlation to clinicopathological data U. Reichelt1, T. Dohrmann2, A. Quaas1, J.T. Kaiﬁ2, P.G. Schurr2, S.J. Gros2, S. Thieltges2, E.F. Yekebas2, A. Marx1, R. Simon1, J.R. Izbicki2, G. Sauter1 1 Institut für Pathologie und 2 Klinik und Poliklinik für Allgemein, Thorax und Viszeralchirurgie, Universitätsklinikum Hamburg-Eppendorf Aims: Epidermal Growth factor Receptor (EGFR) is a protein involved in progression of many cancer types and an important therapeutic target. The aim of our study was to rule out its amplification, expression and mutation status in a large set of esophageal cancers. Methods: Tissue samples from 245 esophageal cancers on a tissue microarray (108 adenocarcinomas (ADC) and 137 squamous cell cancers (SQCC)) and 117 matched metastasis were analyzed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). A subset of cancers was also sequenced for EGFR exon 18–21 mutations. Results: High-level amplification (EGFR/centromer 7 signal ratio ≥3) with more than 6 EGFR copies per tumor cell was seen in 10/108 (9.3%) of adenocarcinomas and in 6/137 (4.4%) of squamous cell cancer. Amplification was always linked to overexpression. Amplification was unrelated to survival, grading, pT or pN stage. EGFR mutations were not found in 60 analyzed cases. Conclusions: EGFR amplification often occurs in a significant fraction of esophageal adenocarcinoma. The strong concordance of EGFR status in primary and metastatic tumor (p≤0.0001) may further argue for the potential utility of anti-EGFR medications and might be worth further investigation in EGFR expressing esophageal cancer.

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Abstracts Sa-051 Prognostic and Predictive Impact of Glucose Regulated Protein GRP78 (BIP) Expression in Esophageal Adenocarcinomas R. Langer, K. Ott1, M. Feith2, H.J. Wester3, H. Höﬂer1,4 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München 1 Chirurgische Klinik, Universitätsklinikum Heidelberg 2 Chirurgische Klinik und Poliklinik und 3 Nuklearmedizinische Klinik und Poliklinik, Klinikum rechts der Isar der TU München

Sa-053 Combined analysis of Rac1, IQGAP1, Tiam1 and E-cadherin expression in gastric cancer A. Walch1, S. Seidl2, S. Rauser1, J. Deplazes2, R. Langer2, C. von Weyhern2, M. Sarbia3, M. Feith4, S. Gillen4, H. Höﬂer1,2, B. Luber21 GSF-Forschungszentrum, Institut für Pathologie, Neuherberg 2 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München 3 Institut für Pathologie Krankenhaus Lichtenberg, Berlin 4 Chirurgische Klinik und Poliklinik, Klinikum rechts der Isar der TU München

Aims: To evaluate the prognostic and predictive impact of Glucose Regulated Protein GRP78 (BiP) expression in adenocarcinomas of the esophagus. Methods: Gene expression analysis was performed by using quantitative realtime RT-PCR after mRNA extraction from formalin-fixed, paraffin-embedded tissue. Protein expression was determined by immunohistochemistry. For prognostic impact of GRP78 expression, primary resected carcinomas (n=137) and normal esophageal tissue (n=10) were investigated. Expression patterns were correlated with pathologic features (pT, pN, G) and patients´ overall survival. In order to determine the predictive value of GRP78, expression levels in pretherapeutic, preoperative biopsies of locally advanced carcinomas treated by neoadjuvant CTX (n=45) were correlated with histopathologic, metabolic (assessed by FDG-PET) and clinical tumor response to CTX. Results: In primary resected carcinomas GRP78 mRNA and protein expression correlated inversely with tumor stage (p<0.05) and differentiation (p<0.05) and there was a trend for a survival benefit for patients with higher GRP78 mRNA levels (p=0.07). In tumors treated by neoadjuvant CTX, responders had higher pretherapeutic GRP78 mRNA levels and none of the patients with a pretherapeutic 3–4 fold overexpression of GRP78 mRNA (n=6) showed nonresponse to CTX, but this differences did not reach statistical significance (p=0.08). Conclusions: Increased expression of GRP78 may be responsible for controlling local tumor growth in early tumor stages of primary resected easophageal adenocarcinomas. In addition, overexpression of GRP78 may also be associated with tumor response to CTX due to increased cellular stress in neoadjuvant treated adenocarcinomas of the esophagus.

Aims: Rho GTPases are a family of major regulators of E-cadherin-mediated cell adhesion that are implicated in the carcinogenic process by deregulated expression of the family members itself or of upstream modulators or downstream effectors. The aim was to determine the expression and prognostic significance of Rac1 IQGAP1, Tiam1 and E-cadherin in gastric adenocarcinomas. Methods: Gastric carcinomas of 76 patients were investigated immunohistochemically in a tissue microarray study for expression of Rac1, IQGAP1, Tiam1 and E-cadherin. Correlations with clinical and follow-up data were examined. Results: Moderate or strong reactivity for Rac1 was observed in 46% and for Tiam1 in 56% of tumors. Expression of IQGAP1 was present in 59% and of Ecadherin in 87% of tumors. Rac1 and E-cadherin expression were not related to prognosis, while trends pointing to favorable prognosis of patients with Tiam1 expression and a lack of IQGAP1 expression were observed. Expression of Rac1 was positively linked to IQGAP1 expression and tended to be inversely associated with expression of E-cadherin. Conclusions: Deregulated expression of the investigated markers in gastric cancer was observed, either upregulation (for Rac1 and IQGAP1) or downregulation (for Tiam1 and E-cadherin) occurs. Presence of Tiam1 and lack of IQGAP1 were related to prognosis.

Sa-052 HER-2/neu is homogeneously ampliﬁed in primary and metastatic gastric cancer A. Marx, L. Tharun, J. Muth, U. Reichelt, R. Simon, J.R. Izbicki1, E. Yekebas1, J.T. Kaiﬁ1, M. Mirlacher, G. Sauter Institut für Pathologie und 1 Klinik für Chirurgie, Universitätsklinikum Hamburg-Eppendorf Aims: To evaluate the potential applicability of trastuzumab we examined the pattern of Her2/neu amplification in primary and metastatic gastric carcinomas. Methods: A tissue microarray (TMA) was constructed from 169 primary gastric adenocarcinomas, 69 lymph node metastases and 4 hematogenic metastases. Slides were analyzed for HER2 amplification and overexpression, using FDA approved reagents for immunohistochemistry (Hercep TestTM; DAKO) and Fluorescence in situ Hybridization (FISH, PathVysionTM; Vysis-Abbott). Results: HER2 amplification was seen in 27 (16%) of 169 adenocarcinomas of the stomach. Amplification was typically high-level with more than 10 HER-2 copies per tumor cell. The analysis of 3–16 large sections from 8 highly amplified cancers and corresponding metastases did not reveal any heterogeneity of HER2 amplification. Conclusions: Her2/neu is homogeneously amplified in gastric carcinomas and their metastases. Attempts to assess the utility of trastuzumab in HER2 amplified gastric cancers may be justified.

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Sa-054 Inﬂuence of a tumor-associated extracellular E-cadherin mutation on activation of Rho GTPase Rac1 J. Deplazes1, M. Fuchs1, P. Hutzler2, H. Höﬂer1,2, B. Luber1 1 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München 2 Institut für Pathologie, GSF-Forschungszentrum Neuherberg Aims: Rac1 is activated in response to homophilic ligation of E-cadherin. Somatic mutations affecting the adhesive function of E-cadherin are frequently observed in diffuse-type gastric cancer. The aim was to determine the activation of Rac1 signaling in relation to a deletion of exon 8 of E-cadherin. Methods: MDA-MB-435S cells transfected with wild-type or mutant E-cadherin were analysed by Western Blot analysis, immunoprecipitation and and pull down-Rac1 activation assay. Results: We observed lower Rac1 activity levels in cells transfected with mutant E-cadherin as compared to the wild-type protein, indicating that the mutation affects Rac1 “outside-in signaling”. Modulating Rac1 activity by the use of dominant-negative or constitutively-active mutants of Rac1 revealed that cell adhesion in the presence of the mutation was restored by forced Rac1 “inside-out signaling”. Consistent with the lower Rac1 activity levels in response to the E-cadherin mutation, complex formation between Rac1 and its downstream effector IQGAP1 was reduced. Our previous observation that exon 8 deletion results in enhanced cell motility and that forced cell-matrix attachment counteracts this effect raised the possibility that the activity level of Rac1 controls cell migration. Here we demonstrate that motility-enhancement by mutant E-cadherin depends on low Rac1 activity. Conclusions: We define Rac1 as a molecular link between the decrease in cell adhesion and motility-enhancement observed in response to the mutation of E-cadherin.

Sa-055 EGFR regulates RhoA-GTP dependent cell motility in E-cadherin mutant cells A.R. Mateus1,2, R. Seruca1,3, J.C. Machado1,3, G. Keller2, M.J. Oliveira1, G. Suriano1,3, B. Luber2 1 IPATIMUP, Porto, P 2 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München 3 Faculdade de Medicina, Porto, P Aims: Gastric cancer associated E-cadherin germline missense mutations lead to significant functional consequences. The aim was to characterize the effect of four E-cadherin germline missense mutations on the interaction with EGFR. We challenged the hypothesis that E-cadherin mutations perturb its ability to bind to EGFR, leading to activation of EGFR, triggering activation of downstream effectors. Methods: Stably transfected CHO cells expressing wild-type or mutant Ecadherin were analysed by Western Blot, immunoprecipitation and wound healing assay. Results: We verified that missense mutations localized in the extracellular domain of the protein (T340A and A634 V) exhibited reduced stability of the EGFR/E-cadherin heterodimers in contrast to germline mutations localized at the cytoplasmatic domain of the protein (P799R and V832 M). We observed that cells expressing E-cadherin extracellular mutants displayed increased levels of phosphorylated EGFR upon ligand stimulation, when compared with cells expressing wild-type E-cadherin or intracellular mutants. We showed that upon treatment of E-cadherin extracellular mutant cells with the EGFR inhibitor, the increase of RhoA activation is abrogated and accompanied by decreased migratory behaviour. Conclusions: We demonstrate that E-cadherin-dependent EGFR activation contributes to enhanced cell motility, in a mechanism involving RhoA activation.

Sa-056 Immunohistochemical expression of KIT cellular eﬀectors in GIST is associated with prognosis: a TMA study L. Tornillo1, I. Zlobec1, S. Dirnhofer1, L.-G. Kindblom2, F. Haller3, L. Terracciano1 1 Institut für Pathologie, Universitätsspital Basel 2 Department of Pathology, Sagrenska Academy University of Göteborg, S 3 Institut für Pathologie, Universitätsklinikum Göttingen Aims: Gastrointestinal stromal tumors (GISTs) are mostly characterized by alterations of KIT or PDGFRA, which are the target of therapy with imatinib. The understanding of the signaling pathways downstream KIT may help to identify effector molecules that could be targeted with other drugs. We have studied the immunohistochemical expression of such signaling intermediates in a large series of GISTs. Our aims were: 1) to determine the feasibility of the immunohistochemistry (IHC) to study the expression of these molecules; 2) to correlate their IHC expression with pathologic features and survival. Methods: 507 cases of GIST were collected from the Institutes of Pathology of the Universities of Basel, Naples, and Göteborg and were used to build a tissue microarray (TMA). IHC staining for pp44/42, AKT1, pAKT, p70s6, pp90SRK, p42MAPK, mTOR, nTOR was performed. The fraction of positive cells was determined for each case and the result was correlated with the risk classification suggested by Fletcher and with survival. Results: There was a significant association between pp44/42 (p=0.008), AKT1 (p=0.001), pAKT (p=0.024) and p70s6 (p=0.028)and the risk classification of Fletcher. In the survival analysis AKT1 expression was associated with better prognosis in the whole series and in the low-risk group of patients. Conclusions: The expression of signaling intermediates may help in stratifying GIST patients in prognostic subgroups and therefore may identify which cases could benefit of targeted therapy. This work has been supported by the Novartis Stiftung für medizinisch-biologische Forschung

Sa-057 Pre-Imatinib Gastrointestinal Stromal Tumors with a Distinct Biphasic Pattern: CGH ﬁndings Indicate a Clonal Progression A. Agaimy, F. Haller1, B. Gunawan1, P. Wünsch, L. Füzesi1 Institut für Pathologie, Klinikum Nürnberg 1 Abteilung Gastroenteropathologie, Universitätsklinikum Göttingen Aims: To date, there are no data on the cytogenetic clonal evolution in preimatinib gastrointestinal stromal tumors (GIST) showing a distinct biphasic pattern. Methods: We evaluated six primary GISTs (three women and three men, mean age 66 yrs) by morphological analysis, and mutation analysis of KIT and PDGFRA as well as CGH. Results: Tumor size ranged from 42 mm to 200 mm (mean 106 mm) and were high-risk (n=4), intermediate risk (n=1) and low risk (n=1) according to the NIH consensus criteria. Tumors showed a mixed epithelioid-spindle cell histology (n=5), and pure epithelioid cell morphology (n=1). All cases expressed CD117 and variably CD34. Direct sequencing of KIT and PDGFRA revealed the same KIT (n=5) or PDGFRA (n=1) mutation in both components in all cases. CGH showed a mean of 8.5 and 3.5 chromosomal aberrations per tumor for high- and low-grade component, respectively. Clonal origin of both components could be observed but with different chromosomal progression. –14q was detected in 4/5 and 6/6 of low- and high-grade components, respectively. Loss of 22q was detected with almost equal frequency. Other common chromosomal alterations having frequently been associated with the higher risk component and/or metastasis included –1p/–1 (5 of 6), -–9p/–9 (4 of 6), –13q (4 of 6), –3p/–3 (3 of 6), +8q (3 of 6). Conclusions: Our results indicate that the different histological variants and phenotypic diversity in GISTs represent a selective clonal progression resulting from different chromosomal alterations during the course of the disease.

Sa-058Double resistance to imatinib and AMG 706 caused by multiple acquired KIT exon 17 mutations in a gastrointestinal stromal tumor F. Grabellus, P. Ebeling1, K. Worm, S.-Y. Sheu, G. Antoch2, K.W. Schmid Institut für Pathologie und Neuropathologie, 1 Innere Medizin (Tumorforschung) und 2 Institut für Diagnostische und Interventionelle Radiologie und Neuroradiologie, Universitätsklinikum Essen In the majority of gastrointestinal stromal tumors (GIST) its tumorigenesis is related to activating oncogenic mutations in genes coding for the receptor tyrosine kinases KIT (CD117) or platelet-derived growth factor receptor alpha (PDGFRα). The tyrosine kinase (TK) inhibitors imatinib was recently shown to be highly effective in GIST treatment. Although the majority of patients with advanced disease initially benefit from imitanib, a high percentage of patients subsequently develops acquired imatinib-resistance, often due to secondary mutations. We report on a patient with an advanced GIST in whom the development of a dual secondary resistance to both imatinib and the novel multikinase inhibitor AMG 706 was caused by multiple acquired KIT exon 17 point mutations. The occurrence of secondary exon 17 mutations in the region which codes for the A-loop of the TK2-domain of KIT seems to be one of the crucial events in the development of resistance of GISTs to different multikinase inhibitors. The understanding of the nature and timing of molecular events in GIST and its interaction with different tyrosine kinase inhibitors is a major challenge in the treatment of resistant tumors. The identification of specific secondary KIT mutations may provide important information for an adapted second line treatment with alternative targeted therapies.

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Abstracts Sa-059 European Society of Medical Oncology (ESMO) Clinical Re-commendations for Gastrointestinal Stromal Tumors – rele-vant for pathologists? E. Wardelmann1, H.-U. Schildhaus1, S. Merkelbach-Bruse1, P. Reichardt2, P. Hohenberger3, R. Büttner1 1 Institut für Pathologie, Universitätsklinikum Bonn 2 Klinikum für Innere Medizin, Medizinische Onkologie, Helios-Kliniken Bad Saarow 3 Sektion Chirurgische Onkologie und Thorax-Chirurgie, Chirurgische Universitätsklinik, Universitätsklinikum Mannheim

Sa-061 Type and density of immune cells within Barrett’s cancer predict clinical outcome S. Rauser, U. Jütting1, S. Tschernitz, D. Veith, K. Ott2, M. Feith3, R. Langer4, H. Höﬂer4, A. Walch Institut für Pathologie und 1 Institut für Biomathematik und Biometrie, GSF Neuherberg 2 Chirurgische Klinik, Universitätsklinikum Heidelberg 3 Chirurgische Klinik und 4 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München

Aims: Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract. Despite their low frequency as compared to gastrointestinal carcinomas with an incidence averaging 1.5/100,000/year they have changed the view on the relevance of the predictive value of pathologic evaluation by immunohistochemistry and molecular methods. Methods: In october 2007, an international gremium of 41 interdisciplinary experts for GIST discussed new ESMO guidelines for diagnosis, treatment and follow-up of these tumors. Results: Tumor samples should be fixed in formalin (Bouin fixation should be avoided because of impairment of molecular analysis). Frozen tissue collection was encouraged, preferentially with a patient’s informed consent. Tumor site, maximal tumor diameter and the relation to gastrointestinal structures should be recorded. CD117 expression should be proven by immunohistochemistry being aware of the possibility of false positive CD117 staining due to antigen retrieval. Mitotic count should be expressed per 50 HPF. Mutational analysis is strongly recommended in all GIST because of its predictive and therapeutic value. Conclusions: The molecular analysis of GIST plays an increasing role in the prediction of prognosis and treatment response as recommended by the new ESMO guidelines and should be available and standardized also in Germany.

Aims: The role of tumour-infiltrating immune cells in Barrett’s cancer (BCA) is unknown. The aim of this study was to examine its prognostic impact on the outcome of patients with primarily resected BCAs as well of BCA patients after neoadjuvant chemotherapy. Methods: The levels of the adaptive immune markers CD3, CD8 and CD45RO were analysed by immunohistochemistry and image analysis in 119 primarily resected BCA patients as well in 90 BCA patients, who underwent neoadjuvant chemotherapy. The findings were correlated with pathological and clinical parameters including patient outcome. Results: The presence of high levels of tumour-infiltrating immune cells was statistically significantly correlated with prolonged survival in primarily resected BCA patients (Overall survival [OS] CD3: P=0.0059; OS CD45: P=0.0152) as well in BCA patients, who underwent chemotherapy (OS CD3: P=0.0119; OS CD45: P=0.0318; OS CD8: P=0.0163). Moreover, high numbers of immune cells were associated with therapy response. Conclusions: Signs of an immune response are associated with prolonged survival both within primarily resected BCAs and within BCA patients who underwent neoadjuvant chemotherapy. These data support the hypothesis that the adaptive immune response influences the behaviour of human tumours. In situ analysis of tumour-infiltrating immune cells may therefore be a valuable prognostic tool in the treatment of BCA.

Sa-060 The prognostic signiﬁcance of the Epidermal Growth Factor (EGFR) Signalling Pathway in Barrett’s Cancer S. Rauser, B. Luber1, U. Jütting2, H. Braselmann3, B. Hilber, D. Veith, A. Voss, M. Feith4, R. Langer1, H. Höﬂer1, A. Walch Institut für Pathologie, GSF-Forschungszentrum Neuherberg 1 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München 2 Institut für Biomathematik und Biometrie und 3 Institut für Molekulare Strahlenbiologie, GSF-Forschungszentrum Neuherberg 4 Chirurgische Klinik, Klinikum rechts der Isar der TU München

Sa-062 The biological signiﬁcance of EGFR and HER2 in Barrett’s Cancer after neoadjuvant chemotherapy D. Veith, S. Rauser, B. Luber1, U. Jütting2, K. Ott3, M. Feith4, M. Motschmann, R. Langer1, H. Höﬂer1, A. Walch GSF-Institut für Pathologie, Neuherberg 1 Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar der TU München 2 Institut für Biomathematik und Biometrie, GSF Neuherberg 3 Chirurgische Klinik, Universitätsklinikum Heidelberg 4 Chirurgische Klinik, Klinikum rechts der Isar der TU München

Aims: The objectives of this study were to examine the expression status of EGFR and related downstream signalling molecules as well EGFR gene copy numbers in Barrett’s cancer (BCA). Methods: Tissue microarray sections of 118 BCA patients were analysed for the expression status of EGFR, phospho-EGFR, phospho-Akt and phospho-MAPK using immunohistochemistry (IHC). EGFR gene copy number was evaluated using Fluorescence in situ hybridization (FISH). The findings were correlated with pathological and clinical parameters including patient outcome. Results: High protein expression of EGFR and elevated EGFR gene copy number were statistically significantly associated with poorer overall (EGFR protein: P<0.0001; EGFR gene copy number: P=0.0316) and disease specific (EGFR protein: P<0.0001; EGFR gene copy number: P=0.0276) survival. Multivariate analysis showed that both overexpression and elevated gene copy number of EGFR were independent prognostic factors. Patients with phosphorylated Akt had a better clinical outcome compared to patients without activation of Akt (DFS: P=0.0246; OS: P=0.0305). No prognostic significance was found for phospho-EGFR, phospho-MAPK, centromere 7 and ratio of EGFR/centromere 7. Conclusions: A subset of BCA patients manifests both EGFR overexpression and EGFR gene copy number alterations and this is associated with a poor clinical outcome, suggesting a biological role of these alterations.

Aims: The role of EGFR and HER2 in Barrett’s cancer (BCA) patients after neoadjuvant chemotherapy is unknown. The aim of this study was to examine the impact of EGFR and HER2 on patient outcome of BCA after chemotherapy and as molecular factors for therapy resistance. Methods: We compared EGFR and HER2 status in resection specimen of BCA patients without (n=124) and with (n=97) neoadjuvant chemotherapy. Tissue microarray sections were analysed for the expression status of EGFR and HER2 using immunohistochemistry. EGFR and HER2 gene copy number was evaluated by Fluorescence in situ hybridization. Findings were correlated with pathological and clinical parameters including patient outcome. Results: BCA after neoadjuvant chemotherapy showed an increase for gene copy number changes within the interval of 4–6 (EGFR, p=0,005; HER2, p<0,0001). In contrast, there was a decrease for the gene copy number changes in the interval of 2.5–4 (EGFR, p=0,017; HER2, p<0,001). Gene copy number changes of EGFR were associated with therapy resistance (p<0,01). EGFR overexpression was associated with poorer overall (p=0,0017) and disease free (p=0,004) survival in BCA patients after neoadjuvant chemotherapy. Conclusions: Alterations of EGFR and HER2 play a biological role in BCA patients after neoadjuvant chemotherapy and may be important molecular markers for therapy resistance.

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Sa-063 Functional characterization of recombinant human acyl-CoA synthetase 5 variants N. Gassler, K. Raupach, J. Kopitz1 Institut für Pathologie, Universitätsklinikum Aachen 1 Pathologisches Institut, Universitätsklinikum Heidelberg Aims: Acyl-CoA synthetase isoform 5 (ACSL5) is abundantly expressed in human small intestinal mucosa acting in long-chain acyl-CoA synthesis. Recently, the ACSL5delta20 splice form has been molecular characterized, but little is known about its biochemical behaviour. Methods: Using a cDNA cloning approach, human ACSL5 and ACSL5delta20 were isolated from small intestinal mucosa and cloned in pQE-30Xa. Recombinant proteins were purified from E. coli M15 with Ni+-affinity chromatography. Functional characterization was performed with different biochemical techniques. Results: Both membrane proteins were expressed without forming inclusion bodies and were soluble in Tris-HCl, 0.1% sodium cholate, 1% triton X-100, and 1 mM EDTA. Digestion experiments revealed that the 6xHis-tag did not affect enzyme function. Monitoring pH dependency of enzyme activity ACSL5 displayed a clear bimodal behaviour with peak activities at pH 7.5 and pH 9.5, whereas ACSL5delta20 lost this characteristic and showed a more uniform performance over a broad pH spectrum. The data was substantiated by applying Lineweaver-Burk analysis to the different pH-optima. Conclusions: In human intestine, enzymatic activity of ACSL5 is probably regulated by differential splicing.

Sa-064 Analysis of LIM-only protein FHL2 in sporadic and hereditary colon cancer: new insights into tissue remodeling N. Friedrichs1, L. Gullotti2, R. Schuele3, R. Buettner1 1 Institut für Pathologie, Universitätsklinikum Bonn 2 Dipartimento di Scienze Farmacologiche, Palermo, I 3 Center for Clinical Research, Freiburg Aims: This study analyses, whether prometastatic factor TGFBeta1 stimulates FHL2 protein expression in fibroblasts activating progression of colorectal cancer by tissue remodeling. Methods: Western blot and immunofluorescence assays analysed the interaction of TGFBeta1 and FHL2 in embryonal fibroblasts. Cell migration assays displayed the migratory capacity of FHL2 wild type versus knock out fibroblasts. In addition, 55 sporadic and 57 HNPCC cases were analysed immunohistochemically using the markers TGFBeta1, FHL2 and alpha-SMA. Results: In vitro analyses of embryonal fibroblasts demonstrated an induction of FHL2 mRNA and protein expression by TGFBeta1. FHL2 wild type fibroblasts migrated faster than FHL2 knockout fibroblasts, illustrating the importance of FHL2 for cell mobility. In western blot, FHL2 protein expression was higher in colon adenoma/carcinoma tissue compared to matched normal colon tissue. In sporadic colon cancer but not in HNPCC a highly significant correlation between TGFBeta1 expression of tumour cells and FHL2 expression of tumour stromal cells was noted in the tumour invasion front. Sporadic colon cancer cases with FHL2 overexpressing stromal cells showed increased metastasis. Conclusions: TGFBeta stimulates FHL2 mRNA and protein expression in tumour and stromal cells. Overexpression of FHL2 in stromal cells was linked with metastasis in sporadic coloncer but not in HNPCC and might therefore serve as a new prognostic marker in sporadic colorectal cancer.

Sa-065 The PEA-15 protein regulates apoptosis, proliferation, and motility in colorectal carcinoma cells V. Funke1,2, K. Grund1,2, G. Gdynia1,2, S. Macher-Göppinger1, O.D. Wiestler2, P. Schirmacher1, W. Roth1,2 1 Pathologisches Institut, Universitätsklinikum Heidelberg 2 Deutsches Krebsforschungszentrum, Heidelberg Aims: The PEA-15 protein is a multifunctional phosphoprotein involved in various signaling pathways which determine survival, proliferation, and migration of cancer cells. Here, we investigated the cellular functions of PEA-15 in colorectal cancer. Methods: PEA-15 expression was analyzed by generation of a tissue microarray comprising 90 colorectal carcinoma specimens and immunohistochemistry. Assays for the measurement of apoptosis, cytotoxicity, proliferation, cell cycle, clonogenicity, migration, and adhesion were performed after transient or stable expression of PEA-15 in colon cancer cells. β-catenin and cytoskeletal proteins were examined by immunofluorescence microscopy. Results: PEA-15 was expressed in the majority of human colorectal cancer specimens. Increased expression of PEA-15 in colorectal carcinoma cells resulted in a strong protection from cell death induced by cytotoxic drugs (cisplatin, 5-FU) or by the death ligand TRAIL. Moreover, serum withdrawal-mediated apoptosis was substantially decreased in PEA-15-over-expressing colorectal carcinoma cells. PEA-15 caused a long-lasting phosphorylation and activation of the MAP kinase ERK1/2. Further, cytoskeletal components, morphology, adhesion, and clonogenicity were profoundly regulated by PEA15 expression levels. Conclusions: The PEA-15 protein is a regulator of apoptosis resistance, proliferation, and motility in colon cancer cells. Thus, PEA-15 might play a crucial role in chemoresistance, progression, and epithelial-mesenchymal transition in colorectal cancer.

Sa-066 Regulation of the EGFR ligands AREG and BTC through a MAPK and DNA methylation-dependent mechanism C. Sers, S. Fonfara, R. Kuner1, A.-S. Kelm, M. Dietel, R. Schäfer, W. Weichert Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte 1 Deutsches Krebsforschungszentrum, Heidelberg The EGFR tyrosine kinase has become one of the most interesting therapeutic targets in human NSCLC and colorectal carcinoma. While patients harboring EGFR abnormalities clearly respond to EGFR-targeted therapies, a fraction of patients remain refractory and little information on reliable biomarkers for therapy response is available. Aims: To further improve the understanding of EGFR function we investigated the regulation of EGFR ligands amphiregulin (AREG) and betacellulin (BTC) Methods: A genome-wide screen was used to identify genes deregulated in human colorectal cancer via oncogenic signaling and DNA methylation. DNA methylation-dependent activation of AREG and BTC was confirmed using real-time PCR and ELISA. Expression analysis in human colorectal tissue was investigated by immunohistochmistry. Results: AREG and BTC are highly expressed in tumor cell lines with active DNMT1. Genetic or pharmacological DNMT1 depletion, or inhibition of MAPK suppressed AREG and BTC. In colorectal carcinomas, AREG expression is correlated with tumor stage and is detected in 40% of the patients. Co-staining of AREG and EGFR suggested a correlation between AREG expression and membrane localization of the EGFR. Treatment of cells showing different AREG expression with EGFR inhibitors, indicates a differential sensitivity correlating with AREG expression. Conclusions: EGFR ligands AREG and BTC show an unusual regulation via DNA methylation and MAPK signaling. Their expression in colorectal tissues might indicate that EGFR sensitivity is partially dependent on ligand expression, which can be modulated using DNA methylation or pathway inhibitors.

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Abstracts Sa-067 Expression of EGFR, k-Ras and PTEN at the tumor center and the invasion front of colorectal adenocarcinomas S.E. Baldus, C. Luszak, K. Reinwald, E. Bollschweiler1, H.E. Gabbert Institut für Pathologie, Universitätsklinikum Düsseldorf 1 Klinik für Viszeral- und Gefäßchirurgie, Universitätsklinikum Köln Aims: Various signalling pathways related to cellular proliferation and survival are activated downstream of EGFR, including the Ras/ Raf/MEK/ERK as well as PI3 K/PTEN/AKT pathways. However, it was not elucidated up to now if the expression of these effectors in tumor cells at the tumor center or the invasion front represents an important factor in tumor progression. Therefore, we investigated if the degree of expression of EGFR, k-Ras and PTEN at the tumor center or the invasion front correlates with pathological parameters. Methods: Tissue microarrays containing tissues cores derived from the tumor center as well as the invasion front of 219 colorectal adenocarcinomas were investigated immunohistochemically. The patients did not receive any targeted therapy. Monoclonal anti-bodies directed against EGFR, k-Ras as well as PTEN and the EnVision/HRP system (DakoCytomation) were applied. Results: About 15% of the carcinomas showed a strong expres-sion of EGFR at the tumor center or the invasion front. On the other hand, most tumor cells were k-Ras and PTEN positive at both sites. Strong EGFR expression at the tumor center (but not at the invasion front) correlated with increased pT stage, but did not have prognos-tic impact. Strong expression of PTEN at the tumor center correla-ted significantly with worse survival in uni- and multivariate analysis. Conclusions: An increased EGFR expression at the tumor center is associated with local tumor progression, whereas PTEN positivity represents an independent prognostic marker in colorectal adenocarcinoma. Its role as predictor of response to EGFR-directed targeted therapies should be studied.

Sa-068 Intra-arterial methylene blue injection improves lymph node harvest in colon cancer specimens B. Märkl, T. Kerwel1, H. Jähnig, K. Wünsch, H. Spatz1, U. Hebick, H. Arnholdt Institut für Pathologie, Klinikum Augsburg 1 Allgemein-,Viszeral- und Transplantationschirurgie, Klinikum Augsburg Aims: A minimal number of 12 investigated lymph nodes (LN) is recommended by the UICC for the staging of colorectal cancer. Other authors demand numbers of up to 30 LN. In contrast, several studies show that even the recommendations of the UICC are not fulfilled in practice. Recently we found that intra-arterial Injection of methylene blue improves lymph node detection in colorectal specimens. Methods: We performed a prospective, randomized study where we compared 25 colon specimens using the new method (methylene group) with 25 cases using standard dissection technique (unstained group). The methylene injection was performed in the pathology laboratory immediately after receiving the specimen in fresh state. After overnight fixing, dissection was performed in both groups by manual technique. Additionally a fat clearance technique was applied afterwards. Results: The mean LN numbers differed highly significantly between the groups with 19±10 and 35±18 (p <0,001) in the unstained and the methylene groups, respectively. The difference between the groups increased further after fat clearance (mean: 24 vs. 42). Adequate staging with a minimal number of 12 LN was reached in all but one case of the methylene group after primary dissection. In contrast, 6 cases from the unstained group showed adequate staging only after additional fat clearance, 2 cases remained inadequate even after additional embedding of fat. Upstaging (IIA–IIIB) after fat clearance occurred in one case of the unstained group. Conclusions: Intra-arterial methylene blue injection is a novel, very simple and cost effective method to improve lymph node harvest in colon specimens.

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Sa-069 Span80 vesicle with brand-new lectin ESA induces speciﬁc apoptosis in Colon26 tumor cell line in vivo T. Miyazaki1,4, K. Kato2, F. Moriki2, H. Osawa2, Y. Fukuta2, K. Sakayama3, M. Nose4 1 Abt. für Zelluläre und Molekulare Pathologie, Deutsches Krebsforschungszentrum Heidelberg 2 Department of Materials Science and Biotechnology 3 Department of Orthopaedic Surgery und 4 Department of Pathology, Ehime University, J Aims: A novel liposome with sorbitan monooleate (Span80) as major constituent has been developed and reported(1). The vesicle, immobilized with Eucheuma serra agglutinin (ESA) and polyethyl-ene glycol (PEG), abbreviated as EPV, induced apoptosis in carcinoma cell lines in vitro. In this study, we aimed to elucidate the effect of EPV administration on tumor selectivity and induced apop-totic signaling pathways in vivo. Methods: Colon 26 murine carcino-ma cells were transplanted subcutaneously into 4-week-old female BALB/c mice followed by administation of EPV intravenously after 2 weeks. The tumors were harvested 3, 6 and 12 hours after injection. Morphological analyses, including apoptosis detection by TUNEL were carried out. Also, accumulation of 125I labeled EPV was evaluated. Apoptotic signal pathways were analysed by PCRarray and Western blot. Results: EPV administration suppressed tumor growth up to one fourth in size and induced marked apoptoses in tumor cells. Apoptotic promotion factors, such as Bad, Bak1 and Bax as well as tumor suppressor Trp53 were upregulated. Caspases were upregulated as well as activated. The apoptotic signal transducers upstream of caspase 9 as well as those down-stream were upregulated at 3 and 6 hours after the administration respectively. Furthermore, EPV targeted tumor cells selectively. Conclusions: These studies revealed that EPV could induce tumor selective caspase-dependent apoptosis in vivo. () Fukuda Y. et al. Anticancer Drugs. ():–, .

Sa-070 Colorectal Polyps in HNPCC- (Lynch-) Syndrome – Impact on Carcinogenesis and Screening Strategies B. Oberschmid, C. Engel1, J. Rüschoﬀ2, A. Müller3, R. Büttner4, N. Friedrichs4, P. Kahl4 Institut für Pathologie Universitätsklinikum Leipzig 1 Informatik, Universität Leipzig 2 Institut für Pathologie Nordhessen, Kassel 3 Chirurgische Klinik, Universitätsklinikum Göttingen 4 Institut für Pathologie, Universitätsklinikum Bonn und German HNPCC Consortium Aims: In carcinomas, analysis of mismatch repair (MMR) gene expression and microsatellite instability (MSI) are valuable tools to identify persons at risk for HNPCC. In precursor lesions, however, currently no validated screening strategy is available. Methods: Therefore, the prevalence of different polyp types was analysed in HNPCC patients using i.) the prospective German HNPCC study group data set with 1950 documented polyps and ii.) polyp samples of reference pathology (Bonn, Kassel). iii.) Finally, the MMR-deficient phenotype was tested in 71 adenomas of 36 HNPCC patients followed over a 5-year period. Results: The vast majority of polyps in the German HNPCC data registry are traditional adenomas (AD: 80.3%) and hyperplastic polyps (HP: 19.5%) with only 5 (0.2%) serrated adenomas (SA). The same holds true in the reference pathology collective (2 out of 120 polyps were SA, 49 HP, 69 AD in the Kassel series and in the Bonn series 3 out of 167 polyps were sessile serrated adenomas (SSA), 5 SA, 40 AD and 119 HP). In the 5-year follow-up study 7/31 (22.5%) adenomas that developed synchronously or metachronously distant to the MSIH carcinoma showed neither MSI nor loss of MMR gene expression. Conclusions: Serrated pathway polyps are rare in HNPCC and not all traditional adenomas in proven Lynch-syndrome exhibit MSI or loss of MMR gene expression. Early testing of precancerous lesions in individuals “suspect-

ed” of HNPCC needs careful data interpreta- tion shedding light on the importance of family history and further research on the early molecular steps of carcinogenesis in HNPCC.

Sa-071 CBX7: a new prognostic marker in colorectal cancer? L. Tornillo1, V. Carafa1, A. Lugli1, D. Baumhoer1, P.L. Pallante2, S. Sacchetti2, A.O. Ferraro2, A. Fusco2, L. Terracciano1 1 Institut für Pathologie, Universitätsspital Basel 2 Department of Cellular and Molecular Biology “Luigi Califano”, “Federico II” University, Naples, I Aims: Polycomb group proteins are important controller of cell fate that are involved in the genesis of different types of cancer. CBX7 is a component of the Polycomb repressive complex 1 that has been shown to have a role in the genesis and progression of different tumours. Our aim was to characterize the expression of CBX7 in colorectal cancer (CRC) in relationship to the clinicopathologic characteristics of the tumors. Methods: We have quantified the expression of CBX7 in colon carcinoma cell lines and in colon carcinoma surgical specimens by semiquantitative and real time RT-PCR. Afterwards, we have performed immunohistochemistry (IHC)and FISH analysis on a series of 1420 colorectal cancer in tissue microarray (TMA) format Results: The RT-PCR experiments showed a decrease of expression of in CRC, with a fold-change ranging from –2.1 to 37.0 (average –4.8). Loss of IHC expression corrrelated with pT category (p=0.0013). In the univariate survival analysis loss of CBX7 was correlated with shorter survival (p=0.0056). In the FISH analysis, 128 cases (12.3%) were polysomic or amplified. In the survival analysis, polysomic and/or amplified cases showed a longer survival both in the univariate (p=0.0017) and in the multivariate analysis (p=0.0094, RR 0.145) with pT and pN. Conclusions: CBX7 expression seems to have a role in the genesis of CRC and to be inversely related to the prognosis. Moreover, the increase of expression in a subgroup of CRCs may be partly due to genomic alterations (amplification and/or polisomy), a mechanism that has been shown to be important in other tumors. Restoration of CBX7 activity may be a a possible therapeutic strategy in subgroups of CRCs

Sa-072 Detection of molecular markers in pre-treatment biopsies of patients with rectal cancer – eﬀects of neoadjuvant radio/chemotherapy and predictive value S. Lassmann1, J. von Brocke1, U. Jütting2, D. Mattern1, C. Weissenberger3, F. Makowiec4, H. Geddert5, M. Henke3, U. Hopt4, M. Werner1 1 Institut für Pathologie, 2 Klinik für Strahlenheilkunde 4 Chirurgische Klinik, Universitätsklinikum Freiburg 3 Institut für Biomathematik und Biometrie, GSF-Forschungszentrum, Neuherberg 5 Institut für Pathologie, St.Vincentius-Kliniken Karlsruhe Aims: To evaluate the predictive value of 6 molecular markers for the response to neoadjuvant radio/chemotherapy (nRCTx) in patients with rectal cancer. Methods: Archival tissues of matched pre-treatment biopsies (pre-Bx) and resection specimens of 25 rectal cancer patients treated by nRCTx were subjected to microdissection of tumour cells and quantitative RT-PCR analysis of Survivin, Cyclin-D1, TS, Mcl-1, Bcl-xl and STK15 mRNA expression. The mRNA levels of the pre-Bx were correlated to pathological tumour regression (pCR
Survivin mRNA expression in pre-Bx was significantly associated with OS (p=0.0365). Conclusions: The 6 selected molecular markers in pre-treatment biopsies do not allow predicting the pathological response of tumour cells to nRCT. However, STK15 and TS mRNA expression may be linked to clonal selection or resistance of tumour cells to nRCT. Finally, Survivin expression in pre-treatment biopsies predicts survival of rectal cancer patients treated by nRCT and surgery.

Sa-073 Germline mutation rates of SMAD4 and BMPR1A in patients with Juvenile Polyposis Syndrome (JPS) W. Back, S. Loﬀ1, M. Stolte2, S. Aretz3 Pathologisches Institut, Universitätsklinikum Mannheim 1 Abteilung für Kinderchirurgie, Universitätsklinikum Gießen-Marburg 2 Institut für Pathologie, Klinikum Bayreuth 3 Institut für Humangenetik, Universitätsklinikum Bonn Aims: Up to now germline mutations of SMAD4 and BMPR1A haven been reported in patients with JPS. We aimed to investigate mutations and genetic defects in these two genes among a group of patients with this rare syndrome. Methods: 80 unrelated patients from different institutions have been analyzed for mutations by direct sequencing and multiplex ligation-dependent probe amplification. Also clinical findings and the histological appearance of polyps have been evaluated. Results: Overall genetic mutation rates for these two genes in JPS were together 60%. Among the SMAD4 mutations one fourth were large deletions whereas for BMPR1A the portion of large deletions was about one fifth of the genetic alterations detected. Conclusions: JPS is still a genetic disease with 40% negative findings, also applying new methods of mutational analysis. The search for other genes involved in the pathogenesis of this polyposis disorder must therefore go on. Nevertheless mutational analysis of JPS patients should incude the search for large deletions in these already known genes. Among mutation-positive JPS cases there is considerable variation of the histopathological classification of polyps especially at first clinical presentation.

Sa-074 High Volume Macroscopic and Histopathologic Work-Out of Neoadjuvant treated Rectal Cancer for Risk Stratiﬁcation – Micrometastatic Lymph Node Disease, Perineural Invasion and Tumor Regression H. Rothe, T. Sprenger1, H. Becker1, T. Liersch1 Zentrum Pathologie und 1 Abt. für Allgemein- und Viszeralchirurgie, Universitätsklinikum Göttingen Aims: Pathologic quality assessment of locally advanced rectal cancers (cUICC stages II/III) n=34 after preoperative 5-Fu based longterm chemoradiotherapy (CT/RT) followed by high performance standardized low anterior (LAR) and abdominoperineal resection (APR)(all R0 resections) with total mesorectal excision(TME). Methods: Postsurgical macroscopic quality grading of mesorectal circumference, extended subtotal gross cutting and subsequent extended histopathologic diagnostic of tumor and mesorectal/perirectal soft tissue, detection of metastatic and micrometastatic lymph node (LN) disease and regional occurrence of perineural invasion. Relation of tumor regression grading to tumor stage. Results: 1. Postsurgical circumferential quality grading was better for LAR than for APR. 2. Mean number of lymph nodes in neoadjuvant treated rectal cancer after CT/RT was 39. 3. Single LN micrometastasis was diagnosed in 10 of 34 patients (29,4%).4. Perineural invasion occurred in 5 of 34 patients (14,7%) 5. Postoperative histopathological tumor stage did not correlate to regression grading. Conclusions: Neoadjuvant CT/RT of rectal cancer followed by high performance surgery with TME challenges adequate and enhanced pathological diagnostic procedures. Despite various regression features, reliable staging Der Pathologe · Supplement 1 · 2008

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Abstracts is possible by skilful evaluation. Additionally gained tumor related informations as micrometastases in LN and perineural invasions should be discussed as candidates for an individualized postoperative systemic therapy. An efficacy analysis of these facts and regression grading with respect to time to recurrence and overall survival is ongoing.

Sa-075 Is the Prognostic Signiﬁcance of Lymphatic and Venous Invasion in Colorectal Cancer Related to Vessel Location? C. Langner, V.S. Pollheimer, M.J. Pollheimer, R.A. Lindtner, P. Kornprat1, A. Schlemmer2, P. Rehak1 Institut für Pathologie, 1 Universitätsklinik für Chirurgie und 2 Institut für Medizinische Informatik, Statistik und Dokumentation, Medizinische Universität Graz Aims: To assess the prognostic impact of intra- and/or extramural lymphatic and/or venous invasion in colorectal cancer (CRC). Methods: The slides of 381 randomly selected CRCs were re-evaluated with respect to tumour stage and grade and presence of intra- and/or extramural lymphatic and/or venous invasion. Data were correlated with outcome in both uni- and multivariate analysis. Results: Lymphatic and venous invasion was observed in 126/381 (33%) and 87/381 (23%) patients and was significantly associated with tumour stage (p<0.001) and grade (p<0.001). Intramural (L1a), extramural (L1b) as well as both intra- and extramural lymphatic invasion (L1ab) was seen in 44%, 6% and 50% of cases. Intramural (V1a), extramural (V1b) as well as both intra- and extramural venous invasion (V1ab) was seen in 16%, 62% and 22% of cases. Regarding progression-free survival, venous and lymphatic invasion proved prognostic significance in both univariate (p<0.001) and multivariate (p<0.001) analysis. In addition, extramural lymphatic invasion (compared with intramural invasion) proved to be a significant prognosticator in univariate (p<0.001) and multivariate (p=0.01) analysis, whereas presence of extramural venous invasion (compared with intramural invasion) demonstrated prognostic significance only in univariate analysis (p=0.02). Conclusions: Patient outcome was significantly associated with presence of angioinvasion. Moreover, extramural lymphatic permeation proved to be a new independent prognostic variable (compared with intramural lymphatic permeation) and should thus be recorded during routine pathological workup of CRC specimens.

Sa-076 Rapid and accurate Diagnosis of Human Intestinal Spirochaetosis by Fluorescence in situ Hybridization (FISH) A. Moter1, D. Schmiedel1, H.-J. Epple2, C. Loddenkemper3, R. Ignatius1, A. Petrich1, H. Stein3, T. Schneider2, U.B. Göbel1 1 Institut für Mikrobiologie, Charité – Universitätsmedizin Berlin, Campus Mitte 2 Klinik für Gastroenterologie und 3 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin Aims: Human intestinal spirochaetosis (HIS) is characterized by overgrowth of the large intestine by spirochaetes of the genus Brachyspira. At present microbiological diagnosis of HIS is hampered by the fastidious nature and slow growth of Brachyspira spp. and therefore unsatisfactory. Fluorescence in situ hybridization (FISH) allows visualization of single bacterial cells within their natural environment and identification of the microorganisms. The aims of the present study were to (i) design a FISH probe covering all Brachyspira spp., (ii) evaluate FISH as a tool for the diagnosis of HIS, and (iii) acquire further information about the phylogenetic structure within the genus Brachyspira. Methods: We analyzed intestinal biopsies from five patients with clinically suspected HIS and positive results in histopathology and culture. PCR amplification and sequencing of the complete 16S rDNA gene of the spirochaetes was performed and a Brachyspira -specific 16S rRNA directed FISH-probe was designed and evaluated.

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Results: The 16S rDNA sequences obtained clustered in the group of Brachyspira aalborgi, B. pilosicoli, and previously unpublished Brachyspira phylotypes. FISH of the biopsy samples resulted in unequivocal strong signals of brush-like formations at the crypt surfaces. It allowed simultaneous visualization of single spirochaetes and identification of the microorganisms as Brachyspira spp. Conclusions: FISH provides a fast and accurate technique to visualize and to identify intestinal spirochaetes in tissue sections and therefore represents a valuable tool for diagnosis of HIS.

Sa-077 Invasion associated upregulation of inﬂammatory NF-κB targets in colorectal cancer D. Horst, L. Kriegl, T. Kirchner, F. Hlubek Pathologisches Institut, Universitätsklinikum München (LMU) Aims: Invasion is the hallmark of colorectal cancer (CRC) progression. Inflammation and activation of NF-κB signalling has been shown to promote CRC invasiveness. The aim of this study was to identify selective NF-κB target gene expression at the tumor center (TC) and the invasive front (IF) in colorectal carcinomas which may contribute to tumor progression and invasion. Methods: The IF, TC and normal mucosa were microdissected from CRC specimens. Gene expression profiling using Affymetrix HG-U133A chips was performed. Upregulated and differentially expressed genes between normal mucosa, the IF and the TC were functionally grouped. Genes of interest were validated via immunohistochemistry on paraffin embedded tumors. Results: We found 510 genes differentially expressed between the IF and the TC. 54 of them were associated with immune response in general and 24 with inflammation. Of the 9 NF-κB target genes differentially expressed between the IF and the TC, 3 genes were already upregulated in the TC compared to normal mucosa and exhibited enhanced expression at the IF. 6 genes were specifically overexpressed at the IF only. These include proteoglycan-1 and tenascin which were immunohistochemically validated. While tenascin was expressed by tumor cells and inflammatory stromal cells, proteoglycan-1 was only expressed by the latter. Conclusions: Here we show a regional distinct expression of NF-κB target genes at the invasion front and tumor center of CRC. Additional immunohistochemial validation indicated that the stromal reaction at the IF is distinctly different from that in the TC. Our results imply that an effective inhibition of NF-κB signalling could be an important strategy to suppress tumor progression of CRC.

Sa-078 An integrated approach combining gene expression proﬁling, computational analysis and conventional molecular techniques for studying signal transduction in colorectal cancer cells K. Jürchott, R. Schäfer Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte Aims: During the last decades, significant progress was made in analysing oncogene addiction in model systems. In the clinical situation, several genetic alterations accumulate and distinct oncogenic pathways cooperate to achieve the full malignant potential. The aim of this project was to identify the impact of a specific signaling pathway (RAS-RAF-MEK-ERK) on the transcriptome of colorectal cancer cells harbouring mulitple intrinsic oncogenic alterations. Methods: We used an integrated approach combining transcriptional profiling, small molecule inhibitors targeting signaling pathway effectors, and computational prediction of regulatory elements in promoters of co-expressed genes with chromatin-based and cellular assays. Results: We identified MEK/ERK-dependent groups (signal-regulated transcriptional modules) of proliferation-associated genes commonly up-regulated in colorectal cancers. These genes demonstrated an overrepresentation of E2F- and NFY-binding sites within the predicted promoter regions, suggesting a crucial role for factors interacting with these DNA motives. To obtain functional evidence and to validate the prediction, we demonstrated a MEK/

ERK-dependent regulation of a model promoter containing a NFY-binding site. Using EMSA and ChIP-assays, we identified YB-1 as the candidate transcription factor binding to the NFY-motive. Reduction of YB-1 protein levels by siRNA significantly compromised the proliferation of a colorectal cancer cell line. Conclusions: We conclude that YB-1 is an important factor mediating the response of tumor cells to a sustained intrinsic oncogenic activation of the RAS-RAF-MEK-ERK-Pathway.

Sa-079 Expression of Melanocyte Diﬀerentiation Antigens (MDAs) Melan-A, Tyrosinase, and gp100 in Malignant Melanoma of the Ano-Rectal Mucosa A. Jungbluth1, D. Frosina1, M. Weiser2, K.-J. Busam3 1 Ludwig Institute for Cancer Research, New York, USA 2 Dept. of Surgery und 3 Dept. of Pathology, Memorial Sloan-Kettering Cancer Center, New York, USA Aims: Malignant melanomas of the anorectal mucosa are rare tumors carrying a poor prognosis, which partially differ from their cutaneous counterparts on a molecular level. Antibodies to melanocyte differentiation antigens (MDAs) are standard diagnostic markers; MDAs also serve as targets for vaccine-based immunotherapy of cutaneous melanoma; however, little data is available about their expression pattern in mucosal melanomas of the anorectum. Methods: 13 cases of MMARM were available. Immunohistochemistry was done using the following mAbs (to the following antigens): A103 (Melan-A/ MART-1, previously generated in our lab), T311 (tyrosinase, previously generated in our lab), HMB45 (gp100). Tumor staining was graded as follows: focal (<5%); + 5–25%, ++ >25–50%, +++ >50–75%, ++++ >75%. Results: A103, T311 and HMB45 were positive in all tested cases displaying immunoreactivity in >75% of the tumor in a majority of the analyzed lesions. Only one case revealed focal immunostaining for A103 and another case focal immunoreactivity for HMB45. Conclusions: MDAs are established markers for cutaneous melanomas and serve as T-cell targets in the immunotherapy of cancer; we recently developed mAbs A103 and T311 for their analysis. The present study demonstrates a high incidence and a homogeneous expression pattern of Melan-A, tyrosinase and gp100 in malignant melanoma of the anorectal mucosa comparable to their expression in cutaneous melanomas. Our study shows the usefulness of MDAs as diagnostic and therapeutic antigens in this subtype of melanoma.

Sa-080 Maspin promotes invasiveness in colorectal cancer cells M. Bettstetter, I. Schardt, B. Gröschl, A.K. Bosserhoﬀ, F. Hofstädter, W. Dietmaier Institut für Pathologie, Universitätsklinikum Regensburg Aims: Maspin is overexpressed in CRC, especially at the invasion front, in dedifferentiated tumors and in disseminated tumor cells. We have shown earlier, that maspin expression is associated with microsatellite instability (MSIH) and poor prognosis in colorectal tumors. Here we analyzed the effect of maspin on growth, morphology, cell motility and invasiveness of colorectal cancer cells. Methods: We generated both stable Maspin overexpressing andez76w maspin-suppressed clones of the CRC cell lines SW480 and SW48, respectively. The modulations of expression were verified by real-time PCR, western blotting, immunocytochemistry and fluorescence microscopy. The effects of maspin-expression-modulations were analyzed by XTT cell proliferation tests, Boyden chamber motility assays and invasion assays through artificial basement membrane. Results: Maspin expression was associated with decreased proliferation rates and the occurrence of motility structures as pseudopodia and filopodia. Overexpression resulted in a switch from a monolayer to a spheroid growth, clotted growth form with increased intercellular adhesion and in decreased adhesion to cell culture treated polystyrol. In the normally highly expressing

cell line SW48 maspin suppression resulted in the loss pseudopodia. Both, tumor cell motility and invasiveness were positively correlated with Maspin expression. Invasiveness was increased by recombinant Maspin and was inhibited by an anti-maspin-antibody. Conclusions: In colorectal cancer cells Maspin promotes an aggressive growth behaviour by enhancing cell motility and promotes invasiveness at the cell surface.

Sa-081 M. Whipple – Unusual manifestation in the liver S. Kriener, F. Hartmann1, A. Stoßberg1, M.-L. Hansmann, K. Engels Institut für Pathologie, Universitätsklinikum Frankfurt am Main 1 Abt. Interne Medizin, St. Marien-Hospital Frankfurt am Main Aims: M. Whipple is a rare infection caused by Tropheryma whipplei. Common clinical symptoms are loss of weight, fever, diarrhoea, and polyarthritis. We present a 38 year old male patient with the above listed symptoms and a hepatosplenomegaly. The initial diagnosis was a suspected malignant lymphoma. Methods: Initially multiple serum tests to exclude viral infections and CTscans were performed. These were followed by biopsies of a para-aortal lymph node, the liver, and the duodenum. Results: Acute hepatotrophic viral infections and multiple other infections and autoimmune disorders were excluded. The blood showed an infectious anaemia and elevated liver enzymes. Histologically an granulomatous inflammation with fatty droplets was detected in a abdominal lymph node and the liver. The epitheloid cells of the granulomas contained PAS-positive (after diastase digestion) Ziehl-Neelsen-negative micro-organisms, which were also found in the following duodenal biopsy. The diagnosis of M. Whipple was established. The consecutive therapy started with 2 g Ceftriaxon (Rocephin) i.v. for 14 days followed by oral 160 mg Trimethoprim plus 800 mg Sulfamethoxazol (Cotrim) twice a day, which has to be administered for at least one year. The clinical symptoms decreased after the initiation of this therapy. Following neurological examinations did not reveal a manifestation of the disease in the central nervous system. Conclusions: M. Whipple is a rare infection which may simulate many other diseases such as a malignant lymphoma. This is the first case showing Tropheryma whipplei within the epitheloid cells in granulomas of the liver.

Sa-082 Rare PIK3CA hotspot mutations in hepato-biliary carcinomas M.-O. Riener, M. Bawohl, P.-A. Clavien1, W. Jochum Institut für Klinische Pathologie und 1 Klinik für Viszeral- und Tranplantationschirurgie, UniversitätsSpital Zürich Aims: Somatic mutations of the PIK3CA gene, which encodes for the p110α catalytic subunit of phosphatidylinositol-3-kinase (PI3 K), are frequent in various cancer types. The majority of mutations cluster at hotspots within exons 9 and 20, which encode for the helical and kinase domains of p110α. PIK3CA mutations in bile duct and gallbladder carcinomas have not been reported yet. Our aim was to screen a large series of hepato-biliary carcinomas for PIK3CA hotspot mutations. Methods: We analysed 118 carcinomas of the biliary tract and the liver (45 cholangiocarcinomas, 23 gallbladder carcinomas, 50 hepatocellular carcinomas) for PIK3CA hotspot mutations using polymerase chain reaction and direct DNA sequencing. Results: PIK3CA missense mutations were found in one of 45 cholangiocarcinomas (E545 K, 2%), one of 23 gallbladder carcinomas (E542 K, 4%) and one of 50 hepatocellular carcinomas (H1047R, 2%). PIK3CA mutations were associated with the expression of the PI3 K downstream targets phosphorylated 4E-BP1 and eIF4-E in carcinoma cells. All three mutations represent hotspot mutations, which also occur in other cancer types. Conclusions: These results indicate that somatic PIK3CA mutations contribute to the frequent activation of the PI3 K/AKT pathway in carcinomas of the biliary tract and liver.

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Abstracts Sa-083 Survivin is upregulated during liver regeneration in rats and humans and is associated with hepatocyte proliferation J. Wohlschlaeger, K.J. Schmitz, S. Nadalin 1, G.C. Sotiropoulos1, B. Levkau2, H.A. Baba Institut für Pathologie und Neuropathologie, 1 Klinik für Allgemein- und Transplantationschirurgie und 2 Institut für Pathophysiologie, Universitätsklinikum Essen Aims: Survivin regulates cell division and suppresses apoptosis. Liver regeneration is a complex process involving both, proliferation and apoptosis. The role of survivin is not well elucidated and no data exist in humans. Methods: 70% liver resection was used to investigate liver regeneration in rats. Survivin was identified by means of RT-PCR, Western blotting and immunohistochemistry. Proliferation and apoptosis were quantified. Liver biopsies from 33 patients who underwent living donor liver transplantation were used to study survivin immunoexpression, proliferation and apoptosis within the first 17 days after transplantation. Seven healthy donors served as controls. Results: Survivin was significantly upregulated after 24 h to 72 h in rat liver regeneration and showed a significant correlation with proliferation but not with apoptosis. In humans survivin was nearly absent in donor and reperfused liver tissue but increased significantly after 5 to 7 days after transplantation and correlated with proliferation but not with apoptosis. In contrast to animals hepatocyte proliferation in humans did not return to control levels. Conclusions: Survivin is upregulated in human and rodent liver regeneration and correlates with proliferation but not with apoptosis. The lower survivin expression and proliferation in humans suggests a prolonged liver regeneration process.

Sa-084 Epigenetic modiﬁcation of tumour suppressor genes in ﬁbrolamellar carcinoma (FLC) W. Tränkenschuh, K. Metzig, B. Hasemeier, P. Flemming1, H. Kreipe, U. Lehmann Institut für Pathologie, Medizinische Hochschule Hannover 1 Institut für Pathologie am AKH Celle Aims: Fibrolamellar hepatocellular carcinoma is a rare variant of hepatocellular carcinomas that occurs in younger patients without obvious underlying liver disease. It provides the opportunity to study epigenetic changes in tumour suppressor genes compared to a morphologically inconspicuous surrounding liver parenchyma in contrast to the cirrhotic or virus-infected background in hepatocellular carcinoma. Methods: Six tumour suppressor and cell cycle control genes (RASSF1A, Cyclin D2, GSTπ1, APC, SOCS-1 and CDH-1) were examined for promoter methylation in carcinoma samples from 17 patients (age 13 to 64 yrs) and compared with the patients tumour free liver parenchyma. Genomic DNA was extracted from manually microdissected liver biopsies (0,5 to 18 years old) and analysed using real-time MSP and Pyrosequencing. Results: 34/36 biopsies provided evaluable results. 7.5% of measurements gave discrepant results using both methods in parallel. Whereas aberrant hypermethylation of the E-cadherin gene (CDH1) was not found in FLC, hypermethylation of the RASSF1A and the cyclinD2 gene is – as in HCC – quite frequent (50, and 39%, respectively). Conclusions: Also in FLC aberrant hypermethylation is quite frequent, displaying gene-specific differences and differences in comparison to HCC. Real-time MSP and Pyrosequencing provide in most instances very similar results but for some cases clear discrepancies were detected.

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Sa-085 Patterns and prognostic value of chromosomal imbalances in primary liver cancers identiﬁed by comparative genomic hybridization B. Gunawan, K. Homayounfar1, S. Cameron2, B. Sander, S. Uecker, T. Lorf1, H. Becker1, L. Füzesi Abteilung Gastroenteropathologie 1 Abteilung Allgemein- und Viszeralchirurgie 2 Abteilung Gastroenterologie und Endokrinologie, Universitätsklinikum Göttingen Aims: Little is known about molecular cytogenetic changes in cholangiocarcinoma (CC) and combined hepatocellular-cholangiocarcinoma (cHCC/CC). Although more cytogenetic data exist on hepatocellular carcinoma (HCC), only limited information is available on the prognostic value of chromosomal imbalances. Methods: The chromosomal profiles in HCC (48 cases), CC (22 cases) and cHCC/CC (7 cases) were examined by comparative genomic hybridization (CGH) with subsequent prognostic evaluation of chromosomal findings. Results: In HCC, gains at 8q and 1q as well as losses at 8p, 4q, 6q, 13q, and 16q were the most frequent chromosomal imbalances. In CC, by far the most frequent imbalances were losses at 6q and 3p, and to a lesser extent losses at 9p, 14q and 13q as well as gains at 1q, 7q, 7p and 8q. The cases of cHCC/CC shared similar chromosomal profiles with HCC including gains at 1q and 8q and losses at 8p as the most frequent alterations. In patients with R0- resected HCC, more than 4 chromosomal gains were associated with poorer overall survival (p=0,009). Conclusions: CC is characterized by a distinct chromosomal pattern marked by combined losses at 6q and 3p, whereas similar genetic events including gains at 1q and 8q may underlie the tumorigenesis of HCC and cHCC/CC. In HCC, the number of gains detected by CGH may prove an additional prognostic parameter for predicting overall survival.

Sa-086 Prostaglandin E2 synthases (mPGES-1 and -2) as possible therapeutic targets in human hepatocellular carcinoma (HCC) M. Breinig, B. Göppert, P. Schirmacher, M.A. Kern Pathologisches Institut, Universitätsklinikum Heidelberg Aims: The increasing incidence of HCC, the oftentimes fatal outcome and the lack of effective treatment options for HCC accentuate the need for novel therapeutic approaches. We and others were able to demonstrate that abrogation of altered PGE2 synthesis via inhibition of Cyclooxygenase (COX)-2 could represent an effective therapeutic strategy, since COX-2 inhibitors exerted antitumorigenic effects in in vitro as well as in vivo models of HCC. Since PGE2 seems to be the major protumorigenic factor, we now wanted to address whether PGE2-synthases might serve as novel target structures downstream of COX-2. Methods: COX-2, mPGES-1 and -2 expression in HCC tissues was analyzed by Tissue Micro-Arrays (TMAs) and by Western Immunoblot in HCC cell lines. mPGES-2-specific siRNA was used to reduce mPGES-2 expression. Western Immunoblot analysis was employed to confirm mPGES-2-knockdown. Functional effects of mPGES-2-knockdown were analyzed by MTTassays (viability) and FACS (cell cycle and apoptosis). Results: mPGES-1 and -2 expression was significantly increased in cirrhotic liver and poorly differentiated HCCs. Successful siRNA-knockdown of mPGES-2 reduced cell viability in HCC cell lines, which was associated with alterations in cell cycle distribution and apoptosis. Conclusions: Based on our results, mPGES-2 might represent a novel therapeutic target in HCC. Since mPGES-1 is also overexpressed in HCC, and since recent findings suggest that COX-2 inhibitors can exert cardiovascular side-effects, inhibition of both PGE2-synthases should be advantageous over the general blockade of prostaglandin synthesis by COX-2 inhibition.

Sa-087 Degenerative changes of the periprosthetic membrane as a possible reason for prosthesis loosening N. Koleganova1, G. Krohmer2, P. Hagjicostas3, B. Fink4, I. Berger1 1 Pathologisches Institut und 2 Chirurgische Klinik, Universitätsklinikum Heidelberg 3 Schwarzwald-Baar Orthopädische Klinik 4 Orthopädische Klinik Markgroningen Aims: The aim of the present study was to perform a comparative evaluation of septic and aseptic interface membranes, assessing histological features, inflammatory infiltrate, and expression of inflammatory cytokines. Methods: Septic and aseptic interface membranes from 102 patients were examined by histology, histochemistry, and immunohistochemistry (tissue arrays). The cell subpopulations were characterized by quantification of CD3, CD4, CD8, CD20, and CD163 positive cells. Additionally, a semiquantitative evaluation of inflammatory cytokines (TNFα, TGF-β1, IL-1, IL-6, CRP, MMP-1, MMP-6) was performed to complete the analysis of inflammatory infiltrates. Results: The histological analysis revealed three different types of aseptic interface membranes: wear particle, degenerative, and mixed type. The expression of inflammatory molecules did not differ between septic and wear particle interface membranes. Significantly lower expression of cytokines, MMPs and CRP was observed, however, in degenerative interface membranes compared to other types. No expression of TNFα was observed in the degenerative interface membranes. Over 88% of patients with degenerative interface membranes had had a clinical record of osteoarthritis. Conclusions: Aseptic interface membranes were represented by wear particle, degenerative and mixed type. The expression of inflammatory factors in wear particle type is similar to this in septic membranes and can contribute to the bone destruction and prosthesis loosening. These factors seem not to play a major role in the degenerative membranes.

Sa-088 5-ﬂourouracil based chemotherapy in conjunction with oxaliplatin is associated with sinusoidal dilatation S. Kandutsch1, M. Klinger2, S. Hacker2, F. Wrba1, R. Punzengruber3, T. Grünberger2 1 Klinisches Institut für Pathologie und 2 Universitätsklinik für Allg.Chirurgie, Medizinische Universität Wien 3 Abteilung für Innere Medizin, LKH Amstetten Aims: The purpose of this study was to assess chemotherapy associated liver injury and to compare between effects of orally and intravenous administered 5-flourouracil on liver parenchyma Methods: Liver specimen of 50 patients with liver metastases from colorectal cancer receiving neoadjuvant capecitabine (Xeloda®) plus oxaliplatin (XELOX) or 5-fluorouracil, leucovorin plus oxaliplatin (FOLFOX4) for six cycles and of 13 chemonaive Patients subjected to liver resection were analysed by two senior pathologists blinded for pretreatement. Steatosis and cocomittant necroinflammation was graded based on Kleiner et al. Sinusoidal injury was interpreted according to Brandt et al. Results: Liver specimens from three groups of patients with equally distributed sex, age and body mass index (BMI) were compared in this study. We found no difference in grading of steatohepatitis among the chemotreated groups and between chemonaive and chemotreated liver parenchyma. Chemotherapy showed an independent effect on fibrosis stage. Furthermore, age and chemotherapy were independently associated with sinusoidal dilation. Centrilobular vein fibrosis correlated with administration of chemotherapy. Conclusions: 5-flourouracil based chemotherapy in conjunction with oxaliplatin does not correlate with the developement of steatosis and steatohepatitis. Age and oxaliplatin predispose to the development of sinusoidal dilation, therefore caution must be taken in old patients treated with oxaliplatin.

Sa-089 Relationship between laboratory data and liver histology in primary biliary cirrhosis (PBC) U. Drebber, J.J.M. Müller, E. Klein, H.U. Kasper1, K. Schardt, M. Odenthal, H.P. Dienes Institut für Pathologie, Universitätsklinikum Köln 1 Institut für Pathologie, Clemenshospital Münster Aims: The study was carried out in order to characterize histopathological lesions in primary biliary cirrhosis (PBC) and subtypes and to assess relationships between histopathological lesions and biochemistries. Methods: Liver biopsies, which had been performed on 252 patients who fulfilled at least two of the diagnostic criteria of PBC were investigated. A laboratory data base was established on each patient retrospectively. Histopathological characterization was performed. Relationships between histopathological features and biochemistries were calculated statistically. Results: Combining the data of 252 patients, a PBC group, consiting of an AMA positive and negative subgroup, and an overlap group were defined, each with a female preponderance of more than 90% and with higher activities of AST in the overlap group. Histopathological changes were characteristic in more than 80%. Periductal concentric fibrosis, lobular granuloma formation and steatosis were frequently remarkable. Correlations were found between ALT activity and modified hepatitis activity index (mHAI) in the overlap group and the AMA positive group. A positive significant relationship was demonstrated between mean AST activity and portal fibrosis for the AMA positive group. A highly significant positive link was seen between mean concentration of bilirubin and stage of portal fibrosis. A trend towards a higher AST/ALT ratio in histologal stages 3 and 4 compared with stages 1 and 2 was seen in the PBC group. The AP activity was influenced by the interaction between the disease stage and ductular metaplasia. Conclusions: Biochemistries reflect only in part the degree of severity of histopathological lesions in PBC. Histopathology reveals comorbidities in a high percentage.

Sa-090 Aberrant P-cadherin expression in biliary tract carcinomas and precursor lesions M.-O. Riener, B. Pestalozzi1, P.-A. Clavien2, W. Jochum Institut für Klinische Pathologie, 1 Klinik für Onkologie und 2 Klinik für Viszeral- und Tranplantationschirurgie, UniversitätsSpital Zürich Aims: P-cadherin (CDH3) belongs to the family of classic cadherins which control cell adhesion, motility and invasive growth of tumor cells. P-cadherin expression is deregulated in various tumor types. The aims of our study were to investigate P-cadherin expression in carcinomas and precursor lesions of the biliary tract, and to evaluate the potential usefullness of P-cadherin in the diagnosis of biliary dysplasia. Methods: Using immunohistochemistry, P-cadherin expression was analysed in a large series of extra- and intrahepatic cholangiocarcinomas (CCA), gall bladder carcinomas (GBC) and bile ducts/gallbladders with inflammation and epithelial dysplasia. P-cadherin expression in CCA was correlated with clinico-pathological parameters. Results: P-cadherin expression was observed in 29/51 CCA (57%) and 21/34 GBC (62%), and was associated with a higher pT stage (p=0.028), tumor grade (p=0.043) and reduced disease free-survival (p=0.0277) after resection in CCA. Epithelial dysplasia in large bile ducts of 19/21 (90%) patients, but not in 4/4 gallbladders, also showed P-cadherin expression. Biliary epithelium in normal bile ducts (n=51) and gallbladders (n=34) was consistently negative for P-cadherin. Large bile ducts with inflammation displayed P-cadherin expression in 1/21 (5%) case. Conclusions: Our results indicate that P-cadherin is upregulated early during carcinogenesis in the biliary tract and that P-cadherin may represent a usefull marker for dysplasia in large bile ducts.

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Abstracts Sa-091 The anti-apoptotic protein Mcl-1 is important for hepatocyte integrity and is up-regulated in hepatocellular carcinoma A. Weber, B. Vick1, P.R. Galle1, H. Schulze-Bergkamen1 Institut für Klinische Pathologie, UniversitätsSpital Zürich 1 I. Medizinische Klinik, Universitätsklinikum Mainz Aims: To evaluate the role of the anti-apoptotic Bcl-2 family member Mcl-1 in liver and liver diseases, in particular in hepatocellular carcinoma (HCC). Methods: MCl-1 expression was studied at the mRNA (RT-PCR) and protein level (western blot, immunohistochemistry) in HCC cell lines and primary human liver tissues including HCC. Conditional hepatocyte-specific knockout mice for Mcl-1 (mcl-1-/-) were created. Liver function was studied serologically and liver histology was analyzed, in particular in response to liver cell damage. Results: Mcl-1 was highly expressed in HCC cell lines and revealed higher expression levels in human HCC tissue compared to corresponding non-neoplastic liver tissue. Suppression of Mcl-1 sensitized hepatocellular carcinoma cells to apoptosis. Mcl-1-/- mice show reduced liver size and weight, elevated basal liver enzymes and higher sensitivity towards Fas-induced apoptosis. Conclusions: The phenotype of hepatocyte-specific mcl-1-/- mouse underlines the important role of Mcl-1 for hepatocyte integrity. In addition, this model is a valuable tool for studying the role of apoptosis in a variety of liver diseases like liver failure, hepatitis and hepatocellular carcinoma. The high expression of Mcl-1 in HCCs suggests that Mcl-1 is an important factor for the apoptosis resistance of human HCC. Therefore, Mcl-1 constitutes a potential target for HCC therapy.

Sa-092 FoxM1 is diﬀerentially expressed in non-neoplastic liver and hepatocellular carcinoma M.P. Bihl1, I. Zlobec1, C. Cillo1, L. Tornillo1, L. Terracciano1 1 Institut für Pathologie, Universitätsspital Basel Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. The Forkhead Box (Fox) M1 transcription factor was described as crucial for the development of hepatocellular carcinomas. Study: Expression of genes in non-neoplastic and neoplastic liver from the same patients was compared using an Affymetrix Human Genome 133A 2.0 microarray (n=3). The 23 and 20 highly upregulated and downregulated genes respectively in HCC were selected for predicting the presence of transcription factor binding sites in DNA sequences situated between –1800 and +200 bp from the transcription starting site (TESS : http://www.cbil.upenn.edu/ cgi-bin/tess/tess). The FoxM1 binding site was highly represented in genes found to be upregulated in HCC and this binding site was located 1200 bp downstream of the transcriptional start site. Using a tissue microarray approach containing cirrhotic (101), HCC (210) and normal liver (94) tissue, we detected the cytoplasmic and nuclear immunohistochemical expression of FoxM1. Interestingly, while in cirrhotic and normal liver tissue expression of FoxM1 was identical (66% cytoplasmic and 9% nuclear), this pattern was different in HCC (21% cytoplasmic and 13% nuclear). The cytoplasmic FoxM1 reduction observed in HCC compared to non-neoplastic liver was significant (p<0.001). The percentage of tissues that stained positively for FoxM1 in the nucleus was 53, 30.5, and 16.6% for stage II (n=34), stage III (n=36) and stage IV (n=12) respectively. Conclusions: The FoxM1 transcription factor is likely to be activated in HCC. The strong reduction of FoxM1 in the cytoplasm of HCC cells may reflect a phosphorylated state of FoxM1 that translocates into the nucleus and therefore activates the cell cycle. Finally, FoxM1 activity seems to be progressively bypassed in advanced stage HCC.

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Sa-093 Urokinase-type-plasminogen activator-receptor (uPAR) gene ampliﬁcation, uPAR-mRNA, and uPAR expression in pancreatic carcinoma and pancreatic intraepithelial neoplasia (PanINs) R. Hildenbrand1,3, M. Niedergethmann2, P. Ströbel1, A. Marx1, H. Allgayer4,5, C. Schleger1 1 Pathologisches Institut und 2 Chirurgische Klinik, Universitätsklinikum Mannheim 3 Institut für Pathologie, Bonn-Duisdorf 4 Experimentelle Chirurgie, Universitätsklinikum Mannheim 5 Deutsches Krebsforschungszentrum Heidelberg Aims: The urokinase-type plasminogen activator (uPA) and its receptor (uPAR) are expressed by normal and malignant cells and are important in many processes including extracellular matrix turnover, invasion, cell migration, proliferation and metastasis. However, the uPA/uPAR system has not been systematically studied in pancreatic carcinoma and its precursors. Methods: uPAR was analyzed in 50 pancreatic carcinomas and 70 PanIN lesions (26 PanIN3-, 16 PanIN2, 28 PanIN1-lesions) by FISH, immunohistochemistry and in-situ hybridization. uPA-levels were determined by ELISA and were correlated with the rate of proliferation and apoptosis. The functional significance of increased uPA for proliferation and apoptosis was functionally demonstrated by suppression of uPA in pancreas-carcinoma celllines by antisense-uPA siRNA. Results: An amplification of the uPAR gene was found in 26/50 pancreas carcinomas, 17/26 PanIN3-lesions and in 7/16 PanIN2-lesions. High level amplifications were detected only in carcinomas and PanIN3 lesions. 96% of carcinomas and 100% of PanIN3 lesions showed strong expression of the uPAR by immuhiostochemistry and in-situ hybridization. The level of uPA showed a positive correlation with cell proliferation and an inverse correlation with apoptosis both in situ and in vitro. Conclusions: Changes of the uPA/uPAR signalling system appear to be critically involved in the pathogenesis of pancreatic neoplasia and in tumor progression from low to high grade lesions, making uPAR a promising target for novel therapeutic strategies in a dismal tumor.

Sa-094 Proteomics of Pancreatic Cancer – A Review I. Gräntzdörﬀer1, S. Carl-McGrath1, M.P. Ebert2, C. Röcken1 1 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte 2 II. Medizinische Klinik, Universitätsklinikum München (LMU) Aims and Methods: We summarized 15 studies applying proteomics to the analysis of pancreatic cancer in order to correlate the current status of knowledge and point out the limits of studies and methods, with the aim of creating helpful criteria for future studies. Results: All studies demonstrate that a range of biological samples are suitable for proteomic analyses. But comparison of different studies is problematic, due to the diversity of methodologies, sample sources and characterization of patient populations. For example, the sample size of study population ranged from 2 to 115. Serum, plasma, pancreatic tissue and juice were used as sample source. Six different methodologies were used for investigation. In the eight studies which identified the proteins, 118 similar proteins were found in only two, 33 in three, and only five in four of these studies. At last, reproducibility between studies has rarely been investigated and no investigation has compared the different methods of proteomic research. Conclusions: The results of this review have shown that more stringent requirements concerning the design and the analysis of future studies should be implemented. These include an adequate patient number, obligatory histological examination of tissues, appropriate control groups, identification of proteins and peaks, validation of differential expression using independent cohorts and/or a second methodology, and finally demonstration of result reproducibility. This will hopefully lead to the discovery of prognostic and predictive biomarKers that help to improve prognosis of pancreatic cancer patients.

Sa-095 Early Undiﬀerentiated Pancreatic Cancer with Osteoclast-like Giant Cells: Direct Evidence for Ductal Evolution F. Bergmann1, E. Herpel1, C. Michalski2,3, I. Esposito1,3, H. Friess2, P. Schirmacher1 1 Pathologisches Institut, Universitätsklinikum Heidelberg 2 Chirurgische Klinik, Universitätsklinikum Heidelberg, current address: Klinikum rechts der Isar der TU München 3 current address: Klinikum rechts der Isar der TU München Aims: The histogenesis of undifferentiated (anaplastic) carcinomas of the pancreas which may contain osteoclast-like giant cells has been discussed with great controversy. Based on histomorphological, immunohistochemical, ultrastructural, and molecular examinations, it has been concluded that these tumors originate from epithelial cells, mesenchymal cells, undifferentiated precursor cells, or stem cells. To date, early stage anaplastic carcinomas have not been described. Methods: An early stage undifferentiated pancreatic carcinoma with osteoclast-like giant cells, which was incidentally detected in a pancreatitis specimen, was investigated histomorphologically and immunohistochemically. Results: Within an area measuring 0.8 cm in diameter, pancreatic intraepithelial neoplasia (PanIN) lesions with severe dysplasia (PanIN3) were detected in association with invasive ductal adenocarcinoma and transition into anaplastic carcinoma with osteoclast-like giant cells. This transition was accompanied by a loss of immunohistochemically detectable expression of cytokeratins and E-cadherin, and a gain of vimentin expression. Conclusions: Our findings for the first time document initial steps in the evolution of an anaplastic pancreatic carcinoma, providing evidence for a ductal origin. We therefore suggest that the tumor should be considered an anaplastic variant of ductal adenocarcinoma.

Sa-096 Class I histone deacetylases in pancreatic cancer – eﬀects of selective inhibition in vitro and expression in vivo A. Röske, G. Kristiansen1, S. Darb-Esfahani, A. Noske, A. Buckendahl, B. Müller, M. Dietel, V. Gekeler2, M. Boehm2, T. Beckers3, C. Denkert, W. Weichert Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte 1 Institut für Klinische Pathologie, UniversitätsSpital Zürich 2 Nycomed AG, Konstanz 3 Oncotest GmbH, Freiburg Aims: Recently, several studies reported a strong functional link between histone deacetylases (HDAC) and the development of cancer. However, comparably little is known on expression patterns and function of specific HDAC isoforms in pancreatic cancer. Methods: HDAC inhibition in pancreatic cancer cell lines was done with vorinostat and valproic acid. The number of viable cells was determined with XTT tests, apoptotic cells were measured by FACS analysis. The subcellular localisation of the NF-κB subunit RelA was assessed by immunofluorescence. Expression of RelA and HDAC isoforms in tissue was investigated by immunohistochemistry. Results: Treatment of pancreatic cancer cell lines with HDAC inhibitors led to a significant dose dependent growth reduction of up to 88% depending on the concentration, cell line and substance used. Anti-HDAC treatment disrupted IL-1β induced nuclear translocation of RelA. Synergistic effects of HDAC inhibitors and high doses of gemcitabine were apparent. In addition, class I HDACs were overexpressed in pancreatic cancer tissue and high expression correlated with nuclear translocation of RelA. Conclusion: Pharmacological HDAC inhibition could serve as a novel tool in the treatment of pancreatic cancer. Effects of HDAC inhibition in pancreatic cancers might be partly due to a disruption of aberrant NF-κB signalling.

Sa-097 Pancreatic Endocrine Tumors Display Fundamental Diﬀerences in Neural Invasion Behavior F. Bergmann1, G. Ceyhan2, E. Herpel1, R.J. Rieker1,3, I. Esposito1,4, H. Friess2, P. Schirmacher1, M.A. Kern1 1 Pathologisches Institut und 2 Chirurgische Klinik, Universitätsklinikum Heidelberg current address: Klinikum rechts der Isar der TU München 3 current address: Medizinische Universität Innsbruck 4 current address: Klinikum rechts der Isar der TU München Aims: Neural invasion has been recognized to represent an important prognostic factor in pancreatic ductal adenocarcinoma. However, in pancreatic endocrine tumors (PETs), where neural invasion represents an important diagnostic feature, systematic investigations of the mode and extent of neural invasion have not yet been carried out. Methods: Neural invasion was analyzed in a total of 53 morphologically, immunohistochemically, and clinically well-characterized PETs (12 well-differentiated endocrine tumors of uncertain behavior, 36 well-differentiated endocrine carcinomas, and 5 poorly-differentiated endocrine carcinomas). Results: Neural invasion was found in 68% of PETs, being only detected within the tumor boundaries and not reaching beyond the tumor invasion front. Neural invasion significantly correlated with the grade of malignancy but occurred irrespective of functional activity, hormone phenotype, or histomorphology. The expression of epidermal growth factor receptor and nerve growth factor seemed to be associated with the frequency of neural invasion. Conclusions: In contrast to pancreatic ductal adenocarcinoma, neural invasion in PETs is only found within the tumor boundaries. Because it does not reach beyond the tumor invasion front, this may explain the low rates of local relapses following tumor resection in PETs despite the high frequency of neural invasion.

Sa-098 Tra2β is highly expressed in ductal pancreatic cancer and its nuclear expression in corresponding normal ductal epithelium predicts poor patient outcome A. Kurz1, K. Wagner2, F. Makowiec3, C. Bauer2, E. Stickeler4, A. zur Hausen2 1 Medizinische Klinik I, 2 Institut für Pathologie, 3 Chirurgische Klinik und 4 Frauenklinik, Universitätsklinikum Freiburg Aims: Alternative splicing represents an important regulatory mechanism of gene expression. Tra2β, member of the extended family of serine-argeninerich (SR) splicing factors, regulates alternative splicing of exons with C/A-rich enhancer sequences. Biological important genes (e.g. CD44, VEGF, insulin receptor, BRCA2, Syk) are alternatively spliced and contain these C/A-rich sequences. Methods: We investigated expression levels of Tra2β in a matched pair analysis of tumor samples and normal tissues of patients with invasive pancreatic cancer by qRT-PCR (n=6) and immunohistochemistry (IHC; n=58). Results: Tra2β transcript expression was two fold upregulated in ductal pancreatic carcinomas (n=6). IHC revealed high Tra2β expression in 36, and low Tra2β expression in 22 pancreatic carcinomas. Tra2β expression within the tumor tissue did not have impact on patient survival. Of the corresponding histological normal ductal epithelium 24 revealed weak and 18 revealed strong nuclear expression of Tra2β. Of interest, high nuclear Tra2β expression in the corresponding normal ductal epithelium correlated significantly with poor patient survival. Conclusions: Our data support a biological relevance for Tra2β expression in pancreatic cancer. Tra2β expression might have a potential as a marker of patient outcome in pancreatic cancer. In addition, high Tra2β expression levels possibly reflect changes in alternative splicing patterns in pancreatic cancer.

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Abstracts Sa-099 Molecular pathogenesis of ﬁbroblastic / myoﬁbroblastic tumors of the gastrointestinal tract H.-U. Schildhaus, T. Cavlar, E. Binot, R. Büttner, S. Merkelbach-Bruse, E. Wardelmann Institut für Pathologie, Universitätsklinikum Bonn Aims: The group of fibroblastic and myofibroblastic tumors consists of a wide variety of benign and malignant mesenchymal tumors Among them are tumors which occur exclusively in the gastrointestinal tract (GIT), e.g. inflammatory fibroid polyps. True fibroblastic sarcomas are rare, but intermediate tumors, as locally aggressive (e.g., desmoid-type fibromatosis) or rarely metastasizing lesions (e.g., solitary fibrous tumors) represent a major diagnostic and therapeutic problem. Little is known about the mechanisms of molecular tumorigenesis in these enitities to date. Methods: A total of 61 fibroblastic/myofibroblastic tumors of the GIT and the peritoneum were included in this study, among them inflammatory fibroid polyps, calcifying fibrous tumors, solitary fibrous tumors, inflammatory myofibroblastic tumors, desmoid-type fibromatoses, as well as low and high grade sarcomas with predominantly fibroblastic differentiation. The tumors were characterized immunohistochemically, and mutational analyses of KIT, PDGFRA and β-Catenin were performed. Gene translocations or amplifications were investigated by using the fluorescence in situ hybridisation. Results: The group of fibroblastic/myofibroblastic tumors of the GIT includes obviously reactive lesions (as calcifying fibrous tumors) which harbour no detectable molecular alterations, as well as tumors with typical changes, as β-Catenin mutations in desmoids. Most fibroblastic sarcomas in this study proved to be dedifferentiated liposarcomas with the typical mdm2-amplification. Conclusions: Different molecular analyses which are useful in the differential diagnosis of fibroblastic/myofibroblastic tumors of the GIT are presented.

Sa-100 Low grade ﬁbrosarcoma simulating an intraabdominal ﬁbromatosis I. Petersen, C. Schewe1, A. Pascher2, A. Stege1, M. Pacyna-Gengelbach1, M. Dietel1, D. Katenkamp Institut für Pathologie, Universitätsklinikum, Jena 1 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte 2 Klinik für Allgemein-, Viszeral- und Transplantationschirurgie, Charité – Universitätsmedizin Berlin, Campus Virchow Aims: To present an unusual case of a 41 year old male patient with multiple peritoneal and intraabdominal tumor manifestations. Methods: Clinical and radiological investigations were followed by laparoscopy. Single tumors were excised and assessed by microscopy and immunohistochemistry (IHC) along with ancillary methods (FISH, CGH, RT-PCR/ DNA sequencing). Results: By laparoscopy, hundreds of macroscopically well circumscribed tumor nodules of 1 to 3 cm size were detectable at the abdominal wall and the omentum. MRI showed an additional larger mesenterial/retroperitoneal tumor mass. Two nodules were excised and revealed histologically a spindle cell tumor of low to moderate cellularity and abundant stroma with minor cytological atypia and inconspicious mitotic activity, but at least focal infiltrative growth. The microscopic pattern was compatible with a low grade fibromyxoid sarcoma. Translocation diagnostics by FISH and RT-PCR/DNA sequencing, however, did not show the reported t(7;16) (q32–24;p11) translocation with fusion of the FUS/CREB3L1 genes. CGH revealed DNA gains on chromosomes 7 and 19 in both tumors with a divergent pattern of additional overrepresentations on 8, 11 and 16p confirming genetic heterogeneity and malignancy. Conclusions: The morphological diagnosis of a low grade fibrosarcoma with intraperitoneal metastases was supported by molecular pathology. The case nicely demonstrates the power and limitations of ancillary methods in sarcoma diagnostics being applied in conjunction with an experienced histopathological assessment.

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Sa-101 Gastrointestinal involvement in mastocytosis: histologic patterns and correlation with detection of KIT D816V K. Sotlar, P. Valent1, H.-P. Horny2 Pathologisches Institut, Universitätsklinikum München (LMU) 1 Innere Medizin I, Abteilung für Hämatologie und Hämostaseologie, Medizinische Universität Wien 2 Institut für Pathologie, Ansbach Aims: In patients with systemic mastocytosis (SM), besides the bone marrow, the gastrointestinal (GI) tract may also be affected. Determination of the frequency and distribution of involvement of the GI tract mucosa by mast cells (MC) in patients with (suspected) SM, and the correlation of histologic findings with the KIT mutation status of codon 816. Methods: Fiftytwo patients with gastrointestinal symptoms with (n=26) or without (n=26) known mastocytosis were analyzed by immuno-histochemistry using antibodies against tryptase, CD117/KIT, and CD25. In addition, tissue samples from all patients were examined for the presence of codon 816 mutations in KIT. Results: In almost all cases without known SM, MC were loosely distributed, round, and, neither displayed CD25 nor KIT D816 V. In contrast, in patients with known SM, the most frequent histological pattern was a diffuse increase of loosely scattered often spindle-shaped mast cells that displayed CD25 and KIT D816 V in 85% (22/26) of cases. In only 6 SM patients, compact infiltrates of CD25+ MC were found. A diffuse compact or band-like (subepithelial) infiltration mimicking chronic inflammation was only found in a single case. Conclusions: The GI tract is involved in the majority of patients with SM. While KIT D816 V is usually detectable, most of these patients present with an infiltration pattern of loosely scattered atypical (CD25+) mast cells, whereas compact or even destructive infiltrates are only detected in very few cases.

Sa-102 Cytomegalovirus PCR of Biopsies from Inﬂammatory Bowel Disease C. Schäfer1, H. Hannig1, J. Brandes2, M. Reinshagen2, K. Donhuijsen1 1 Institut für Pathologie und 2 Medizinische Klinik I, Städtisches Klinikum Braunschweig Aims: An acute activity of inflammatory bowel disease (IBD) may be simulated and /or aggravated by a viral superinfection. Such complication has to be clarified before a immunocompromised therapy is done. Which relevance in this relationship does a cytomegalovirus (CMV) PCR have? Methods: An acute activity of IBD was histologically diagnosed in 127 bowel biopsies in the years 2004–2007. The deparaffinized slides were investigated by a CMV-PCR. DNA-isolation was isolated after Proteinase-K-digestion by a magnet-beads-based method. The amplification was done as a real time PCR with the Light-Cycler (Fa. Roche). Results: Among 127 biopsies with an acute inflammation investigated by PCR 120 biopsies were negative and 7 biopsies had positive CMV-PCR results, i.e. in 5.5 percent of the investigated biopsies. Histological criteria of CMV-infections as nuclear inclusions could not be detected. The percentage of positive CMV-PCR-results seems to increase with the number of investigated biopsies in a given patient. Conclusions: A viral superinfection with CMV can simulate or aggravate an acute phase of an IBD. A CMV detection by PCR on biopsies can be important for the decision for an immunomodulated therapy.

Sa-102a Expression and signiﬁcance of hypoxia-inducible factor-1 alpha (HIF1α) in colorectal adenomas and adenocarcinomas N. Simiantonaki, M. Taxeidis, C. Jayasinghe, C.J. Kirkpatrick Institut für Pathologie, Universitätsklinikum Mainz Aims: To investigate the significance of the transcription factor hypoxia-inducible factor-1 (HIF-1α) during colorectal carcinogenesis and in tumor progression we examined its expression in precursor lesions constitute the con-

ventional adenoma–carcinoma pathway and the so-called serrated pathway as well as in non-metastatic and metastatic adenocarcinomas. Methods: HIF-1α expression has been investigated in hyperplastic polyps (HPP), sessile serrated adenomas (SSA), low-grade (TA-LGD) and highgrade (TA-HGD) conventional adenomas as well as in colorectal carcinomas (CRC) by immunohistochemistry. Results: In non-neoplastic colonic tissue, HPP and TA-LGD HIF-1α was not expressed. Perinuclear protein accumulation and nuclear expression of HIF-1α were shown in the surface epithelium of SSA and TA-HGD. In the CRC a significant HIF-1α overexpression compared to the precursor lessions was observed but a significant correlation with the metastatic status was not found. In the tumor tissue about 50% of the malignant cells showed a nuclear expression of HIF-1α in each case. An increased distribution of nuclear immunostaining was found around necrotic areas and in the vicinity of peritumoral inflammation. In agreement with these findings, treatment of 8 CRC cell lines with the potent inflammatory factor lipopolysaccharide (LPS) also stimulated HIF-1α expression. Conclusions: HIF-1α may play an important role in carcinogenesis of CRC. In addition to hypoxia, peritumoral inflammation seems to be also associated with HIF-1α. In this context LPS, the endotoxin of ubiquitous colonic bacteria is a non-hypoxic stimulator of HIF-1α.

Sa-102b Isolation of colonic tumor-derived endothelial cells (CTDEC): Eﬀects of hypoxia on their VEGFR-1 and 2 expression N. Simiantonaki, C. Jayasinghe, R. Michel-Schmidt, C. J. Kirkpatrick Institut für Pathologie, Universitätsklinikum Mainz Aims: Growth and progression of colorectal carcinomas (CRC) is dependent on the vasculature of the tumor microenvironment. Tumor-derived endothelial cells differ functionally from the normal counterpart. For this reason, we isolated microvascular endothelial cells from human CRC (CTDEC) and compared them with endothelial cells from normal tissue (HCMEC). Since hypoxia is a universal hallmark of carcinomas, we examined its effects on CTDEC of five individuals in comparison with the corresponding HCMEC, in respect to expression of the two important vascular endothelial growth factor (VEGF) receptors VEGFR-1 and 2. Methods: CTDEC were isolated from surgically removed human CRC. VEGFR expression analyses were performed using ELISA. Results: Under hypoxic conditions VEGFR-1 expression levels of CTDEC remained unchanged in all five individuals. In contrast, in HCMEC hypoxia induced in three cases an upregulation of VEGFR-1. Under normoxic conditions in 2/5 cases VEGFR-2 was significantly upregulated in CTDEC in comparison to the corresponding HCMEC. Following exposure of CTDEC to hypoxia a markedly downregulation of VEGFR-2 in four cases was observed. Conclusions: Comparative studies of CTDEC with HCMEC will be useful for understanding the progressive behavior of CRC in response to hypoxia. The unchanged VEGFR-1 expression pattern of CTDEC under normoxia and hypoxia could be a marker for their “hypoxia-resistance”. Since downregulation of VEGFR-2 can block induction of endothelial cell apoptosis, the lower VEGFR-2 levels in CTDEC under hypoxia could imply decrease of endothelial apoptosis and consequently promotion of tumor angiogenesis.

Sa-102c Isolation of human colonic microvascular endothelial cells (HCMEC) and their VEGFR-1 and 2 expression in response to hypoxia: Comparative study with HUVEC C. Jayasinghe, N. Simiantonaki, R. Michel-Schmidt, K. Peters, M.I. Hermanns, C. J. Kirkpatrick Institut für Pathologie, Universitätsklinikum Mainz Aims: Biological characteristics of large-vessel endothelial cells (EC), such as HUVEC, may differ significantly from microvascular EC. For future studies of inflammatory and neoplastic diseases of the colon, we established a method for isolation of human colonic microvascular EC (HCMEC). Since

inflammation and cancer are often accompanied by oxidative stress we examined its effects on HCMEC of five individuals in comparison with HUVEC, in respect to protein expression of the two important vascular endothelial growth factor (VEGF) receptors VEGFR-1 and 2. Methods: CTDEC were isolated from the normal mucosa and submucosa of surgically removed human colorectal carcinomas. VEGFR expression analyses were performed using ELISA. Results: Under hypoxic conditions a significant up-regulation of VEGFR-1 on HUVEC was observed. In contrast, in HCMEC the VEGFR-1 expression pattern was inconsistent. Whereas in one case the levels remained unchanged under normoxia and hypoxia, in two individuals hypoxia induced an upregulation and in two cases a downregulation of VEGFR-1 protein. In 4/5 cases VEGFR-2 was significantly downregulated in HCMEC under hypoxia. Two hypoxic HUVEC cultures overexpressed VEGFR-2 whereas three expressed the same levels under normoxic and hypoxic conditions. Conclusions: The different expresion profiles of VEGFR-1 and 2 between HUVEC and HCMEC under normoxia and hypoxia underline the importance of using a functionally adequate microvasculature for the in vitro studies of colonic diseases.

Sa-102d Transactivation of the Epidermal Growth Factor Receptor by Angiotensin II in Human Gastric Cancer Cells S. Kuyumcu, S. Carl-McGrath, C. Röcken Institut für Pathologie, Charité - Universitätsmedizin Berlin, Campus Mitte Aims: The Epidermal Growth Factor Receptor (EGFR) plays a pivotal role in cell proliferation, hypertrophy and migration. Transactivation of the EGFR by G-Protein coupled receptors (GPCRs) has been investigated in various tissues. Previously, we found that the expression of the angiotensin II receptor type 1 (AT1R), a major GPCR, correlates with nodal spread in intestinal type gastric cancer, and that the metalloproteinases ADAM 9, 12 and 15, which are involved in EGFR-ligand-cleavage, are upregulated in gastic cancer. We here investigated if AngII transactivates EGFR via its receptor in the gastric cancer cell lines MKN28, MKN45, N87 and AGS. Methods: EGFR-expression was determined by RT-PCR and Western Blot in the gastric cancer cell lines MKN28, MKN45, N87 and AGS. The receptor activation as well as the involved enzymes and ligands were investigated in cell culture experiments followed by immunoprecipitation, immunoblotting and enhanced chemiluminescence. Results: The EGFR is highly expressed at both mRNA and protein level in all investigated cell lines. Transactivation of the EGFR and the involvement of AngII in this process have been verified. Conclusions: AngII was found to transactivate the EGFR in gastric cancer cell lines, thereby promoting cancer cell migration and proliferation. This leads to the conjecture that the correlation of high AngII receptor expression with poorer prognosis may be in part due to the EGFR transactivation induced by AngII via its GPCR. Indeed, inhibition of AngII receptor signalling by ACE inhibitors or AT1R-antagonists might become an important supporting therapy for gastric cancer.

Sa-103 Acute appendicitis as an in situ model to analyse the dynamic processes of peritoneal inﬂammation C. Brochhausen, V. Schmitt, F. Bittinger, C.J. Kirkpatrick Institut für Pathologie, Universitätsklinikum Mainz Aims: Inflammation and surgical interventions represent major causes of peritoneal adhesion formation. In this process mesothelial cells (MC) play a major role due to their fibrinolytic properties and also in leucocyte recruitment. Unfortunately there are few human in situ models for the analysis of the morphological and functional changes during peritoneal inflammation to study pathomechanisms of adhesion formation. Acute appendicitis is the most frequent inflammatory bowel disease which presents in different stages and affects the serosal membrane.

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Abstracts Methods: Frozen sections from different stages of appendicitis – 6, 12, 24–48 and more than 72 hours after onset of clinical symptoms were analysed histomorphologically, immunohistologically and ultrastructurally. Results: Normal MC covered the peritoneal cavity as a flat monolayer, ultrastructurally with numerous microvilli and expression of the cell adhesion molecule ICAM-1 at the apical surface. During inflammation MC become more cuboid to round shaped with diminished number of microvilli. Furthermore, MC could be found within fibrin clots and in contact with leucocytes. These cells show at the entire surface a strong ICAM-1 and VCAM-1 expression. In ulcero-phlegmonous appendicitis (24–48 hours) a second layer of MC could be observed at the interface with the fibrin clot. Conclusions: The present data elucidate the active role of MC during peritoneal inflammation in acute appendicitis and offer a suitable in situ model to analyse the pathomechanisms of peritoneal inflammation. These data are currently being used to compare in vitro data with the in situ situation and with results of animal studies.

Sa-104 Remesothelialization and adhesion prevention by a polylactidebased membrane (SupraSeal®) in rats C. Brochhausen1, V. H. Schmitt1, B. Krämer2, T. Rajab2, C. Wallwiener2, M. Wallwiener2, D. Wallwiener2, H. Planck3, C.J. Kirkpatrick1 1 Institut für Pathologie, Universitätsklinikum Mainz 2 Frauenklinik, Universitätsklinikum Tübingen 3 Institut für Textil- und Verfahrenstechnik, Denkendorf Aims: Intraperitoneal adhesions represent a major problem after abdominal surgery and intraabdominal inflammation with serious clinical complications. Thus, great efforts have been made to prevent the development of intraperitoneal adhesions. In the present study we analysed the remesothelialization of a lactide-based copolymer (SupraSeal®) in rats. Methods: SupraSeal® was implanted in standardized peritoneal defects in a rat adhesion formation model. After 14 days the peritoneal wall with the barrier and surrounding peritoneum were explanted. The barrier material and the interface to intact peritoneum were analysed histomorphologically and by scanning electron microscopy (SEM). Results: After 14 days the barrier was attached on the side of the peritoneal defect with marked shrinking and folding. Only minor smooth adhesive strands located at the suture material could be observed. Histologically, a minor inflammatory reaction with multinucleated giant cells were seen. The mesothelial cells (MC) in the surrounding tissue were mostly flat without signs of activation. In SEM analysis the surface of the biomaterial was completely covered by a single layer of almost resting MC. Conclusions: The present polylactide-based membrane revealed good antiadhesive effects. The minor adhesions were in close contact to the suture material. In this context, further investigations are required regarding the role of suture material or suture inducing ischaemia as a possible reason for adhesion formation. The rapid remesothelialization of the barrier could explain the good anti-adhesive effects, a conclusion which is under further investigation.

Poster: Uropathologie Sa-105 Urinay cytology in after kidney transplantation M. Gajda1, E. Schulze1, O. Reichelt2, H. Wunderlich2, U. Ott3, J. Gerth3, J. Schubert2, G. Wolf3, I. Petersen1 1 Institut für Pathologie, 2 Klinik für Urologie und 3 Klinik für Innere Medizin III, Universitätsklinikum Jena Aims: To assess the diagnostic value of urinary cytological examinations in the context of immunosuppression after kidney transplantation because these patients are at higher risk of tumor development and viral infection.

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Methods: The study was performed between the years 2002 and 2004 and included two groups. The first consisted of 355 urine specimens from 107 patients being sampled on days 3, 6 and 9 after renal transplantation. A second group of 253 samples were taken in the context of clinical suspicion of Polyoma virus infection. Results: In the first group 91 samples revealed atypical cells. In the second group, decoy cells were visible in 15 specimens from 3 patients in which Polyoma virus infection was confirmed by PCR or external review of the cases. Cancer cells were not detected. Conclusions: Ischemia and reperfusion injury were the most likely causes for atypical cells in the first group. Our study confirmed the fact that kidney recipients are in danger for reactivation of Polyomavirus infection and is supportive for performing urinary cytological evaluation in the surveillance of patients after kidney transplantation and immunosupression.

Sa-106 Age-related Early Onset Of epithelial-to-mesenchymal transition in experimental renal transplantation J.H. Braesen1, U. Hoﬀ2, M. Nieminen-Kelhae2, Y. Linde2, M. Loew2, B. Hegner2, F.C. Luft2, D. Dragun2 1 Institut für Pathologie, Universitätsklinikum S-H, Campus Kiel 2 Nephrologie, Charité – Universitätsmedizin Berlin, Campus Virchow, Mitte und Buch Aims: Epithelial-to-mesenchymal cell transition (EMT) has been im-plicated in chronic renal injuries. We aimed at determining the role of EMT in delayed graft function of older renal allografts. Methods: We studied age-matched young vs. old rat donors in a model of renal transplantation with allogeneic versus syngeneic strain combinations. Grafts were harvested 1, 2, 5, and 7 days post-transplant for analysis. Results: We found clear, age-related differences in epithelial responses to postischemic injury. Greater functional impairment in older grafts during the first post transplant week accompanied delayed recovery of tubular epithelium and transient signs of epithelial dedifferentiation (loss of cytokeratin and apical megalin, and de novo vimentin expression). The effects were independent of the immunologic background and leukocyte infiltration. Young allografts showed no increase in dedifferentiation of tubular epithelium. Loss of apical megalin in cortical tubules of older organs was associated with greater low-molecular-weight proteinuria, implicating changes in epithelial polarity and function. TGF-β gene expression increased continuously in all groups during the entire time period. Conclusions: Our data implicate a reduced regenerative capacity in old kidneys and a greater propensity to EMT. Early onset of EMT may be related to greater susceptibility to TGF-β. Low-molecular-weight proteinuria could serve for detection of early EMT. Minimization of cold preservation time and tubulotoxic immunosuppressive agents, especially in old-to-old graft

Sa-107 Mixed immune cell population in intimal arteritis of renal allografts N. Kozakowski, G. Böhmig1, A. Soleiman, M. Exner2, K. Nagy-Bojarszky, H. Regele Klinisches Institut für Pathologie, 1 Klin. Abt. für Nephrologie und Dialyse, Universitätsklinik für Innere Medizin III und 2 Klinisches Institut für Medizinische und Chemische Labordiagnostik, Medizinische Universität Wien Aims: Vascular rejection is characterised by infiltration of the arterial intima of renal allografts by immune cells, traditionnally assumed to be lymphocytes, reflecting the common use of anti-lymphocytic drugs for therapy. A recent study however found in a small number of cases that the majority of intimal immune cells would be macrophages rather than T-cells. Thus we investigated the proportion of T-cells and macrophages in a larger number of unselected cases of vascular renal allograft rejection.

Methods: Immunohistochemical double labelling with antibodies against CD68 and CD3 was performed on paraffin sections from 42 biopsies with vascular rejection diagnosed between 1997 and 2003. Results: The CD68/CD3 ratio ranged from 0,4 to 8,25. Predominance of monocytes was associated with re-transplantation but not with C4d deposits or PRA-levels. The type of infiltrating immune cells did not depend on age of donors or recipients, duration of cold ischemia or number of mismatches. Kaplan-Meier analysis revealed no statistically significant difference in transplant outcome and patient survival between groups. Conclusions: Intimal arteritis is always composed of a mixed population of monocytes/macrophages and lymphocytes. Predominantly monocytic infiltration of the intima was associated with re-transplantation but not with indicators of humoral response and had no clear-cut influence on renal function and graft survival.

Sa-108 Is high salt intake in pregnancy responsible for altered kidney morphology in the oﬀspring? N. Koleganova1,2, G. Piecha1,2, M.-L. Gross1, A. Geldyyev1, L.E. Becker1,2, J. Müller3, E. Ritz2 1 Pathologisches Institut, 2 Medizinische Klinik, Abteilung Nephrologie und 3 Medizinische Klinik, Abteilung Kindernephrologie, Universitätsklinikum Heidelberg Aims: Intrauterine malnutrition may predispose the offspring to developing hypertension, obesity, diabetes, and cardiovascular disease in adulthood. We aimed at investigating whether dietary salt intake during pregnancy would alter kidney development in the offspring. Methods: Sprague-Dawley rats were fed low (0.15%), normal (1.3%), or high (8.0%) salt diet during pregnancy and weaning. The offspring were separated from mothers at 4 weeks of age and maintained on the same or low salt diet. Systolic blood pressure was controlled by tail pletysmography and intraaortally. Albumin excretion was measured by specific ELISA. Kidney morphology was assessed at 7 and 12 weeks postnatal. Results: No significant differences in blood pressure were observed between the groups. Albumin excretion was significantly higher in offspring on high salt compared to those on normal and low salt. The number of glomeruli was significantly lower in the offspring of mothers fed high salt compared to normal salt and tended to be lower in the offspring of mothers fed low salt. The mean glomerular volume was significantly higher in male offspring of mothers fed high salt and maintained on high (6.19±1.50 106mm3) or low salt (6.84±1.35) compared to offspring of mothers on normal (4.79±1.27) or low salt (4.62±1.08). The same differences were observed in females. Conclusions: Both high and low salt intake during pregnancy leads to lower nephron number in rat offspring. This changes seem not to affect blood pressure significantly. The effects of high prenatal salt load are not reversed by later salt restriction.

Sa-109 Case-report: A 16 year-old female patient with manifest Fabry’s disease and mesangioproliferative IgA-glomerulonephritis M. Knoess1, P. Knoess1, A. Schwarting2, M. Beck, E. Wandel2, M. Otto1, J. Kriegsmann1 1 Zentrum für Histologie, Zytologie und Molekulare Diagnostik, Trier 2 I. Medizinische Klinik und 3 Pädiatrische Klinik, Universitätsklinikum Mainz Aims: We report on a case of a 16-year-old female patient with manifest Fabry’s disease, mesangioproliferative IgA-glomerulonephritis and focal fibrosis of the glomerular capsule. Methods: Case report and review of literature. Results: The patient presented with preexisting Fabry’s disease, recurrent macroscopic hematuria and acanthocytosis of 16%, chronic persistent proteinuria and intermittent blood pressure peaks.

Histopathological examination of the renal tissue by light microscopy demonstrated the typical foamy cytoplasm of glomerular cells. Furthermore, focal fibrosis of the glomerular capsule was detectable. Electron microscopy revealed osmiophilic lamellar bodies in podocytes. Additionally, light microscopy showed mild increase in mesangial cell number and mesangial matrix with a fine granular reaction in immunohistochemical analysis with antibodies against IgA. Furthermore, mesangial electron dense deposits were detectable in electron microscopy. Conclusions: Fabry’s disease may be rarely associated with other renal diseases. We described the combination of Fabry’s disease and IgA-nephritis.

Sa-110 Occurrence of disseminated tumor cells within the periphal blood is a prognostic factor in renal cell carcinoma K. Blümke, H. Heynemann, S. Göbel1, A. Meye2, M. Kotzsch3, H. Taubert1, P. Fornara, S. Hauptmann1 Klinik für Urologie und 1 Institut für Pathologie, Universitätsklinikum Halle/Saale 2 Klinik für Urologie und 3 Institut für Pathologie, Universitätsklinikum Dresden Aims: Disseminated tumor cells (DTCs) are the soil of distant metastases. Therefore, presence of DTCs is discussed to be a negative prognostic factor in a variety of malignant tumors. Methods: We have established an immunomagnetic cell separation procedure (autoMACS) that allows the sensitive detection of DTCs of renal cell carcinoma (RCC) patients. In this study, 233 blood samples from 154 RCC patients were investigated for occurrence of DTCs by autoMACS technique and immunocytochemical cytokeratin staining. We correlated the presence of DTCs to tumor size, occurrence of metastases and tumor grade. Results: Ninety-six of the 233 blood samples (41.2%) originating from 81 of 154 RCC patients (52.6%) carried cytokeratin positive tumor cells. The occurrence of DTCs correlated significantly with lymph node status (P<0.001) and occurrence of distant metasta-ses (P<0.01). RCC patients with disseminated tumor cells in the peripheral blood at the time of primary tumor resection had a sig-nificant shorter overall survival rate than patients without DTCs, even within a multivariate analysis: We could confirm this both in KaplanMeier analysis (P<0.05) and in Cox’s regression hazard analysis (P<0.05). Conclusions: This study indicates that the presence of DTCs in RCC at the time of primary surgery is an independent negative prognostic factor.

Sa-111 Expression of the tight junction protein Claudin-1 in Renal Cell Carcinoma F. Fritzsche1, B. Oelrich2, M. Dietel1, K. Jung2, G. Kristiansen3 1 Institut für Pathologie und 2 Institut für Urologie, Charité – Universitätsmedizin Berlin, Campus Mitte 3 Institut für Klinische Pathologie UniversitätsSpital Zürich Aims: Claudins belong to a family of transmembrane proteins that have been identified as components of tight junction strands and that are commonly dysregulated in neoplasia. We evaluated the expression of the cell adhesion protein claudin-1 in renal cell carcinoma on a large thoroughly characterized patient cohort with follow-up data. Methods: 359 patients undergoing tumour nephrectomy between 1992– 2005 at the Charité Universitätsmedizin Berlin were included in the study. A tissue microarray with matched tumour/normal pairs was constructed and immunostainined with anti-claudin-1 (Invitrogen). Results: Claudin-1 was found in 28.7% (n=102) of all renal cancer cases. Stratified for histological type only 21% (n=60/281) of clear cell carcinoma showed positivity versus 76% (n=25/33) of papillary and 40% (n=4/10) of chromophobe tumours. Claudin-1 expression was significantly associated with tumour grading and positive nodal status. In univariate analysis expression of claudin-1 was associated with significantly shortened patient survival.

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Abstracts Conclusions: Claudin-1 is absent in the majority of clear cell renal cell carcinomas. If present, Claudin-1 expression was associated with markers of unfavourable prognosis and was a significant prognosticator for shortened patient survival.

Sa-112 Expression analysis of Caspase-8 inhibiting genes in renal cell carcinomas – a prominent role of ARC S. Heikaus, T. Kempf, C. Mahotka, H.E. Gabbert, U. Ramp Institut für Pathologie, Universitätsklinikum Düsseldorf Aims: Activation of the initiator Caspase-8 participating in the extrinsic as well as the intrinsic pathways of apoptosis is under tight control of multiple antiapoptotic regulators like ARC, FlipS, FlipL and PED/PEA-15. However, nearly nothing is known about the expression of Caspase-8 and its antiapoptotic regulators especially in renal cell carcinomas (RCCs) in vivo. Therefore, the aim of the present study was to analyse and compare the in vivo expression of Caspase-8, ARC, FlipS, FlipL and PED/PEA-15 to identify which of these genes might be crucial for apoptosis resistance in RCCs. Methods and Results: Our investigation was performed in primary tumour tissue of 48 moderately (G2) or poorly (G3) differentiated RCCs and in corresponding non-neoplastic renal tissues by real-time PCR. Thereby, relative mRNA expression of Caspase-8, FlipS, FlipL and PED/PEA-15 was significantly increased only in early tumour stages (pT1 and pT2) compared to nonneoplastic renal tissues. In contrast, ARC mRNA expression was significantly increased in all tumour stages without differnces between pT1, pT2 and pT3 carcinomas. Furthermore, the relative mRNA expression ratio between ARC and Caspase-8 was significantly increased in advanced RCCs (pT3) when compared to pT1 and pT2 carcinomas. In contrast, the relative mRNA expression ratio between FlipS, FlipL or PED/PEA-15 and Caspase-8 remained constant. Conclusions: In conclusion, our analysis revealed that ARC is the only Caspase-8 inhibiting regulator being constantly overexpressed in RCCs when compared to non-neoplastic renal tissue. In consequence, the balance between antiapoptotic ARC and proapoptotic Caspase-8 is the only one to be gradually disturbed during tumour progression of RCCs, resulting in a relative increase of antiapoptotic ARC over Caspase-8 expression, contributing to impaired apoptosis and multidrug-resistance of RCCs.

Sa-113 VHL isoform-speciﬁc regulated cell surface proteins in Renal Cell Carcinoma G. Boysen, B. Wollscheid1, D. Bausch-Fluck1, V.D. Luu, P. Schraml, W. Krek2, H. Moch Institut für Klinische Pathologie, UniversitätsSpital Zürich 1 Institut für Molekulare Systembiologie und 2 Institut für Zellbiologie, ETH Zürich Aims: Proteomic and transcriptomic expression profiling of tumor cells result in complex data sets. Bioinformatics is used in Systems Biology to extract functional information from these data. The von Hippel-Lindau (VHL) tumorsuppressor gene is somatically inactivated in the majority of renal sporadic clear cell RCCs (ccRCC). VHL encodes two protein isoforms pVHL30 and pVHL19. Different functions are proposed for these protein isoforms due to differences in their subcellular localizations. As membrane proteins represent potential cancer drug targets the aim of this study was to identify cell surface proteins that become upregulated due to loss of pVHL19 or pVHL30. Methods: By applying LC-MS/MS expression profiling of cellular membrane proteins was assessed from human ccRCC VHL null 786-O and the pVHL re-expressing stable transfectants 786-O-pVHL19 and 786-O-pVHL30. Using a Low Density Array approach and Affymetrix genechips, expression patterns of the identified proteins were further validated on the RNA level. Results: Several novel pVHL isoform-specific regulated candidates were identified. In silico classification categorized these proteins mainly in the EGFR-, Cadherin- and G-protein signalling pathways.

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Conclusions: In summary, the combination of quantitative proteo- and transcriptomics allowed the identification of cell membrane proteins that are concertedly and separately regulated by the two pVHL isoforms. The results of our study will lead to a better understanding of the biological consequences of a deregulated VHL-pathway in ccRCC. This knowledge will finally contribute to improve diagnosis and therapeutical strategies of renal cancer.

Sa-114 Expression of Transcription Factor 2 (TCF-2) is a relevant prognostic parameter in metastasized renal cell carcinoma G. Assmann1, T. Kirchner1, M. Castro2, A. Hennig2, C.G. Stief3, W. Zimmermann2, A. Buchner3 1 Pathologisches Institut, 2 Labor für Tumorimmunologie und 3 Urologische Klinik und Poliklinik, Universitätsklinikum München (LMU) Aims: The clinical course of patients with metastasized renal cell carcinoma (mRCC) shows a high range in the overall survival, even within groups of patients with similar tumor stage. In a previous expression profiling study using laser microdissection and oligonucleotide microarrays, we have identified TCF-2 as a potentially prognostic factor. In this study we analysed the expression of TCF-2 on RNA and protein level. The results were correlated with the survival data of the patients. Methods: Samples of normal renal tissue (n=6), RCC primary tumor (n=57) and RCC metastases (n=59) were analysed by quantitative RT-PCR. Immunohistochemistry was performed so far in a subset of the tissue samples (n=18) using a monoclonal antibody against TCF-2. Results: TCF-2 was part of a prognostically relevant three gene signature in a previous microarray study (Affymetrix GeneChip® HG U133 Plus 2.0). The RT-PCR data show a significant decrease in TCF-2 expression correlated with malignant transformation (normal renal tissue >primary tumor >metastasis; p<0.0001). There is also a correlation between high TCF-2 expression in primary tumor and better prognosis. TCF-2 immunohistochemistry shows a strong and specific nuclear staining in tumor cells both of the primary tumors and of mRCC. No differences in the staining intensity are detectable. Conclusions: The expression of TCF-2 is a relevant prognostic factor in mRCC with a significant decrease of the RNA expression level correlated with malignant transformation. TCF-2 immunohistochemistry shows a strong nuclear staining both in primary tumors and metastases. There is no correlation between the RNA expression level of TCF-2 and the staining intensity.

Sa-115 Predictive value of pathological and immunohistochemical parameters for recurrence and survival in pT1 bladder cancer S. Bertz1, S. Denzinger2, S. Link1, R. Stoehr3, M. Fritsche2, W. Wieland2, F. Hofstädter1, A. Hartmann3 1 Institut für Pathologie und 2 Klinik für Urologie, Universitätsklinikum Regensburg 3 Instiut für Pathologie, Universitätsklinikum Erlangen Aims: To evaluate several methods for subdividing pT1 bladder cancers into groups of microinvasion and advanced invasion and to detect immunohistochemical markers correlating with risk of recurrence, progression to muscleinvasive disease and survival. Methods: pT1 tumors of 308 patients were revaluated histomorpho-logically by three pathologists according to the WHO classifications of 1973 and 2004. Tumors were classified according to invasion depth in relation to the L. musc. muc. (pT1a/b) and to the area of invasive tumor (<1,=1,>1 HPF). Additionally IHC with CK20, MIB-1, p53 and MCM-2 was performed on tissue microarrays. Results: 69 cases showed recurrence: 52 (75.4%) >1HPF, 7 (10.1%)=1HPF, 10 (14.5%) <1HPF. 62 cases showed progression: 50 (80.6%) >1HPF, 10 (16.1%)=1HPF, 2 (3.2%) <1HPF. There was no correlation of the pT1a/b classification with any of the parameters. Absence of CIS, papillary growth pattern and pushing/ trabecular invasion pattern were significantly associated with low progression risk and good survival. Low proliferation (MIB-1<15%-

p>0.001)) and negativity for p53 (p=0.021) was significantly correlated with survival, but not recurrence. Aberrant expression of CK20 (>5% or total loss) was associated with both recurrence (p<0.01) and poor survival (p<0.0019). Conclusions: MIB-1-, p53- and CK20-expression are negatively correlated with survival but only CK20 with recurrence. Histopatholo-gical parameters (CIS, growth and invasion pattern and the su-classification of pT1 tumors by area of invasion (1HPF) indepen-dent from the L. musc. muc. are simple and reliable parameters for risk stratification of pT1 bladder cancer patients.

formed blood vessels although RCC cells can principally synthesize IDO as demonstrated by in vitro stimulation with interferon-γ. A highly significant inverse correlation between the density of IDO-positive microvessels and the content of proliferating Ki67-positive tumor cells in primary and metastatic RCC was found (p=0.004). Conclusions: IDO in endothelial cells might limit the influx of tryptophan from the blood to the tumor or generate tumor-toxic metabolites thus restricting tumor growth and contributing to overall survival.

Sa-116 Automated analysis of immunohistochemical staining of clear cell renal cell carcinoma tissue microarrays T.J. Fuchs, P.J. Wild1, N. Wey1, T. Lange, P. Fürnstahl2, W. Einhäuser, H. Moch1, J. Buhmann Institut für Computational Science und Kompetenzzentrum für Systemphysiologie und metabolische Krankheiten, ETH Zürich 1 Institut für Pathologie, UniversitätsSpital Zürich 2 Institut für Bildverarbeitung, ETH Zürich

Sa-118 Stage and grade-dependent loss of components of the HLA class I antigen processing machinery (APM) in renal cell carcinoma (RCC) C.G. Hammerschmied, B. Walter1, H. Moch2, K. Junker3, H. Blaszyk4, R. Hinze5, A. Hartmann, B. Seliger6 Pathologisches Institut, Universitätsklinikum Erlangen 1 Lehrstuhl für Urologie, Universitätsklinikum Regensburg 2 Institut für Klinische Pathologie, UniversitätsSpital Zürich 3 Lehrstuhl für Urologie, Universitätsklinikum Jena 4 Institute of Pathology, University of Vermont, USA 5 Institut für Pathologie, Klinikum Schwerin 6 Institut für Medizinische Immunologie, Universitätsklinikum Halle

Aims: Validation of the clinical importance of candidate genes or proteins requires large-scale analysis of human tissues. To date, this analysis constitutes an important bottleneck in the process of discovery because tissue analysis by the conventional slide-by-slide strategy is slow and expensive. To overcome these limitations, tissue microarray (TMA) technology has been developed and allows for the simultaneous analysis of up to 1,000 tissue samples in a single experiment. However, manual quantitative TMA scoring can also be very time consuming and prone to error. To facilitate and standardize this laborious task we propose a novel approach for the automated analysis of the Ki-67 (MIB1) proliferation index, using a renal cell carcinoma TMA. Methods: A semi-supervised method, which is based on an iterative morphological algorithm and a soft-margin support vector machine, detects and segments renal cell carcinoma nuclei with high precision and reliability. The quality of automated nuclei detection is determined by comparison with ground-truth data of more than 2000 cell nuclei which have been labeled independently by pathologists. Results: Quantitative results show that the proposed cell nuclei detection and segmentation system outperforms existing algorithms and approaches the performance of experienced clinical pathologists. Conclusions: Our automatic, high throughput analysis system for tissue microarray analysis promises new avenues for the discovery of biomarkers to detect and prognose renal cell cancer.

Sa-117 Expression of indoleamine 2,3-dioxygenase in tumor endothelial cells correlates with long term survival of renal cell carcinoma patients C. Weiler, R. Riesenberg1, R. Kammerer1, W. Zimmermann1 Pathologisches Institut und 1 LIFE-Zentrum – Labor für Tumorimmunologie, Universität München (LMU) Aims: The inflammatory enzyme indoleamine 2,3-dioxygenase (IDO) participates in immune tolerance and tumor immune escape processes by degradation of the essential amino acid tryptophan and formation of toxic catabolites. In this study, we analyzed the role of IDO in tumor growth and disease progression in patients with clear cell renal cell carcinoma (RCC). Methods: Expression of IDO mRNA was analyzed by quantitative reverse transcription-PCR in 55 primary, 48 metastatic RCC and 32 normal kidneys. Western blot and immunohistochemistry was used to semiquantitatively determine IDO protein in a subset of tumor samples, in RCC cell lines and microvessel endothelial cells. IDO expression was correlated with expression of the proliferation marker Ki67 in tumor cells and survival of tumor patients. Results: More than 75% of the clear cell RCC in comparison to normal kidney contained elevated levels of IDO mRNA which correlated with their IDO protein content. Low IDO mRNA levels in primary tumor represented an unfavorable independent prognostic factor. Immunohistological analyses revealed that IDO is nearly exclusively expressed in endothelial cells of newly

Aims: HLA class I abnormalities are frequently found in tumor cell lines and malignant tissues and in some cases can also be correla-ted with metastatic phenotype and the clinical outcome of patients. So far there are only limited data available on the expression of components of the MHC class I APM in RCC. Methods: Using tissue microarrays immunohistochemical analysis of 351 formalin-fixed, paraffin-embedded RCC of clear cell, papillary or chromophobe subtype was performed using antibodies directed against the APM components LMP2, LMP7, TAP1, TAP2, tapasin, beta-microglobulin, calnexin, calreticulin, ERp57, ERAP1 and the HLA class I heavy chain. Results: Tumors of low UICC stages were significantly associated with more frequent nuclear negativity for LMP2 and less frequent membranous negativity for ERp57 than tumors of high UICC sta-ges. Tumors of low UICC stages demonstrated significantly more cytoplasmatic negatives for Erp57 and more frequently negative for LMP2 than high grade tumors. Conclusions: RCC demonstrated UICC stage and nuclear grade dependent loss of APM components. These findings emphasize the need for further evaluation of the underlying molecular mechanisms and indicate an important role of APM alterations in RCC.

Sa-119 Prognostic value of Tumor Necrosis Factor-Related Apoptosis-inducing ligand (TRAIL) receptors in renal cell cancer S. Macher-Göppinger1, S. Aulmann1, N. Wagener2, A. Haferkamp2, E. Herpel1, F. Autschbach1, P. Schirmacher1, W. Roth1,3 1 Pathologisches Institut und 2 Urologische Klinik, Universitätsklinikum Heidelberg 3 Deutsches Krebsforschungszentrum Heidelberg Aims: The differential expression of the TRAIL receptors (TRAIL-R1–4) determines the susceptibility of cells to apoptosis induced by the death ligand TRAIL. Here, we studied a possible association between the expression of TRAIL-Rs and the prognosis of patients with renal cell carcinomas (RCCs). Methods: Expression of TRAIL-Rs was examined by immunohistochemistry using a multiple tissue array containing RCC tumor tissue samples and corresponding normal tissue samples from 838 patients. The effect of TRAIL-R expression on disease-specific survival was assessed using univariate analysis and multivariate Cox regression analysis. Results: High TRAIL-R2 expression levels were associated with high-grade RCCs (p=.0001) and correlated negatively with disease-specific survival (p=.02). In contrast, low TRAIL-R4 expression was associated with high-stage RCCs (p=.0005) as well as with the incidence of distant metastasis (p=.005) and correlated negatively with disease-specific survival (p=.01). The combined expression of high TRAIL-R2 levels and low TRAIL-R4 levels defined Der Pathologe · Supplement 1 · 2008

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Abstracts a subgroup of patients with a significant shorter survival (p=.001). Clear-cell RCCs exhibited a lower expression of both TRAIL-R2 and TRAIL-R4 than the other histopathological subtypes of RCCs (p<.0001). Conclusions: High TRAIL-R2 and low TRAIL-R4 expression levels are associated with a worse disease-specific survival in patients with RCCs. The assessment of TRAIL-R expression offers valuable prognostic information that could be used to select patients for adjuvant therapy.

Sa-120 Lung metastases of renal cell carcinoma show similar characteristics compared to primary tumours and they express survivin, XIAP, and mortalin M. Meinhardt, P. Schneider1, A. Meye1, A. Rolle2, O. Holotiuk3, M. Wirth1, G. Baretton Institut für Pathologie und 1 Klinik für Urologie, Universitätsklinikum Dresden 2 Fachkankenhaus Dresden-Coswig 3 Gemeinschaftspraxis Pathologie Dresden-Neustadt Aims: Investigations on renal cell carcinoma usually concentrate on primary tumours. This study is based on lung metastases of renal cell carcinoma. Well established parameters of renal cell carcinoma like grading, proliferation, and expression of CD 10 were compared in metastases. Additionally, the immunohistological expression of anti-apoptotic markers was examined. Methods: 84 lung metastases of clear cell renal carcinomas were resected between 1999 and 2004. Cores were punched and arranged in tissue micro arrays. Slides were stained H&E and antibodies against CD10, Ki67, survivin, XIAP, and mortalin were used for immunohistochemistry. Results: Metastases were classified as well, moderately, and poorly differentiated in 20.7% (G1), 63,9% (G2), and 15.4% (G3), respectively. Grading correlated with overall survival. The average proliferation index (Ki 67) was 13,5%. A strong majority of the cases showed an expression of CD 10, survivin, XIAP, and mortalin. Conclusions: Grading in metastases is similar to primary renal cell carcinomas. The proliferation rate in metastases is expectedly higher than in primaries. The anti-apoptotic proteins survivin, XIAP and mortalin probably are expressed in primaries and metastases alike. Therefore, investigations of metastases with criteria used so far only for primary tumours, appear valid and valuable.

Sa-121 Array Based micro-RNA Proﬁling of Renal Cell Cancer Identiﬁes Differentially Expressed miRNAs with Diagnostic Potential G. Kristiansen, H.-J. Mollenkopf1, W. Nietfeld2, C. Grimm2, I. Wagner1, M. Jung3, K. Jung3 Institut für Klinische Pathologie, UniversitätsSpital Zürich 1 Max Planck Institut für Infektionsbiologie Berlin 2 Max Planck Institut für molekulare Genetik Berlin 3 Urologische Klinik, Charité – Universitätsmedizin Berlin Aims: Recently, short non-coding micro-RNAs have been identified as important regulators of gene expression. We aimed to identify differentially expressed miRNAs in renal cell cancer by comparing the miRNA profiles of tumor tissue with adjacent normal tissue. Methods: 12 pairs of fresh frozen matched normal and tumor tissue from renal cell carcinoma were analysed using human miRNA-microarrays encoding probes for 470 human and 64 human viral microRNAs (Agilent Technologies). Selected miRNAs were further confirmed via TaqMan miRNA PCR assays (Applied Biosystems). Results: 34miRNAs displayed a differential expression >2 fold in malignant to non-malignant samples: 14 miRNAs were up- and 20 were downregulated in clear cell carcinoma. An unsupervised hierarchical cluster analysis of differentially expressed miRNAs demonstrated a good discrimination of normal and tumor samples. 16 miRNAs showed a fold change differences >4, among these were the oncogenic miR-155 and miR-210, the latter comprising a putative hypoxia response element. The comparison of differential expression

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measured by microarrays and quantitative real time PCR demonstrated an excellent rate of concordance. Conclusions: Renal cell cancer is characterised by significant changes in miRNA profiles. The miRNA expression profiles allow a clear molecular discrimination between renal cell carcinoma and normal tissue.

Sa-122 Growth inhibitory eﬀects of paclitaxel (Taxol®) alone and in combination with curcumin on an osseous metastasis of human renal cell carcinoma in vitro P. Reinecke1, C. Peters1, T. Kalinski2, H.E. Gabbert1 1 Institut für Pathologie, Universitätsklinikum Düsseldorf 2 Institut für Pathologie, Universitätsklinikum Magdeburg Aims: Human renal cell carcinomas (RCCs) are highly resistant to radiotherapy and conventional chemotherapy. Recently, paclitaxel (Taxol®) has been shown to inhibit growth of RCCs in vitro. Therefore, we analyzed the chemotherapeutic potential of paclitaxel on our established cell line N109, derived from an osseous me-tastasis of a human RCC. In addition, we examined the role of the phytochemical curcumin on the growth inhibition which is known to act as a modulator of the Multidrug Resistance Phenotype. Methods: MTT assay, immunofluorescence microscopy, and flow cytometry. Results: 1. Significant dose-dependent growth inhibition and pacli-taxel-induced morphological alterations were observed in the cell line N109. 2. No significant dose-dependent growth inhibition was observed after exposure to curcumin alone. 3. A synergistic effect of growth inhibition was detected after treatment with both, paclitaxel and curcumin. 4. The cell line N109 shows a pronounced P-glycoprotein expression. 5. After exposure to paclitaxel (Taxol®) no modulation of the P-glycoprotein expression level was observed. Conclusions: This study demonstrates that paclitaxel can effec-tively inhibit the growth of an osseous metastasis of human renal cell carcinoma in vitro whereas a pronounced synergistic inhibition effect in combination with the phytochemical curcumin became evident.

Sa-123 Rare genomic EGFR-ampliﬁcation in renal cell carcinoma is associated with advanced tumour stage J. Neumann, E. Herrmann1, E. Korsching, H. Buerger2, S. Bierer1, W. Böcker, C. Wülﬁng1, E. Eltze, H. Schmidt Gerhard Domagk Institut für Pathologie und 1 Klinik und Polyklinik für UroIogie, Universitätsklinikum Münster 2 Institut für Pathologie, Paderborn Aims: The epidermal growth factor receptor (EGFR) expression is frequent detected in renal cell carcinoma (RCC) and associated with reduced progression-free and overall survival rates. The aim of the present study was to evaluate if the amplification of the first CA repeat in the first intron of EGFR is responsible for the overexpression in RCCs. Methods: Based on our previous immunohistochemical analyses of EGFR on tissue micro array (n=183) we investigated 47 RCCs with EGFR overexpression and six with complete negative EGFR status using fresh frozen tissue. Thereof 47 were clear cell, 6 papillary and one sarcomatoid type ranging from pT1 to pT3. For the detection of genomic amplification of EGFR we used a quantitative gene dosage PCR of the first CA repeat in the intron one. Results: Genomic amplifications of the EGFR were found in 4 of 47 RCCs (9%) with an immunohistochemical overexpression. None of the immunohistochemical negative RCCs showed an amplification. All 4 cases with an amplification were tumour stage pT3 and clear cell carcinomas. Conclusions: Genomic amplifications of the EGFR-gene are rare events in renal cell carcinomas associated with elevated stage. Therefore only 9% of the RCCs with EGFR protein overexpression could be explained by genomic amplification. These results implicate that in the majority of RCCs other regulatory effects are involved in EGFR expression, like the length of the investigated CA repeat previously shown in mamma carcinoma. Future studies

should evaluate if patients with a genomic EGFR amplification benefit from molecular based therapies against the EGF-Receptor.

Sa-124 Decoy Receptor 3 predicts survival in renal cell cancer S. Macher-Göppinger1, S. Aulmann1, N. Wagener2, B. Funke1, A. Haferkamp2, F. Autschbach1, P. Schirmacher1, W. Roth1,3 1 Pathologisches Institut und 2 Urologische Klinik, Universitätsklinikum Heidelberg 3 Deutsches Krebsforschungszentrum Heidelberg Aims: Decoy Receptor 3 (DcR3) is a soluble protein that binds to and inactivates the death ligand CD95L. Here, we studied a possible association between DcR3 expression and prognosis in patients with renal cell carcinomas (RCCs) Methods: A multiple tissue microarray containing RCC tumor tissue samples and corresponding normal tissue samples from 838 patients was generated. DcR3 expression was examined by immunohistochemistry. The effect of DcR3 expression on disease-specific survival and progression-free survival was assessed using univariate analysis and multivariate Cox regression analysis. DcR3 serum levels were determined by ELISA. Results: High DcR3 expression was associated with high-grade (p=.005) and high-stage (p=.048) RCCs. DcR3 expression correlated negatively with disease-specific survival (p=.0004) and progression-free survival (p=.0003) in univariate analyses. A multivariate Cox regression analysis retained DcR3 expression as an independent prognostic factor that outperformed the Karnofsky performance status. In patients with high stage RCCs expressing DcR3, the two-year survival rate was 20%, whereas in patients with DcR3-negative tumors, the survival rate was 56% (p=.00016). Moreover, DcR3 serum levels were significantly higher in patients with high-stage RCCs (p=.00001). Conclusions: DcR3 expression is a powerful independent predictor of RCC progression and mortality. Therefore, the assessment of DcR3 expression levels offers valuable prognostic information that could be used to select patients for adjuvant therapy.

Sa-125 Combined analysis of FGFR3 status and UroVysion test exactly deﬁnes high-grade and high-stage urothelial bladder tumors P.J. Wild, T.J. Fuchs1, I. Steiner2, S. Frigerio, D.R. Zimmermann, B. Padberg, E.C. Zwarthoﬀ3, R. Stoehr4, F. Hofstaedter2, G.O. Kristiansen, S. Denzinger5, M. Provenzano6, T. Hermanns6, H.H. Seifert6, T. Sulser6, V. Roth1, J.M. Buhmann1, H. Moch, A. Hartmann4 Institut für Pathologie, UniversitätsSpital Zürich 1 Instute for Computational Science, ETH Zürich 2 Institut für Pathologie, Universitätsklinikum Regensburg 3 Dept. of Pathology, Rotterdam, NL 4 Institut für Pathologie, Universitätsklinikum Erlangen 5 Abt. für Urologie, Universitätsklinikum Regensburg 6 Abt. für Urologie, UniversitätsSpital Zürich Aims: To investigate the prognostic and predictive impact of FGFR3 mutation status and UroVysion fluorescence in situ hybridization (FISH) analysis in patients with urothelial bladder cancer, and to estimate the diagnostic power for the detection of bladder cancer in urine using UroVysion FISH, FGFR3 analysis and cytology as predictors. Methods: Expression of CK20, Ki-67 and p53 was analyzed by immunohistochemistry and chromosomal alterations using multicolor FISH (UroVysion) on tissue microarrays of bladder cancer (n=255). FGFR3 status was studied by SNaPshot mutation detection. Additionally, urine samples of 91 patients were collected and matched with their biopsies. Results: 70% of urothelial neoplasms showed polysomy of at least one chromosome. A relative p16 deletion was found in 46% of cases. Histologic grade, growth pattern and FGFR3 status were independent prognostic factors for survival. Given the prognostic impact of grade, a model consisting of UroVysion FISH and FGFR3 status predicted high grade and stage equally well compared to the molecular grade proposed by van Rhijn et al.

Conclusions: UroVysion FISH plus FGFR3 status reached a high accuracy in predicting stage and grade of bladder cancer and could be used as non-invasive methods in urine.

Sa-126 Her-family tyrosine kinase receptor and ligands expression as well as proliferative cellular nuclear activity (PCNA) in chronic cystitis, nephrogenic metaplasia and neoplastic tubular bladder lesions M. Abbas1, D. Atkins2, S. Mansy1 , S. Störkel2 1 Theodor Bilharz Research Institute Kairo, ET 2 Institut für Pathologie der UWH und Helios Klinikum Wuppertal Aims: Urothelium undergoes proliferative and metaplastic changes to a variety of stimuli, notably inflammation and irritation. The study is focussing on the expression of Her-family receptors and ligands in inflamed urothelium, metaplastic and neoplastic tubular urothelial lesions with respect to their proliferative activity. Methods: 36 paraffin-embedded tissue samples were investigated immunohistochemically with antibodies against Her-family receptor members, ligands EGF, TGF-alpha and Heregulin according to standard protocols and 4-tailed scoring system. Proliferative activity is assessed using PCNA Antibody which applied on the same tissue samples. Results: Inflammatory samples showed expression of Her-3 and Her-4 in 63% (3+) while EGF and TGF-alpha ligands expression was 25%. Nephrogenic metaplasia showed expression of Her-4 in 85% (3+), of EGF in 85% and of TGF-alpha in 82%. Invasive adenocarcinoma showed expression of Her-2 in 50% (3+), of Her-3 in 50% (3+), of Her-4 in 75% (3+), of EGF in 75% and of TGF-alpha in 88%. PCNA is strongly expressed in both inflamed urothelium and adenocarcinoma (87.5%) while is expressed in 46% of nephrogenic metaplasia. Conclusions: Low level expression of ligands in inflamed urothelial lesions compared to increased expression in both nephrogenic metaplasia and adenocarcinoma points to the role of ligand expression in different pathophysiological pathways occurring in urothelium as well as the role of proliferative activity in directing these changes. High expression level of Her-2 in adenocarcinoma in comparison with other examined lesions determines its role in carcinogenesis and invasion.

Sa-127 Expression of Her-family members and their ligands EGF, TGF-alpha and Heregulin in normal urothelium and diﬀerent urinary bladder carcinoma variants M. Abbas 1, D. Atkins2, S. Mansy1 , S. Störkel2 1 Theodor Bilharz Research Institute Kairo, ET 2 Institut für Pathologie der UWH und Helios Klinikum Wuppertal Aims: The study is focussing on the expression of Her-family members and their ligands in CIS, papillary non-invasive urothelial carcinoma (UC) and invasive UC variants with respect to grade and stage and in normal urothelium of different ages. Methods: Paraffin-embedded tissue samples from 88 patients (German and Egyptian) were investigated immunohistochemically with antibodies against Her-family members and their ligands EGF, TGF-alpha and Heregulin according to standard protocols and 4 tailed score. Results: Expression of EGFR in normal aging urothelium (>70 Ys) is in 19%(3+), CIS is in 20%(3+) and squamous cell carcinoma (Sq.C.Ca) with or without Schistosomiasis is in100%(3+). Her-2 is expressed in invasive UC in 50%(3+) and in 50%(2+) and adenocarcinoma in 50%(3+) and in 25%(2+), correlated with high grade and stage. Her-3 and Her-4 are expressed in low and high grade non-invasive UC in 90%(3+) and in 91%(3+) respectively. EGF ligand is 100% expressed in low grade non-invasive UC and in 73% of high grade non-invasive UC. TGF-alpha is found in 81% of aging urothelium (>70Ys), in 70% of CIS, in 64% of high grade non-invasive neoplasms, in 67% of UC, in 88% of adenocarcinoma and in 90% of Squamous cell carcinoma. Conclusion: Similar expression of EGFR and TGF-alpha in aging normal urothelium (>70Ys) and CIS lesions warrants further investigation to predict Der Pathologe · Supplement 1 · 2008

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Abstracts the molecular changes of aging urothelium. The Expression of TGF-alpha in high grade non-invasive UC, CIS and invasive UC variants denotes its vital role in carcinogenesis and invasion. Variation in receptor expression is associated with the morphological phenotypical changes of bladder cancer while ligand expression is correlated with proliferation and aggression.

Sa-128 Interobserver diﬀerences in the diagnosis of stroma invasion in bladder cancer (pTa/pT1) S. Minner, J. Köllermann, S. Zuber, G. Sauter Institut für Pathologie, Universitätsklinikum Hamburg-Eppendorf Aims: Recognition of early invasion (pT1) in bladder cancer is challenging and interobserver differences between pathologists a significant issue. Moreover, histological staging in superficial bladder cancer is crucial for subsequent therapy. Earlier studies investigating tumours that were initially diagnosed before 1996 showed interobserver discrepancies between 25 and 35%. The aim of this study was to evaluate whether the same variations still apply today. Methods: All sections from 101 bladder carcinomas staged as pT1 in the Department for Pathology in Hamburg between 2001 and 2007 were reviewed by two pathologists. Results: From 101 tumours originally staged as pT1, 90% (91) were classified as pT1 and 10% (10) were downstaged to pTa. Among the 10 tumours donwnstaged to pTa, 6 were graded as G2 and 4 were graded as G3. Reasons for diagnostic differences were primarily the false assessment of tangential sectioning and endophytic growth patterns of non-invasive tumour parts. Conclusions: Our results confirmed that recognition of minimal stroma invasion in bladder cancer remains a difficult issue. However, the results of this study were considerably better than those of comparable studies between 1988 and 2003. We conclude that the correct staging in superficial bladder cancer (pTa/pT1) may have substantially improved in recent years perhaps due to the awareness of the importance of differentiating between pTa and pT1 tumours.

Sa-129 Androgen ablation therapy results in increased p65, c-Myc and Androgen Receptor expression in prostate cancer in vivo L. Kenner1,2, M. Schlederer2, P. Mazal1, A. Haitel1, M. Susani1, J.A. Schmid2 1 Klinisches Institut für Pathologie, Medizinische Universität Wien 2 Ludwig Boltzmann Institut für Krebsforschung (LBI-CR), Wien Aims: Developing digital imaging analysis and protein quantification in vivo. Investigating the role of Androgen Receptor (AR) and NFkB in prostate carcinogenesis. Methods: Target gene expression analysis in human prostate cancer samples on tissue arrays before and after androgen ablation therapy using immunofluorescence microscopy with “Tissue FACS” analysis and immunohistochemistry. Quantification of fluorescence intensities was performed using TissueQuest™ and ImageJ. Macros for automated image analysis of epithelial structures were designed. We established mouse models for prostate cancer by prostate specific loss of PTEN (Pb-Cre+ PTENf/f ). Mouse tumor samples were evaluated as described for human samples. Results: Quantification with TissueQuest™and ImageJ sofware enables reproducible and high throughput protein quantification. We established an epithelial specific protein quantification for cell recognition. AR, p65 and c-Myc were upregulated in prostate carcinomas after androgen ablation therapy. Pb-Cre+ PTENf/f mice develop prostate cancers after 2 months. They show increased AR and p65 protein expression. Conclusions: Quantification with TissueQuest™ and ImageJ software enables reproducible and high throughput protein quantification. Androgen ablation therapy results in high stimulation of c-Myc, AR and p65 expression. These are also consistently overexpressed in prostate cancers of the Pb-Cre+ PTENf/f mice.

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Sa-130 Classiﬁcation of subgroups in 963 prostate cancers belonging to a progenitor like cell lineage E. Eltze, A. Semjonow1, B. Rademacher, W. Böcker, I. Buchwalow, H. Schmidt, E. Korsching Institut für Pathologie und 1 Klinik und Polyklinik für UroIogie, Universitätsklinikum Münster Aims: Basal cells as the proliferative compartment of the prostatic acinus produce a spectrum of basal cell lesions. Basal cell carcinoma usually adenoid cystic type is predominantly known to have an indolent course with local infiltrative behaviour. The interesting point for us was, to define and compare characteristics of basal lesions with respect to stem cell models. Methods: Using the tissue microarray technique, 963 prostate carcinomas of normal histological types were evaluated by immunohistochemistry using 12 different antibodies – amongst others the cytokeratins(CK) CK 5/6, CK 14, CK 8/18 and CK19. Bootstrap cluster analysis and interaction analysis was applied to the immunohistochemical data to reveal dependency patterns. Results: The cytokeratin expression in prostate cancer showed 7% CK 5/6, 95% CK8/18, 80% CK19 and 28% CK 14 positive tumours. The expression profiles correlated significantly (p≤0.001). No correlation could be found between CK expression and stage, Gleason grading, preoperative PSA or early PSA recurrence. Bootstrap clustering underlines the relation of CK 5/6 with CK 14, MIB1 and BCL2. Another stable cluster is formed between NKX3A and ApopTag. The interaction analysis revealed a synergistic action of all mentioned cytokeratins concerning a raising order of factors with LOXL2 and SNAIL as outstanding partners. Conclusions: Tumours with conventional histological type include a subgroup with an expression pattern typical for basal cells. This basal subtype comprises markers typically associated with stem or progenitor cells. The permutation approaches show a fine structure in the data set which gives clues for a group specific reanalysis.

Sa-131 Amyloidosis of the seminal vesicles: Does it protect from invasion by prostate cancer ? A. Erbersdobler, T. Schlomm1 Institut für Pathologie, Universitätsklinikum Hamburg-Eppendorf 1 Martiniklinik Hamburg Aims: Amyloidosis of the seminal vesicles is an unusual finding in prostatectomy specimens. The deposits are usually localized and asymptomatic. However, the question arises whether this disorder may affect extension of prostate cancer into the seminal vesicles. Methods: We identified 77 cases of seminal vesilcle amyloidosis out of a cohort of 6575 prostatectomy specimens surgically removed because of clinically localized prostate cancer. All cases were confirmed by a congo stain. Mean thickness of the amyloid band was measured in each case and correlated with clinical and pathological parameters. The frequency of seminal vesicle involvement by prostate cancer in the amyloidosis cases was compared with the percentage of pT3b classifications in the large cohort. Results: Mean age of the patients with amyloid deposits was 64 years (range: 52 to 73 years), which was slightly higher than the mean age in the large cohort (62 years). Mean thickness of the amyloid band did not correlate with patients´age, pre-operative PSA value, weight of prostate glands, or Gleasonscore and T-classification of prostate cancers. In the amyloidosis group, six cancers invaded the seminal vesicles (7.8%), which was not significantly lower than the percentage of seminal vesicle involvement by cancer in the large cohort (9.2%, P=0.6735). Conclusions: The pathogenesis of seminal vesicle amyloidosis is still poorly unterstood, but the disorder does not seem to provide an absolute or relative protection from seminal vesicle involvement by prostate cancer.

Sa-132 Expression and prognostic relevance of Annexin A3 in prostate cancer J. Köllermann1, T. Schlomm2, G. Schwall3, R. Simon1, H. Huland2, G. Sauter1, A. Schrattenholz2 1 Institut für Pathologie und 2 Martiniklinik, Universitätsklinikum Hamburg-Eppendorf 3 ProteoSys AG, Mainz Aims: In a previous protein expression study we could show that Annexin A3 (ANXA3) expression was significantly and inversely associated with prostate cancer. The aim of the present study was to thoroughly analyze ANXA3 expression and its potential as a prognostic marker in a large set of benign, preneoplastic and neoplastic prostate tissue samples. Methods: Immunohistochemistry-based ANXA3 expression was analyzed on 1589 prostate cancers obtained from radical prostatectomy specimens and smaller subsets of benign epithelium and high grade prostatic intraepithelial neoplasia (PIN) brought on a tissue microarray format. Median PSA followup was 33 months. Results: All samples of benign epithelium and PIN showed ANXA3 expression with PIN lesions showing a decreased staining intensity compared to benign epithelium (p<0.001). In cancer ANXA3 expression was essentially reduced, resulting in a negative staining rate of 27.2%, which correlated with increasing pT-stage and Gleason grade (p<0.0001). ANXA3 status in cancer was shown to be an independent adverse prognostic factor by multivariate analysis and was able to substratify the large group of intermediate risk patients (n=969) into high and low risk subgroups. Conclusions: ANXA3 represents a promising candidate tissue marker which when combined with the standard prognostic parameters harbors the potential to provide a more precise prediction of prognosis in the individual patient.

Sa-133 Expression and prognostic relevance of Axl and Gas6 in prostate cancer E. Stenzel, M. Müller1, R. Willers2, A. Ullrich3, H.E. Gabbert, R. Engers Institut für Pathologie und 1 Urologische Klinik, Universitätsklinikum Düsseldorf 2 Institut für Statistik, Universität Düsseldorf 3 Max-Planck-Institut für Biochemie, Martinsried Aims: Gas6 is a ligand for the Axl receptor tyrosin kinase and the Gas6-Axl system has been implicated in important functions such as oncogenesis, cell survival, proliferation, migration and cell adhesion. Since very little is known about the role of Axl and Gas6 in prostate cancer, we investigated their expression and prognostic relevance in a representative cohort of prostate carcinomas (PCas). Methods: Semiquantitative immunohistochemistry. Results: Expression of Gas6 and Axl was investigated in benign prostate, high-grade prostatic intraepithelium neoplasia (HG-PIN) and PCas of 79 and 91 R0-resected radical prostatectomy specimens, respectively. By semiquantitative immunohistochemistry Gas6 and Axl proved to be significantly overexpressed in both HG-PIN (p<0.001) and PCas (p<0.003) when compared with their expression levels in benign secretory epithelium. Axl overexpression in PCa correlated significantly with high pT stage, high Gleason scores and strong overexpression of Tiam1, Cathepsin D, and B-Myb, respectively. Gas6 overexpression correlated significantly with strong overexpression of Rac and B-Myb. However, no significant prognostic impact of Axl and Gas6 expression could be found. Conclusions: Gas6 and Axl are significantly overexpressed in PCas and HGPIN suggesting a role in prostate cancer development and providing potential targets for new therapeutic approaches. Correlations of Axl and Gas6 overexpression with distinct molecular alterations in PCas might provide new clues to the molecular mechanisms of Gas6-Axl-dependent signaling and functions.

Sa-134 Reduced expression of HLA class I antigen processing molecules in prostate cancer R. Stöhr, M. Burger2, W. Wieland2, A. Hartmann, B. Seliger1 Institut für Pathologie, Universitätsklinikum Erlangen 1 Institut für Medizin. Immunologie, Universitätsklinikum Halle-Wittenberg 2 Lehrstuhl für Urologie, Universitätsklinikum Regensburg Aims: HLA class I abnormalities are frequently found in cell lines and malignant tissues and in some cases can also be correlated with metastatic phenotype and the clinical outcome. So far there are only limited data available on the expression of components of the HLA class I antigen processing machinery (APM) in prostate cancer. Methods: The protein expression of HLA class I processing mole-cules LMP 2,7, of the TAP1 and TAP2 submits, tapasin, calnexin, calreticulin, ERp57, HLA class I heavy chain and β2-microglobulin was investigated using immunohistochemistry on a Tissue Micro-array comprising 59 primary prostate tumors and autologous normal prostate epithelium and correlated to histopathological and clinical data. Results: No differences in the protein expression were detected for TAP1, β2-microglobulin and HLA class I heavy chain. All other com-ponents frequently showed a reduced or even total lack of protein expression in prostate cancers compared to autologous normal pro-static epithelium. Moreover, reduced expression of calnexin and the MHC class I heavy chain was correlated with early disease recur-rence also after prostatectomy. Reduced protein levels of calnexin were associated with Gleason score ≥7. Conclusions: Thus, protein expression analysis of components of the HLA class I antigen processing machinery in prostate cancer re-vealed expression abnormalities in most tumors analysed. These findings emphasize the need for further evaluation of the molecular mechanisms resulting in such deficiencies and indicate an important role of APM alterations in prostate cancer.

Sa-135 Defective genomic imprinting of the Insulin-like growth factor 2 (IGF2) gene locus in patients with prostate cancer M. Kirchner1, B. Kneitz2, E. Gerharz2, D. Belharazem1, C. Sauer1 C. Bolenz3, M.S. Michel3, A. Marx1, P. Ströbel1 1 Pathologisches Institut, Universitätsklinikum Mannheim 2 Urologische Klinik und Poliklinik, Universitätsklinikum Würzburg 3 Urologische Klinik, Universitätsklinikum Mannheim Aims: Prostate carcinomas (PCA) are slowly growing tumors in a hormonesensitive tissue. Alterations of the insulin/insulin-like growth factor (IGF) signaling cascade may contribute to the pathogenesis of PCA. Expression of the IGF-2 gene is regulated by genomic imprinting, an epigenetic modification that results in the silencing of the maternal allele. Methods: We examined DNA from peripheral blood cells of 144 individuals with a history of histologically proven PCA and radical prostatectomy and of 181 healthy males using Restriction Fragment Length Polymorphism (RFLP) and ELISA to determine disorganisations of the IGF-2 imprinting and blood serum levels of IGF-2. Results: PCA patients were more frequently heterozygous for an apa1-sensitive IGF2 polymorphism than control persons (144/72 PCA patients (50%) vs. 181/73 (40,3%), p=0.09) Bi-allelic IGF2 expression (i.e. loss of imprinting) was more frequent and marked in PCA patients than in controls. The IGF-2 serum levels of PCA patients (PSA-negative at the time of this analysis) were significantly increased compared to controls (p<0.05). There was a significant correlation (p=0.02) between imprinting status and IGF-2 serum level in healthy persons, which was lost in individuals with former PCA. Conclusions: Our data suggest a genetically determined relaxation of the imprinting of the IGF-2 locus in patients with PCA. Whether these findings indeed result in constitutively increased IGF-2 blood serum levels and represent a potential risk factor not only for PCA, but also other growth-hormone sensitive tumors, needs further analyses.

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Abstracts Sa-136 ADAM9 Expression is a Signiﬁcant and Independent Prognostic Marker of PSA Relapse in Prostate Cancer G. Kristiansen, M. Jung1, A. Tölle1, P. Wild, A. Hartmann2, K. Wassermann3, A. Rabien1, M. Dietel3, C. Pilarsky4, D. Calvano4, R. Grützmann4, F.R. Fritsche3, K. Jung1 Institut für Klinische Pathologie, UniversitätsSpital Zürich 1 Urologische Klinik, Charité – Universitätsmedizin Berlin, Campus Mitte 2 Institut für Pathologie, Universitätsklinikum Erlangen 3 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte 4 Klinik und Poliklinik für Viszeral-, Thorax- und Gefäßchirurgie, Universitätsklinikum Dresden Aims: ADAM9 has been implicated in tumour progression of various tumours including prostate cancer. We aimed to determine ADAM9 expression on protein and mRNA level in a larger cohort of prostate cancer cases and to correlate the findings with clinical pathological parameters including PSA relapse times. Methods: 198 clinical pathologically characterised prostate cancer cases were immunostained for ADAM9. For 25 additional cases ADAM9 mRNA of microdissected tumour and normal tissue was analysed via quantitative RT-PCR. Results: ADAM9 was significantly overexpressed in prostate cancer in comparison to normal tissue on mRNA and protein level. Immunohistochemically, we found ADAM9 protein expression significantly associated with shortened PSA relapse-free survival times in univariate and multivariate analyses. Conclusions: ADAM9 is overexpressed in prostate cancer cases and is an independent prognostic marker of PSA relapse-free survival following radical prostatectomy, which clearly warrants further confirmational studies to verify its role of a prognostic marker.

Sa-137 Dual Role of the Androgen Receptor in the VEGF-A Regulation of Prostate Cancer M. Muders, H. Zheng, D. Tindall, K. Datta Urology Research, Mayo Clinic Foundation, USA Aims: Androgen plays a critical role in controlling the growth and survival of prostate cancer cells. Androgen depletion therapy usually achieves significant clinical responses in the early stages of the disease. However, under the selective pressure of androgen withdrawal, prostate cancers progress to an androgen-independent disease. This project evaluates the role of the androgen receptor in regulation of VEGF-A production in androgen dependent and androgen independent prostate cancer cells. Methods: The syngenic human cell lines LNCaP (androgen dependent) and LNCaP C4–2 (androgen depletion independent) were used. VEGF-A transcription was tested after androgen withdrawal with a VEGF-A/luciferase reporter construct. The role of the androgen receptor was evaluated by RNA interference. Results: Withdrawal of androgen in androgen dependent (AD) cells leads to a significant reduction of the VEGF-A transcription. In contrast, withdrawal of androgen leads to a significant increase in VEGF-A transcritption in the androgen independent (AI) cells. Knocking down the androgen receptor (AR) with short interfering RNA reduces the VEGF-A promoter activity in the AD cells. In contrast, AR-siRNA increases the promoter activity in the AI cells. Mechanistic studies are on their way. Conclusions: Our results suggest a dual role of the AR in the regulation of VEGF-A transcription in prostate cancer. AR activity increases VEGF-A expression in AD cells but it reduces VEGF-A expression in AI cells. This emphasizes the importance of different strategies in targeting the AR according to the stage of disease.

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Sa-138 TFF3 expression deﬁnes TMPRSS2-ERG fusion negative prostate cancers S. Perner1,2, F. Demichelis1, A. Sboner, S. Setlur, M.A. Rubin1 for the Swedish and US Consortium 1 Pathology, Brigham and Women’s Hospital, Boston, MA, USA 2 Institut für Pathologie, Universitätsklinikum Ulm Aims: By combining compelling clinical and gene expression data, we showed the existence of a relevant prostate cancer (PCa) subclass defined by TMPRSS2-ERG fusion. Using expression array profiling on a large cohort of primary PCa, we aimed to identify a gene expression signature, distinguishing TMPRSS2-ERG fusion PCa as a discrete molecular entity. The aim of this study was to develop an immunohistochemical marker serving as a substitute for TMPRSS2-ERG fusion negative PCa. Methods: We interrogated a total of 354 localized PCa cases from a Swedish Watchful Waiting cohort by the DASL gene expression array, comprising >600 genes. By means of t-test (q-value cut-off: 0.005), genes were identified as being significantly associated with TMPRSS2-ERG fusion as determined by FISH. Two-tailed p-values were adjusted for multiple hypotheses testing. Hierarchical clustering was computed using average linkage and Pearson correlation as distance metric. Immunohistochemistry was performed with a TFF3 monoclonal antibody. Results: One of the top ranked genes that showed up as being over expressed in fusion negative PCa and as highly down regulated in fusion positive PCa was trefoil factor 3 (TFF3), a member of a family of polypeptides encoded by a cluster of genes on chromosome 21. We discovered an inverse association between TMPRSS2-ERG fusion status and TFF3 expression which was identified on the transcriptome level and also conserved on the protein level as detected by immunohistochemistry. Conclusions: We thus believe that TFF3 plays an important biological role in fusion negative PCa and that the TFF3 antibody is a helpful surrogate marker in defining fusion negative PCa.

Sa-139 TMPRSS2-ERG fusion in a German prostate cancer cohort S. Perner1,4, M. Schoppe, E. Stenzel, M. Müller2, R. Willers3, H.E. Gabbert, M.A. Rubin1, R. Engers Institut für Pathologie, Universitätsklinikum Düsseldorf 1 Well Cornell Medical Center, New York, USA 2 Urologische Klinik und 3 Institut für Statistik, Universitätsklinikum Düsseldorf 4 Institut für Pathologie, Universitätsklinikum Ulm Aims: Fusion of the androgen-regulated TMPRSS2 gene to ERG, a member of the ETS family of transcription factors, has been reported to occur frequently in prostate cancer (PCa). The aim of the present study was to analyze the incidence and prognostic impact of TMPRSS2-ERG fusion in a German PCa cohort. Methods: Fluorescence in situ hybridization (FISH) assays were applied to test for TMPRSS2-ERG fusion on tissue microarrays, composed of 255 tissue cores from 97 patients. The cohort is restricted to R0-resected tumors without any adjuvant or neoadjuvant therapy. Results: FISH analysis was assessable in 74 tumors (76.3%). Of these, 41 tumors (55.4%) exhibited TMPRSS2-ERG fusions, whereas 33 tumors (44.6%) were negative. In 26 (63.4%) positive tumors, fusion resulted from intronic deletion between ERG and TMPRSS2, whereas in 15 tumors (36.6%) fusion was due to insertion. TMPRSS2-ERG fusions significantly correlated with pT stage (p=0.048) and perineural invasion (p=0.02) as indicators of aggressiveness, but not with other important clinico-pathological and molecular characteristics. Finally, neither the occurrence nor the type of TMPRSS2-ERG fusions (fusion through deletion vs. fusion through insertion) was significantly associated with disease recurrence, as reflected by the time course until PSA failure. Conclusions: TMPRSS2-ERG fusions occur frequently in PCa and mostly result from intronic deletions. TMPRSS2-ERG fusions do not predict PSA fail-

ure, but their prognostic impact in terms of disease-specific survival remains to be determined.

Sa-140 Transcriptional Proﬁling of Transurethral Resection Samples Provides Insight into Molecular Mechanisms of Hormone Refractory Prostate Cancer O. Stoss1, P. Albers3, T. Henkel1, J. Rüschoﬀ2, P. Middel2 1 TARGOS Molecular Pathology GmbH, Kassel 2 Institut für Pathologie Nordhessen 3 Institut für Urologie, Klinikum Kassel The molecular mechanisms for hormone resistant prostate cancer progression still remain elusive, mainly due to the limited availability of corresponding tissue. Aims: As transurethral resection (TUR) is a common palliative therapy of patients with hormone resistant prostate cancer (HRPC), we aimed to demonstrate that TUR samples can be used to identify significantly affected biological pathways during the switch to hormone refractory prostate cancer using oligonucleotide microarray analysis. Methods: Samples of 7 HRPC patients were compared to tissues from 9 hormone-sensitive prostate cancer patients. All tissue samples had been pathologically evaluated. Only macrodissected prostate samples with at least 70% tumor content were used for RNA extraction. RNA quality was controlled using the Bioanalyzer Nanochip (Agilent Technologies, Palo Alto). Expression analysis was performed on Affymetrix oligonucleotide arrays. Results: Unsupervised clustering based on the molecular signature of TUR samples is able to distinguish HRCP hormone-sensitive prostate cancer. We identified 30 molecular pathways that are significantly deregulated between both subgroups. Among the most affected pathways, we could identify transcripts coding for the proteasome as well as for oxidative phosphorylation and ATP-synthesis.Transcriptional profiling was validated by immunohistochemistry. Conclusions: TUR samples are an interesting alternative for biopsies for transcriptional profiling of HRPC. Targeting energy metabolism and protein synthesis is a promising approach for hormone resistant prostate cancer.

Sa-141 Ki67 Labelling Index is a prognostic factor in pre-operative biopsies of prostate cancer S. Savic1, T. Zellweger2, S. Günther1, I. Zlobec1, E. Curschellas3, H.J. Rüfenacht3, G. Sauter4, H. Moch, M.J. Mihatsch1, L. Bubendorf1 1 Institut für Pathologie, Universitätsspital Basel 2 Abteilung Urologie, St. Claraspital Basel 3 Institut für diagnostische Histopathologie, Basel 4 Departement Pathologie, UniversitätsSpital Zürich 5 Institut für Pathologie, Universitätsklinikum Hamburg-Eppendorf Aims: To test the prognostic utility of Bcl2, p53 and Ki67 immunohistochemistry in prostate biopsies. Methods: Prostate biopsies of 279 men who subsequently underwent radical prostatectomy were prospectively analyzed for Gleason score, number and percentage of positive cores (NPC, PPC), total percentage of biopsy tissue with tumor (TPT), maximum tumor percentage per core (MTP), and expression of Ki67, Bcl-2 and p53. We examined the association between these preoperative parameters with histomorphologic features and biochemical recurrence after radical prostatectomy. Results: Stage pT3 was independently predicted by TPT, Gleason score by biopsy Gleason score, MTP, and Ki67 labelling Index (LI), seminal vesicle invasion by Ki67 LI, large tumor diameter by PPC, and positive margins by MTP, respectively. Biopsy Gleason score, NPC (1 vs. >1), TPT (<7% vs. ≥7%), and Ki67 LI (≤5% vs. >5%) were significant predictors of biochemical recurrence. KI67 LI was superior to Gleason score as a prognostic factor in the subgroup with low TPT (<7%). Conclusions: Pre-operative Gleason score, NPC, TPT and Ki67 LI significantly predict the risk of biochemical recurrence after radical prostatectomy.

Analysis of Ki67 LI should be routinely considered in all positive prostate biopsies as it provides prognostic information that may be superior to the morphologic parameters.

Sa-142 MALDI imaging to identify and characterize prostate cancer K. Schwamborn, M. Reska, G. Jakse1, R. Knüchel, R.M. Caprioli2, R.C. Krieg, A. Wellmann Institut für Pathologie und 1 Urologische Klinik, Universitätsklinikum Aachen 2 Department of Biochemistry, Vanderbilt University, Nashville, USA Aims: Prostate cancer is the most common malignancy in men in Germany but its pathogenesis is still poorly understood. However, a crucial role in tumor progression is ascribed to the tumor micro-environment. Matrix assisted laser desorption ionization (MALDI) imaging is a promising new technique which allows for visualization of spatial distribution of protein expression within intact non-lysed tissue samples. Methods: Cryostat sections of prostate tissue with (n=11) and without carcinoma (n=11) were mounted onto conductive glass slides (Bruker Daltonics, Bremen) and evenly spray coated with matrix solution. Mass spectra were acquired on every measuring point of a virtual grid overlaid on the sample using a Reflex IV MALDI-TOF-MS (Bruker). Following MS analysis matrix was removed by organic solvents and sections were H&E stained for pathological appraisal. Results: Based on different statistical evaluation strategies non-cancerous and cancerous samples could be discriminated with an overall cross validation, sensitivity and specificity up to 88%, 85.21% and 90.74%, respectively. Additionally, differences between normal and tumor stroma were discovered. Conclusions: MALDI imaging enables scanning for unlimited proteins in parallel on a molecular scale. Identifying proteins differentially expressed between normal and cancerous tissue might elucidate development and progression of prostate cancer.

Sa-143 Periostin is strongly overexpressed in Prostate Cancer Stroma and is associated with shorter PSA relapse-free survival Times F.R. Fritzsche, P. Wild, K. Jung1, T. Hermanns2, A. Soltermann, P. Schraml, H. Moch, G. Kristiansen Institut für Klinische Pathologie, UniversitätsSpital Zürich 1 Urologie, Charité – Universitätsmedizin Berlin, Campus Mitte 2 Urologie, UniversitätsSpital Zürich Aims: Periostin (osf2) is a secreted protein that was originally described to regulate adhesion and differentiation of osteoblasts. Recently, we found periostin in solid tumors using a proteomic approach and could confirm its expression in tumor stroma. We aimed to determine periostin expression in a clinically characerized cohort of prostate cancer cases and to correlate the findings with clinical pathological parameters including PSA relapse times. Methods: A tissue micro array was constructed as a screening tool, comprising 93 prostate cancer cases, half of which suffered a PSA relapse. Immunohistochemistry was evaluated semiquantitatively applying an immune reactive score (IRS). Results: Periostin was significantly overexpressed in prostate cancer stroma in comparison to normal tissue, in some cases virtually rimming single prostate cancer glands. Periostin expression did not correlate to clinico-pathological parameters (age, pT stage, Gleason score, pre-op PSA) but was significantly associated with biochemical relapse on univariate analysis (median 78 vs. 36 months, p=0.045). Conclusions: Periostin is highly expressed in prostate cancer stroma, irrespective of clinico-pathological parameters and is associated with significantly earlier biochemical relapse which clearly warrants further confirmational studies to clarify its prognostic or diagnostic role in surgical pathology.

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Abstracts Sa-144 Saturation biopsy improves preoperative Gleason scores of prostate cancers P. Kahl1, S. Wolf1, A. Adam1, R. Vorreuther2, G. Solleder2, R. Büttner1 1 Institut für Pathologie, Universitätsklinikum Bonn 2 Abteilung Urologie, Evangelisches Waldkrankenhaus Bonn Aims: To evaluate the differences between conventional needle biopsy (CB) and saturation biopsy (SB) techniques regarding Gleason score, tumor-stage and insignificant prostate cancer. Methods: We analyzed data from a total number of 240 patients, a main group of 185 patients with an extensive prostate needle biopsy technique (SB) all of which received more than12 biopsies (n=185), and a control group of patients all of which received less than 12 biopsies (n=55). Results: There was no significant difference in tumour stage between the two groups. In the main group the Gleason score of the biopsy was confirmed in 19.5%, in the control group in 23,5% according to the prostatectomy specimen. Upgrading after the operation was found in 56.7% in the main group and 60% in the control group. Downgrading after the operation was found in 23.9% in the main group and 16,3% in the control group. Importantly, only 33.5% of the prostate carcinomas of the main group, but 45.4% of the control group impaired by more than one Gleason point in the prostatectomy specimen. There was no significant difference between the two types of biopsy-techniques in detecting insignificant carcinomas. Conclusions: The advantage of the extensive prostate needle biopsy technique (SB) is a significantly better preoperative prediction of the Gleason score of the prostatectomy specimen. Both techniques fail to detect insignificant prostate cancer. As a disadvantage the extensive biopsy technique is more timeconsuming and costly.

Sa-145 Role of the Rho-like GTPase Rac3 in prostate carcinogenesis A. Maaßen, S. Ziegler, J.G. Collard1, H.E. Gabbert, R. Engers Institut für Pathologie, Universitätsklinikum Düsseldorf 1 The Netherlands Cancer Institute, Amsterdam, NL Aims: Rac3 is one out of three isoforms of the Rho-like GTPase Rac, but so far, only little is known about its functional role. Recently, we found that Rac3 and its activator Tiam1 are significantly overexpressed in most prostate carcinomas (PCas) and high-grade PIN (HG-PIN) lesions and that both Tiam1 and Rac3 overexpression predict an aggressive clinical course. Here, we investigated the role of Rac3 in prostate cancer development. Methods: Stable gene transfection, proliferation assay, adhesion assay, migration assays, transformation assays, immunoblotting. Results: In comparison to mock-transfected control cells, overexpression of constitutively active V12-Rac3 in benign prostatic epithelial cells (BPH1) resulted in decreased cell-cell and cell-substrate adhesion (p<0.001) and consequently strongly stimulated cell migration (p=0.004) in two different migration assays. V12-Rac3 had no significant effects on cell proliferation, but strongly induced oncogenic transformation as shown in both the soft agar and colony formation assay. In contrast, empty vector and wild-type (wt)Rac3 failed to transform BPH1 cells in vitro. On the molecular level, V12-Rac3 strongly activated c-Jun-N-terminal kinase (JNK) and induced expression of the invasion-promoting matrix metalloproteinase 9, but had no effect on expression and activities of p38 and p70-S6 kinase. Conclusions: Constitutive activation of Rac3 induces oncogenic transformation of benign prostatic epithelial cells in vitro and affects several cell biological functions towards a more aggressive phenotype. Given the fact that Rac3 is overexpressed in most HG-PIN lesions and prostate carcinomas our results suggest a major role of Rac3 signaling in the development of prostate cancer.

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Sa-146 Declining diagnostic value of high grade PIN in prostatic biopsies J. Vosbeck1,2, S. Hailemariam2, L. Bubendorf1 1 Institut für Pathologie, Universitätsspital Basel 2 Kantonales Institut für Pathologie, Liestal Aims: The detection of isolated high-grade prostatic intraepithelial neoplasia (HG-PIN) in prostatic biopsies has long been considered an important risk factor for a concomitant adenocarcinoma. Given an estimated risk of 30%, re-biopsy within 6 months has usually been recommended. However, the diagnostic importance of HG-PIN has recently been questioned. Here, we investigate the diagnostic relevance of isolated HG-PIN in the biopsy material submitted to our institutes of pathology during the past 10 years. Methods: 125 patients with a diagnosis of isolated HG-PIN were identified in our database from 1995 to 2005. All histological slides were re-evaluated by the authors according to standardised criteria, and compared to the result of re-biopsies. Immunohistochemical examinations were performed for AMACR, Bcl-2 and Ki-67. Data were compared with a series of HG-PIN that had an adenocarcinoma in another simultaneously obtained core. Results: 39 cases (31%) did not meet our stringent review criteria for HGPIN and were excluded from further investigations. A re-biopsy was done in 46 of the remaining 86 patients. Three of these re-biopsies (7%) showed an adenocarcinoma, and 11 (24%) showed isolated HG-PIN. 32 re-biopsies (69%) were inconspicuous. The expression pattern of molecular markers was not significantly associated with adenocarcinoma in a simultaneous or subsequent biopsy. Conclusions: The diagnostic value of isolated HG-PIN in a contemporary PSA-screened population is low, questioning the necessity of a short-term re-biopsy. AMACR, Bcl-2 or Ki-67 are not helpful for better predicting the risk of adenocarcinoma.

Sa-147 Early detection of prostate cancer using DNA methylation analysis of the GSTP1 gene in histopathologically negative prostate biopsy specimens: potential to improve the clinical routine? W. Haedicke1, J. Distler2, R. Lesche2 1 MVZ Vorpommern GmbH, Pasewalk 2 Epigenomics AG, Berlin Aims: Aberrant DNA methylation is among the earliest and most frequent molecular alterations found in the development of cancer. The Glutathione S transferase p (GSTP1) gene has been reported to be methylated in more than 90% of prostate cancer patients. Prostate cancer is diagnosed by histo-pathological analysis of prostate biopsies. Consequently, it has been suggested that a combination of histo-pathological analysis and DNA methylation analysis of GSTP1 might have the potential to improve the diagnosis of prostate cancer. Methods: 157 prostate biopsy specimens of patients with histologically verified adenocarcinoma were analyzed to establish the assay methodology. For DNA extraction and bisulfite treatment from prostate biopsy specimen an optimized pre-analytical workflow was used. GSTP1 methylation was measured in the exon 1 region of the gene using HeavyMethyl (HM) real-time PCR technology. Results: Highly sensitive, quantifiable detection of GSTP1 methylation is possible using single core prostate biopsy specimens. The optimized preanalytical workflow resulted in sufficient DNA for downstream analysis in more than 90% of the clinical specimens. An update on clinical validation of GSTP1 methylation analysis in histopathologically negative prostate biopsy specimens will be presented. Conclusions: DNA methylation analysis may serve the pathologist as a versatile tool to improve early diagnosis of clinically asymptomatic prostate cancer.

Sa-148 Correlation of modiﬁed Gleason Grading of prostate carcinoma with stage, age, serum PSA,and tumor extent in needle biopsy and radical prostatectomy specimens B. Helpap1, L. Egevad2 1 Abteilung Pathologie, Hegau-Bodensee-Klinikum Singen 2 International Agency of Research on Cancer (IARC) Lyon, F Aims: At an ISUP (International Society of Urological Pathology) consensus conference in 2005, the Gleason grading system for prostatic carcinoma underwent its first major revision.The aim of this study was to investigate the correlation of tumor grade with stage, age, serum PSA, and biopsy tumor extent using the conventional © and modified (m) Gleason grading system. Methods: More than 3000 consecutive needle biopsy (NBX) and radical prostatectomy (RP) specimens of prostate carcinoma were collected between 1995 and 2000 graded mit © Gleason grading and between 2006 and 2007 graded with (m) Gleason grading. Results: After (m) grading the maximal Gl.score(Gs) in needle biopsies shifted from Gs. 6 to Gs.3+4=7a. Gs 2–4 could not be observed. After RP pT2 carcinomas correlated with Gs 3+4=7a, pT3 tumors correlated with Gs.4+3=7b and higher. The agreement between Gs of NBX and RP increased from 58% after © grading to 80% after (m) grading. Both © and (m) Gleason grading correlated with age, serum PSA, percent positive biopsies, and percent cancer length. In 2006–2007, the patients were in average younger and more biopsy cores were taken per patient. Serum PSA and percent prositive cores were lower than in 1995/2000, indicating a stage shift downward, but the Gs were nevertheless higher. Conclusions: Conventional and modified Gleason grading both correlated with age,PSA and cancer involvement in needle biopsies. With modified Gleason grading there is a grade shift upwords despite the downstaging that has been observed in recent years.

Sa-149 Is there Replacement of Podocytes by Bone Marrow-derived Stem Cells in Puromycin Nephropathy? J.U. Becker1, C. Meyer-Schwesinger2, A. Raabe3, C. Lange3, E. Kobayashi4, H.H. Kreipe1, F. Thaiss2 1 Institut für Pathologie, Medizinische Hochschule Hannover 2 III. Medizinische Klinik, Universitätsklinikum Hamburg-Eppendorf 3 Institut für experimentelle Radioonkologie und Radiobiologie, Universitätsklinikum Hamburg-Eppendorf 4 Center for Molecular Medicine, Jichi Medical School, Tochigi, J Aims: Adult podocytes are virtually incapable of replication. Loss of podocytes leads to focal and segmental glomerulosclerosis, a final common pathway leading to nephron loss in many glomerular diseases. Replacement of podocytes by bone marrow derived cells has already been shown in mice. Also in humans, immigrating progenitor cells seem to be able to differentiate into podocytes. We examined in a rat model of specific podocyte damage, if and to what extent podocytes are replaced by bone marrow-derived stem cells. Methods: Puromycin aminoglycoside nephropathy (PAN) was induced in rats grafted with green fluorescent protein (GFP)-positive bone marrow. Kidneys were examined by double immunohistochemical staining for WT-1 and GFP for bone marrow graft-derived podocytes. Results: No GFP-positive podocytes were found in the PAN group or the untreated controls up to 60 days after induction of PAN. Conclusions: These results indicate, that bone marrow derived stem cells do not seem to contribute to podocyte regeneration after podocyte injury in the experimental model of PAN.Further research is needed to show, if pharmacologic stimulation can drive podocyte replacement by bone marrow derived stem cells.

Sa-150 Coculture with murine Podocytes induces Neotranskription of Podocyte Markers in human mesenchymal Stem Cells J.U. Becker1, C. Lange2, U. Lehmann1, I. Tossidou3, H. Haller3, H.H. Kreipe1, M. Schiﬀer3 1 Institut für Pathologie, Medizinische Hochschule Hannover 2 Institut für experimentelle Radioonkologie und Radiobiologie, Universitätsklinikum Hamburg-Eppendorf 3 Klinik für Nieren- und Hochdruckerkrankungen, Medizinische Hochschule Hannover Aims: Adult podocytes are virtually incapable of replication. Loss of podocytes leads to focal and segmental glomerulosclerosis, a final common pathway leading to nephron loss in many glomerular diseases. Replacement of podocytes by immigrating progenitor cells, e.g. human mesenchymal stem cells (hMSCs) could be therapeutically interesting to slow down nephron loss. Methods: To investigate, if hMSCs are able to differentiate into podocytes, we cocultivated them with immortalized murine podocytes. RNA was harvested and examined by Realtime-PCR with human-specific primers for Podocytespecific Nephrin-, Podocin- and Synaptopodin transcripts. Results: RT-PCR only of cocultures showed transcripts of human Nephrin, Podocin and Synaptopin. Conclusions: These results could indicate that murine podocytes are able to induce podocyte differentiation in human mesenchymal stem cells. Further research is needed to exclude fusion of murine podocytes and hMSCs and to delineate mechanisms how this differentiation is induced.

Sitzung: AG Gynäko- und Mammapathologie So-001 Microcalciﬁcation-associated lesions in mammography screening – Results from the Reference Centre Münster D. Hungermann1,3, C. Kersting1,3, S. Weigel2,3, W. Heindel2,3, W. Böcker1,3, T. Decker1,3 1 Institut für Pathologie und 2 Institut für Radiologie Universitätsklinikum Münster 3 Referenzzentrum Mammographie, Münster Aims: To evaluate the diagnostic impact of microcalcifications (MC) in minimal invasive biopsies (MIB) in mammography screening, the underlying lesions in different B-Categories were analysed. Methods: 2048 MIB of lesions radiologically classified as BIRADS 4/5 were analysed. Results: Overall malignancy rate of MIB was 47,5%. Out of 868 MIB (42%) which were positve for microcalcifications, 34% were malignant (B4 or B5, 298/868), most of them DCIS (160/298). 45% (392/868) were categorised as B2, mostly fibroadenomas with MC. Wheras the overall B3-rate was 12%, 20%( 172/868) of MC positive MIB belonged to this category. Among these, flat epithelial atypia and atypical proliferation of ductal type were the most prevalent lesions, mostly associated with blunt duct adenosis, microcysts and scleradenosis which carried the targeted MC. Conclusions: Although the malignancy rate in MIB performed for microcalcifications is lower than the overall malignancy rate, there is a high frequency of DCIS, which represents 81 % of all screening detected DCIS. In general, lesions detected by assessment of MC represent a broad, challenging spectrum, underlining the demand of special trained breast screening pathologists.

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Abstracts So-002 AKAP12 / Gravin – a candidate tumor suppressor gene in breast cancer is inactivated by epigenetic mechanisms M. Blankenburg1, S. Seitz2, I. Petersen3, S. Scherneck1, W. Hofmann4 1 Max Delbrück Centrum für Molekulare Medizin, Berlin 3 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte 3 Institut für Pathologie, Universitätsklinikum Jena 4 Berufsverband Deutscher Humangenetiker e.V., Berlin Aims: The aim of this work was to analyse the role of AKAP12/Gravin (Akinase anchoring protein) in breast cancerogenesis. Methods: To evaluate the expression of AKAP12 in human breast tumours, we performed a large-scale expression analysis using cancer profiling and chip arrays, northern blot analysis and semi-quantitative RT-PCR. Breast tumors and their matching normal breast tissues were analysed for LOH, mutations and methylation. In addition, transfection studies of AKAP12 in breast cancer cell lines were carried out. Results: Our results indicate that loss of AKAP12 expression is a common event in breast cancer. AKAP12 promoter methylation was found in 52% of the breast tumors. Although loss of heterozygosity was found in 35% of tumors, structural mutations in the coding or promoter region of the gene were not observed. Transfection of the gene into AKAP12 deficient breast cancer cell lines resulted in an increase of gene expression and a reduced anchoragedependent growth and a reduction of the invasive potential of the transfected cells. Conclusions: Our data showing a reduced AKAP12 expression in several breast tumor cell lines and tumor samples as well as its ability to retard cell growth and invasion support an important role of AKAP12/Gravin in breast cancer.

So-003 Nuclear Karyopherin alpha 2 expression predicts poor survival in patients with advanced breast cancer P.J. Wild, O. Gluz1, R. Diallo-Danebrock2, E. Ting2, A. Herr3, S. Mohrmann1, H. Geddert2, A. Rody4, K.L. Schaefer2, S.E. Baldus2, M. Burson5, H.E. Gabbert2, U. Nitz1, C. Poremba2, G.O. Kristiansen, H. Moch, A. Hartmann6 Institut für klinische Pathologie, UniversitätsSpital Zürich 1 Abt. für Gynäkologie und Geburtshilfe und 2 Institut für Pathologie, Universitätsklinikum Düsseldorf 3 Institut für Klinische Genetik, Universitätsklinikum Dresden 4 Abt. für Gynäkologie und Geburtshilfe, Universitätsklinikum Frankfurt am Main 5 Dept. of Pathology, Univ. of Colorado at Denver and Health Sciences Center, Aurora, Colorado, USA 6 Pathologisches Institut, Universitätsklinikum Erlangen Aims: To evaluate the impact of KPNA2 expression on prognosis of patients with high risk breast cancer (HRBC) and response to intensive chemotherapy within the randomized WSG-AM-01 trial. Methods: KPNA2 nuclear expression was measured by immunohistochemistry on tissue arrays of 191 patients randomized to tandem high dose vs. conventional dose-dense chemotherapy in HRBC with >9 positive lymph nodes and correlated with clinical outcome by Kaplan-Meier and multivariate Cox hazard model analysis including molecular subtypes determined by k-clustering. Results: KPNA2 overexpression (39%) significantly correlated with shorter event-free (EFS) and overall survival (OS) in both therapy arms by univariate analysis. Multivariate analysis showed that the overexpression of KPNA2 was an independent prognostic factor of decreased overall survival (HR=1.89, 95%-CI=1.06–3.35, p=0.03). Conclusions: Our data suggest the use of nuclear KPNA2 expression as novel prognostic marker in node-positive breast cancer patients.

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So-004 Variable expression of basal cytokeratins and Topoisomerase II in poorly diﬀerentiated Her2 positive breast carcinomas Z. Varga, P. Schraml, S. Behnke, B. Odermatt, R. Caduﬀ, H. Moch Institut für Klinische Pathologie, UniversitätsSpital Zürich Aims: Triple negative (Her2, hormone receptors) and poorly differentiated breast carcinomas frequently express basal cytokeratins. This phenotype is associated with poor clinical outcome. Topoisomerase II (Top2A) alterations (deletion, amplification) occur in up to 40% of Her2 positive breast tumors with possible therapeutic consequence to adjuvant chemotherapy. Here, we analyzed the presence of Top2A alterations and basal cytokeratin expression in Her2 positive breast cancer. Methods: The expression of basal cytokeratins (cytokeration 5/6) and aberrations of Top2A were analyzed in 42 poorly differentiated Her2 (FISH) positive breast carcinomas. Basal cytokeratins were assessed immunohistochemically, alterations of Top2A were evaluated with FISH technology. 20 of 42 carcinomas were hormone receptor positive. Results: Top2A amplification was found in 15 patients (35%), deletion in 7 patients (17%). 11 cases (26%) showed at least focal expression of cytokeratin 5/6. Aberration of Top2A and expression of basal cytokeratins were similar in both hormone receptor negative and positive cases. Triple positive carcinomas (Her2 andTop2A amplification, cytokeratin 5/6 expression) were identified in 4 patients (10%), 2 of them were also hormone receptor positive. Conclusions: Our results show that basal differentiation is not restricted to triple negative breast carcinomas only. Basal cytokeratin expression and Top2A amplification and/or deletions occur in poorly differentiated Her2 positive breast tumors. Triple positive phenotype (Her2 and Top2A amplification, cytokeratin 5/6 expression) can be seen in both hormone receptor positive and negative patients. The biological importance and the clinical implications of this phenotype are not fully understood yet.

So-005 Systematic discovery and validation of promoter-methylation markers in breast cancer E. Dahl, J. Veeck, E. Noetzel, F. Wiesmann, R. Knuechel Institut für Pathologie, Arbeitsgruppe Molekulare Onkologie, Universitätsklinikum Aachen Aims: Promoter methylation is a fundamental mechanism of gene silencing involved in the development of human malignancies. The availability of the blue print of the human genome in combination with DNA array expression profiling techniques allows a systematic search for genes silenced by promoter methylation in tumors such as breast cancer. These genes may represent valuable tumor markers for early detection of tumor DNA in blood serum, and for prediction of patient prognosis and/or response to chemotherapy. Multi-marker panels based on the analysis of DNA methylation may develop to a prime technology in pharmaco-genomics supporting the design of personalized breast cancer medicine. Methods: Genes significantly downregulated in breast cancer were analy-zed for the presence of CpG islands. Downregulation was confirmed by real time PCR analysis. Methylation specific PCR (MSP) was performed. Re-induction of mRNA expression in cell lines after DNA-demethylation treatment by DAC/TSA served as a prerequisite for further gene analysis in human breast cancer. For these genes, large collections (n>100) of breast cancer specimens with complete clinical follow-up data were analyzed. Biomarker potential was evaluated in correlation analyses of methylation profiles and clinico-pathological patient characteristics. Results: DNA array expression experiments revealed more than 100 genes significantly downregulated in breast cancer of which nearly one third had clearly defined CpG islands in their promoter region. Three genes, SFRP1, ITIH5 and ID4 were selected for further analysis. Promoter methylation of SFRP1 and ITIH5 were clearly associated with dramatically shortened overall survival. Conclusions: The presented discovery strategy was successfully applied to identify novel methylation biomarkers in breast cancer. SFRP1 is an important negative regulator of the WNT signaling pathway and a putative tumor

suppressor. ITIH5 seems to represent a metastasis suppressor gene. While methylated SFRP1 is a pan-tumor marker, methylated ITIH5 is likely to be more tissue-specific. Markers like these genes should be optimally combined to generate highly informative multi-gene methylation-marker panels. Supported by a RWTH Aachen START grant.

So-006 Comparison of Automated Silver Ampliﬁed in situ-Hybridisation (SISH) with Conventional Fluorescence in situ-Hybridisation (FISH) to Determine the HER2-Status of Breast Cancer G. Kristiansen, H. Moch, I. Ellis1, H. Höﬂer2, H. Kreipe3, K. Kölble4, A. Rieger4, M. Dietel4 Institut für Klinische Pathologie, UniversitätsSpital Zürich 1 Department of Histopathology, City Hospital Nottingham 2 Institut für Pathologie, Technische Universität München 3 Institut für Klinische Pathologie, Medizinische Hochschule Hannover 4 Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte Aims: Currently, Her2 status is the most important predictive marker for the estimation of therapy response to perception therapy. However, the ideal technique to determine the Her2 status is a point of discussion since the cheap immunohistochemistry lacks in specificity, whereas the more specific Her2 in situ-hybridisation is more laborious and costly. Methods: Silver in situ-Hybridisation (SISH) is a new detection system that amplifies the amplification signal by a photochemical reaction resulting an easy to evaluate silver deposits. To validate this method paraffin sections from 100 breast cancer cases was subjected to conventional FISH and the novel SISH to determine Her2 status. Results: Between FISH and SISH a rate of concordance of 96% (Kappa 0.754) was found. Particularly, a low inter-observer variability in the interpretation of SISH suggested that SISH is equally reliable in determining HER2 amplification as FISH. Conclusions: We conclude that the Her2 status of breast cancer cases can reliably be detected by a Her2 SISH. Currently, further technical improvements to allow simultaneous detection of HER2 and Centromere 17 are underway.

So-007 Histomorphological and immunohistochemical characteristics of BRCA I germ line mutated breast cancer in Germany P. Ahrens, Y. Freder, M. Heskamp, H. Bruchardt, M. Christgen, H. Kreipe Institut für Pathologie, Medizinische Hochschule Hannover Aims: Evaluation of histological and immunohistochemical surrogate markers in breast carcinomas associated with a BRCA I germ line mutation compared to familial BRCA I negative breast cancer cases in Germany. Methods: Correlation of histomorphology, immunohistochemistry and BRCA I status in familial breast carcinomas. Results: Invasive breast carcinomas associated with a BRCA I germ line mutation are usually high grade ductal carcinomas with either an atypical medullar or solid-trabecular growth pattern and a high proliferation index as detected by Ki-67 immunohistochemistry. As a constantly observed histomorphological feature they exhibit a moderate to severe T-lymphocytic inflammation of the adjacent acinar tissue which is variously affected by a broad lobular struma cancerisation. Furthermore these tumors are more likely to show polycyclic margins than diffuse infiltrative borders. The tumors are triple negative and exhibit a high Ki-67 labelling usually exceeding 60%. Familial breast carcinomas lacking a BRCA I germ line mutation seem to encompass the same morphological and immunohistochemical spectra than sporadic breast cancer cases. Conclusions: BRCA I mutated breast carcinomas show different histomorphological and immunohistochemical characteristics. The determination of surrogate markers for these lesions remains an important field of investigation in the near future and will substantially contribute to a better understanding of the biological behaviour of these tumors.

So-008 Comprehensive immunohistochemical analysis of Her-2/neu-oncoprotein overexpression in breast cancer using two diﬀerent antibodies (Dako and Ventana) and correlation to results of Fluorescencein situ-hybridization (FISH) S. Heim1, C. Werhan2, T. Kirchner1, D. Mayr1 1 Pathologisches Institut, Universitätsklinikum München (LMU) 2 Ventana, Strassbourgh Aims: Immunohistochemical analysis of Her-2/neu-oncoprotein overexpression is used as marker for herceptin® therapy. To investigate the sensitivity and specificity of immunohistochemistry we assessed two different antibodies in a large series of breast carcinomas and validated by Fluorescence-in situ-hybridization. Methods: Paraffin-embedded material of 130 patients was available. Immunohistochemical expression of Her-2/neu-oncoprotein using two different antibodies (Dako and Ventana) was analysed. Validation by FISH was performed in all cases with uncertain overexpression (2+) and all cases with different immunohistochemical staining. Results: Same immunohistochemical results were seen in 96/130 (73.8%) cases (22 cases with strong expression=3+, 29 with uncertain overexpression=2+ and 45 with no overexpression=0 or 1+). Two of 10 cases (20%) with oncoprotein-overexpression (3+) detected by Ventana and confirmed by FISH showed no overexpression (1+) by Dako. From 21 cases with 2+ by Ventana, 15 cases (71.4%) had no gene-amplification, but two of these cases (13.3%) 3+ by Dako; three cases showed a low or high gene-amplification, including one case (33%) with failing overexpression by Dako; two other cases were polysome, one could not be analysed. Three negative cases by Ventana were 2+ by Dako; gene-amplification in FISH was not seen. Conclusion: Ventana-immunhistochemistry seems to be more sensitive and specified with a better concordance to Fluorescence- in situ-hybridization, no case failed by immunhistochemistry. The results by Dako were in five cases incorrect, two times false positive and three times false negative, validated with FISH

So-009 Der Schnellschnitt bei Mammatumoren aus der Sicht der Klinik J-U. Blohmer Berlin So-010 Der Schnellschnitt bei Mammatumoren aus der Sicht der Pathologie A. Lebeau Hamburg So-011 Selective intraoperative frozen section analysis of sentinel lymph nodes D. Hungermann1, C. Kersting1, B. Schulte1, J. Tio2, T. Decker1 1 Institut für Pathologie und 2 Frauenklinik, Universitätsklinikum Münster Aims: Although being part of national and international guidelines the role of intaoperative analysis of sentinel lymph node is still controversial. Our aim was the evaluation of reliability of selctive frozen section investigation. Methods: In 137 cases of unifocal invasive Carcinoma of 2–24 mm diameter (median 14 mm) 264 SLN were detected. Lymph nodes were lamellated in 2mm slices. They were carefully examined by macroscopy and by palpation. In case of suspected metastasis, frozen section was performed. Remaining SLN-slices were completely paraffin embeded and examined by step sections with an interval of 500μm. Immunhistochemistry was applicated in invasive lobular carcinomas when conventional histology was negative. Results: In 29 of 137 cases posive SLN were detected. The false negative rate was 2,9%. Secondary axillary dissection was performed in 4,4%. Selective frozen section investigation had a sensitivty of 88% and a specifity of 98% Conclusions: A standardised macroscopic examination of SLN allows a selective application of frozen section with clinically reliable results.

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Abstracts So-012 Der Schnellschnitt bei Uterus- und Ovarialtumoren aus der Sicht der Pathologie A. Schneider Berlin So013 Der Schnellschnitt bei Uterus- und Ovarialtumoren aus der Sicht der Pathologie S. Lax Graz So-014 Topoisomererase IIα α mRNA and protein expression in invasive ovarian carcinoma clinicopathological and prognostic signiﬁcance A. Faggad, S. Niesporek, B. Sinn, R. Wirtz1, J. Sehouli2,3, A. Mustea1,2, W. Weichert, A. Noske, M. Dietel, C. Denkert Institut für Pathologie, Charité – Universitätsmedizin Berlin, Campus Mitte 1 Siemens Medical Solutions – Diagnostics, Leverkusen 2 Klinik für Frauenheilkunde und Geburtshilfe, Charité – Universitätsmedizin Berlin, Campus Mitte 3 Tumorbank Ovarian Cancer – Network, Berlin Aims: The aim of this study was to investigate topoisomerase IIα (Top IIα) expression at the RNA and protein level and to evaluate the relationship between Top IIα expression and clincopathological variables and outcome in patients with untreated invasive ovarian cancer. We attempted to assess the feasibility of measuring Top IIα gene expression in RNA isolated from archival formalin fixed paraffin embedded (FFPE) tissue specimens which are used routinely in pathology laboratories. Methods: We examined 133 FFPE tumor samples taken from patients with primary ovarian carcinoma for the expression of topoisomerase IIα mRNA using real time RT-PCR as well as for protein expression by immunohistochemistry; and we evaluated the correlation of topoisomerase IIα expression with clinicopathological data and patient outcome. Results: Increased topoisomerase IIα expression was observed in higher grade tumors and advanced stage patients. In univariate Kaplan-Meier analysis, patients with higher expression of topoisomerase IIα had a significantly shorter survival. Conclusions: In patients with ovarian cancer, over-expression of topoisomerase IIα correlates with higher grade tumors and advanced stage; furthermore Top IIα expression is a prognostic factor for overall survival.

So-015 Frequent gains of 3q26 in preinvasive and invasive squamous cell lesions of the vulva S. Aulmann, J. Schleibaum1, R. Penzel, P. Schirmacher, G. Gebauer1, H.P. Sinn Pathologisches Institut und 1 Frauenklinik, Universitätsklinikum Heidelberg Aims: Recently, vulvar intraepithelial neoplasia (VIN) has been reclassified into a classical variant, which is related to high risk HPV infection, and a differentiated (or simplex) variant that is associated with chronic inflammation. To further characterise molecular alterations involved in the formation or progression of these lesions, we analysed 3q26 gains and p53 alterations in a series of differentiated and classical VINs and invasive SCC of the vulva. Methods: Using fluorescence in situ hybridisation (FISH), 3q26 gains were evaluated in classical VINs (1-3), VINs of the differentiated type and invasive squamous cell carcinomas of the vulva. In addition, all cases were examined for HPV-DNA, p53 mutations, as well as p16 and p53 protein expression. Results: Gains of chromosome 3q26 were present in none of the VIN1/2 lesions, 57% of VIN3 lesions, 100% of differentiated VINs and in 81% of SCCs. HPVassociated lesions exhibited the typical, strong cytoplasmic p16 accumulation while mutated p53 was only detected in HPV-negative VINs or SCCs and was associated with an overexpression of p53 protein.

Conclusions: Immunohistochemical evaluation of p16 and p53 expression aids in the differential diagnosis of squamous cell alterations of the vulva. However, detection of 3q26 imbalances may be of additional diagnostic value in difficult cases of HPV-unrelated classical VINs and VINs of the differentiated type.

So-016 Estrogen receptor gene (ESR1) ampliﬁcation is frequent in endometrial cancer and endometrial hyperplasia A. Lebeau1*, T.J. Grob1*, F. Holst1, H. Moch2, L. Terracciano3, A. Turzynski4, M. Choschzick1, G. Sauter1, R. Simon1 1 Institut für Pathologie, Universitätsklinikum Hamburg-Eppendorf 2 Institut für Pathologie, UniversitätsSpital Zürich 3 Institut für Pathologie, Universitätsspital Basel 4 Gemeinschaftspraxis für Pathologie, Lübeck *These authors have equally contributed to the study Aims: Recently, we have shown that amplification (AMP) of the ESR1 gene encoding ER α constitutes a major mechanism for ER overexpression in breast cancer. ER α also plays a critical, but diverse and not fully understood role in endometrial cancer. Most endometrial cancers express ER α but only some of them respond to anti-estrogen therapy such as tamoxifen (TAM). On the other hand, TAM therapy of breast cancer constitutes a risk factor for endometrial cancer development. Therefore, we studied ESR1 AMP and ER expression in endometrial cancer and hyperplasia to determine its potential role in carcinogenesis. Methods: 368 endometrial cancers and 35 cases of endometrial hyperplasia were analyzed for ESR1 AMP by FISH. ER expression was determined by immunohistochemistry. Results: FISH revealed ESR1 AMP in 40/176 (23%) cancers and 11/35 (31%) endometrial hyperplasias. Strong ER protein expression (score 7–8 according to Allred) was significantly linked to ESR1 AMP in endometrial cancer (p=0.0036). Conclusions: These data indicate that ESR1 AMP is a frequent mechanism for ER overexpression in endometrial cancer, and suggest an early role for ESR1 AMP in the development of a significant fraction of endometrial cancer. Given the predictive role of ESR1 AMP for TAM response in breast cancer, it will be interesting to investigate the response of ESR1 amplified endometrial cancers to TAM and other anti-estrogenic drugs.

So-017 ERCC1-immunoexpression does not predict platinum-resistance in ovarian cancer S. Stadlmann1, S. Dirnhofer2, U. Güth3, S. Thies2, G. Singer1 1 Institut für Pathologie, Kantonsspital Baden 2 Institut für Pathologie und 3 Abteilung Gynäkologie und gynäkologische Onkologie, Frauenklinik Universitätsspital Basel Aims: Excision repair cross complementing 1 (ERCC1) is a DNA repair gene which is crucial in tailoring cisplatin-based chemotherapy. In ovarian carcinomas, no data on ERCC1 protein expression have been reported so far. Our hypothesis was that ERCC1-protein could act as an indicator for chemoresistance, in order to select patients that would primarily benefit from chemotherapeutics other than platinum-based drugs. Methods: We have investigated the immunoexpression of ERCC1 in tumor tissues from 80 patients with primary ovarian serous carcinomas after conventional platinum-based chemotherapy. ERCC1-protein expression was analyzed using a mouse monoclonal antibody specific for the full-length human ERCC1-protein (1:300 dilution, Clone 8F1, Neomarkers, U.S.A.). Analysis of ERCC1-expression was performed according to Olaussen et al (1).Results: ERCC1 expression was observed in 20.3% of the ovarian carcinomas. ERCC1-immunoexpression in chemoresistant tumors was not significantly different compared to the chemosensitive tumors (P=0.210).. 27.8% of the chemoresistant and 17.8% of the chemosensitive tumors showed ERCC1-expression. ERCC1-expression was not associated with platinum-resistance (P=0.380). Conclusions: ERCC1-protein expression is rare in ovarian cancer and appears not to be a useful tool for the selection of tailored chemotherapy in these patients. [] Olaussen et al., N Engl J Med ;: –

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Autorenregister

Autorenregister Alle Erstautoren mit Programmheftnummern (Geladene Referenten sind kursiv)

A

C

Abbas, M. Sa-126, Sa-127 Adam, P. Sa-006 Adams, H. Fr-138 Agaimy, A. Sa-057 Agelopoulos, K. Fr-021, Fr-100 Ahrens, P. So-007 Andrulis, M. Do-032, Do-055 Arndt, S. Do-089 Assmann, G. Do-067, Sa-114 Aulmann, S. Fr-024, So-015 Aumueller, C. Fr-067 Aust, D. Do-002

Cahyadi, O. Do-025 Chen, Y. Fr-123 Chott, A. Fr-068 Christgen, M. Sa-048

B Baak, J. Fr-032 Back, W. Sa-073 Bagó-Horváth, Z. Fr-129 Baldus, S.E. Do-002, Sa-067 Baretton, G. Sa-040 Baumhoer, D. Fr-102 Becker, J.U. Sa-149, Sa-150 Becker, K.-F. Fr-031 Bektas, N. Fr-114 Benz, K. Fr-078, Fr-079 Berezowska, S. Fr-149 Bergmann, F. Sa-095, Sa-097 Bernd, H.-W. Do-044 Berndt, A. Do-063 Bertolini, J. Do-031 Bertz, S. Sa-115 Bettstetter, M. Sa-080 Bierhoff, E. Do-082 Bihl, M.p. Sa-092 Bläker, H. Do-015 Blankenburg, M. So-002 Blohmer, J-U. So-009 Blümke, K. Sa-110 Bock, O. Do-042 Bode, S. Fr-157, Sa-005 Böhm, T. Fr-130 Böhnke, A. Sa-026 Boysen, G. Sa-113 Braesen, J.H. Sa-106 Brakhage, A. Fr-001 Braunschweig, T. Do-072 Breinig, M. Sa-086 Bremm, A. Do-006 Breuer, E. Fr-022 Brochhausen, C. Fr-010, Fr-088 , Fr-110, Fr-111 Fr-160, Sa-103, Sa-104 Bruder, E. Do-074 Buckendahl, A. Fr-051 Budczies, J. Fr-058 Büttner, R. Sa-019

D Dahl, E. So-005 Davies, J. Sa-037 Dellmann, A. Fr-118 Denkert, C. Fr-050 Deplazes, J. Sa-054 Dienes, H.P. Sa-041 Dierkes, C. Fr-101 Divo, M. Fr-131 Dolznig, H. Do-033 Donhuijsen, K. Fr-076 Drebber, U. Sa-089

E Eltze, E. Sa-130 Engels, K. Fr-059 Erbersdobler, A. Sa-046, Sa-131 Eriksson, M. Fr-151 Esposito, I. Fr-044

F Faggad, A. So-014 Fahr, M.S. Do-073, Do-079 Falkeis, C. Do-077 Fathke, C. Do-005 Fehm, T. Fr-039 Ferlinz, A. Fr-139 Friedrichs, N. Sa-064 Fritzsche, F. Sa-111, Sa-143 Fuchs, T.J. Sa-116 Funke, B. Do-009 Funke, V. Sa-065

Gu, J. Fr-027 Guenther, R. Fr-140 Fr-141 Gunawan, B. Sa-085 Günther, S. Fr-142

H Haag, J. Fr-108 Haedicke, W. Sa-147 Haller, F. Do-016 Haluza, D. Fr-087 Hämmerle, M. Do-091 Hammerschmied, C.G. Sa-118 Hannig, H. Fr-161 Haroske, G. Do-036 Hartmann, E. Do-049 Hartmann, S. Fr-070 Hauptmann, K. Sa-017 Heikaus, S. Sa-112 Heim, S. So-008 Helpap, B. Sa-148 Henkel, T. Sa-020 Henopp, T. Fr-096 Hermanns-Sachweh, B. Fr-112 Hermeking, H. Do-001, Fr-011 Herpel, E. Fr-134, Fr-135 Fr-156 Heywang-Köbrunner, S. Fr-045 Hiemann, N.E. Fr-089 Hildenbrand, R. Sa-093 Hinsch, N. Fr-097 Hipp, S. Fr-034 Hofmann, M. Do-007 Hofstädter, F. Fr-014 Höftmann, J. Fr-060 Höller, S. Do-046 Hopfer, H. Fr-017 Horst, D. Sa-077 Huang, B. Fr-072 Huebner, S. Fr-077 Hufnagl, P. Sa-022 Hummel, M. Do-059 Hungermann, D. So-001, So-011 Hussein, K. Fr-061

I Ihrler, S. Do-068 Ikenberg, K. Fr-152

G

J

Gabriel, B. Fr-033 Gaisa, N.T. Fr-016 Gajda, M. Fr-132, Sa-105 Garbrecht, N. Fr-099 Gassler, N. Sa-063 Gattenlöhner, S. Fr-056, Sa-021 Geissinger, E. Fr-069 Glatz, K. Do-029 Goeppert, B. Do-022 Goldmann, T. Fr-005 Grabe, N. Sa-043 Grabellus, F. Sa-058 Gradistanac, T. Fr-133 Gräntzdörffer, I. Sa-094 Grob, T. Do-075 Groene, H.-J. Fr-013 Groh, A. Fr-009 Gross. A. Sa-045

Jayasinghe, C. Fr-117a, Sa-102c Jin, M. K. Do-050 Jochum, W. Fr-106 Jonigk, D. Fr-163 Jundt, G. Sa-015 Jungbluth, A. Do-092, Sa-079 Junker, K. Sa-039 Jürchott, K. Sa-078

K Kahl, P. Sa-144 Kähler, D. Fr-128 Kain, R. Fr-006 Kalinski, T. Do-028 Kandolf, R. Fr-002 Kandutsch, S. Sa-088 Karpe, B. Fr-080 Katzenberger, T. Do-048

Kayser, G. Sa-023 Kemmerling, R. Fr-062, Fr-065 Kenner, L. Sa-129 Kilic, E. Fr-023 Kirchner, M. Sa-135 Kirchner, T. Fr-052 Klein, C. Sa-001 Kloor, M. Sa-031 Klotzsche, A. Fr-136 Knoess, M. Fr-154, Sa-109 Knöß, P. Fr-093 Köbel, M. Fr-037 Koch, K. Do-051 Koleganova, N. Fr-109, Sa-087, Sa-088 Köllermann, J. Sa-132 Kommoss, F. Fr-036 Koperek, O. Fr-094 Korsching, E. Do-034, Do-038 Köster, J. Do-083 Kozakowski, N. Sa-107 Kreipe, H. Fr-046 Kreisel, M. Fr-107 Kriegl, L. Do-088 Kriener, S. Sa-081 Kristiansen, G. Sa-121, Sa-136, So-006 Krueger, S. Do-003 Kuphal, S. Do-086 Kurrer, M. Fr-073; Fr-074 Kurz, A. Sa-098 Kuyumcu, S. Sa-102d Kwapiszewska, G. Fr-126

L Lage, H. Sa-003 Laimer, D. Do-045 Lang, F. Fr-071 Lang, S. Sa-018 Langer, R. Sa-051 Langner, C. Sa-075 Lassmann, S. Do-014, Sa-072 Lax, S. So-013 Lebeau, A. So-010so-016 Lehmann, U. Fr-057 Leich, E. Do-047 Loddenkemper, C. Fr-004 Longerich, T. Do-019 Lüttges, J. Do-002 Luu, V.D. Sa-009

M Maaßen, A. Sa-145 Macher-Göppinger, S. Sa-119, Sa-124, Madlener, S. Fr-158 Mainka, P. Fr-026 Malz, M. Do-021 Märkl, B. Sa-068 Marx, A. Fr-053 Marx, A. Sa-052 Mateus, A.R. Sa-055 Mayr, D. Fr-043 Meinhardt, M. Sa-120 Metzger, A. Fr-075 Mikuz, G. Fr-054

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Autorenregister Minner, S. Sa-128 Miyazaki, T. Sa-069 Moch, H. Fr-015, Sa-002 Mogler, C. Fr-153 Möller, P. Sa-012 Morawietz, L. Sa-014 Moter, A. Sa-076 Mottok, A. Do-060 Muders, M. Sa-137 Müller, A.M. Do-087, Do-81a, Fr-115 Müller, B. Sa-024 Munding, J. Do-023

N Nährig, J. Fr-047 Neubauer, H. Fr-117 Neumann, J. Sa-123 Noetzel, E. Fr-018

O Oberschmid, B. Sa-070 Otterbach, F. Fr-025 Otto, M. Sa-016

P Pammer, J. Do-084 Penzel, R. Fr-063 Perez-Bouza, A. Do-030 Perner, S. Fr-124, Sa-138, Sa-139 Petersen, I. Fr-003, Sa-100 Petry, C. Fr-048 Pflegerl, P. Do-090 Piecha, G. Fr-083 Poeschl, A. Do-076 Porschewski, P. Fr-150 Porubsky, S. Sa-007 Prall, F. Do-010 Proch, C. Fr-092

R Rau, T. Do-004 Rauser, S. Sa-060, Sa-061 Regele, H. Fr-012 Reichelt, U. Sa-050 Reinacher-Schick, A. Sa-030 Reinecke, P. Sa-122 Reiner, D. Fr-119 Renné, C. Do-057 Reu, S. Do-070 Richter, P. Do-065 Rieker, R. J. Fr-041, Fr-143 Riener, M.-O. Sa-082, Sa-090 Röcken, C. Sa-032 Roessner, A. Do-026 Röhle, A. Do-053 Römermann, D. Do-039, Do-040 Röske, A. Sa-096 Rothe, H. Sa-074 Rupp, C. Fr-144 Rüther, W. Sa-013

S Sassen, S. Fr-020 Savic, S. Fr-145, Sa-141

100 |

Schäfer, C. Sa-102 Schäfer, H. Do-078 Schäfer, K.-l. Fr-155 Schäfer, R. Fr-030 Scheel, S.K. Do-011 Scheil-Bertram, S. Fr-105, Sa-028 Schildhaus, H.-U. Sa-099 Schirmacher, P. Sa-034, Sa-035 Schmidt, H. Do-066, Fr-019 Schmitz, K.J. Do-018 Schnabel, P. Fr-085, Fr-086, Fr-125 Schnall, S. Fr-055 Schneider, A. So-012 Schoner, K. Do-071 Schopf, C. Fr-122, Do-035 Schrader, T. Do-035, Sa-008 Schraml, P. Sa-038 Schröder, C. Fr-162 Schwamborn, K. Fr-066, Sa-142 Sehouli, J. Fr-049 Seitz, S. Fr-159 Sers, C. Fr-029, Sa-066 Sheu, S.Y. Fr-095 Shiozawa, E. Do-056 Simiantonaki, N. Sa-102a, Sa-102b Siebolts, U. Fr-146 Singer, S Sa-011 Sinn, H.P. Sa-042, Sa-044 Slotta-Huspenina, J. Do-052 Soleiman, A. Do-013, Fr-147 Sotlar, K. Do-043, Fr-028, Sa-101 Spangler, B. Do-085 Stadlmann, S. So-17 Staebler, A. Fr-038, Fr-040 Stege, A. Sa-034 Stelniec, I. Sa-047 Stenner, M. Do-062 Stenzel, E. Sa-133 Stocker, G. Do-008 Stöcklein, H. Do-054 Stöhr, R. Sa-134 Stoss, O. Sa-140 Strack, I. Do-017 Straub, B.K. Do-020 Ströbel, P. Sa-033 Sturm, K. Fr-103 Sulzbacher, I. Do-061

T Tavassoli, F.A. Sa-029 Theophile, K. Do-041, Fr-064 Theurillat, J.P. Do-024 Ting, E. Sa-004 Tinguely, M. Sa-010 Tischler, V. Fr-127 Tischoff, I. Do-064 Tornillo, L. Sa-056, Sa-071 Tränkenschuh, W. Sa-084

V Varga, I. Varga, Z. Veeck, J. Veith, D. Vogel, C. Vogel, U.

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Fr-081 So-004 Sa-025 Sa-062 Do-012 Do-069, Fr-116, Fr-137

Vokuhl, C. Do-080 Völker, H.-U. Do-037, Fr-098 Völklein, C. Sa-027 Vosbeck, J. Sa-146

W Wagner, M. Fr-007 Walch, A. Sa-053 Wardelmann, E. Sa-059 Weber, A. Sa-049, Sa-091 Wegener, S. Do-058 Weichert, F. Fr-008 Weichert, W. Fr-042 Weiler, C. Fr-104 Sa-117 Westerhoff, H. Fr-035 Wiegert, S. Fr-148 Wiesmann, F. Fr-113 Wild, P.J. Sa-125, So-003 Wohlschlaeger, J. Fr-082, Sa-083 Wolf, U. Fr-084 Wolff, J.-C. Fr-120

Z Zatloukal, K. Sa-036 Zernecke, A. Fr-090, Fr-091 Zimpfer, A. Do-081, Fr-121 Zwönitzer, R. Do-027

Abstracts der Deutschen Gesellschaft für Pathologie

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