Abstracts Pathologe 2016 · 37:3–156 DOI 10.1007/s00292-016-0169-5 Online publiziert: 4. Mai 2016 © Springer-Verlag Berlin Heidelberg 2016

100. Jahrestagung der Deutschen Gesellschaft für Pathologie e. V. Berlin, 19. bis 21. Mai 2016

Keynotes Multiscale Imaging in translational research KN.01 Multiscale Imaging in Translational Research F. Kiessling* Uniklinik RWTH Aachen, Institut für Experimentelle Molekulare Bildgebung, Aachen, Deutschland The term “imaging” has different meanings in different scientific communities. For example, a pathologist or basic biologist will predominantly think about microscopic techniques, while a radiologist or specialist in nuclear medicine will mostly consider non invasive methods like ultrasound, x-ray, CT, MRI, PET and SPECT. However, in principle imaging encompasses all methods and tools that result in an image. A holistic view on the imaging field is important to fulfill the demands on diagnostic procedures in translational research: On the one hand, many diseases (including cancer) underlie systemic pathophysiological mechanisms making it necessary to investigate the entire organism. On the other hand, the information at whole body and organ level needs to be linked to cellular interactions as well as micro-environmental remodeling processes at tissue level to get a true understanding of diseases and to develop therapeutic strategies. From microscopy, non-invasive optical technologies evolved that reach the mesoscopic scale or even whole body level in rodents and that are even applicable to humans, e. g. to characterize superficial tissues and to improve endoscopy and intraoperative surgery. However, the mesoscopic scale was conquered with MRI, ultrasound and CT as well, and nowadays the transition between resolution scales offered by different imaging modalities is blurred. Multiscale imaging is not only limited to morphology but also offers interesting functional features, e. g. to assess tissue oxygenation, perfusion, metabolism, inflammation, apoptosis as well as many molecular biomarkers. These help us to improve our mechanistic understanding of diseases and may act as imaging biomarkers in clinical routine. Together with omics data such imaging biomarkers will help us to apply systems biology in clinical routine, e. g. to characterize diseases, preselect patients to therapies and to monitor therapeutic success. Finally, multiscale imaging can strongly support image guided interventions, in particular if a whole body examination is directly followed by a surgical resection or a biopsy. In conclusion, the convergence of classical histological and non invasive imaging methods enables a holistic multifunctional view on pathomechanisms, links basic and clinical research, supports drug development and leads to the development of new imaging tools for precision medicine.

The supplement these abstracts are part of is not sponsored by the industry.

Translational Optical Molecular Imaging: from Experimental to Clinical Phase III Evaluation in Oncology – What’s the Role of the Pathologist? KN.02 Translational Optical Molecular Imaging: from Experimental to Clinical Phase III Evaluation in Oncology – What’s the Role of the Pathologist? G. M. van Dam Surgery, Nuclear Medicine and Molecular Imaging and intensive Care, University Medical Center Groningen The main focus of the talk will be an overview of translational optical molecular imaging in various solid tumors: breast cancer, colorectal cancer, esophageal and pancreatic cancer. In particular based on the initial clinical study using a folate-receptor alpha targeted imaging approach in ovarian cancer, more recent developments using therapeutic antibodies, nanobodies, small peptides and smart-activatable probes for intraoperative imaging and drug development will be presented for each tumor type in patients. Newly developed Standard Operating Procedures from the operating theatre into the pathology department will be showcased in order to validate and cross-correlate data from multicenter studies and per tumor type. Besides tumor detection, new developments for treatment of (microscopic) residual disease will be presented as based on targeted photodynamic therapy. In particular the spect of fluorescence imaging into the workflow of the pathologist will be outlined and showcased with clinical studies.

Molecular Cytology: Application and Potential KN.03 Molecular Cytology: Application and Potential F. Schmitt* Laboratoire National de Santé, Dudelange, Luxemburg Currently, the use of molecular techniques is widely accepted on cytology as adjuvant of morphology for diagnosis and prognosis. Moreover, the study of markers of therapeutic response has been helpful in some types of neoplasms. There are many advantages in the use of cytological material to perform molecular studies: ease of obtaining fresh material, ability to check the quality of the material immediately after harvest and better preservation of proteins, DNA and RNA. The possibility of using genomic and proteomic studies in small amounts of material obtained by fine-needle aspiration (FNA) can minimize invasive procedures and allow the monitoring of cancer. Proper specimen processing is of utmost importance maintaining morphology and nucleic acid and proteins integrity. The role of the cytopathologist is mandatory in the collection and Der Pathologe Suppl 1 · 2016

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Abstracts selection of cells. The objective of this presentation is to address the use of molecular techniques to refine diagnosis and to study prognostic and predictive markers in different cytological specimens and also discuss the role of modern cytology in contemporary diagnosis and management of metastatic cancer.

Precision care for prostate cancer KN.04 Precision care for prostate cancer M. Rubin* Weill Cornell Medicine, Institute for Precision Medicine, New York, Vereinigte Staaten von Amerika Prostate cancer remains a leading cause of male cancer death worldwide. The mainstay of therapy for patients with metastatic spread, including castration resistant disease, is hormonal therapy targeting the AR. Enzalutamide and abiraterone are potent AR-targeted therapies approved for the treatment of men with CRPC. While significantly improving survival and quality of life, most patients ultimately develop resistance to these agents. Thus, predictive biomarkers to help distinguish responders from non-responders prior to starting the next line of hormonal therapy are urgently needed. We and others have observed that a subset of resistant tumors show small cell carcinoma or neuroendocrine features on metastatic biopsy (CRPC-NE). This phenomenon may therefore reflect an epithelial plasticity that enables tumor adaptation in response to AR-targeted therapies. Prognosis of CRPC-NE is poor due to late recognition, heterogeneous clinical features, and lack of effective systemic therapies. One major hurdle in the diagnosis and treatment of androgen-independent prostate cancer including CRPC-NE is our lack of understanding of the genetic and epigenetic underpinnings of this aggressive subset. To address this, we interrogated 114 metastatic tumor specimens from 81 individuals including 51 with clinical and histologic features of castration resistant adenocarcinoma (CRPC-Adeno), and 30 with CRPC-NE as confirmed by pathologic consensus criteria; we studied matched normal cells from all patients, multiple tumor biopsies from 17 patients and a single tumor biopsy from 64 patients. We hypothesized that CRPC-NE could be distinguished from CRPC-Adeno based on distinct molecular alterations and that this information could improve upon and supplement the current often challenging diagnostic features reliant on morphology. We also hypothesized that CRPC-NE that develops after therapy arises clonally from a CRPC-Adeno precursor, rather than from selection of pre-existing neuroendocrine clones. Lastly, we hypothesized that AR-independent prostate adenocarcinomas that share CRPC-NE-specific molecular alterations may represent tumors at high risk for progression or in transition towards CRPC-NE.

Tissue Phenomics – Discovering Prognostic and Predictive Histological Phenotypes KN.05 Tissue Phenomics – Discovering Prognostic and Predictive Histological Phenotypes G. Binnig* Definiens AG, Munich, Deutschland Histological phenotypes generally play an essential role in diagnostics, but in particular in oncology there is a growing need for selecting the right patients for the right treatments based on information derived from tis-

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sue slides. Oncology is a rapidly evolving field with many new treatment methods being discovered. To finally cope with the complexity of cancer the treatments of cancer patients is becoming increasingly more complex through the availability of those methods and their combinations. As a consequence diagnostics and the decision, which of those treatments is best for a specific patient at what stage of his or her disease, is certainly not trivial. Tissue Phenomics, TPhx, represents a novel technique to discover relevant patterns in tissue slides that predict drug response or other clinical outcome. This method is based on digital pathology, image analysis and data mining, and is more and more evolving into a comprehensive big data approach. The benefit of TPhx is most obvious for oncology and more specifically -with respect to companion diagnostics- for immunotherapy. Quantified morphological structures combined with the local co-existence of certain cell types carry biological meaning. For immunotherapy the characterization of the tumor with its defense mechanisms as well as that of the state and the configuration of the immune cells turns out to be most important. Profiling in a quantitative form the local tumor ecosystem in a big data approach leads to the discovery of novel histological phenotypes that are difficult or impossible to discover by other means.

History and meaning of classification schemes for pathology KN.06 History and meaning of classification schemes for pathology F. Bosman* Institute of Pathology, University Medical Center, Lausanne, Schweiz Adequate disease classifications are universal: every medical professional should be able to use them in daily patient care, in communicating with peers and patients and in medical research. Credible classifications have to be clinically relevant, based upon understanding of the biology of the disease and rooted in consensus. Disease classifications developed along with growing understanding of the biology of disease and mostly based upon notions of etiology and pathogenesis. They were initiated already in the 19th century but became mainstream when the World Health Organization was created and assumed responsibility for global survey of disease patterns, which required universally applicable classification schemes. This resulted in the International Classification of Disease (ICD) of which we all now use the 10th edition (ICD10). Tumor classification is part of ICD but evolved into a separate field, notably with the evolution of the WHO classification of human tumors and the ICD-O (for oncology) codes. WHO was not the only player in this field: the Armed Forces Institutes of Pathology (better known under its acronym AFIP) initiated the Fascicle series, which are continued by the American Registry of Pathology. Important difference between the Fascicles and the WHO classification of human tumor series (better known as ’Blue Books’ due to the consistently used blue color of the covers) is the consensus notion: each volume of the ’Blue Book’ series has more than 100 authors and its publication is preceded by a consensus meeting in which the main specialists in the field participate. The breakthrough for the Blue Books came when the Director of the International Agency for Research on Cancer (IARC) published the 3rd edition of the Blue Books in a format and with a content extremely useful in daily diagnostic working environment of pathologists. This has been consolidated in the 4th edition and the WHO classification of human tumors now constitutes a solid basis for truly universal tumor classification. With the advent of molecular genetic approaches toward disease (sub)classification it has become obvious that revision of classifications needs to be more efficient than what can be attained in a 10 year revision cycle (the time it took to complete the 4th edition). It is therefore likely that the 5th edition will have an ’electronic’ format, allowing revision of chapters according to need rather than of complete volumes in their order of initial appearance.

This dialog is designed to present selected basics in the radiological and pathological diagnosis of focal liver lesions. Both radiological imaging findings as well as pathological image patterns in primary liver lesions are shown. In addition morphological changes after interventional treatment of liver lesions are discussed.

DGP-Hauptprogramm HP: Radiologie trifft Pathologie DGP01.01 Cross-sectional imaging of focal liver lesion – radiological-pathological correlation

DGP01.03 Radiology meets Pathology: Pancreatic tumors and inflammation A. Schreyer*1, I. Esposito*2

A. H. Mahnken*1, T. Longerich2 Universitätsklinikum Marburg, Klinik für Diagnostische und Interventionelle Radiologie, Marburg, Deutschland, 2Universitätsklinikum, RWTH Aachen, Aachen, Deutschland

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Cross-sectional imaging is a key for accessing focal liver lesions. Dynamic contrast-enhanced imaging allows for a diagnostic assessment of these lesions with a high diagnostic accuracy. Vascular anatomy reflected by contrast-enhancement pattern and the use of specific contrast agents permits a precise non-invasive diagnosis in accordance with recent guidelines. Magnetic resonance imaging (MRI) even provides information on iron content and the presence of fat. In addition diffusion may be assessed, not only improving sensitivity, but also diagnostic accuracy. With these techniques even subtyping of several liver tumor became feasible (e. g. liver adenoma, tumor grading in HCC). With interventional radiology presenting an important part in the treatment of liver lesions, including transarterial as well as percutaneous treatment techniques the assessment of treatment response as well as the identification of specific treatment related side effects became more complex.

Universität Regensburg, Institut für Röntgendiagnostik, Regensburg, Deutschland, 2Universität Düsseldorf, Institut für Pathologie, Düsseldorf, Deutschland

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This presentation intends to introduce pathologists to the most important examination techniques, applied by radiologists to evaluate the pancreas focusing on ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI). Additionally, a dedicated MRI technique to visualize the pancreatic duct (MRCP) will be demonstrated. After an introduction and overview of the radiological imaging methods, we discuss important and difficult pathological and radiological differential diagnoses in dialogue form. We will put special emphasis on the radiological diagnosis of IPMN. Additionally, characteristic radiological signs for the diagnosis of serous and mucinous pancreatic tumors will be explained by showing typical radiological and corresponding pathological images. We will also attempt to demonstrate the difficulties in the differential diagnosis between chronic pancreatitis and pancreas carcinoma, based on radiological and pathological cases. The dialogue will be concluded by discussing

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Abstracts the solid pseudopapillary neoplasm as a rare but prototypical pancreas tumor, being notoriously difficult to diagnose, both radiologically as well as pathologically. The aim of our dialogue between a pathologist and a radiologist is to mutually understand the possibilities and limitations of each disciplines’ methods for the correct diagnosis of pancreatic tumors.

HP: Urotheltumoren DGP02.01 Was will der Urologe vom Pathologen: Aktueller Dialog A. Hartmann*, A. Heidenreich FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland Beim Urothelkarzinom der Harnblase ist die Kommunikation zwischen Urologen und Pathologen wesentlich. Beim nicht-muskelinvasiven Urothelkarzinom spielt dabei die pathologische Einteilung in Tumoren mit niedrigem bzw. hohem Progressionsrisiko eine wichtige Rolle. Für letztere sollte eine frühe Zystektomie im Tumorboard besprochen werden. Pathologische Risikofaktoren für eine hohes Progressionsrisiko sind neben dem Auftreten eines Carcinoma in situ (CIS) eine extensive Stromainfiltration sowie das Auftreten von histologische Subtypen. So gibt es z. B. beim Auftreten einer mikropapillären oder plasmazytoiden Tumorkomponente inzwischen valide Daten, die eine frühe Zystektomie empfehlen. Beim muskelinvasiven Urothelkarzinom ist insbesondere die Schnellschnittdiagnostik bei radikaler Operation wesentlich. Hier ist die Untersuchung des Absetzungsrandes der Ureteren bzw. der Urethra essentiell um die Radikalität der Operation zu gewährleisten. Die Schnellschnittdiagnostik ist hier wichtig, da bei vielen Patienten mit muskelinvasiven Karzinomen der gesamte Urotheltrakt im Sinne einer Feldkanzerisierung betroffen ist und Absetzungsränder mit vollständiger Entfernung des invasiven Tumors und eines CIS erzielt werden sollten. Oft ist eine Nachresektion gut möglich. Wesentlich ist eine histopathologische Diagnostik entsprechend der aktuellen S3-Leitlinie, die in den letzten 2 Jahren erarbeitet wurde, und der WHO-Klassifikation von 2016. Zusätzlich sollten allerdings gut etablierte Risikofaktoren (wie z. B. eine Einteilung des invasiven Urothelkarzinoms in G2 und G3 entsprechend der WHO-Klassifikation 1973) im Dialog zwischen Pathologen und Urologen in der klinisch-pathologischen Konferenz weiter berücksichtigt werden. Die EORTC-Risikokriterien sprechen

beim oberflächlich invasiven Harnblasenkarzinom diesem Grading eine wesentliche Rolle in der Risikoabschätzung zu. Neue prädiktive Marker des Urothelkarzinoms sollten in den Dialog zwischen Pathologen und Urologen in der klinisch-pathologischen Konferenz einbezogen werden. Beispiele dafür sind das Auftreten von FGFR3-Mutationen bei Patienten mit papillären Urothelkarzinomen sowie die neue Einteilung des Urothelkarzinoms in unterschiedliche molekulare Subtypen. Es deutet sich hier an, dass eine molekulare Klassifizierung des Urothelkarzinoms (ähnlich wie sie für das Mammakarzinom seit Jahren etabliert ist), möglicherweise eine Stratifizierung von Patienten in unterschiedliche Risikogruppen ermöglicht.

DGP02.02 Molecular Subtyping in muscle invasive bladder cancer by RT-qPCR predicts disease-specific survival after radical cystectomy R. Wirtz*1, M. Kriegmair2, A. Steidler2, J. Breyer3, T. Gaiser4, C. Bolenz5, A. Hartmann6, P. Erben2 1 Stratifyer Molecular Pathology GmbH, Köln, Deutschland, 2Klinik für Urologie, Universitätsklinikum, Mannheim, Deutschland, 3Universität Regensburg, Klinik und Poliklinik für Urologie, Regensburg, Deutschland, 4 Institut für Pathologie, Universitätsklinikum, Mannheim, Deutschland, 5 Klinik für Urologie, Universitätsklinik, Ulm, Deutschland, 6Pathologisches Institut, Universitätsklinikum, Erlangen, Deutschland

Background.  The hormone receptors ESR1 and PGR as well as HER2 and KI67 are key biomarkers to determine molecular subtypes in breast cancer. Recently, it could be shown that distinction of luminal and basal subtypes is also crucial to predict outcome in urothelial carcinoma of the bladder. We analyzed mRNA expression of ESR1, PGR, HER2 and KI67 using RT-qPCR in FFPE tissues from patients with muscle invasive bladder cancer (MIBC, pT1-4; Nx). Methods.  Gene expression were analyzed retrospectively from FFPE tissues of 92 patients (f = 22; m = 70; median age: 67 [range 45–97]; pT1/2: 23, pT3: 48, pT4: 21) and measured by single step RT-qPCR using RNA-specific TaqMan Assays. Relative gene expression was determined by normalization to two housekeeping genes (CALM2, B2M) and using synthetic in-vitro transcripts of target and housekeeping gene as control using the 40 ΔΔCT method. Prognostic value of singular genes and subtyping algorithm was analyzed by unsupervised partitioning tests, Mann Whitney tests and Kaplan Meier estimates of Disease-specific Survival (DSS). Results.  ESR1 and PGR mRNA expression was higher in comparison with HER2 und Ki67 gene expression (Median ESR1 34.8; PGR: 33.9; Ki67: 37.7; Her2: 36.9). High expression of Her2 mRNA was found in 17 % of patients Fig. 1 I DGP02.02 9 Molecular subtypes and DSS in MIBC after radical cystectomy

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(pts) and associated with good prognosis (57 % vs 7 % DSS after 5 years; p = 0.0001). When combining the categorized values of ESR1, PGR, HER2 & KI67 mRNA expression using a predefined algorithm from the breast cancer setting the subtypes with significantly different survival could be identified: HER2+ (7 % DSS, after 5 years); HER2-/ESR+ (23 % of pts, 20 % DSS, after 5 years); HER2-/ESR-/Ki67+ (48 % of pts, 60 % DSS after 5 years); HER2-/ ESR-/Ki67- (12 % of pts, 100 % DSS, after 5 years; p < 0,0001) (. Fig. 1). Conclusions.  Subtyping by ERS1, PGR, HER2 and KI67 mRNA identifies molecular subtypes with prognostic relevance in MIBC. This may be helpful to identify high risk MIBC pts and pts appropriate for already established targeted therapies. Prospective studies based on this data might be reasonable.

DGP02.03 Meta-analysis and review of clinical, pathological and therapeutic data of 948 urachal carcinomas H. Reis*1, O. Módos2, C. Niedworok3, A. Szendröi2, A. Szasz4, P. Nyirady2, T. Szarvas2, 3 Universitätsklinikum Essen, Universität Duisburg-Essen, Institut für Pathologie, Essen, Deutschland, 2Semmelweis Universität, Klinik für Urologie, Budapest, Ungarn, 3Universitätsklinikum Essen, Universität Duisburg-Essen, Klinik für Urologie, Essen, Deutschland, 4Semmelweis Universität, 2. Abteilung für Pathologie, Budapest, Ungarn 1

Background.  Urachal carcinoma (UrC) is a rare cancer entity accounting for <1 % of all bladder cancer cases. Due to its rarity, clinico-pathological and therapeutic data is limited. We therefore conducted a meta-analysis and review of the literature and data from our institutions. Methods.  A systematic PubMed search was conducted using the terms “urachal cancer” and “urachal carcinoma”. For survival analysis, studies with <20 patients were excluded due to low statistical power. Studies applying characteristic patient selection (e. g. some studies with a focus on chemotherapy, included only patients with present metastasis) have been used only for those analyses they were conducted for. Results.  UrC was more prevalent in men (59 %; 561/948), median age was 51 years and the most frequent symptom was haematuria (74 %, 344/466). Cystoscopy held a positive detection rate of 88 % (217/248) while urine cytology had a low sensitivity of 29 % (30/102). Calcification in the midline was not a mandatory radiologic finding (32 %, 45/142). Adenocarcinoma (ADC) was the most common UrC-type (90 %, 360/401). No immunohistochemical or molecular markers were able to accurately differentiate primary bladder (PB-ADC) from UrC-ADC. The Mayo-staging system exhibited a more balanced stage distribution than the Sheldon system and radical cystectomy did not show a survival benefit compared to partial cystectomy. 21 % (122/572) of patients had synchronal distant metastasis (M+), 13 % (32/239) lymph node metastasis (LN+) and both groups exhibited a similar poor prognosis (< 20 % 5-year survival). In multivariable analyses, Sheldon stage >IIIB, Mayo stage >II, M+, LN+, positive surgical margin status (R+) and ECOG status were independent prognostic factors. The highest response rates were found in the 5-FU and cisplatin-5-FU groups (44 %, 43 %) with lowest progression rate in the cisplatin-5-FU group (14 %). Conclusions.  UrC is a rare malignancy which can be detected by cystoscopy and additional imaging methods. Considering the different clinico-pathological and therapeutic characteristics, differential diagnostic markers for UrC-ADC and PB-ADC are needed. Furthermore, 5-FU containing chemotherapy seems to be superior to cisplatin-based regimens with the lowest progression and highest remission rates for their combination. Given the rarity of UrC, large scale prospective studies comparing systemic therapies can hardly be conducted. This underlines the need for the molecular characterization of UrC to identify targets for tailoring therapy.

DGP02.04 Comprehensive screening of rare histologic variants of urothelial cancer by multiplexed targeted resequencing reveals distinct mutation signatures with potential therapeutic impact S. Bertz*1, E. A. Moskalev1, U. Zinnall1, R. Stöhr1, E. Comperat2, P. Camparo3, A. Lugli4, A. Perren4, M. Toma5, G. Baretton5, N. Gaisa6, R. Knüchel6, L. Cheng7, B. Keck8, B. Wullich8, F. Haller1, A. Hartmann1 FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland, Hospital Pitié Salpetriere, UPMC Paric VI, Institut für Pathologie, Paris, Frankreich, 3Centre de Pathologie Amiens Picardie, Amiens, Frankreich, 4 Universität Bern, Institut für Pathologie, Bern, Schweiz, 5 Universitätsklinikum Carl Gustav Carus, TU Dresden, Institut für Pathologie, Dresden, Deutschland, 6Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, 7Indiana University School of Medicine, Department of Pathology and Laboratory Medicine and Urology, Indianapolis, Vereinigte Staaten von Amerika, 8Universitätsklinikum Erlangen, Klinik für Urologie, Erlangen, Deutschland

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Background.  Rare variants of urothelial carcinoma (UC) include – among others – micropapillary urothelial carcinoma (MPUC), nested type UC (NVUC) and plasmacytoid UC (PUC), all of them associated with poor prognosis. Little is known about the molecular alterations in those rare but clinically aggressive variants, and only few molecular studies exist that respect these specific variants. However, these studies deal with low case numbers and focus on a limited number of genetic alterations. The aim of the present study was to characterize large cohorts of MPUC, NVUC and PUC by comprehensive multiplexed targeted resequencing using a large cancer panel in order to find the most frequent and important mutations and potential therapy targets. Methods.  Archival FFPE tumor tissues of a large cohort of 70 UCs including MPUC, NVUC and PUC were collected from several pathology centres in Germany, France and the USA. Complete histomorphological reevaluation of all cases according to the current WHO and TNM classification was performed by two uropathologists (A. H., S. B.). Multiplexed targeted resequencing of 48 genes was performed using the TruSeq Amplicon Cancer Panel (Illumina, San Diego, USA) on a MiSeq instrument (Illumina). Results.  MPUC, NVUC and PUC showed different mutation signatures. Most MPUC cases harbored a gain-of-function mutation in a receptor tyrosine kinase or associated PI3K/MAPK pathways with potential therapeutic impact. Additionally, the majority of MPUC cases had an inactivating mutation in the TP53 tumor suppressor gene. The mutation signatures of NVUC and PUC variants were different, with less frequent activating mutations in oncogenes, and TP53 mutations only in a subset of cases. Conclusions.  Comprehensive genetic profiling support the opinion that MPUC, NVUC and PUC variants are genetically distinct subtypes of UC, with specific pathway alterations contributing to morphological differ­ ences. Notably, the majority of MPUC cases harbor gain-of-functions in oncogenes that can be potentially addressed by targeted therapies.

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Abstracts HP: Moderne Bildgebung I DGP04.01 Multiparametric prostate MR in the clinical context: challenges, possibilities and limitations H.-P. Schlemmer*1, N. Gaisa2 1 Deutsches Krebsforschungszentrum, Abteilung Radiologie, Heidelberg, Deutschland, 2Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland

Prostate cancer has become the most frequent cancer in men in the industrialized countries and is accordingly associated with high medical and socio-economic impact. The conventional way of diagnosis and treatment selection is mainly based on PSA serum levels and histopathologically derived Gleason Scores of systematically sampled TRUS biopsy specimens. In prostate confined cancers radical prostatectomy has proven to prolong survival, however, avoidable morbidity and costs are feared due to overdiagnosis and overtreatment in a selective but so far unpredictable patient group. Depending on the tumor stage other treatment options like radiotherapy and focal treatments, e. g. thermal ablation with laser or HIFU, are available. In early stage cancer even active surveillance without therapy is possible. Accordingly, accurate diagnosis and patient stratification for choosing the best individual treatment is increasingly challenging. Since prostate cancer is characterized by a notable range of aggressiveness due to tumor multifocality and biological heterogeneity systematically sampled biopsies bear substantial limitations. In this setting multiparametric MR imaging has proven remarkably advantageous for detecting the cancer, characterizing its heterogeneity and aggressiveness, targeting the most aggressive part (the dominant intraprostatic lesion, DIL) by guiding the biopsy needle to that area, assessing the local tumor spread and providing guidance for focal therapy. Meanwhile different MR/TRUS image-fusion biopsy systems are available, further improving the detection rate of clinically significant cancers. Pathologists mainly benefit from MR imaging biopsies regarding reduced specimen loads and precisely mapped and sampled lesions. The additional information retrieved by MR therefore considerably improves individual decisions regarding treatment selection, planning, guidance, monitoring and follow-up. In case of active surveillance functional MR parameters serve as objective and reproducible “biomarkers” for non-invasive monitoring of changes of tumor aggressiveness over time. This lecture will provide insights into the application of multiparametric MR imaging in the management of prostate cancer by highlighting current challenges, possibilities and limitations for radiologists and pathologists.

DGP04.02 3D Morphology meets Molecular Pathology: Chronic Lung Alveolar Dysfunction revisited M. Kühnel*1, J. Wehling2, M. Kellner3, G. Warnecke4, M. Heidrich5, N. Izykowski2, R. Grothausmann1, L. Knudsen1, H. Golpon6, A. Haverich4, F. Länger2, D. Jonigk2 1 Institut für Funktionelle und Angewandte Anatomie, Medizinische Hochschule, Hannover, Deutschland, 2Institut für Pathologie, Medizinische Hochschule Hannover, Hannover, Deutschland, 3University of Arizona, College of Medicine, Center for Lung Vascular Pathobiology, Tucson, Arizona, Vereinigte Staaten von Amerika, 4Klinik für Herz, Thorax-, Transplantationsund Gefäßchirurgie, Medizinische Hochschule, Hannover, Deutschland, 5 Laser Zentrum Hannover e. V., Biomedizinische Optik, Hannover, Deutschland, 6Klinik für Pneumologie, Medizinische Hochschule, Hannover, Deutschland

Background.  After lung transplantation survival of patients is severely limited by chronic lung allograft dysfunction (CLAD). CLAD manifests

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in two forms: bronchiolitis obliterans, the fibrotic remodeling of small airways and the restrictive allograft syndrome (RAS), characterized by progressive scaring of the alveolar parenchyma, leading to a 5 year median survival of about 40/10 %, respectively. For both CLAD entities appropriate animal models are lacking, especially regarding disease initiation. However, in explanted lung allografts different stages of these entities coexist including early phases of remodeling. To identify these initial lesions we established an imaging strategy, which allows us to perform 3D imaging of remodeled parenchyma from explanted lungs up to 1 cm3 in size in a life like state and correlate 3D imaging in a next step with molecular expression data. Methods.  Crucial for the analysis was the development of our novel embedding technique called CRISTAL which combines different resins in order to clear the embedded lung samples to full optical transparency. With this development the very same biopsy can be used for 3D tomography using 2 Photon Scanning Laser Optical Tomography (2P-SLOT) followed by preparation of thin sections for histology, immunohistochemistry and mRNA isolation after laser-assisted microdissection. These techniques allow a morphological and molecular comparison of different pulmonary diseases. Results.  3D imaging at a resolution of 10 to 12 µm combined with high resolution 2-photon imaging at a resolution of <1 µm allowed to visualize CLAD manifestations BO and RAS in human lung samples from explanted lung transplants. We identified individual remodelling stages and describe their morphological complexity through endogenous contrast measurements and thickness analysis. These 3D results of individual BO lesions and RAS areas were then correlated with conventional histology and gene expression. Particularly the differences in MMP expression levels in BO versus RAS correlate well with the respective structural entities. Conclusions.  In summary, embedding of biopsies in clearing resin allows for the correlation of high resolution 3D information with histology, immunohistochemistry and mRNA expression in CLAD manifestations BO and RAS and illustrates how differences in MMP expression profiles account for morphological differences in BO and RAS entities.

DGP04.03 Non-invasive molecular imaging of kidney fibrosis J. Ehling*1, 2, B. Klinkhammer1, Q. Sun1, M. Baues2, R. Knuechel1, F. Kiessling2, J. Floege3, T. Lammers2, P. Boor1, 3 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, 2Institut für Experimentelle Molekulare Bildgebung, RWTH Universitätsklinikum, Aachen, Deutschland, 3Abteilung für Nephrologie, RWTH Universität, Aachen, Deutschland 1

Background.  Renal fibrosis, which is characterized by the excessive deposition of extracellular matrix (ECM) proteins by activated (myo-)fibrobloblasts, is the best predictor and common end-point of virtually all progressive chronic kidney diseases (CKDs). Although millions of patients worldwide suffer from CKDs, no specific imaging protocols or agents are available for non-invasively monitoring renal fibrosis. Thus, the aim of our efforts was to establish novel contrast agents and molecular imaging techniques for monitoring renal fibrosis in experimental murine models of CKD. Methods.  Two murine models of kidney fibrosis of different etiology were used: unilateral ureteral obstruction (UUO) and unilateral ischemia/reperfusion injury (IR). The deposition of ECM proteins during disease progression was visualized in vivo using collagen- and elastin-specific probes enabling T1-weighted molecular magnetic resonance imaging (MRI) and computed tomography/fluorescence-mediated tomography (CT/FMT) hybrid imaging. The latter multimodal approach was also used for peptide-based targeted imaging of the plated-derived growth factor receptor beta (PDGFR-ß), a marker of renal (myo-)fibroblasts. In vivo findings were validated by conventional methodologies including qRT-PCR, Western blotting, immunohistochemistry and 2-photon microscopy.

Results.  In vivo optical CT/FMT hybrid imaging revealed a 3-fold higher accumulation of Cy7-labeled CNA35, a collagen-specific contrast agent, in fibrotic kidneys compared to healthy contralateral kidneys in mice. A comparable tendency was found for the elastin-specific contrast agent ESMA compared to the non-specific gadolinium-containing MR contrast agent (Gd-DTPA): 24 hours after i. v. injection, R1 relaxivity values based on T1 mapping MRI sequences revealed a significant difference in the medulla of fibrotic vs. healthy kidneys. Finally, the highest diagnostic power was found for PDGFR-ß targeted imaging using in vivo CT/FMT hybrid imaging: fibrotic kidneys showed a 12-fold higher probe accumulation compared to contralateral controls. Conclusions.  We have established specific molecular imaging protocols for non-invasively monitoring renal fibrosis using molecular MRI and CT/ FMT hybrid imaging. Our data lay the basis for the establishment of three novel clinically translatable imaging probes facilitating the non-invasive diagnostic imaging of renal fibrosis.

HP: Moderne Bildgebung II DGP04.04 Towards new protocols and guidelines for diagnosis and treatment of GIST S. Schönberg* Universitätsklinikum Mannheim, Institut für Klinische Radiologie und Nuklearmedizin, Mannheim, Deutschland GIST (Gastrointestinal stromal tumour) is a rare disease with a high potential for metastases and an average life expectancy of < 3 years. The MITIGATE project that is co-funded by an EC grant (FP7 602306) will develop and validate an integrated closed-loop molecular environment for minimally invasive treatment of patients with metastatic GIST resistant to the current medication class (tyrosine kinase inhibitors, TKI). This personalised treatment concept combines novel strategies for biopsy, inline tissue analysis, molecular tumour characterisation, theranostics by PET & MRI technologies and companion radiopharmaceuticals followed by assessment of biodistribution, dose calculation and measurement of therapeutic effectiveness. In its 2nd year of operation the project has already achieved several promising results. A novel endoscopic biopsy system coupled to a tissue dissociation device was developed. GIST subtype classification based on mass spectrometry could show that TKI-resistant & -responsive cells were separable with 100 % accuracy. Immunocompromised GIST animal models were established to evaluate potential radiotracers. Small molecule and peptide derived precursors targeting GIST biomarkers (e. g. KIT, GLP-2, NT-1, DOG-1) for the visualisation of lesions and therapy response were synthesized and are currently being tested. Standard procedures to radiolabel peptides with 68Ga were developed for the synthesis of radiopharmaceuticals. 23Na MRI and sequential MRI/PET protocols for GIST visualisation have been developed and validated in GIST animal models. A concept clinical study combining MITIGATE’s developments is set to start in 2016.

DGP04.05 Response evaluation of tumors after neoadjuvant treatment – views from the nuclear physician/the pathologist A. Buck*1, E. Wardelmann*2 1 Universitätsklinikum Würzburg, Klinik und Poliklinik für Nuklearmedizin, Würzburg, Deutschland, 2Universitätsklinikum Münster, Gerhard-DomagkInstitut für Pathologie, Münster, Deutschland

Pathologists are increasingly faced with the challenge to work up tumor specimens after neoadjuvant treatment. Depending on the tumor response this approach results in diagnostic limitations concerning subtyping, evaluation of tumor necrosis prior to therapy and grading. Positron emission tomography (PET) allows molecular imaging of tumors. Major characteristics of tumor biology such as cellular metabolism, proliferation, hypoxia, angioneogenesis or receptor expression can be visualized and quantified. Since several years, non-invasive response evaluation tools are being introduced to measure effects of neoadjuvant radio-/chemotherapy on changes in tumor metabolism. Promising results have been demonstrated for head and neck tumors, gastrointestinal stromal tumors (GIST) and colorectal cancer. Our presentation will include the introduction of novel developments and trends in PET and hybrid imaging such as PET-CT/PET-MR for response evaluation. We will discuss how these innovative tools enable both nuclear physician and pathologist to report their findings as precisely as possible. A major focus will be the localisation and quantification of vital tumor residues by combining imaging results and pathological work-up and how procedures in pathology are influenced by neoadjuvant treatment regimens.

DGP04.06 Image-guided drug delivery for personalized (nano-) medicine T. Lammers* Universitätsklinik RWTH Aachen, Experimentelle Molekulare Bildgebung, Aachen, Deutschland Nanomedicines are 1–100(0) nm-sized carrier materials designed to improve the biodistribution of systemically administered (chemo-) therapeutics. By delivering drug molecules more efficiently to pathological sites, and by preventing them from accumulating in potentially endangered healthy tissues, nanomedicines are able to beneficially affect the balance between efficacy and toxicity. Prototypic examples of nanomedicines are liposomes, polymers and micelles, and these formulations primarily rely on the Enhanced Permeability and Retention (EPR) effect for efficient target site accumulation. The EPR effect, however, is notoriously known to be highly variable, both in animal models and in patients. To address this high variability in EPR, we are developing probes and protocols for image-guided drug delivery, which can be used to preselect patients, and to individualize and improve tumor-targeted nanomedicine treatments.

HP: Hodentumoren DGP05.02 Recent results of Testicular Tumor Biology L. Looijenga* Erasmus MC, Pathology, Laboratory of Experimental Patho-Oncology, Rotterdam, Niederlande Testicular germ cell tumors comprise a heterogeneous group of neoplasms, with different origin and pathogenesis. The newest WHO classification (2016) recognizes three types, defined as GCNIS-related (Germ Cell Neoplasia In Situ), being the seminomas and nonseminomas, as well as two variants of the GCNIS-none related. The GCNIS-related germ cell tumors are malignant, referred to as Germ Cell Cancer (GCC). They originate from a totipotent embryonic germ cell. GCNIS, as well as all seminoma and embryonal carcinoma cells are characterized by a nuclear presence of the pluripotency marker OCT3/4 (POU5F1), a highly informative diagnostic marker. Seminomas (and GCNIS) are positive for SOX17 and embryonal carcinomas for SOX2. Individuals are at a higher risk for GCC if part of the Y chromosome (GBY) is present in the karyotype. In addition, presence of KITLG (SCF) is detected in the earliest stage of GCNIS forDer Pathologe Suppl 1 · 2016

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Abstracts mation, able to distinguish delayed matured germ cells (in young patients) from pre-GCNIS. All the mentioned parameters are highly informative, although determined based on histological evaluation of gonadal tissue. A new level of risk stratification has been provided based on Genome Wide Association studies (GWAS) indicating that a selection of Single Nucleotide Polymorphisms (SNPs) are associated with GCC. Interestingly, these map to a limited number of highly relevant pathways, including gonadal development (DMRT1), embryonic germ cell proliferation and maintenance (KITLG, SPRY4, TERT,BAK1, etc). Data will be presented that this information can be informative in a clinical context for risk stratification. A unifying model will be presented in which a delicate interaction between the genomic constitution and (gonadal) micro-environment (referred to GENVIRONMENT) is the actual determinant for the risk of an individual to develop a GCC. Functional studies on the role of P53 identified that a selected set of embryonic-specific microRNAs, being the miRs 371-3 and 302/367 clusters are highly expressed in the various types of GCC, including the precursor lesion GCNIS. The only exception is the fully differentiated element teratoma. These miR are informative as molecular serum markers to identify patients with a GCC at the moment of diagnosis, as well as during clinical follow up. The current data based on a quantitative detection protocol will be presented, showing the outperformance of this test compared to the currently used standard biomarkers AFP and hCG.

HP: Uropathologie und Technik DGP06.01 Transcript clusters with prognostic potential for prostate cancer revealed by next-generation sequencing M. Toma*1, M. Muders1, S. Christ-Breulmann2, S.-H. Puppel2, T. Buschmann2, K. Reiche2, 3, 4, M. Specht2, C. Bertram2, M. Friedrich2, S. Binder5, C. Blumert2, J. Hackermüller2, 3, 4, M. Kreuz6, M. Löffler7, S. Fuessel8, M. Fröhner8, M. Wirth8, F. Horn2, 5, G. Baretton1 Universitätsklinikum Carl Gustav Carus, Institut für Pathologie, TU Dresden, Dresden, Deutschland, 2Fraunhofer Institut für Zelltherapie und Immunologie, Diagnostische Abteilung, Leipzig, Deutschland, 3Universität Leipzig, Abteilung Computerwissenschaften, Leipzig, Deutschland, 4 Helmholtz Zentrum für Umweltforschung, Halle/Leipzig, Deutschland, 5 Medizinische Fakultät, Universität Leipzig, Institut für Klinische Immunologie, Leipzig, Deutschland, 6Medizinische Fakultät, Universität Leipzig, Insitut für Medizinische Informatik, Statistik und Epidemiologie, Leipzig, Deutschland, 7Institut für Medizinische Informatik, Statistik und Epidemiologie, Universität Leipzig, Leipzig, Deutschland, 8Klinik und Poliklinik für Urologie, Universitätsklinikum Carl Gustav Carus, TU Dresden, Dresden, Deutschland 1

Background.  Prostate cancer (PCa) is the most common malignant tumour in men. Although first assays for risk assessment are available, the choice of whether or not to use interventional treatment for low-risk disease remains difficult. The Fraunhofer-led RIBOLUTION consortium conducted an unbiased transcriptome-wide expression study in PCa tumour tissues. Methods.  We analysed the transcriptomes of prostate tumour and tumour-free samples using strand-specific, paired-end long-RNA next-generation sequencing (NGS) and microarray studies. Fresh frozen tissues were obtained from patients underwent radical prostatectomies for PCa. The follow-up time was at least seven years. The patient cohort included distinct risk groups according to Gleason score, lymph node metastases, and survival. RNA was isolated from cryosections, quality-controlled for tumour cell contents. 64 samples were subjected to NGS at a depth of 200 million reads per sample. Subsequently, 256 prostate samples were studied using customized microarrays covering transcripts differentially expressed in the sequencing cohort as well as all RNAs annotated in public databases. To retrieve prognostic biomarker signatures, the data obtained

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were analysed using an unsupervised clustering approach utilizing self organizing maps (SOM) in combination with Cox-Proportional-Hazards regression models. Results.  A high numbers of transcripts differentially expressed between the PCa risk groups were identified. We defined also several clusters of transcripts that exhibit strong prognostic potential according to the Kaplan-Meier analysis. A biomarker signature generated from these clusters yielded an area under the ROC curve (AUC) value of 0.80 for specificity and sensitivity. As a comparison, the transcripts used for two commercially available prognostic PCa tests yielded AUC values of 0.67 and 0.66, respectively, in our cohort. To study the pathway correlation of the clusters, we knocked down the androgen receptor, STAT3, and MAP kinase pathways by RNA interference in PCa cell lines. The results demonstrate that the transcript clusters with prognostic potential relate to distinct biological networks. Conclusions.  Our study reveals RNA transcripts clusters from PCa that exhibit strong prognostic potential. By combining these clusters, a biomarker signature was generated that discriminated survival from PCa-specific death with high specificity and sensitivity. This signature might provide a basis for the development of a prognostic test for PCa.

DGP06.02 Quantification of Prostate Cancer Cell-Specific Changes in Heterogeneity During Tumor Progession L. De Vargas Roditi*1, A. Jacobs2, W. Macnair1, P. Wild3, B. Bodenmiller2, M. Claassen1 1 Institute of Molecular Systems Biology, ETH Zürich, Zürich, Schweiz, 2Institut für Molekulare Biologie, Universität Zürich, Zürich, Schweiz, 3Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz

Background.  Sequencing and proteome profiling efforts in the past few years have revealed widespread genetic and proteomic heterogeneity of solid tumors. Prostate cancer is characterized by multiple genomic alterations leading to the complex nature and heterogeneity of the disease. Intra-tumor heterogeneity can impair precise molecular analysis of tumors and interfere with biomarker qualification, treatment personalization as well as drive therapy resistance, highlighting the importance of characterizing tumor heterogeneity. Using single cell mass cytometry, we developed a computational approach that uses the distinct phenotypic profiles of prostate cancer and adjacent non-tumor cells to characterize and quantify the changes in heterogeneity at different stages of prostate cancer. Methods.  Experiments with single-cell resolved readouts, such as mass cytometry, provide dozens of parameters simultaneously at the single-cell level, for thousands of cells. However, the high dimensionality and the biologically induced variability of such data constitute a challenge for data analysis and require development of computational approaches for interpretation of the results. Results.  We analyze tissue sections from surgically removed prostates using mass cytometry with a comprehensive set of 40 antibodies to jointly identify surface markers, enzymes, transcription factors and functional readouts. Cells derived from prostatectomy specimens were analyzed with mass cytometry and the readout was the quantification of dozens of proteins for thousands of single cells. This data occupies a certain volume in the high dimensional space of proteins measured. This volume constitutes a quantitative measure for cell population heterogeneity and can be computationally estimated via a set of hyper-ellipsoids that are fit to cover the minimal spread of the data. Comparison of high-dimensional volume of prostate tissue data revealed that cells from cancer samples exhibit a significantly higher volume than cells from non-tumor samples. Conclusions.  Here, we present for the first time an analysis of prostate tumor cell heterogeneity by mass cytometry coupled to a quantitative assessment of heterogeneity. Our results show that immune cells, stromal cells and heterogeneous tumor cells can be comprehensively described.

This work demonstrates how single cell phenotypic profiles of prostate tumor and non-tumor cells, taken from surgically removed prostates, can be used to quantify the varying heterogeneity during tumor progression.

DGP06.03 Uncovering the proteome signature of metastatic and non-metastatic advanced clear cell renal cell carcinoma in FFPE tissue samples B. Heckelmann1, C. A. Jilg2, M. Föll3, M. L. Biniossek3, W. Schultze-Seemann2, U. Wetterauer2, M. Werner1, 4, 5, O. Schilling3, 4, V. Drendel*1 Institut für Klinische Pathologie, Department für Pathologie, Universitätsklinikum, Freiburg, Deutschland, 2Klinik für Urologie, Universitätsklinikum, Freiburg, Deutschland, 3Institut für Molekulare Medizin und Zellforschung, Albert-Ludwigs-Universität, Freiburg, Deutschland, 4Deutsches Konsortium für translationale Krebsforschung (DKTK), DKFZ, Heidelberg, Deutschland, 5Tumorregister CCCF Freiburg, Universitätsklinikum, Freiburg, Deutschland

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Background.  Outcome for patients with advanced clear cell renal cell carcinoma (ccRCC) is mainly determined by the presence of metastases. Processes leading to metastasis in ccRCC are still not fully understood. This study characterizes the proteome signature of primary ccRCC tumors from patients with clinical metastases compared to those without clinical metastases to identify proteins that may promote or suppress metastasis formation. Methods.  Two cohorts of primary ccRCCs (pT3a category, Fuhrman grading 2–3) were compared by quantitative proteomic analysis. Group A (n = 6) presented with synchronous regional lymph node and distant metastases at the time of nephrectomy while group B (n = 6) did not and remained relapse-free over a median follow up of 6 years. Formalin-fixed paraffin-embedded (FFPE) tissue samples were labeled using stable isotopic dimethylation for quantification and analyzed by liquid chromatography-tandem mass spectrometry. Data were queried for enriched pathways utilizing DAVID Bioinformatics Resources 6.7 and Ingenuity Pathway Analysis. Results.  In total 2190 proteins were quantified. Group A, in comparison to group B, presented increased abundance of proteins associated with cellular growth and proliferation, survival and cellular movement. This enrichment analysis includes, for example, Cofilin, CAP1 and CAPZB which are more abundant in group A. They are main components of cytoskeleton modulation and thus involved in cell motility. Overexpression of EEF1G and valosin containing protein (VCP) was detected which are important components promoting protein synthesis and protein folding. The latter is mainly involved in degradation of misfolded proteins, cell division and preventing apoptosis. Conclusions.  We successfully applied quantitative proteomic analysis of FFPE tissue samples of ccRCCs. Proteins involved in pathways of cell survival, proliferation and cell motility were overexpressed in primary ccRCC tissue in group A giving rise to metastasis formation. With our results we were able to characterize the metastatic potential in primary tumor tissue based on its proteome signature.

DGP06.04 Imaging Mass Spectrometry in Prostate Cancer – Looking beyond Histology K. Schwamborn*1, P. Wild2, R. Caprioli3 Institut für Pathologie, München, Deutschland, 2Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz, 3Department of Biochemistry, Nashville, Vereinigte Staaten von Amerika 1

Background.  Imaging Mass Spectrometry (IMS) is a molecular analytical technology that enables the visualization of the spatial distribution and the relative abundance of tissue specific protein/peptide expression profiles in correlation with histological features. By comparing benign prostatic hyperplasia, prostate cancer, hormone refractory prostate cancer, and prostate cancer metastases tissue microarray (TMA) samples we aimed to identify disease related markers. Methods.  Four different TMAs were subjected to on-tissue tryptic digestion and IMS comprising tissue cores from the following samples: 115 benign prostatic hyperplasia, 535 prostate adenocarcinomas, 57 hormone refractory prostate adenocarcinomas, and 46 metastases. Briefly, sections were mounted onto conductive glass slides and underwent paraffin removal well as antigen retrieval. On-tissue digestion was achieved by spotting trypsin onto the tissue in an array pattern using a Portrait 630 reagent multi-spotter. Following digestion, matrix was spotted directly onto the array of tryptic spots. Samples were analyzed utilizing an UltrafleXtreme MALDI-TOF/TOF mass spectrometer. Additionally, MS/MS measurements of selected peptides were acquired. Data analysis was performed by using the ClinProTools 2.2 and FlexImaging 2.1 software. Results.  On-tissue tryptic digestion of prostate tissue revealed on average hundreds of tryptic peptides in the mass range from m/​z 600–3000. Supervised classification analysis comparing prostate cancer and benign prostatic hyperplasia resulted in a classifier composed of 25 peptides that could predict the class of cancer and benign samples achieving a sensitivity and specificity of 94.8 % and 81.1 %, respectively. Similarly, utilizing a different classifier, 94.5 % of all hormone naïve and 70.4 % of all hormone refractory prostate cancer samples could be classified correctly. In total 73 peptides form 34 different proteins could be identified. For example, fatty acid binding protein, epidermal, glutathione S-transferase P and heat shock protein beta-1 were differentially expressed between prostate cancer and benign hyperplasia. Immunhistochemical validation analyses are ongoing. Conclusions.  These results could provide insight into the discovery of novel biomarkers for prostate cancer.

DGP06.05 Targeting intracellular EGFR trafficking as a new therapeutic approach in prostate cancer P. Hönscheid*1, S. Dutta2, M. Stanton2, A. Schulz1, S. Zeiler1, S. Schellenburg1, G. Baretton1, K. Datta2, M. Muders1 Institut für Pathologie des Universitätsklinikum Carl Gustav Carus, TU Dresden, Dresden, Deutschland, 2Abteilung für Biochemie, University of Nebraska Medical Center, Omaha, Nebraska, USA, Vereinigte Staaten von Amerika

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Background.  Targeting endocytosis pathways in cancer cells might represent an interesting treatment option in oncology. EGFR is frequently overexpressed in cancer including prostate cancer. In our previous studies we have discovered a novel role of Neuropilin-2, a non-tyrosine kinase receptor, in receptor endocytosis. Neuropilin-2 is expressed and is correlated with shorter disease free survival in prostate cancer. In this study we investigate the effect of blocking EGFR endocytosis by targeting Neuropilin-2. Methods.  Two metastatic prostate cancer cell lines (PC3 and DU145) were used in our mechanistic studies. Both cell lines express high levels of Neuropilin-2 and its ligand VEGF-C. Neuropilin-2 expression was depleted by RNAi. EGFR signaling was monitored by immunoblotting against EGFR, ERK and pERK. Trafficking experiments were performed by executing confocal fluorescence microscopy using antibodies against EEA1, EGFR and pEGFR. Cell death and apoptosis was measured by standard methods like FACS and immunoblotting for cleaved PARP. Results.  NRP2 depletion blocked the transport of cell surface EGFR inside the cell. The accumulation of functionally active EGFR in intracellular EEA1 positive endocytic vesicles led to aberrant ERK activation, a downstream target of EGFR. Interestingly, aberrant ERK activation resulted in

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Abstracts increased apoptosis as measured by cleaved PARP and increased overall cell death. Similar results could be observed in pancreatic cancer cell lines. Conclusions.  The inhibition of intracellular EGFR trafficking by targeting Neuropilin-2 triggers cell death in prostate cancer cells. Therefore, these mechanisms are potentially useful in future therapy approaches not only for prostate cancer.

DGP06.06 Quantifying thousands of proteins in prostate tissue specimens T. Guo*1, Y. Zhu1, P. Wild2, R. Aebersold1 Institut für Molekulare Systembiologie, ETH Zürich, Zürich, Schweiz, 2 Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz

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Background.  Current measurements of protein abundance for clinical specimens are dominantly performed by antibody-based methods. These mostly offer semi-quantitative estimation of protein abundance and cover only a fraction of the wide dynamic range of the protein expression. Furthermore, only a limited number of proteins is actually measured in clinical practice due to the high cost for each protein measurement, availability of antibodies, and the insufficient sample amount allowing for parallel protein measurements.Recent advances in proteomic technologies are changing this situation. Methods.  Here, we demonstrate that the abundance of clinically relevant proteins can be precisely determined across hundreds of prostate tissue samples by integration of pressure cycling technology, SWATH-MS, synthetic isotope labeled peptides, and intelligent computer software including an expert system. Results.  The quantity of prostate specific antigen (PSA), for instance, expressed in tumors was precisely determined, along with the abundance of thousands of other proteins in a single, massively parallel measurement which consumed as little as 0.3 mg tissue (wet weight). We next applied the methodology to study protein expression in 105 prostate tissues for disease classification. Conclusions.  Our data show that the methodology offers precise quantitative proteomic data from tumor tissues for making clinical decisions.

Interview zur Historie der DGP: „Jung“ trifft „Alt“ DGP07 Historical talk show: „young“ meets „old“ Moderator.  T. Braunschweig (Aachen) Participants.  Anne-Sophie Meyer (Heidelberg), K. Steinestel (Münster), F. Hofstädter (Regensburg), H. K. Müller-Hermelink (Würzburg/Kiel), H. Nizze (Rostock) Aims.  The 100th meeting of the German Society of Pathology (DGP) prompted the idea of a panel discussion with young and senior society members about differences in the perception of pathology over the past decades until the present. In this talk, the historical aspects against the background of the 100th anniversary of the DGP will be exemplified in a story-based form. Topics.  The following points will be topics of an inter-generation discussion: 55Pathology: doctrine for understanding disease or simple clinical tool? 55„It is a great time to be a young pathologist“ (Thomas M. Grogan, pathologist and founder of Ventana Medical Systems, Tucson, Arizona): Differences and similarities in the development of histological training of residents in pathology in the course of time 55Personal DGP-memories of a former East German pathologist (e. g. DGP-meetings in Koblenz 1989, Münster 2001, Rostock 2004) 55„German speaking“ pathologists abroad

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55From autopsy to next generation sequencing – development of diagnostic techniques in pathology 55Famous women in German pathology: e. g. Martha Schmidtmann 1892–1981. Conclusions. To diagnose, to investigate, and to explain human diseases remain the valued task triad also for modern pathology with its new methods. For pathologists and the more than one hundred years old DGP, the continual integration of molecular and other forward-looking ideas ensures their position in future medicine.

HP: Nierentumoren DGP08.01 Nierentumoren: Was braucht der Urologe vom Pathologen? C. Stief*1, H. Moch*2 LMU München, Klinik für Urologie, München, Deutschland, UniversitätsSpital Zürich, Zürich, Schweiz

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C. Stief: Pathohistologische Information bei Nierentumoren wird in 3 Szenarien benötigt: Nierentumor-Biopsie: Durch die weite Verbreitung von Ultraschall und Schnittbildgebung wie CT und MRT nimmt die Diagnose von kleinen soliden oder gemischt cystisch-soliden Raumforderungen der Niere seit ca 20 Jahren ständig zu. Da der Anteil gutartiger Tumore bei den Befunden unter 3 cm mit über 30 % überproportional hoch ist, sind viele Kliniken dazu übergegangen, hier eine Nierenbiopsie durch Pathologen anzustreben. Intraoperativer Schnellschnitt: Seit ca 15 Jahren ist offensichtlich, daß die Organerhaltung in der Nierentumorchirurgie im Vergleich zur kompletten Resektion der Tumortragenden Niere signifikante Überlebensvorteile erzielt. Eine Schnellschnittbeurteilung des Pathologen bzgl der Tumorfreiheit des Absetzungsrands ist hier entscheidend. Definitive Beurteilung des Nierentumors: Trotz der Verbesserung der Diagnostik werden bei einem Drittel der Erstdiagnosen synchrone Metastasen des Nierenzellkarzinoms festgestellt. Bei einem weiteren Drittel der Betroffenen treten metachrone Metastasen in der Folge auf. Die systemische Therapie ist abhängig von der Pathohistologie des Primärtumors. Der Urologe braucht vom Pathologen eine diff.Klassifikation des Nierenzellkarzinoms in die verschiedenen Tumortypen. H. Moch: Im Januar 2016 ist eine neue WHO-Klassifikation des Nierenzellkarzinoms erschienen. Die erste Konsens-basierte S3-Leitlinie Nierenzellkarzinom von 2015 empfiehlt, dass sich der Befundbericht der Pathologie nach der 2016 WHO-Klassifikation richtet. Das WHO/ISUP-Gradierungs-System gilt für klarzellige und für papilläre Nierenzellkarzinome und ersetzt das Fuhrman-Grading. Zusätzlich sollte der prozentuale Anteil von Tumornekrosen im Befundbericht erwähnt werden. Neue WHO-anerkannte epitheliale Tumortypen sind das tubulozystische Nierenzellkarzinom, Nierenzellkarzinome assoziiert mit einer erworbenen zystischen Nierenerkrankung, das klarzellig-papilläre Nierenzellkarzinom, SDH-defiziente Nierenzellkarzinome und das Hereditäre Leiomyomatose- und Nierenzellkarzinom-Syndrom (HLRCC)-assoziierte Nierenzellkarzinom. Das Karzinoid der Niere wird neu als neuroendokriner Tumor der Niere bezeichnet. Das papilläre Nierenzellkarzinom sollte weiterhin in Typ 1 und 2 eingeteilt werden, obwohl es häufig Mischformen gibt. Papilläre Tumoren mit einem WHO/ISUP Grad 1–2 ohne Kapsel können bis zu einem Durchmesser von 1,5 cm als papilläres Adenom bezeichnet werden.

DGP08.02 MET expression and copy number status in clear-cell renal cell carcinoma: prognostic value and potential predictive marker S. Macher-Göppinger*1, 2, R. Penzel2, V. Endris2, K. Tagscherer3, S. Pahernik4, M. Hohenfellner4, H. Gardner5, P. Schirmacher2, W. Roth1 Institut für Pathologie, Universitätsmedizin, Mainz, Deutschland, Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, 3 Deutsches Krebsforschungszentrum (DKFZ), KKE Molekulare Tumorpathologie, Heidelberg, Deutschland, 4Urologische Klinik, Universitätsklinikum, Heidelberg, Deutschland, 5AstraZeneca, Translationale Medizin, Waltham, Vereinigte Staaten von Amerika 1

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Background.  Antiangiogenic therapy in RCCs has substantially improved patient outcome with advanced clear-cell renal cell carcinoma (RCC), but complete remission is uncommon and many tumors eventually develop resistance. Mechanistic, preclinical, and early clinical data highlight c-Met (hepatocyte growth factor receptor) as a promising target for RCC therapeutic agents. Methods.  We have examined MET expression and frequency of MET gene copy gains in a large, hospital-based series of renal cell carcinomas with long-term follow-up information. Results.  Protein levels and copy number increase of MET showed a positive correlation. Out of 572 clear-cell RCC, only 17 % were MET-negative by immunohistochemistry, whereas 32 % showed high protein levels. MET amplification (defined as >4 copies/nucleus) was seen in 7 % of tumors. This proportion increased up to 17 % in lymphnode positive and 20 % in high-grade carcinomas. Furthermore high MET expression and increase of MET copy numbers was associated with an unfavorable patient outcome. Conclusions.  Our data reveal that clear-cell RCC with MET upregulation show an aggressive biologic behavior and MET gene copy gains are evident in a substantial percentage of patients with high-grade carcinomas and metastasized disease. Besides prognostic significance, diagnostic assessment of MET characteristics may be of predictive value to guide targeted therapy against MET signaling in patients with clear-cell RCC.

DGP08.03 Retrospective analysis identified TFE3 and TFEB translocation renal cell carcinomas but no anaplastic lymphoma kinase (ALK) translocation carcinoma A. Zimpfer*1, W. H. Barthel2, B. Schneider2, C. Geier1, I. Petersen1, H. Zettl3, M. Maruschke4, O. W. Hakenberg5, A. Hartmann6, A. Erbersdobler2 Institut für Pathologie, Universitätsklinikum Jena, Jena, Deutschland, Institut für Pathologie, Universitätsmedizin, Rostock, Deutschland, 3 Klinisches Krebsregister, Universität Rostock, Rostock, Deutschland, 4Helios Hanseklinikum Stralsund, Urologische Klinik, Stralsund, Deutschland, 5 Abteilung Urologie, Universität Rostock, Rostock, Deutschland, 6FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland 1

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Background.  The World Health Organization (WHO) has classified renal cell carcinoma (RCC) associated with Xp11.2 translocation and TFE3 gene fusion (Xp11RCC) as a distinct entity which predominantly affects young patients. However, more cases in older patients have also been identified. As histomorphology overlaps with other RCC entities, this tumour entity seems to be underdiagnosed. In the upcoming WHO classification, RCC with t(6;11) translocation is defined as a new entity and anaplastic lymphoma kinase (ALK) translocation carcinomas introduced as provisional entity. We aimed to identify translocation RCC with TFE3 or TFEB or ALK gene fusion in a large cohort of RCC patients and show possible clinicopathological correlations. Methods.  523 renal tumours were evaluated by tissue microarray, including 322 clear cell (cc) RCC, 110 papillary (p) RCC, 47 chromophobe (cp) RCC, 38 oncocytomas, and 6 RCC, unclassified. Immunohistochemistry for TFE3, Cathepsin K and Alk was performed to identify potential translocation RCCs. Only a distinct moderate to strong nuclear or cytoplasmatic/membranous staining in at least 10 % of tumour cells was regarded as positive. Break apart fluorescence in situ hybridisation (FISH) for TFE3 and ALK translocations was done in 28 TFE3 and 6 Alk positive cases. To define Xp11 and ALK translocations, only two signal diameters distance between split signals were considered as positive. Cut-off levels were 15 % for TFE3 and 20 % for ALK gene translocations. Results.  Strong nuclear TFE3 expression was seen in 24 ccRCC, 2 pRCC, 1 cpRCC and 1 RCC, unclassified. Among these, 16 ccRCC, 1cpRCC and 1 pRCC were TFE3 FISH positive. 1 ccRCC and 1 cpRCC was positive for Cathepsin K, but negative for TFE3. Both cases were reclassified as t(6;11) translocation RCC. 6 cases with Alk expression were FISH-negative. In an additional three-colour break-apart ALK-FISH, gene copy number gains

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Abstracts were found in all 6 cases. Clinicopathological analysis showed high grade morphology and higher pT stage in 17/20 and 12/20 TFE3 and TFEB cases, respectively. Conclusions.  After immunohistochemistry and FISH, in 20 (3.8 %) of RCC cases reclassification was necessary. The majority of these cases were high grade and high stage tumours. No single ALK translocation was found when applying the strict evaluation criteria used in break apart ALK-FISH analysis with a cut-off level of 20 %. ALK gene copy number gains might be the cause for Alk overexpression in some RCC.

HP: Tissue Engineering DGP09.01 „Lost in Translation“ – The Impact of Pathology to Regenerative Medicine and Tissue Engineering C. Brochhausen* REPAIR-Lab, Institut für Pathologie, Universität Regensburg, Regensburg, Deutschland Great tissue damages due to wounding, chronic and degenerative diseases as well as tumour resections lead to irreversible loss of functional human tissue or even to the loss of whole organs. Until today the loss of tissue and organs could only be treated by transplantation or prosthesis with the risk of functional restrictions and consecutive illness. In the light of an aging population degenerative and chronic inflammatory diseases especially of the musculo-skeletal system represent a challenge for patient care. At the interface between medical science, the life and the biomaterial science innovative strategies were developed and will be further optimized to trigger not only the restoration of a micro-anatomical structure but also the physiological function of the lost tissue. Until today the failure of such implanted constructs in up to 20 % of the cases represent a relevant clinical problem. A crucial parameter for the successful use of Tissue Engineering constructs and strategies from Regenerative Medicine is the histo-pathological situation of the target-tissue, because this gives the microenvironment for implanted scaffolds, cell-scaffold constructs and growth factor delivery-devices respectively. Since there is a significant interaction between the implantation site and the implanted constructs, the cellular and humeral arsenal of the implantation site has a crucial impact to the effectiveness of such strategies. The aim of all these strategies is to stimulate the invasion and differentiation of stem cells, vessels of fibroblasts from the edges of the transplantation site beneath the differentiation and maturation of implanted cells. Clinical applications in orthopaedic surgery could clearly demonstrate that pathology could play a crucial role to stratify patients for the different tissue engineering based treatment options. In the interdisciplinary cooperation between the bio-medical, the lifeand the basic science together with pathology a significant impact could be given to the proper translation of Tissue Engineering and Regenerative Medicine from bench to bed site. In that view pathology could play a crucial role to optimize patient care not only in orthopaedic but also in trauma and cancer-surgery to personalize Tissue Engineering strategies in clinical use.

DGP09.02 Bone Tissue Engineering – from cell culture towards large animal models S. Neuss-Stein* Institut für Pathologie und IBMT-ZMG, RWTH Universitätsklinikum, Aachen, Deutschland Large bone defects are a major clinical problem, since in up to 40 % of the patients, autologous bone grafts are not available. Therefore, implants made of natural or synthetic biomaterials are a promising alternative. Based on defect size, shape and volume, as well as on patient associated complications, degradable biopolymers (e. g. poly lactic acid), metals (e. g. titanium), ceramics (e. g. calcium phosphates), or thermoplastic polymers (e. g. polyaryletherketones) are transplanted. Although these biomaterials are already used in the clinics as bone substitutes, they have disadvantages, such as reduced biomechanics and/or failed osseointegration. To overcome such limitations, complex tissue engineered constructs are produced and analyzed for their suitability as bone substitutes. Human mesenchymal stem cells (MSC) are the natural precursor cells of osteoblasts and are shown to be a promising cell source for bone tissue engineering. Besides a suitable scaffold material and MSC, endothelial cells have to be co-cultured and induced to form capillary-like structures to allow for access to the already existing vessels after transplantation. MSC and endothelial cells both support each other in osteogenic differentiation and vessel formation, respectively. Using mechanoreactors, bone formation can be triggered without any differentiation factors in the culture medium. After adjusting all these single elements in cell culture experiments, success of bone formation could be demonstrated via alizarin red staining, electron microscopy, 2-Photon Microscopy, detection of transcripts and proteins related to osteogenic differentiation (e. g. RunX2, alkaline phosphatase, Collagen I, osteocalcin) and prove of functionality in vivo. In several projects, we successfully established tissue engineered bone constructs based on polymers or ceramics seeded with MSC (± endothelial cells). In addition, we performed first large animal studies to prove functionality of our constructs. Here we used Rhoen sheep and transplanted PEKK-based constructs for 12 weeks into calvarial defects either cell-free, or seeded with autologous ovine MSC (undifferentiated or differentiated towards osteoblasts). Related to the cell seeding, no difference in the volume or quality of newly formed bone after 12 weeks was observed. However, we could show that PEKK is conductive for attachment, growth and differentiation of human and ovine MSC.

DGP09.03 Recapitulation of the hematopoietic bone marrow niche using β-tricalcium phosphate-based scaffolds M. S. Ventura Ferreira1, H. Fischer2, T. H. Brümmendorf3, R. Knüchel1, B. L. Ebert4, R. Schneider-Kramann*1 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, Zahnärztliche Werkstoffkunde und Biomaterialforschung, RWTH Aachen, Aachen, Deutschland, 3Abteilung für Hämatologie und Onkologie, RWTH Universität, Aachen, Deutschland, 4Department of Hematology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Vereinigte Staaten von Amerika 1

2

Background.  Bone marrow (BM) niches are often inaccessible for controlled experimentation due to their difficult accessibility, biological complexity and three-dimensional (3D) geometry. Methods.  Here, we report the development and extensive characterization of a BM model composed of cellular and structural components with increased potential for hematopoietic recapitulation at ectopic transplantation sites. Cellular components included mesenchymal stromal cells (MSC) and ckit+ hematopoietic stem and progenitor cells. Structural components included 3D β-tricalcium phosphate (β-TCP) scaffolds comple-

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mented with matrigel or collagen I/III gels for the recreation of the osteogenic/extracellular character of native BM. Results.  In vitro, β-TCP/Matrigel® combinations pre-seeded with MSC robustly maintained proliferation, osteogenic differentiation, matrix remodeling capacities as well as self-renewal and maintenance of c-kit+ hematopoietic stem and progenitor cells. In vivo, scaffolds promoted recruitment of trilineage hematopoietic cells to sites of ectopic transplantation, vascularization and soft tissue formation including osteogenesis. Conclusions.  Our tissue engineered bone marrow system is a powerful tool to explore the regulatory mechanisms of hematopoietic stem and progenitor cells and their microenvironment for a better understanding of hematopoiesis in health and disease.

DGP09.04 Characterization of Biomaterial-Tissue Interactions in their Three-Dimensional Context: An in vivo Study W. Wagner*1, 2, M. Benckendorff3, F. Barsch3, A. Ysasi4, S. J. Mentzer4, C. J. Kirkpatrick3, M. Ackermann2, C. Brochhausen1 1 REPAIR-Lab, Institut für Pathologie, Universität Regensburg, Regensburg, Deutschland, 2Institut für Funktionelle und Klinische Anatomie, Universitätsmedizin, Mainz, Deutschland, 3REPAIR-Lab, Institut für Pathologie, Universitätsmedizin, Mainz, Deutschland, 4Harvard Medical School, Laboratory for Adaptive and Regenerative Biology, Boston, Vereinigte Staaten von Amerika

Background.  Synthetic biomaterials are widely used for the treatment of chronic and acute wounds. They play an increasingly vital role in regenerative medicine. However, the adaptations and interactions of biological tissue at implantation site with biomaterial of complex architecture are difficult to characterize in their three-dimensional (3D) context. Methods.  This morphometric study uses computerized quantification of synchrotron radiation x-ray tomographic microscopic (SRXTM) data to evaluate the interplay of a filamentous synthetic copolymer, mainly based on DL-lactic acid, with porcine host tissue. Complementary analysis and cross-validation were performed by light- and electron microscopy. One SRXTM- reconstruction of the materials prior to implantation, and 30 days after implantation were analyzed. The filaments were segmented in 3D using thresholding and suitable morphological operators. In the post-implantation volume image, biomaterial-induced multinucleated giant cells (BIMGC) have been identified based on their 3D-texture. Results.  SRXTM allows for non-destructive 3D analysis of biomaterial on a sub-micron level. Thereby parameters defining filamentous biomaterial, such as filament-diameter, -volume-density, -porosity, -directionality and -clustering, are now available for quantification without stereological detriments (. Fig. 1). SRXTM provides a sufficient spatial resolution (voxel-edge-length: 325 nm) to visualize and quantify single BIMGCs in their spatial extend and relation to the synthetic filaments (2/4). Cell and collagen fiber directionality are assessable in 3D and can be correlated to filament alignment (. Fig. 1). Conclusions.  In the present study we demonstrate that SRXTM allows qualitative and quantitative 3D investigation of biomaterial-host tissue interactions with sufficient spatial resolution to detect single cells. BIMGCs can be identified in their volumetric morphological extend with high sen-

Fig. 1 I DGP09.04 7 Filamentous synthetic biomaterial. Filamentous synthetic biomaterial pre- (1A) and post- (1B) implantation. 1C: Filament diameters remain unchanged (21.6 ± 5.0 μm vs. 23.3 ± 6.9 μm, respectively). The 3D host tissue directionality (1C) can be color coded in relation to the biomaterial (blue). 2: 3D reconstrucion of implanted biomaterial (white), host tissue (green) an BIMGC (purple). 3: Corresponding structures in conventional Goldner’s trichrome stained histology section. Regarding 3D-morphologic volume, BIMGCs measure mostly below 60,000 μm3 4A/B Der Pathologe Suppl 1 · 2016

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Abstracts sitivity. Cross-validation with histologic tissue samples and electron micrographs correlate well with synchrotron imaging results and provide complementary insights into interactive and adaptive processes.

DGP09.05 Pitfalls in patient derived xenograft models (PDX): lessons from a case report S. Schölch1, P. Hönscheid2, G. Folprecht3, J. Weitz1, G. Baretton2, M. Muders*2 Klinik und Poliklinik für Viszeral- Thorax- und Gefäßchirurgie, Universitätsklinikum Carl Gustav Carus, Dresden, Deutschland, 2Institut für Pathologie, Universitätsklinikum Carl Gustav Carus, Dresden, Deutschland, 3 Medizinische Klinik I, Universitätsklinikum Carl Gustav Carus, Dresden, Deutschland 1

Background.  Patient derived xenograft models are powerful models to execute patient specific in vivo and ex vivo drug screens. One advantage is the preservation of tumor heterogeneity and tumor stroma. To test the possibility of a highly experimental therapy we transplanted tissue of a 21 year old patient with metastasized colorectal cancer into the subcutaneous tissue of 8–12 weeks old nude mice. Methods.  PDX models were done by implanting colorectal cancer tissue in the flank of the mice. Histology was performed by executing HE staining. Immunohistochemistry includes CD3, CD20, CD45 and Ki67 staining. In situ CISH was performed to detect EBV. ALU gene detection was performed by PCR. Results.  Three weeks post transplantation histology resembles the morphology of the parent cancer tissue. In contrast, in some mice a lymphoid neoplasm could be detected. Firstly, we tested the origin of these neoplasms and found an amplification of the human ALU gene. Accordingly, the lymphoid neoplasm is of human origin. In a next step, we could detect a strong expression of CD45 and CD20 but no expression of CD3 which resembles a B-cell lymphoma. To test for the association with the Epstein-Barr Virus we tested for the presence of EBV-encoding RNA by in situ hybridization. We found a strong expression of EBV-RNA. Conclusions.  In conclusion, in immunocompromised nude mice the reactivation of EBV could lead to EBV associated B-cell lymphomas that could be mistaken for tumor derived from the parent cancer tissue. Studies using these systems to elucidate the sensitivity to therapy options or to evaluate the biology of these tumors could be severely hampered by this fact. Therefore, the evaluation of histology is of utmost importance in all PDX models.

DGP09.06 Optimization of biofabricated 3D alginate based hydrogels for studying tumor cell dissemination R. Detsch1, P. Lennert*2, T. Zehnder1, J. Ivanovska2, T. Forgber1, A. Hartmann2, A. Boccaccini1, R. Schneider-Stock2 FAU Erlangen-Nürnberg, Institut für Werkstoffwissenschaften, Erlangen, Deutschland, 2FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland 1

Background.  Over the past decade hydrogels have become an important class of biomaterials since they are able to mimic the extracellular matrix. Alginate and gelatin, which are naturally occurring biopolymers, are extensively used for many biomedical applications. Visual representation of proliferation inside the hydrogels is the key challenge for the development of biofabricated constructs as 3D cell culture models. Previously we have established an in vitro system for cancer research purposes that has the possibility to mimic the structural architecture, composition and biological functions of the extracellular matrix. Still there were limitations regarding the maintenance of the gel scaffold. Thus we aimed to improve the long term stability of the gel without changing its chemical composi-

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tion. Here we used Ba2+ instead of Ca2+ as gelation ions to reduce the degradation driven by ion exchange. Methods. CaCl2 and BaCl2 were used as gelation solutions for the alginate di-aldehyde gelatin crosslinked hydrogel system. The difference induced by the gelation process was examined by nanoindentation measurements to evaluate the changes in the gel stiffness. The biocompatibility of the two gelation ions was investigated by WST-8 cell viability tests and a life/dead staining. We examined the adhesion characteristics of the cells by RT-PCR for different integrin subunits. Phenotypic evaluation was done by Western Bloting for specific mesenchymal and epithelial markers. The formation of cell agglomerates and outgrown cell layers on the surface of the constructs was evaluated by electron microscopy. Results.  We observed that cells from the ADA-GEL matrix possessed better proliferation and optimal cellular networks through the material compared to cells growing in pure alginate. Maintenance of the gel scaffold could be successfully prolongated to 14 days. Compared to pure alginate, ADA-GEL shows degradation during the incubation period, which could also be controlled by choice of the divalent cations for gelation. BaCl2 improves the stiffness and also the stability during the incubation compared to CaCl2. Conclusions.  Therefore, we have further developed and optimized the 3D hydrogel composition to study cellular functions of colon cancer cells dependent on the stiffness of the extracellular matrix. We strongly believe that this system will be applicable as a suitable 3D tumor model to study a variety of aspects of tumor cell dissemination and metastasis.

HP: Prostatakarzinom DGP11.01 Prostatakarzinom: Was braucht der Urologe vom Pathologen? C. Stief* LMU München, Klinik für Urologie, München, Deutschland Pathohistologische Information beim Prostatakarzinom wird in 3 Szenarien benötigt: Prostata-Biopsie, intraoperativer Schnellschnitt und definitive Beurteilung des Prostatapräparats Prostata-Biopsie: Trotz signifikanter Fortschritte der Bildgebung, und insbesondere der multiparametrischen MRT, ist die Prostatabiopsie in 2016 noch zur Diagnosesicherung und und Festlegung des Managements erforderlich. Entscheidend ist hier der Tumoranteil pro Stanzzylinder, das Gleasongrading der einzelnen Stanzzylinder und insbesondere beim Gleason7Summe-Karzinom die Verteilung der Gleason 3 und 4-Anteile. Weiterhin, falls biopsiert oder mitgetroffen, eine mögliche extrakapsuläre Tumorausdehnung und eine Samenblasenbeteiligung. Intraoperativer Schnellschnitt: Aufgrund der außergewöhnlich dünnen Prostatakapsel, der häufigen peripheren Lage der Tumore mit früher Kapselperforation und des Verlaufs der autonomen Nerven unmittelbar auf der Prostatakapsel lateral und dorsal ist der Erhalt dieser wichtigen autonomen Fasern bzw. die Resektion dieser zusammen mit der Prostata oft schwierig zu beurteilen. Hier glauben mache Autoren, daß der intraoperative Schnellschnitt helfen kann. Definitive Beurteilung des Prostatapräparats: Um den Patienten nach der Op bzgl. seiner Prognose und möglicher notwendiger adjuvanter Therapien adäquat beraten zu können weiteren, ist eine genaue pathohistologische Beurteilung vonnöten. Hier sind Tumorvolumen, größter Tumordurchmesser, Gleason-Summe und eine mögliche Kapsel- und Samenblasenbeteiligung wichtig. Weiterhin die Beurteilung des Absetzungsrands, die Lange eines möglichen positiven Absetzungsrands und dessen Ausdehnung. Die Bedeutung genetischer/molekularer Marker ist beim Prostatakarzinom noch nicht etabliert.

DGP11.02 Gleason Grading System, Intraductal Carcinoma of the Prostate, and Beyond: What is the Most Predictive Pathological Factor of Patient Outcome T. Tsuzuki* Japanese Red Cross Nagoya Daini Hospital, Departement of Pathology, Nagoya, Japan Background.  The Gleason grading system, which was developed by Dr. Donald F. Gleason, is one of the most important predictive factors of patient outcome. The end points of the original Gleason grading system were clinical recurrence and cancer specific survival. Because it was relatively simple and reasonably reproducible, it had been widely used among pathologists and urologists. In 2005, the International Society of Urological Pathology introduced a new modified Gleason grading system to adjust to the current practice based on prostate-specific antigen (PSA) screening, and follow up and unify several criteria for each grading. Further modification ensued in 2014 to improve its reproducibility and predict patient outcomes more preciously. Intraductal carcinoma of the prostate (IDC-P) was initially introduced by Kovi. Subsequently, McNeal et al established the criteria for IDC-P with invasive prostate carcinomas and showed that IDC-P was an independent predictive factor of PSA failure. Guo et al proposed another set of criteria for IDC-P without the component of invasive prostate carcinomas. The presence of them predicts high-grade prostate carcinomas in such cases. Recently, this speaker’s group demonstrated that the presence of IDC-P according to the McNeal criteria is the only predictive pathological factor in patients with radical prostatectomy and distant metastasis at presentation. This speaker provides a brief overview of the 2014 modified Gleason grading system, as well as IDC-P, and demonstrate its usefulness and limitations.

7 years. Benign prostatic hyperplasia and normal prostates from radical cystoprostatectomies served as controls. RNA was isolated from cryosections that were quality-controlled for tumour cell contents. 64 samples were subjected to strand-specific RNA next-generation sequencing at a depth of 200 million reads per sample. Biomarker candidates were subsequently validated in 256 prostate samples by customized microarrays that covered all differentially expressed transcripts derived from the sequencing study as well as all RNAs annotated in public databases. Standard in vitro cell cultures of LNCaP cells were used to further characterize the biological function of selected lncRNAs. Results.  We discovered more than 2000 genes that showed a significant differential expression between tumour and control samples (false discovery rate < 0.01). Among them, several novel lncRNAs exhibited high diagnostic specificity and sensitivity (area under the ROC curve (AUC) >0.9) and outperformed established markers like PCA3. These RNAs were also detected in urine samples of patients, allowing a non-invasive measurement. RNA interference of a lncRNA that we name TAPIR (tumor-associated proliferation-inducing RNA) resulted in a complete proliferation arrest in LNCaP cells, suggesting that this lncRNA is essential for cancer cell growth. TAPIR expression was found to be elevated in several other cancers and represents an important previously undiscovered oncogenic RNA. Conclusions.  We found several novel lncRNA biomarkers with a high potential for the development of a precise urine-based and non-invasive test for early PCa diagnosis. The data suggest an oncogenic function at least of one of the discovered lncRNAs (named TAPIR) rendering it a promising therapeutic target for PCa.

HP: Digitale Pathologie DGP12.01 Digital Pathology: from an experimental state into routine practice

DGP11.03 Next generation sequencing discovers a novel proliferation inducing long non coding RNA (named TAPIR) for urine based early prostate cancer diagnosis and targeted therapy M. Muders* , M. Toma , S. Christ-Breulmann , S.-H. Puppel , T. Buschmann2, 3, K. Reiche3, 4, 5, M. Specht2, 3, C. Bertram2, M. Friedrich2, S. Binder2, 6, C. Blumert2, J. Hackermüller2, 4, 5, M. Löffler2, 7, M. Fröhner2, 8, S. Füssel2, 8, M. Wirth2, 8, F. Horn2, 3, G. Baretton1, 2 1, 2

1, 2

2, 3

2, 3

1 Institut für Pathologie, Universitätsklinikum Carl Gustav Carus, Dresden, Deutschland, 2The Fraunhofer Ribolution Consortium, Leipzig, Deutschland, 3 Fraunhofer Institut für Zelltherapie und Immunologie (IZI), Leipzig, Deutschland, 4Abteilung für Computerwissenschaft, Universität Leipzig, Leipzig, Deutschland, 5Helmholtz Zentrum für Umweltforschung, Halle, Deutschland, 6Institut für Klinische Immunologie, Medizinische Fakultät, Universität Leipzig, Leipzig, Deutschland, 7Institut für Medizinische Informatik, Statistik und Epidemiologie (imise), Universität Leipzig, Leipzig, Deutschland, 8Klinik und Poliklinik für Urologie, Universitätsklinikum Carl Gustav Carus, Dresden, Deutschland

Background.  Long non-protein coding RNAs (lncRNAs) are characterized by a tissue- and disease-specific expression pattern and represent promising biomarker candidates. As part of the Fraunhofer RIBOnucleic acid-based diagnostic soLUTIONs consortium we identify and validate novel biomarkers that has been detected by Next Generation Sequencing (NGS). Therefore, we conducted an unbiased transcriptome-wide expression study to characterize novel lncRNAs that have potential to act as biomarkers and molecular targets for prostate cancer. Methods.  All studies have been executed on fresh frozen tissue. Tumor and matched tumor-free human tissue samples were obtained from radical prostatectomies of prostate cancer patients with a follow-up of at least

M. Nap* Nap Pathology consultance, Berg en Terblijt, Niederlande Background.  Only a decade ago digital pathology was not considered able to replace conventional microscopes in routine diagnosis of histoand cytopathologists. This technique was admired for the possibilities it provided for teaching, panel discussions and consultations by experts on complex cases. However, it took until late 2013 before the CAP launched a document with guidelines for the individual pathologist and digital diagnostic activity in routine. In 2005 our first scanner from 3DHistech entered the lab. We made our technical staff familiar with the daily use and began to train as pathologists in viewing and reporting selected cases with a final check of the diagnosis on “the real thing” the conventional slide, by a colleague who did not judge the digital slide. After creating confidence and expertise in a safe way, we upgraded and gradually there were no limitations on what kind of diagnostic case could be reported from a digital slide. However, the use of digital slides for primary diagnosis was left to the preference of the individual pathologist. This resulted in a situation where only 2/4 pathologists signed out digitally. Digital images were used by all pathologists in tumor boards and in comparing the results of IHC stainings. In 2012 we deployed a fully integrated workflow and a closed loop was installed from receipt of a specimen until final sign out. Images were stored for a period of 12 months and than deleted. The use of digital pathology did not result in prolonged turnaround or increased diagnostic revisions. Quantification of IHC labeling of tumor characteristics has profited substantially from the developments in image analysis and this can now be carried out in centralized positions for companion diagnostics at for instance a company like HistoGeneX and others. Remote consultation on complex cases was already done in the very beginning of digital pathology. However, the reverse, routine diagnostic activity delivered in bulk by a remote consultant on a virtual platDer Pathologe Suppl 1 · 2016

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Abstracts form has only just begun. Recently an initiative was launched together with the department of pathology at the Rigshospitalet in Kopenhagen. After obtaining all necessary registrations and permissions we started on a weekly basis with remote reporting. The use of coded texts in combination with detailed diagnostic terminology from SNOMED enabled the work across country and language borders in a safe way. More complex cases are now referred back to the local expert pathology staff.

DGP12.02 Automatic and objective quantification of immune cell infiltrate in the tumor microenvironment using digital pathology G. Litjens1, L. Aprupe1, M. Zauser2, K. Safferling1, T. Sütterlin1, F. Lasitschka3, B. Lahrmann4, N. Grabe*1 Hamamatsu TIGA Center, University of Heidelberg, Heidelberg, Deutschland, 2Deutsches Konsortium für Translationale Krebsforschung und DKFZ, Heidelberg, Deutschland, 3Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, 4Steinbeis Center for Medical Systems Biology, Heidelberg, Deutschland

1

Background.  The objective measurement of immune cell infiltrates in immunotherapy trials is currently hampered through a lack of automation, standardization and various existing methods of counting. This is especially challenging in prostate cancer due to the cancer’s undefined invasive margin and complex morphology. Therefore, comparable data on the role of immune cell infiltration is currently lacking. Various tools and image analysis algorithms have been proposed to provide better, quantitative data, but up till now resulted in varying and unstandardized results. Robust and reproducible image analysis is thus of the utmost importance in immunotherapy and an important field of research. Methods.  Deep Learning offers the chance of obtaining highly robust tissue analysis independent of staining variation. We present AUGURA, a novel Deep Learning based platform for automated, objective and standardized analysis of the tumor micro-environment. We show that the tumor micro-environment of prostate carcinoma can be fully automatically identified and segmented based on AMACR/p63 double stained tissue specimens in combination deep convolutional neural network (DCNN). Through virtual multiplexing of consecutive slides stained for CD3, CD4 and CD8 immune cell counts in differing regions of the tumor and its micro-enviroment could be evaluated. We applied this methodology in slides from 26 patients. Results.  We found that the DCNN in AUGURA was able to classify the key prostate tissue components, such as benign glands, malignant glands and stroma automatically. For each patient, an individual tumor micro-environment of flexible size was automatically generated based on the digital classification of the tissue. Virtual multiplexing then allowed us to propagate the digital annotation of the tumor and it’s micro-environment to CD3+, CD4+ and CD8+ stained tumor slides, yielding a fully automatic spatial analysis of immune cell infiltrates. Counts were in agreement with average of human immune cells counts and outperforming off-the-selfsoftware. Conclusions.  DCNN can overcome staining variations up till now hindering robust and high-throughput biomarker evaluation. The results show an important leap in automation of measurement of the tumor microenvironment and give a perspective on how to overcome some of the challenges pathology as a discipline is facing in the age of immunotherapy.

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DGP12.03 The key role of workflow control for the implementation of digital pathology G. Haroske*, M. Mörz Institut für Pathologie des KH Dresden-Friedrichstadt, Dresden, Deutschland Background.  Although the scanning techology for microscopic slides has been known for more than 15 years, its practical use in daily routine is still on the very beginning. Fast and reliable scanners enabled their increasing use in teaching, but not yet in consultation and primary diagnostics. So far the scanning is not handled as a process in the pathology laboratory by most of the pathology systems,leading to an interrupted workflow with delays and additional expenses. Methods.  Performance statistics of routine pathology were evaluated in different phases of digital workflow control over more than 10 years in a medium-sized institute of pathology. Three phases were defined: 1. Uncontrolled workflow with digital dictation, digital macrophotography and a “classic” pathology software system 2. As in #1, but added by digital microphotograpy at few pathology workstations 3. Digital workflow control including digital dictation and digital photography. In a pilot study at the end of the evaluation period the additional benefits of slide scanning were estimated. Results. In the period between 2006 and 2015 a decrease of turnaround-time of roughly 40 % was seen. Alone the effects of a (sub)total digital workflow control contributed about half of that effect. The implementation of slide-scanning did not add further acceleration so far, but enabled some additional functionality for improving quantitative reporting. This was achieved without an explicit committment of the pathology software to standards in workflow control and with still leaving a few laboratory processes out of the control. Milestones and key elements of workflow management are reported in detail. Conclusions.  Without a close linkage with digital workflow control the slide scanning will not be successfully used in routine settings of pathology. All processes both in the laboratory and in the diagnostics have to be checked (and changed, if necessary) for being fit in a streamlined pathology workflow. The implementation of scanners into the routine diagnostics will enforce those essential development leading to increased productivity and quality.

DGP12.04 Proliferative tumor heterogeneity across the spectrum of thymic and pulmonary neuroendocrine tumors as assessed by digital pathology B. W. Grießmann*1, C.-A. Weis1, A. Warth2, H. Bohnenberger3, P. Ströbel3, A. Marx1 Institut für Pathologie, Universitätsmedizin, Mannheim, Deutschland, Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, 3 Institut für Pathologie, Univeristätsmedizin, Göttingen, Deutschland

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Background.  Despite differences on the genetic level, thymic and pulmonary neuroendocrine tumors (TNETs, PNETs) are currently classified according to identical histological criteria as typical (TC) and atypical carcinoids (AC), large cell neuroendocrine carcinomas (LCNEC) and small cell carcinomas (SCC). So far, the site of origin (thymus vs. lung) depends on clinical staging. Therefore, we aimed to compare TNETs and PNETs regarding their mitotic and proliferative activity and their degree of heterogeneity (i. e. frequency and distribution) by digital image processing. Methods.  In cooperation with the NCT tissue bank (Heidelberg) and the University Medical Center Göttingen we studied 143 PNETs and 25 TNETs. By immunohistochemistry, expression of phospho-Histone H3 (H3) and Ki7 served as mitotic and proliferation markers, respectively. The analysis was performed using a custom-written and implemented algorithm in MATLAB (Mathworks, Natick USA.) on three representative

Fig. 1 I DGP12.04 7 a), b) Ki67-Indexes for TNET and PNET c), d) Different distributions with equal histograms regions on each digitalized slide, creating gridded and continuous heat maps of the proliferative and mitotic activity. Results.  Reaching a Ki67-Index of 10 % or more allows a reliable distinction between the high-grade NET (LCNEC and SCC) from the poorly proliferating TC and AC with a sensitivity of 93.5 % and a specificity of 98.0 % for TNET and a sensitivity of 85.2 % and a specificity of 87.2 % for PNET. Further examination will reveal how these entities differ in the heterogeneity of distribution, aiming to define, detect and objectify proliferative and mitotic ‘hot spots’. Conclusions.  Our findings show that discrimination between TC and AC on the one hand and LCNEC and SCC on the other hand is possible for both TNET and PNET regarding the highly significant difference in the quantity of proliferation. However, this analysis does not allow inferring the distribution of proliferative hotspots, which demonstrates the need of spatial distribution analysis (. Fig. 1). Since TNETs and PNETs overlap in proliferative activity, the degree of heterogeneity of mitotic and proliferative distribution might help to distinguish TNETs from PNETs. Such new data will be presented.

HP: Ärztinnen: Zukunftsperspektive für die Pathologie – Dialog DGP13 Ärztinnen: Zukunftsperspektive für die Pathologie A. Bühren* Praxis für Psychosomatik, Murnau am Staffelsee, Deutschland 1898 stimmte der XXVI. Deutsche Aerztetag trotz vielfältiger Befürchtungen zu, dass auch Frauen Medizin studieren dürfen, unter der Annahme, dass davon nur wenige Gebrauch machen würden. Heute konkurrieren die Fachgebiete um die ca. 65 % weiblichen und nur noch ca. 35 % männlichen Berufseinsteiger. Die aktuelle sogenannte Generation Y = Why hat unabhängig vom Geschlecht hohe Erwartungen und Ansprüche an die Vereinbarkeit ihrer Berufstätigkeit mit ihrer Freizeitgestaltung und ihren familiären Auf-

gaben, an geregelte Arbeitszeiten und Bürokratieabbau, und an Struktur und Qualität ihrer Weiterbildung. Die Attraktivität des Fachgebietes Pathologie kann gestärkt werden, z. B. durch: 55Entlastung von Bürokratie zugunsten Forschung, Fortbildung, pünktlichem Dienstschluss 55Arbeitszeitkompatible, arbeitsplatznahe, qualitativ hochwertige Kinderbetreuung 55Gesetzeskonforme Gestaltung möglichst wunschgerechter Arbeitsplätze für Schwangere und Stillende 55Haushaltsnahe Dienstleistungs- und Serviceangebote 55Schnuppertage für Studierende bei Pathologie-Kongressen 55Ausbau Mentoring mit Rollenvorbildern 55Transparentere und gleichberechtigtere Karrierewege und Berufungen Niedergelassene: Unterstützung für Beteiligung am Notärztlichen-/Bereitschaftsdienst. Ärztinnen haben neue Fragestellungen in der Forschung, z. B. Gendermedizin. Sie arbeiten effektiv und haben ausgeprägte kommunikative Kompetenzen: Interdisziplinär, interkollegial, in Führungspositionen. Die Erwartungen der jungen Ärzte- und Ärztinnengeneration entsprechen einer gesamt gesellschaftlichen Entwicklung. Die Vorarbeit für das nötige Umdenken innerhalb der traditionell konservativen, hierarchisch ausgerichteten Medizin haben die Ärztinnen geleistet bzw. leisten müssen. Eine Umfrage des Hartmannbundes unter Medizinstudentinnen und jungen Ärztinnen zeigt 2014 ernüchternd: Rund 50 % streben eine Position als Oberärztin oder Chefärztin an, gleichzeitig sind zwei Drittel davon überzeugt, dass sie nicht die gleichen Chancen auf eine Karriere haben wie ihre männlichen Kollegen. Berufungskommissionen sind bis heute ganz überwiegend männlich besetzt und die Verfahren verlaufen intransparent. Die erste Ordinaria für Frauenheilkunde und die erste Lehrstuhlinhaberin für Viszeralchirurgie wurden erst 2001 berufen. Zuvor war die erste C4- Professorin im Fach Neuropathologie 1990 berufen worden: Marika Kiessling (1951–2012). Als erste Ordinaria für Pathologie wurde 2003 Professor Dr. med. Ruth Knüchel-Clarke an die RWTH Aachen berufen.

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Abstracts Aktuelle Habilitationen I DGP14.01 Prognostic and predictive biomarkers for metastatic colorectal cancer J. Neumann* Ludwig-Maximilians-Universität München, Pathologisches Institut, München, Deutschland Background.  In colorectal cancer (CRC) distant metastases determine significantly overall survival and prognosis. Biomarkers that correlate with the occurrence of distant metastases are therefore of great clinical importance for risk stratification and treatment decisions. Methods.  Potential biomarkers were analysed in various collections of CRC patients with different patterns of distant spread (liver, peritoneum and CNS). Furthermore, the biomarkers were analysed in matched pairs of primary tumours and metastases. Results.  We could show that CRC with microsatellite instability (MSI), detected by loss of hMLH1 expression, exhibit a very low risk of distant metastases. In contrast microsatellite stable (MSS) CRC (positive hMLH1 expression), activation of the Wnt/β-catenin signaling pathway and a strong expression of the cancer stem cell marker CD133 showed a very high risk for liver metastases. From these observations, a twostep algorithm based on three immunohistochemical markers could be established, allowing to identify CRC with either a very high or a very low risk of distant spread. In further studies increased expression of target genes of the Wnt/β-catenin signaling pathway, such as the cancer stem cell marker SOX2 and the micro-RNA miR34, correlated with increased risk for distant spread. In addition, we could demonstrate that deregulation of the Wnt/β-catenin signaling pathway and the expression of cancer stem cell markers such as CD133 and CD44 were associated with liver metastasis but not with peritoneal carcinomatosis or hematogenous metastasis to the CNS. 60 % of CRC patients showed mutations in at least one or more genes of the EGFR signaling pathway. KRAS and BRAF mutations were mutually exclusive, whereas mutations in the PIK3CA gene showed large overlap with other alterations in the EGFR-signaling pathway. Comparative analysis of primary tumors and matched metastases revealed a 100 % concordance for KRAS, BRAF, PIK3CA- (exon 20) and AKT mutations, whereas alterations in the PIK3CA gene (exon 9) and immunohistochemistry for EGFR and PTEN showed high rates of discordant results. Conclusions.  In summary, we able to demonstrate that by selection of appropriate biomarkers CRCs can be subtyped with respect to their risk for distant spread. Furthermore, these biomarkers correlate with different patterns of distant metastases in CRC. Thus, from these results it can be concluded that for the different patterns of distant spread in CRC different molecular mechanisms have to play a role.

DGP14.02 Epigenetics and genetics along the serrated colorectal carcinogenesis point backwards to phenotypic changes useful for daily diagnostic in colonoscopic screening programs T. T. Rau*1, 2 Pathologisches Institut, Friedrich-Alexander-Universität ErlangenNürnberg, Erlangen, Deutschland, 2Pathologisches Institut, Universität Bern, Bern, Schweiz

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Background.  The serrated pathway to colorectal cancer accounts for approximately 15 % of malignancies of the colon. It is associated with the oc-

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currence of interval carcinomas during surveillance programs and a high level of sporadic microsatellite instability. After terminological issues have been solved regarding the distinction between the entities hyperplastic polyps, traditional serrated and classical adenoma as well as sessile serrated adenoma with and without dysplasia, the backbone for genetic analysis has been formed. Methods.  In several study-groups of different compositions, we were able to standardize lesions according to the entities of consensus meetings and recently according to the new WHO classification. Strong efforts were undertaken to increase herein the number of the exceedingly rare number of SSA-D, which as “caught in the act” lesions rapidly progress to cancer. Taken the low frequency of truly dysplastic serrated lesions into account the investigated collectives were distilled out of n > 10,000 polyps and investigated for epigentetic events like MLH1, p16 methylation as well as KRAS/BRAF mutations and quantitative morphological analysis including digital pathology approaches. Results.  The genetics and epigenetic events especially of micro-dissected SSA-D argue for the WHO proposed progression model along the serrated route to colorectal cancer. From a clinical perspective the delay of more than 12 years for an SSA to develop dysplasia is remarkable. The mutual exclusive BRAF and KRAS mutations argue against the former concept of a mixed polyp. The gain of MLH1 hypermethylation in the SSA-D areas is one of the best molecular markers to reflect the rapid progression and is accompanied by certain morphological details already visible in H&E sections. Furthermore, digital pathology approaches allow a quantification of the serration patterns. Conclusions.  The current concept of a progression from certain hyperplastic polyps to SSA, the gaining of dysplasia in SSA-D to the complete development of sporadic MSI colorectal cancer can be underlined with the found molecular events. Redirecting the genotype to a phenotype including quantification options and digital pathology might lead to useful automatable image analysis for the time consuming screening of colorectal polyps in daily routine.

DGP14.03 Molecular characterization of pancreatic acinar neoplasms in comparison with other exocrine and neuroendocrine pancreatic neoplasms F. Bergmann* Pathologisches Institut, Universität Heidelberg, Heidelberg, Deutschland Background.  Pancreatic acinar cell carcinomas (PACs) are biologically aggressive neoplasms whose treatment options are very limited. The molecular mechanisms of the initiation and progression of PACs are largely not understood, and precursor lesions have not been identified. The aim of this study was a genetic and molecular characterization of PACs to define similarities and differences between PACs and other exocrine pancreatic tumors, especially pancreatic ductal adenocarcinomas (PDACs), as well as pancreatic neuroendocrine neoplasms (PNENs). A focus of the analyses was to identify putative therapeutic targets. Furthermore, we aimed to characterize acinar cell cystadenomas by means of molecular analyses to clarify the controversially discussed nature (neoplastic vs. reactive) of these benign acinar lesions. Methods.  PACs, PDACs (including variants), PNENs, and acinar cell cystadenomas were investigated. Technically, among others, comparative genomic hybridization, expression, mutational, and epigenetic analyses, as well as functional in vitro assays were used. Results.  We show that PACs display a microsatellite stable, chromosomal unstable genotype, characterized by recurrent chromosomal imbalances that clearly discriminate them from PDACs and PNENs. Based on CGH findings, several candidate genes could be identified. Thus, for example, dysregulation of DCC seems to represent an early event in PACs. On the other hand, genes that are frequently mutated in PDACs were shown to play no significant role in PACs. Distinct phenotypes between PACs,

PDACs and PNENs were also detected on the epigenetic level. Several therapeutic targets could be identified in PACs and other pancreatic neoplasms, including EGFR, L1CAM, and HSP90. Moreover, L1CAM was shown to play a significant role in the tumorigenesis of PDACs. Functional analyses in PNENs revealed promising anti-tumorigenic effects using EGFR- and HSP90-inhibitors, affecting the cell cycle and, in case of HSP90, regulating several other oncogenes. Finally, acinar cell cystadenomas were shown to represent non-clonal lesions, suggesting that they do not represent neoplasms. Conclusions.  Our investigations provide new insights in the molecular mechanisms underlying the initiation and progression of PACs, opposing the more frequent ductal carcinogenesis. Beyond distinctive genotypes, common therapeutic targets structures do occur in PACs, PDACs and PNENs that should be further evaluated in vivo. Precursor lesions of PACs remain to be identified.

DGP14.04 Tumour spread and epigenetically regulated genes in bladder cancer N. Gaisa* Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland Background.  The work describes bladder tumourigenesis in two ways: first, a clinico-histological and molecular characterization of the tumour spread and, second, expamples of epigenetically regulated genes in tumour progression. Methods.  Cumulative discussion of the results of six independent scientific publications. Results.  “Field cancerization” and “tumour cell seeding/migration” are the two main hypotheses used for explaining syn- and metachronous tumours in the urinary bladder/tract. By detailed histological mapping of completely embedded cystectomy specimens we found a single tumour focus in nearly 2/3 of the bladders accompanied by surrounding pre-invasive Carcinoma in situ in 85 % (of these cases). We substantiated our findings by two studies analyzing TP53-mutations in various tumours sites. Identical TP53-mutations argued for a clonal relationship of the tumour foci. The second part discusses the mechanism of gene silencing by epigenetic DNA-methylation. We analyzed the DNA-methylation of three genes (ITIH5, ST6GAL1 and OASIS/CREB3L1), which had been found downregulated in bladder cancer by RNA-expression analysis. Most importantly, epigenetically silenced ITIH5 was associated with early relaps in pT1 HG tumours and showed functionally an enhanced invasive-metastatic phenotype in tumour cells. Likewise the epigenetic inactivation of OASIS/CREB3L resulted in invasive-metastatic properties, suggesting an initially tumour suppressive role of CREB3L1. Additionally, we identified HTRA3 as a potential downstream target of CREB3L1. Conclusions.  Explaining the occurrence of multiple tumours in the urinary bladder our results rather support the intraepithelial migration theory than a cancerogenic field effect. Furthermore, we showed that DNA-methylation is an additional mechanism of inactivating putative tumour suppressive genes during bladder tumour progression.

Aktuelle Habilitationen II DGP14.05 Metabolome analysis of solid tumors: contributions to bioinformatics and translational tumor research J. Budczies* Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland Background. The metabolome is defined as the complete set of small-molecule chemicals in a biological sample. Metabolomics is the science of the analysis of these molecules. Methods.  Gas chromatography combined with time-of-flight mass spectrometry (GC-TOF-MS) was used to investigate frozen tissue samples of ovarian cancer, colon cancer and breast cancer. Bioinformatic methods were developed to integrate metabolomics data with other -omics level data and to interpret metabolic changes in context of the metabolic network of enzymatic reactions. Results.  It was shown that metabolic profiles are different in cancer and non-caner tissues and are capable to separate between ovarian cancer and borderline tumors of the ovary, colon cancer and normal colon mucosa as well as breast cancer and normal breast tissues. The method PROFILE clustering was developed to exploit biochemical pathway knowledge and to order metabolites with respect to the position in the network of enzymatic reactions. Further, METAtarget was developed to analyze metabolic changes of product-substrate pairs as surrogate for the activity of enzymatic reactions. In an analysis of the breast cancer metabolome, it was shown that the metabolic profiles strongly correlated hormone receptor (HR) status, but not with HER2 status. Beta-alanine accumulation and down-regulation the beta-alanine catalyzing enzyme 4-aminobutyrate aminotransferase (ABAT) were shown to positively correlate with the aggressiveness of breast cancer. Further, the ratio of glutamate to glutamine (GGR) was introduced as new biomarker for breast cancer. Based on the GGR, 88 % of HR- tumors and 56 % of HR+ tumors were glutamate-enriched compared to normal breast tissues. Therefore, evaluation of the recently developed new specific glutaminase inhibitors appears to be promising in HR- tumors and in selected HR+ tumors. Conclusions.  The pioneering work of GC-TOF-MS analysis of large tissue cohorts has shown that metabolomics is a stable and reproducible method for biomarker research complementary to genetics, transcriptomics, proteomics and other methods of systems biology.

DGP14.06 Predictive tissue-based and molecular patient stratification in hematooncology M. Andrulis*1, 2 Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, Institut für Pathologie, Ulm, Deutschland

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Background.  The treatment options of haematological neoplasia have been dramatically broadened by new targeted cancer therapies in the last decade. Success of targeted therapies critically depends on efficient biomarker-based patient stratification. The aim of the present work was to establish and validate new tissue-based and molecular biomarkers for hematological neoplasia. Methods.  Histology, Immunohistochemistry using mutation-specific, phosphorylation-specific and glycosylation-specific primary antibodies, Tissue-Micro-Arrays, sequencing, cell culture. Results.  The immunohistochemistry using mutation-specific IDH1 R132 and BRAF V600E antibodies proved to be sensitive and specific method detection of mutation at protein level. BRAF V600E immunohistochemistry was confirmed to be reliable for diagnosis of hairy cell leukemia and for Der Pathologe Suppl 1 · 2016

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Abstracts detection of this mutation in multiple myeloma (MM). The identification of BRAF V600E mutated MM allowed to stratify the patients for therapy with BRAF inhibitors and to demonstrate the efficacy of this treatment in MM. Analysis of heat shock proteins (HSP90 and HSP70) and their interactions with major oncogenic signaling pathways paved the way for patient stratification strategy for clinical use HSP-inhibition in MM. Investigation of aberrant MUC1 glycosylation revealed groups of MM patients that are likely to benefit from MUC1-targeting antibodies. Conclusions.  Taken together, our findings stress the significance of tissue-based and molecular predictive biomarkers for patient stratification for targeted therapies.

DGP14.07 New prognostic and predictive markers of breast carcinoma D. Wachter* Pathologisches Institut, Erlangen, Deutschland Background.  Breast carcinoma represents a heterogenic and complex disease. Recently, 4 molecular subtypes with different clinical implications were defined. Because molecular analyses are too expensive at the moment, these subtypes are determined immunohistochemically in routine practice with the Ki-67 index playing a significant role. Methods.  The prognostic and predictive value of immunohistochemically determined molecular subtypes using a Ki-67 cut-off of >13 % was examined. Additionally prognostic value of the Ki-67 index in the primary tumor of metastatic breast carcinoma was examined. Furthermore other predictive markers were identified and heterogeneity of the Luminal B subgroup was investigated. Results.  Using a Ki-67 cut-off of >13 %, 2.9 % of Luminal subtype cases with low proliferative index compared to 8 % of cases with high proliferation achieved complete pathological response (pCR). In contrast 14.3 % of low-proliferative triple-negative Carcinomas and 49.2 % of high-proliferative triple-negative carcinomas showed pCR. Using a Ki-67 cut-off of 36–40 % for Luminal Type Carcinomas resulted in 3 % pCR in cases with low proliferation and 18.9 % pCR in cases with high proliferation. Additionally we could show that the molecular subtypes reveal different prognosis and that patientis with triple-negative or HER2 enriched carcinomas with pCR have better prognosis than patients without pCR. The Ki-67 index of the primary tumor has prognostic significance even in the metastatic setting especially in interaction with the expression of the progesterone receptor. CK5 and CK18 expression of the tumor cells lowers the probability of pCR. 20 % of Luminal B carcinomas show neuroendocrine differentiation. Conclusions.  The Ki-67 index of breast carcinomas has prognostic and predictive value even in the metastasic setting. The molecular subtypes should be divided further to achieve more personalized therapies with better prognosis.

DGP14.08 Degenerative DNA changes in Osteoarthritis S. Söder* FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland Background.  Osteoarthritis (OA) is a common disease affecting an increasingly ageing society. In contrast to secondary forms of OA caused by excessive wear and tear of the joint or by other illnesses, the etiology of the more frequent primary forms remains rather enigmatic. So far several factors were identified which are involved in the development of OA. Among these, catabolic cytokines, especially TGF beta and TNF alpha, seem to be essential, as they can induce matrix degradation by activating matrix metalloproteinases (MMPs). In this study we aimed to identify the initial events of OA and to delineate further factors involved in its pathogenesis.

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Methods.  Human cartilage and isolated human chondrocytes derived from normal and osteoarthritic tissue were compared on mRNA level with cDNA-arrays and real time PCR and on protein level with immunohistochemistry, Western blots, ELISA and FACS analysis. DNA alterations were determined by the Comet-assay and southern blotting. Results.  The down regulation of the anabolic cytokine OP1/BMP7 by antisense nucleotides reduces expression of proteoglycans in chondrocytes. Their depletion in cartilage matrix results in a reduced Matrix integrity. This suggests an involvement of OP1 in cartilage homeostasis. We found a low expression of IL1 in both OA and normal cartilage, consistent with a role of catabolic cytokines in the homeostasis of cartilage. In OA there was a significantly stronger activation of the IL-1 signal transduction pathways, making OA cartilage more susceptible to damage induced by exogenous IL-1 from synovial fluid or other sources. MMP-9 expression was detected in cartilage but at too low a level to be of importance for OA. A clear difference between chondrocytes derived from normal and OA-tissue was found in respect of DNA-damage. The induction of a level of DNA-damage in normal chondrocytes, comparable to that in OA derived chondrocytes, resulted in a similar phenotype with discoordinated gene expression showing typical properties of senescence. Conclusions.  The pathogenesis of primary OA is complex involving different cytokines and MMPs from the cartilage and is influenced by the surrounding tissues. DNA damage can induce a senescent phenotype in chondrocytes and can be an explanation for at least some alterations found in primary OA and might be even one of the initial events in its pathogenesis of OA.

Ungarisch-Deutscher Workshop – Neue Technologien in der Pathologie DGP-INT01.01 Old pathology with new faces – diagnostic and research possibilities in the modern pathology C. Toth* Universitätsklinikum Heidelberg, Institut für Pathologie, Heidelberg, Deutschland Recent developments in human (molecular) pathology are reshaping this discipline and thus our approach to therapy and diagnosis. DNA and RNA based testing methods are becoming crucial diagnostic tools not only in research activities but also in a wide variety of diagnostic processes. Different from the morphology-based traditional diagnostic histopathology, molecular pathology is based on standardized methods and molecular evidences. This presentation elaborates the most recent concepts and techniques of molecular pathology and explains how molecular pathology can assist cancer diagnosis, treatment, and prognostication of hematologic and solid neoplastic diseases. Some of the common techniques used in molecular pathology (i. e. Next Generation Sequencing – NGS), followed by a brief discussion of technologies with great future potentials in molecular diagnostics (DNA chip technology and proteomics) will be introduced. Next-generation sequencing is a massively parallel, high-throughput DNA sequencing technique, which will change our approach to tumor molecular profiling including diagnostic tests and tumor biology research. Next Generation Sequencing allows testing simultaneously a large number of clinically targetable hotspots, allowing the selection of the appropriate treatment for each patient. NGS technologies reduced sequencing cost by orders of magnitude and significantly increased the throughput, making whole-genome sequencing a possible leading to better understanding of genetic changes in carcinogenesis However, the benefits offered by implementation of Next Generation Sequencing come with a number of challenges which must be adequately

met before they can be transformed from research tools to routine molecular pathology diagnostics (i. e. extensive validation for diagnostic purposes).

DGP-INT01.02 Differential expression of miRNAs in hepatic cirrhosis and focal nodular hyperplasia G. Lendvai, E. Konstek, T. Szekerczses, G. Lotz, Z. Schaff, A. Kiss* 2. Abteilung für Pathologie, Semmelweis Universität, Budapest, Ungarn Introduction.  Hepatic cirrhosis develops as a response to chronic injury and the resulting nodular microscopic structure with fibrotic tissue produced in excess seems to surround the remaining liver parenchyma. Nevertheless, focal nodular hyperplasia (FNH), a benign proliferating reaction of the liver to a localized vascular malformation, also shows a nodular microscopic appearance that is similar to cirrhosis. Our aim was to compare the expression of selected miRNAs in these two liver diseases. Methods.  Relative expression levels of miR-17-5p, miR-18a, miR-21, miR34a, miR-122, miR-140, miR-195, miR-210, miR-214, miR-221, miR-222, miR-223, miR-224 and miR-328 were determined in 67 RNA samples isolated from 30 cirrhotic and 22 FNH cases, altogether with 15 normal formalin-fixed paraffin-embedded liver tissues using TaqMan MicroRNA Assays. The cirrhosis samples were obtained upon orthotopic liver transplantation from patients suffering from chronic hepatitis C (HCV) or alcoholic liver disease (ALD). Results.  Increased miR-21, miR-34a, miR-224 and decreased miR-17-5p and miR-221 levels were found in cirrhosis (P < 0.01), whereas elevated miR34a and miR-224, and reduced miR-17-5p, miR-18a, miR-210, miR-222 and miR-223 expression were observed in FNH (P < 0.03) in comparison to normal liver. In respect to cirrhosis etiology, miR-224 was elevated and miR-17– 5p and miR-221 were reduced in chronic HCV and ALD samples (P < 0.01) as compared with normal liver. Furthermore, the levels of miR-18a, miR-21, miR-195, miR-210 and miR-222 were decreased in FNH (P < 0.02) in comparison to cirrhosis and, in particular, to chronic HCV samples. Conclusion.  The increased miR-34a and miR-224 and the decreased miR17–5p levels may reflect molecular events that are common in cirrhosis (with chronic viral hepatitis background) and FNH. In addition, downregulation of miRNAs seem to be more frequent in FNH.

DGP-INT01.03 Mid-infrared imaging as a promising tool to characterize cell lines E. Kontsek*1, S. Gergely2, A. Salgó2, Z. Schaff1, A. Kiss1 Semmelweis University, 2nd Department of Pathology, Budapest, Ungarn, Budapest University of Technology and Economics, Department of Applied Biotechnology and Food Science, Budapest, Ungarn

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Introduction.  The goal of our project is to develop a spectroscopy based diagnostic tool, revolutionizing medical diagnostics, especially cancer identification. Life science associated mid- and near-infrared based microscopic techniques are developing exponentially, especially in the past decade. This is a potential non-destructive approach to investigate malignancies. Aims.  Our goal was to observe detectable differences by infrared imaging between tumorous cell lines, since cell culture models are the first step to study complex biological systems, finally the pathological alterations of the human. Material and methods.  A-375 melanoma cell line, HT-29 colon tumour cell culture and MDA-MB-231 breast cancer cell line were selected. The effect of paraformaldehyde and ethanol fixation was investigated by transflectance mid-infrared imaging under dye-fee condition. Further, selection of spectra was carried out by mean transmittance optimization and principal component analysis. Results.  Fixation methods by each cell lines could be identified and separated by mathematical handling of the spectra. Further, using the same fixation method the infrared imaging features and their analysis enabled

us to differentiate the various tumorous cell lines from each other. The best results were found in the fingerprint region (1800–750 cm–1). Conclusions.  Mid-Infrared imaging could be used to differentiate cell lines and might be a promising tool to image tumors in vivo in animal models. This is a key step to the final goal that this dye-free technique could be used during intraoperative surgical procedures. Altogether, our results project the feasibility of infrared imaging to identify different tissues under in vivo conditions. Therefore, the technique might be applied to judge the resection margins of malignancies. Acknowledgement.  This work was supported by OTKA grants K101435 and K108548 from the National Scientific Research Foundation.

DGP-INT01.05 Cervical cancer cells downregulate tissue pathway inhibitor2 (TFPI2) in tumor-associated fibroblasts A. Fullar1, G. Lendvai2, K. Rada1, K. Karászi1, E. Regös1, A. Kiss2, G. Sobel3, K. Baghy1, I. Kovalszky*1 Semmelweis University, 1st Department of Pathology and Experimental Cancer Research, Budapest, Ungarn, 2Semmelweis University, 2nd Department of Pathology, Budapest, Ungarn, 3Semmelweis University, 2nd Department of Obstetrics and Gynecology, Budapest, Ungarn 1

Background.  TFPI2 a Kunitz type serine protease inhibitor was described in 1994. The protein is abundantly expressed in full term placenta and several other tissues such as kidney, liver, and pancreas. The protein resides in the extracellular matrix and binds heparin/HS with high affinity. It cleaves plasmin, trypsin, chymotrypsin, and components of extrinsic clotting cascade. In the last ten years more and more evidence were gathered, indicating the role of TFPI2 in the inhibition of tumor invasion. These data resulted in the conclusion that TFPI2 acts as a tumor suppressor molecule. In our studies comparison of normal fibroblasts to cervical cancer derived cancer associated fibroblasts (CAF) mRNA microarray revealed, that the expression of TFPI2 is twelve times lower in the latter, being the most downregulated mRNA of CAFs. Aims.  Our aim was to clarify if cancer cells are capable to downregulate TFPI2 expression in normal and cancer associated fibroblast and if they do so, what is the mechanism they utilize for that purpose? Methods.  Promoter methylation, of the gene and miR profile of tumor cells, as well as normal and tumor associated fibroblast were studied. Results.  Immunohistochemistry revealed that surgically removed cervical cancer specimens are negative for TFPI2 protein. Western blot of primary CAF cultures showed considerable, significant decrease of protein expression, as well, although the extent of decrease was different in individual samples of origin. Direct co-culture of cervical cancer cells with normal, TFPI2 expressing, and tumor associated fibroblast proved the potential of cancer cells to downregulate the protease in the fibroblasts. Promoters of TFPI2 in cancer cells were fully methylated, however, only modest promoter methylation was found in CAF samples. As an alternative option for mRNA inactivation miR regulation has been expected. Studying a series of miRNA, upregulation of miR-23a, miR17–5-p, miR-18-a and mir613-3p went parallel with the decrease of TFPI2, indicating, their possible role in the regulation of TFPI2 expression. It is a further question if and how tumor cells are able influence TFPI2 activity of CAFs? Exosome mediated transfer, or paracrine mechanisms can be considered for the further experiments.

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Abstracts DGP-INT01.06 Applications of Mass Spectrometry in Pathology M. Kriegsmann* Institut für Pathologie, Ruprecht-Karls Universität Heidelberg, Heidelberg, Deutschland Mass spectrometry (MS) is a versatile analytical tools used in various areas of medical diagnostics and basic research. MS has been successfully applied to study microbiological colonies, plants, insects, vertebrates including whole animals, human cells and tissues. The method allows to investigate genes, proteins, lipids, carbohydrates and metabolites. Whereas the analysis of genes and peptides by MS has found its way into routine diagnostics, other techniques such as MS Imaging that link the detection of numerous molecules with information about their spatial distribution in cells or tissues promise to have great potential in the near future. In this presentation general aspects of the MS technique are elucidated and possible applications in pathology are highlighted.

DGP-INT01.07 Cancer Cell Invasion at the Cancer Host Interface and its clinic-pathological aspects in periampullary carcinoma A. Csanadi*1, T. Krauss2, K. Enderle-Ammour1, M. Bader1, S. Timme1, M. Kuehs1, 3, O. Schilling4, 5, T. Brabletz6, M. Werner1, 3, 5, U. F. Wellner7, P. Bronsert1, 3, 5 1 Department für Pathologie – Ludwig-Aschoff-Haus – Institut für Klinische Pathologie, Universitätsklinikum Freiburg, Freiburg, Deutschland, 2Klinik für Radiologie, Universitätsklinikum Freiburg, Freiburg, Deutschland, 3 Comprehensive Cancer Center, Tumorbank Freiburg, Freiburg, Deutschland, 4 Institut für Molekulare Medizin und Zellforschung, Albert-LudwigsUniversität, Freiburg, Deutschland, 5Deutsches Krebsforschungszentrum, Zelluläre und molekulare Pathologie, Heidelberg, Deutschland, 6Nikolaus Fieber Zentrum für Molekulare Medizin, Universität Erlangen-Nürnberg, Erlangen, Deutschland, 7Klinik für Chirurgie (UKSH), Campus Lübeck, Lübeck, Deutschland

Cancer cell invasion takes place at the cancer-host interface and is a basic requirement for tumor progression and distant metastasis. Tumor budding, a conventional histological correlate of epithelial to mesenchymal transition (EMT) describes the progress of cancer invasion and is directly associated with patients overall survival. Pancreatic cancer (PDAC) is characterized by elevated EMT activity, high tumor budding and a consequent poor patients outcome. Reasons for poor patients outcome are the late clinical detection, distant metastases and a challenging resection. Thereby the resection-status plays a crucial role in histo-pathological interpretation. Actual German guidelines recommend the concept of the close resection margin (CRM). Nevertheless, PDAC tumor- biology is also associated with a diffuse stromal reaction. Here we illuminate the interactions between EMT, tumorbudding and tumor-cell-migration in a three dimensional context and transfer these results onto a novel perception of PDAC resection margin interpretation. Two series of strictly serial tissue slices were prepared. Thereof, two independent research approaches were taken. The first series of tissue slides was used for 3D reconstruction of tumor buds (TB), second series aimed at the characterization of morphology, after staining pancytokeratin (PCK) ZEB1 and e-Cadherin (ECad). Cell morphology and expression of ZEB1 and ECad were assessed at the single tumor cell level. ZEB1 and E-cadherin were evaluated separately. Next, a PDAC cohort comprising 90 patients was histological reviewed for tumor stromal at the mesopancreatic resection margin (S-Status) as present or absent (S+/S0) and correlated with clinic-pathological and radiological data. Our results demonstrate that the majority of PDAC tumor buds observed in pathological routine diagnostics are 2D artifacts. We also provide evidence for collective cancer cell migration as the relevant mode of local human adenocarcinoma cell invasion. For mesopancreatic PDAC

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resection margins, a complete removal of tumor cells and concomitant fibrotic stroma seems to be a determinant of curative resection in PDAC and can be preoperative predicted by radiological cross-sectional imaging.

DGP-INT01.08 The newly developed claudin-1 based dual-staining test highly correlates with oncogenic transformation in cervical samples T. Szekerczés*1, M. Benczik2, Á. Galamb3, R. Koiss4, G. Lotz1, B. Járay1, A. Kiss1, C. Jeney2, Z. Schaff1, G. Sobel3 Semmelweis University, 2nd Department of Pathology, Budapest, Ungarn, CellCall Ltd., Budapest, Ungarn, 3Semmelweis University, 2nd Department of Obstetrics and Gynecology, Budapest, Ungarn, 4Saint Stephen Hospital, Department of Obstetrics and Gynecology, Budapest, Ungarn 1

2

Background.  Cervical cancer is the fourth most common cancer in women worldwide. It is the ninth most common cause of tumor deaths among women in Hungary. Pap smear is a cytological test designed to detect abnormal cervical cells. The method is highly specific, however, only of moderate sensitivity. There is a need to develop new auxiliary diagnostic methods to increase the sensitivity and improve efficiency of cytological diagnostics. Aims.  We demonstrated increased expression of claudin-1 in cervical squamous intraepithelial neoplasia and invasive carcinoma. This protein is one of the major components of tight junction structure. Our aim was to develop a claudin-1 based dual-staining method featuring as good specificity and sensitivity as of CINtec Plus (Roche), which is a p16ink4a- and Ki67 dual expression test Material and methods.  For the study, 2845 cervical samples from a randomized group of women were collected by gynecologists and were kept in preservation solution for subsequent liquid-based cytology, which is an accepted alternative to conventional Pap test. Immunochemical reactions were performed on 1342 liquid based smears including both CINtec Plus test and Claudin-1/Ki-67 test. The two methods were compared statistically by a McNemar’s and Kappa test. Results.  1101 samples provided acceptable results. 899 liquid based cytological analyses detected normal stage and 202 showed premalignant or malignant stage. The positivity obtained by either the CINtec Plus or Claudin-1+Ki-67 tests corresponded well to cytological evaluation. 202 Claudin-1+Ki-67 positive cases displayed the following cytological distribution: 41/165 (24.8 %) ASCUS and LSIL; 18/30 (60 %) CIN 2; 3/4 (75 %) CIN 3 and 3/3 (100 %) of invasive cervical carcinoma. The specificity (79.9 %) and sensitivity (68.8 %) of CINtec Plus test was comparable with the specificity (84.9 %) and sensitivity (64.9 %) of Claudin-1+Ki-67 test. There was no statistically significant difference between the two tests. Conclusion.  Our newly developed claudin-1 based dual-staining test showed comparable specificity and sensitivity with the results of CINtec Plus test. Therefore, the new method might prove to be the basis for a new auxiliary method for cytodiagnostics and to be applied as triage test in HPV detection. Furthermore, claudin-1+Ki-67 test might assist gynecologists in decision making for patients with CIN2 status.

Junges Forum Pathologie DGP03.01 ESP Residents Committee M. Pollheimer* Medizinische Universität Graz, Institut für Pathologie, Graz, Österreich Im Mai 2015 nahmen AssistenzärztInnen der ESP-Mitgliedsländer in Brüssel an der ersten Sitzung des „ESP Residents Committee Meetings“ teil. VertreterInnen aus folgenden Ländern waren anwesend: Belgien, Bosnien & Her-

zegowina, Griechenland, Italien, Kosovo, Kroatien, Niederlande, Österreich, Polen, Russland, Serbien, Slowenien, Tschechien, Tunesien, Türkei und Ungarn. Der Grund ein Residents Committee zu gründen basierte auf dem Ziel die Gesellschaft zukunftsorientierter zu machen und „frischen Wind“ in die Gesellschaft zu bringen. Die Mitglieder des Residents Committees sollen ihr Land vertreten und via Online-Foren und Telekonferenzen miteinander in Verbindung bleiben und sich regelmäßig bei internationalen Tagungen treffen. Idealerweise sollte es eine/​n VertreterIn aus jedem Mitgliedsland geben. Dabei ist eine enge Verbindung mit der entsprechenden nationalen Gesellschaft sehr wichtig. Als Vorsitzende wurden Dr. Aleksandra Starzyńska (Polen), und ihr Vertreter, Dr. Rui Caetano Oliveira (Portugal), gewählt. Ein wichtiges Ziel des ESP Residents Committees besteht in einer Verknüpfung von „schlechter entwickelten“ Ländern an die besser entwickelten europäischen Länder. Dementsprechend wurde ein Schwerpunkt in Bezug auf die Umsetzung von Austauschprogrammen zu Ausbildungszwecken innerhalb Europas gesetzt. Ein Plan ist derzeit in Entstehung, der den Austausch von AssistenzärztInnen innerhalb der ESP-Mitgliedsländer erleichtern soll. Oft ist dies speziell für die „schlechter entwickelten“ Länder schwierig. Ein weiterer Schwerpunkt wurde in Bezug auf eine Harmonisierung des Ausbildungsplans zum/​r Facharzt/-ärztin für Pathologie innerhalb der ESP-Mitgliedsländer gesetzt. Um eine bessere Öffentlichkeitsarbeit zu erreichen, wurde vorgeschlagen, die ESP-Homepage neu zu designen bzw. zu verbessern. Im Moment werden Ideen und Entwürfe gesammelt. In diesem Zusammenhang soll auch ein Logo für das ESP Residents Committee entstehen. Weiters arbeiten wir an der Umsetzung einer digitalen Lernplattform, die es in Ausbildung stehenden ermöglichen soll, eine Fallsammlung mit typischen Fällen (vor allem Basics) aus allen Fachdisziplinen zu mikroskopieren.

DGP10.01 Virtual microscopy: computergames instead of microscopy M. Nap* Nap Pathology consultance, Berg en Terblijt, Niederlande Computers and especially computer games have for long become part of our daily life. What began as a way to relax or fill the empty moments with a simple game has evolved to serious business in which virtual environments have been created to train highly specialized activities in almost every professional area. Histopathologists have become familiar with computers mainly at the level of reporting texts, retrieving patient histories from geographical remote areas, monitoring the laboratory process by the use of a LIS. But this has not led to any kind of “gaming”. With the introduction of whole slide images (WSI) the option of gaming in the sense of practicing pathology diagnosis and competing with each other without direct clinical consequences came within reach. Yet there was another aspect in the ever evolving field of histopathology that became of interest: Quantitative pathology. More and more arguments have become available that it is important to estimate the relative contribution of various elements in tumor growth patterns and reactions of the immune system to classify the tumor phenotype and eventually its genotype in relation to the possible success of therapeutic interventions. It is there, in the area of “estimations” that computer games can possibly have a function in training. Estimating surface percentages of a tissue section, taken by tumor cells, the relative number of big and small tumor cells, the relative contribution to the cellularity in the image by cells from the immune system and last but not least the percentage of tumor cells that express a single marker or co-express a combination of characteristics. With the emerging field of companion diagnostics the pathologist of tomorrow will be faced with the request of not only rendering a correct and detailed diagnosis but in addition provide information about possible treatment options and the success rate of expensive interventions. At HistoGeneX we have developed a computer game that allows pathologists and those in training or linked to the field to make themselves familiar with and train their capacities for the estimation of surfaces, sizes, phenotypes and numbers. We cannot predict at this moment what the result of gaming in quantitative pathology will be. Will the pathologist beat the image analyzer or vice versa?

Biobanking – Handhabung und Nutzung seltener Biomaterialien (organisiert von den cBMB) Biobanken02.01 Cryobiology and Tissue Banking B. Glasmacher* Leibniz Universität Hannover, Institut für Mehrphasenprozesse, Hannover, Deutschland Cryopreservation allows long-term preservation of viable cells & tissues at ultra-low temperatures. It has major impact for biobanking, reproductive medicine, animal husbandry and conservation of endangered species. Cryopreservation protocols essentially include two methods: conventional, slow freezing and vitrification (rapid freezing). Conventional freezing usually implies a cooling rate of 1 to 2 °C/min and rapid thawing. Vitrification is the process by which a solution rapidly solidifies without formation and growth of ice crystals. The typical freezing processes cause significant damage to biomaterials through ice crystal formation and cellular dehydration in line with the 2-factor hypothesis of freezing injury by Mazur. With the aid of cryoprotectant solutions, biospecimens can be stabilized in the glassy or amorphous state, allowing for long-term cryopreservation at deep temperatures. Effective cryopreservation protocols realized in controlled rate freezers are essential for long-term storage of cells, tissues and organs and – upon thawing – their subsequent clinical/analytical application. Freezing protocols are generally considered as safe, however, putative effects on epigenetic marks have not yet been studied in detail. While post-thaw cell survival rates are normally used to evaluate the success of cryopreservation protocols, increasing evidence suggests that freezing may be associated with deviations from the physiological epigenetic marks with putative long-term effects on the cells and/or their derivatives. The presentation will highlight new findings in this field. Furthermore, improvement of freezing protocols and freezing devices will be presented. Cryovariables including cell specific adapted cooling & thawing rates, directional solidification and induced ice formation via laser and ultrasound will be discussed. The development of new CPAs, reduction of DMSO concentration and the use of ice nucleating proteins will be described. In addition, the method of encapsulation of cells and tissues in alginate beads in order to prevent chilling effects, the adjustment of storage temperatures below -135°C, and studies concerning epigenetic alterations (DNA methylation and histone modification patterns) associated with cryopreservation will be presented. With respect to epigenetic changes, a better understanding of the underlying mechanisms would be beneficial for improving safety and effectiveness of freezing protocols.

Biobanken02.02 Cryopreservation strategies & technical solutions for pluripotent stem cell banking H. Zimmermann*, J. C. Neubauer Fraunhofer-Institut für Biomedizinische Technik IBMT, Sulzbach, Deutschland For a few years now, it has been possible to artificially produce pluripotent stem cells (PSCs) by epigenetic reprogramming from somatic cells. These induced pluripotent stem cells (iPSC) have similar properties as stem cells derived from the inner cell mass of blastocysts, for example differentiation into cells of all three germ layers, but without the ethical restrictions of embryonic cells [1]. This new technique enables the generation of patient- or disease-specific PSCs as model system that will significantly improve the validity of compound screenings. Permanent availability of stem cells or stem cell-derived progenitors is crucial for prospective applications, so that the number of local biobanks increased rapidly in the last years. But only few of them fulfill the demands of the pharmaceutical industry: large numDer Pathologe Suppl 1 · 2016

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Abstracts bers and high diversity of cells, furthermore characterized and catalogued systematically. To achieve this, optimized cryopreservation protocols and innovative biobanking technologies are necessary. Due to the cultivation as three-dimensional cell colonies, PSCs are usually cryopreserved as cell clusters. This often leads to suboptimal survival rates as large ice crystals emerge within the cluster [2]. Therefore, cryopreservation strategies like surface-based vitrification or proteinfree cryomedia have been evaluated. The main problem of conventional biobanks is that during removal of samples from the cryotank all other samples in the storage rack are exposed to warmer room temperature, probably leading to adverse recrystallization processes. Additionally, humidity in the ambient air condenses on the frozen samples, resulting in significant ice formation inside the cryotank. Aside from impairing the readability of labels and barcodes, this fact also increases the risk of bacterial and viral contamination. Hence, an automated stem cell biobank with protective hood system has been developed that avoids sample warming during in- and output. The hood is cooled with dry nitrogen gas, preventing frost formation on the stored samples and contains a gripper for transferring samples to the final storage position. Additionally, the cryotanks are connected via a rail system for the transportation of samples. In summary, we describe here innovative cryopreservation strategies and technical solutions for the efficient storage of PSCs in large-scale biobanks. References 1. Takahashi K, Tanabe K, Ohnuki M, et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors, Cell 131, 861–872 2. Pegg DE (2001) The current status of tissue cryopreservation, Cryo Lett. 22, 105–114

Biobanken02.03 BioPsy – Biobank of Psychiatric Diseases S. Witt*, H. Dukal, C. Hohmeyer, D. Schendel, J. Frank, F. Streit, J. Strohmaier, M. Rietschel Zentralinstitut für Seelische Gesundheit, Abteilung Genetische Epidemiologie in der Psychiatrie, Mannheim, Deutschland Die Abteilung Genetische Epidemiologie in der Psychiatrie untersucht die genetische Basis von psychischen Erkrankungen und Resilienz über die gesamte Lebensspanne. Die Ziele der Forschung sind (i) das Verständnis der zugrundeliegenden biologischen Mechanismen, (ii) die Entwicklung eines biologisch basierten Klassifikationssystems und (iii) die Weiterentwicklung der sogenannten Precision Medicine im Bereich der psychischen Gesundheit. Eine unabdingbare Voraussetzung für die Untersuchung der biologischen Ursachen von psychischen Erkrankungen ist die Verfügbarkeit von geeigneten Biomaterialien. Diese werden vom Molekulargenetische Labor in der laboreigenen Biobank (BioPsy – Biobank of Psychiatric Diseases) mit modernsten Methoden und Standards gesammelt und asserviert. Zu den Biomaterialien zählen u. a. Blut, DNA, mRNA, miRNA, Plasma, Serum, Urin, Haare und Plazenta. Die Biomaterialien stammen von Patienten, Verwandten und Kontrollen, die von der Abteilung selbst, sowie vonZI-internen als auch externen Kollaborationspartnern in national und international geförderten Projekten rekrutiert werden. Die Proben werden barcodiert, pseudonymisiert und über ein Laboratory Information Management System (LIMS) verwaltet. Die Prozessierung des Biomaterials erfolgt teilautomatisiert mit einer DNA-Workstation (Tecan Freedom Evo Plattform). Die Biobank wird nach den neuesten Erkenntnisse hinsichtlich Datenschutz, Ethik und Qualitätsstandards in enger Anlehnung an die Richtlinien der der OECD, des Deutschen Biobankenknotens (GBN), des europäischen Verbunds BBMRI sowie und der TMF e. V. betrieben. Dies umfasst auch die automatisierte Probenerfassung und kontinuierliche Kontrolle und Dokumentation der Lagerungsbedingungen. Um die hohe Qualität des Biobankings fortlaufend zu gewährleisten bilden sich die Mitarbeiterinnen kontinuierlich fort und nehmen aktiv an Veranstaltungen der Biobanking Community teil. Die Abteilung ist Teil der Biobankeninitiative NetBi-omics des Forschungsnetzwerks für die psychische Erkrankungen, mit dem Ziel der Etablierung eines verbundüber-

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greifenden modernen und flexiblen Biobankings für genomische, epigenomische, transkriptomische,proteomische und mikrobiomische Analysen.

Biobanken02.04 Diabetes-associated biobanking to support biomedical research E. Herpel*1, 2, 3, K. Kynast1, 2, F. Lasitschka1, P. Nawroth2, 4 Universitätsklinikum Heidelberg, Pathologisches Institut, Heidelberg, Deutschland, 2Universitätsklinikum Heidelberg, SFB 1118 – Reaktive Metabolite als Ursache diabetischer Folgeschäden, Heidelberg, Deutschland, 3NCTGewebebank, Gewebebank des Nationalen Centrums für Tumorerkrankungen (NCT), Heidelberg, Deutschland, 4Universitätsklinikum Heidelberg, Klinik für Endokrinologie, Stoffwechsel und Klinische Chemie, Heidelberg, Deutschland 1

The increasing prevalence of diabetes and its associated long-term complications as well as the herewith accompanied growing economic burden call for a rapid clinical translation of biomedical research. To meet this condition, the Collaborative Research Center (CRC) 1118-Biomaterialbank (BMB) was established at the Institute of Pathology to collect, characterize, preserve and provide human diabetic specimen and its respective controls specifically to diabetes-associated research proposals that investigate reactive metabolites as cause of diabetic late complications. The BMB bench-to-bedside approach thereby greatly depends on the availability of high-quality human biomaterial and its associated clinical data. Since treatment of diabetes does not primarily demand surgical intervention diabetic specimens are always limited. To counteract this circumstance the BMB constantly preserves and specifically annotates diabetic specimen with all recorded patient-specific ICD-diagnoses, information that is extremely valuable for the interpretation of research results. Beside the provision of tissue slides the BMB performs special stainings (PAS, Trichrome, Sudan Black, Toluidine Blue) to precisely describe pathophysiological-induced morphological adaptions. To more efficiently translate research findings to the clinical setting the BMB establishes IHC-stainings of auspicious, significant diabetes-associated target structures on mouse and human biomaterial of diverse tissue entities and generates PAN-Tissue-Microarrays of approved diabetic mouse models. The consistent structure of the implemented quality standards (SOPs, IT-System, data protection, ethical guidelines, accredited quality management, pathologic-anatomical “exit-control” evaluation of provided biomaterial) and the utilization of platform technologies (TMA construction, generation of sections, IHC, virtual microscopy) enhance the goal-oriented, purposeful usage and compilation of diabetic specimen. The BMB demonstrates that preservation of rare specimen is also particularly relevant in the non-neoplastic field and essentially supports basic research, promotes comprehensive scientific data and fortifies the sustainability for diabetes research. In addition, the herewith increased understanding of how metabolic imbalance triggers diabetes onset and progression and favors diabetic late symptoms might eventually hold some promise for future potential diagnostic and/or therapeutic innovative applications.

Gastvorträge in den Arbeitsgemeinschaften

Assessment and scoring of liver lesions in morbidly obese patients

New strategies in the immune therapy of solid cancer: checkpoints, CARs and TRUCKs AG04.07 New strategies in the immune therapy of solid cancer: checkpoints, CARs and TRUCKs

AG01.12 Assessment and scoring of liver lesions in morbidly obese patients

H. Abken*

P. Bedossa*

Background.  The immune system has the power to control cancer, however, needs specific activation and resistance to repression. The anti-tumor activity is utilized in a number of clinical trials applying ex vivo amplifie, autologous tumor-infiltrating T cells to the cancer patient. On the other hand, the T cell anti-tumor attack in situ is substantially blocked by the repressive checkpoint molecules in the solid tumor tissue; blocking the blockers improved the anti-tumor response, in particular blocking the inhibitory PD-1 or CTLA-4 pathway. Such so-called checkpoint blockade induced substantial tumor regression in solid cancer accompanied by a broad anti-tumor immune response. In order to shape immune cells more specifically towards tumors, patient’s cytolytic T cells from the peripheral blood are ex vivo engineered with a recombinant T cell receptor (TCR) or chimeric antigen receptor (CAR) which recognize a pre-defined antigen on the target cell and drive the activation of T cells upon antigen engagement. CAR T cells substantially reduced the tumor burden in early phase trials and induced spectacular and lasting remissions. However, loss of target antigen make cancer cells invisible to CAR T cells and likely contribute to deadly tumor relapses, which is particularly the case in solid cancers. We discuss recent developments in the fourth generation of CAR T cells, so called TRUCKs, which release inducible IL12 upon CAR engagement in the targeted tumor lesion and thereby boost an innate immune response in order to control such solid cancer lesions.

University of Paris, Hôpital Beaujon, Departement of Pathology, Clichy, Frankreich Background.  Nonalcoholic fatty liver disease (NAFLD) covers a spectrum of histological lesions ranging from steatosis to a complex pattern with hepatocyte injury and inflammation in an appropriate clinical context. The disease has been artificially dichotomized into NAFL (Non Alcoholic Fatty Liver/ steatosis) and NASH (Non alcoholic steatohepatitis)a pattern characterized by association of steatosis with hepatocellular injury and inflammation, but it is increasingly clear that intermediate features may exist. More than NASH, the stage of fibrosis was shown to govern prognosis and for such evaluation, a liver biopsy of adequate size and width is needed. Semi-quantitative scoring systems may partially avoid the limit of classification per category. Scoring systems which have been developed in most chronic liver diseases,have been shown useful by increasing reproducibility between pathologists, creating homogenous group of patients according to their histology, and provide an easier way to compare lesions when repeated biopsies are performed. These histologic scores are not useful in clinical practice but concur to categorize homogeneous group of patients according to their histology. The Fatty Liver Inhibition of Progression (FLIP) consortium is an European group of liver pathologists that propose a new scoring system (SAF score) for assessing the different features of liver pathology in NAFLD. In addition they provide an algorithm (The FLIP algorithm) that allows to segregate the disease in different categories. Today, liver histology is the mainstay for clinical trials. Biopsy is used both for enrollment and for assessing benefit of clinical trials. Endpoints such as reversion of NASH or regression of fibrosis are acceptable but require a clear histological definition based on scoring systems.

Focal Therapy and Template Biopsy for Prostate Cancer

Uniklinik Köln, Klinik für Innere Medizin, Köln, Deutschland

Lynch-associated neoplasia of the female genital tract AG05.07 Lynch-associated neoplasia of the female genital tract T. Longacre*

AG02.01 Focal Therapy and Template Biopsy for Prostate Cancer D. Eberli* UniversitätsSpital Zürich, Klinik für Urologie, Zürich, Schweiz Background.  Precise risk stratification is essential in times where alternative treatment options to radical prostatectomy (RP) such as active surveillance, brachytherapy, radiotherapy and focal treatment of prostate cancer (PC) are available. However, the current concordance of conventional transrectal prostate biopsy (PB) regarding Gleason score (GS) is low, when compared to RP specimens. Multiparametric MRI (mpMRI) of the prostate and fusion transperineal prostate mapping (TPM) biopsy might allow increasing the concordance facilitate the decision making. The additional 3D Information will allow a more precise focal treatment. HIFU uses ultrasound energy to produce a thermal ablation zone within the prostate. The overall goal is to reduce side effects of conventional treatment options; Impotence and incontince. We will discuss the improvements in diagnostics and treatment of prostate cancer over the last years for localized disease.

Stanford University School of medicine, Department of Pathology, Stanford, CA, Vereinigte Staaten von Amerika Background.  Lynch syndrome is responsible for approximately 5 % of endometrial cancers and 1 % of ovarian cancers. The molecular basis for Lynch syndrome is a heritable functional deficiency in the DNA mismatch repair system, typically due to a germline mutation. The most frequent clinically relevant mutations occur in the MLH1, MSH2, MSH6, PMS2, and EPCAM DNA mismatch repair genes. The cumulative risk for endometrial cancer varies depending on the germline mutation, with the highest estimated risk for women with MSH6 germline mutations (up to 70 % by age 70). In contrast, the estimated risk for women with MLH1 or MSH2 germline mutations ranges from 20 to 55 % by age 70 and for PMS2, only 12 % by age 70. Patients with these germline mutations are also at variably increased risk for multiple other malignancies, including carcinomas of the colorectum, stomach, small intestine, pancreas, hepatobiliary tract, and urinary tract. This presentation discusses the rationales and relative merits of current Lynch syndrome screening tests for endometrial and ovarian cancers and provides attendees with an informed algorithmic approach to universal Lynch syndrome testing in gynecologic cancers. Specific tests addressed include mismatch repair protein immunohistochemistry (2 panel versus 4 panel), microsatellite instability by PCR, MLH1 methylation,

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Abstracts and germ line as well as somatic tumor mutational analysis. Differences in colorectal cancer universal testing and endometrial cancer universal testing are emphasized. Pitfalls in test interpretation and strategies to resolve discordant test results are presented. The potential role for next-generation sequencing panels in future screening efforts is also discussed.

Drug resistance, Target Therapy and precision medicine in osteosacoma: The present and the future AG07.05 Drug resistance, target therapy and precision medicine in osteosarcoma: the present and the future M. Serra* Orthopädisches Institut Rizzoli, Labor für experimentelle Tumorforschung, Abteilung für Pharmakogenomik und Pharmakogenetik, Bologna, Italien Background.  High-grade osteosarcoma (HGOS), the most common malignant tumour of bone, is currently treated with pre- and post-operative chemotherapy in association to surgery. New treatment approaches and drugs are warranted to improve prognosis of this neoplasm, since conventional treatments allow to cure about 60–65 % of patients with primary tumours and only 20–25 % of patients with recurrent disease. One of the major clinical problems that limits cure probability of HGOS patients is the presence of inherent or acquired drug resistance mechanisms, which remarkably decrease the drug sensitivity of tumour cells. Due to this, HGOS patients variably respond to conventional treatments and, therefore, there is the need for markers which may be of help for an early indentification of patients with greater or reduced treatment responsiveness, in order to tailor chemotherapy. In the last 20 years, a consistent body of translational and clinical research has been performed with the following aims: 1) validation of markers which may be used to early stratify HGOS patients into different risk groups; 2) identification of new therapeutic targets and targeted drugs that can be proposed as adjuvant to conventional chemotherapeutics to better control the local and metastatic disease; 3) definition of more efficient rescue treatments for relapsed HGOS patients, who did not benefit from first-line regimens; 4) identification of pharmacogenetic biomarkers which may provide the bases for planning personalised treatments aimed to increase the therapeutic benefits and to avoid or limit unnecessary toxicities. This talk will provide an overview of the biomarkers identified so far in HGOS, which appear to be promising candidates for a translation to clinical practice, after further investigation and/or prospective validation. Since several targeted therapies against molecular and immunological markers in HGOS are presently under clinical investigation, it may be speculated that some new treatment approaches may emerge in the next years and that the treatment of this tumour is evolving towards personalised approaches according to the principle of precision medicine.

Die Rolle von Telozyten bei Erkrankungen und Regeneration des Herzens AG09.02 The role of telocytes in cardiac diseases and heart regeneration S. Kostin* Max-Planck-Institut für Herz- und Lungenforschung, Bad Nauheim, Deutschland

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Background.  Our previous studies suggested that an important variable of the progression of contractile dysfunction to terminal heart failure is the imbalance between myocyte cell death and myocyte renewal. For this reason, preventing myocyte cell death and an increasing generation of new myocytes may represent attractive targets in the treatment of human heart failure. Prospective clues to enhance myocardial regeneration are the newly discovered cells termed telocytes, formerly called interstitial Cajal-like cells, which are believed to nurse or “guide” the endogenous and exogenous stem cells for activation and commitment, but they also act as supporting cells for progenitor cells migration toward injured myocardium. The unique structural features of telocytes comprise distinct cellular compartments: (1) cell body (the proper telocyte), (2) very long cellular prolongations (telopodes) and (3) the labyrinthine system made of telopodes. Despite a relatively low quantity (less than 1 % of interstitial cells in the human heart), the intramyocardial telocytes contribute to form a complex three-dimensional network by interacting with cardiomyocytes and surrounding stromal cells via heterocellular cell-to-cell contacts, shedding microvesicles, exosomes or paracrine secretion including microRNAs. We have recently found that telocytes are reduced in the diseased and failing myocardium. Importantly, the imbalance between telocyte proliferation and telocyte death is responsible for the telocytes depletion in cardiac diseases leading to heart failure. We have also demonstrated that telocytes are influenced by the extracellular matrix protein composition such that the telocytes are almost absent in areas of severe fibrosis. It is plausible that the reduction in telocytes in diseased human hearts could participate in the abnormal three-dimensional spatial organization and disturbed intercellular signalling of the myocardium. Decreased telocytes in diseased hearts would also be predicted to alter the property of telocytes to maintain cardiac stem cell niche by decreasing the pool of cardiac stem cells and thereby impairing cardiac regeneration.

Sitzungen der Arbeitsgemeinschaften der DGP AG Gastroenteropathologie AG01.01 The role of autophagy in response to neoadjuvant chemotherapy in esophageal adenocarcinomas O. Adams*1, 2, B. Dislich1, J. Slotta-Huspenina3, K. Becker3, M. Feith4, K. Ott5, M. P. Tschan1, 2, R. Langer1 Experimental and Clinical Pathology, Institut für Pathologie, Universität Bern, Bern, Schweiz, 2Graduate School for Cellular and Biomedical Sciences, Universität Bern, Bern, Schweiz, 3Institut für Allgemeine Pathologie und Pathologische Anatomie, Technische Universität München, München, Deutschland, 4Chirurgische Klinik und Poliklinik, Klinikum Rechts der Isar der Technischen Universität München, München, Deutschland, 5Klinik für Allgemein-, Gefäß- und Thoraxchirurgie, Klinikum Rosenheim, Rosenheim, Deutschland 1

Background.  Autophagy is a cellular degradation process maintaining cellular homeostasis, but is also involved in degeneration, immune response and cancer. Although the role of autophagy in cancer is not completely understood, it is regarded as a potential therapeutic target. Moreover, it may influence resistance to cytotoxic treatment by either supporting tumor cell survival or – in contrast – promoting cell death. Esophageal adenocarcinomas (EAC) are aggressive tumors with high rates of resistance to chemotherapy (CTX). Treatment options for patients with advanced or recurrent disease are limited. We aimed to assess the role of autophagy in CTX response in EAC. Methods.  Immunohistochemistry for autophagy related LC3B and p62 was performed on EAC biopsies (n = 128) prior to a platinum based neoadjuvant CTX (+/– paclitaxel) and post-treatment EAC resection specimens (n = 41). In addition, the EAC cell line OE33 was treated with

paclitaxel and LC3B assessed via Western blotting. Autophagic flux was assessed by the addition of the autophagy inhibitor Bafilomycin A1 (BafA). Results.  LC3B and p62 dot-like staining was observed in varying degrees in EAC samples. Low pretherapeutic LC3B expression was associated with non-response (tumor downstaging; Χ2 = 0.012) and with a worse overall survival in the total group (p = 0.119), which was even more dominant in non-responding patients (p = 0.03). Similar, low p62 expression was associated with worse outcome (p = 0.05) but not significantly linked to tumor response. In matched samples, post-treatment samples had higher LC3B and p62 levels than pre-treatment samples (p = 0.009, p = 0.053). Of note, the prognostic impact of both markers after neoadjuvant CTX was inverse to pretherapeutic conditions with a trend for better survival in LC3B and p62 low expressing tumors after treatment. Preliminary in vitro data indicate that paclitaxel induces autophagy in OE33 as elevated levels of membrane-bound LC3B were observed when co-treated with BafA indicating autophagic flux. Conclusions.  Our preliminary data suggest that pretreatment status of autophagy of EAC may influence response to CTX and patient outcome. However, our preliminary in vitro and ex vivo data suggest that autophagy is also induced by CTX itself and that the nature of autophagic reaction may be linked to tumor response and prognosis as well. Therapeutic modulation of autophagy may influence response behavior to CTX and represent a potential tool for overcoming resistance in EAC.

AG01.02 Expression and putative tumor biological value of PD-L1 and PD-1 in gastric cancer C. Böger*, H.-M. Behrens, S. Krüger, M. Mathiak, C. Röcken Institut für Pathologie, Christian-Albrechts-Universität, Kiel, Deutschland Background.  Antibody therapies targeting the inhibitory checkpoint PDL1 and PD-1 have recently shown promising results in several cancer entities. As PD-L1 positive tumors show higher response rates to anti-PD-L1 therapy, it is supposed that identifying patients with PD-L1 expression may increase the amount of therapy responders. We therefore investigated the expression of PD-L1 and PD-1 in gastric cancer (GC) testing the following hypotheses: are these molecules expressed in GC, are they putatively involved in GC biology and are they prognostically relevant. Methods.  PD-L1 (rabbit monoclonal anti-PD-L1-antibody; clone E1L3N) and PD-1 (mouse monoclonal anti-PD-1-antibody; clone MRQ-22) expression was analyzed by immunohistochemistry in 464 GCs. For PDL1, the immunostaining of tumor, stroma and immune cells was evaluated separately by quantity and intensity, and an immunreactivity score was generated. PD-1 expression was evaluated in intratumoral lymphocytes. The expression of both antibodies was correlated with various clinico-pathological patient characteristics and patient survival. Results.  Membranous PD-L1 expression in tumor cells was found in 140 cases (30.1 %). The amount of PD-L1 positive tumor cells ranged from 0 to 80 % (mean 1.7 %). A weak immunostaining (PD-L1 1+) was found in 36 (7.7 %) cases, a moderate (PD-L1 2+) in 88 (18.9 %) cases and a strong (PD-L1 3+) in 16 (3.3 %) cases. PD-L1 was expressed in 107 (23.0 %) cases in stroma cells and in 411 (88.4 %) cases in immune cells. PD-1 was found in 385 (82.8 %) GCs in intratumoral lymphocytes. A high PD-L1 expression in tumor cells (≥ 2 % PD-L1 1+ or ≥ 1 % PD-L1 2+ tumor cells) was significantly more prevalent in male patients, intestinal and unclassified GCs, EBV-positive, microsatellite instable (MSI), Her2/neu-positive respectively papillary type GCs. Patients with PD-L1 positive GCs showed a significantly better median survival (25.0 vs. 16.0 months; p = 0.018). PDL1 positive tumor cells were localized near the mucosal surface and generally accessible by biopsy in 79 (57.4 %) cases. Conclusions.  PD-L1 and PD-1 are expressed in a substantial amount of GCs, which makes GC an interesting objective regarding future studies of PD-L1/PD-1 targeting therapy. PD-L1 expression in tumor cells is significantly more prevalent in EBV-positive, MSI and papillary type GCs and is associated with a better prognosis. We propose a putative PD-L1 bio-

marker score, which can be tested and validated prospectively in future clinical trials.

AG01.03 Neuroendocrine differentiation of primary hepatic neoplasms N. Waldburger*1, K. H. Weiss2, P. Schemmer3, S. Roessler1, B. Goeppert1, P. Schirmacher1 Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, Abteilung Innere Medizin IV, Universitätsklinikum Heidelberg, Heidelberg, Deutschland, 3Klinik für Allgemein-, Viszeral- und Transplantationschirurgie, Universitätsklinikum, Heidelberg, Deutschland

1

2

Background.  Existence and relevance of primary neuroendocrine tumors of the liver have been debated. Neuroendocrine differentiation in primary hepatic neoplasms has not been thoroughly investigated and its contribution to mixed/intermediate differentiated hepatic neoplasms is unclear. Respective entities are not included in current classification systems. In contrast, primary adenocarcinomas with neuroendocrine differentiation located in other parts of the gastrointestinal system are recognized entities in the WHO classification (e. g. MANEC). Methods.  We screened cohorts of primary neoplasms of the liver (hepatocellular carcinomas (HCC): n = 111, combined hepatocellular-cholangiocarcinomas (HCC-CC): n = 15) for neuroendocrine expression by immunohistochemistry using antibodies against Synaptophysin and Chromogranin A. Respective morphological features were evaluated in detail. Results.  Synaptophysin was positive in 5/15 combined HCC-CC with typical morphology. Furthermore 2/5 Synaptophysin positive combined HCC-CC simultaneously expressed Chromogranin A. 4/111 HCC showed specific expression of the neuroendocrine marker Synaptophysin in absence of Chromogranin A. One specific single case of primary hepatic carcinoma showed neuroendocrine histomorphology but expressed 3 hepatocellular markers (AFP, Arginase 1 und OCH1E5) together with Synaptophysin and Chromogranin A. Conclusions.  This study demonstrates the (rare) existence of neuroendocrine differentiation in primary liver cancer with hepatocellular differentiation (HCC, HCC-CC). Neuroendocrine differentiation appears to be more frequent in liver cancer of mixed/intermediate differentiation suggesting that it may be another facet of mixed differentiation and higher tumor cell plasticity. This lends further evidence for the (rare) occurrence of primary hepatic neuroendocrine tumors. Neuroendocrine differentiation should be considered in future classification systems of liver cancer.

AG01.04 Cellular Apoptosis Susceptibility (CAS) is linked to integrin-β1 and required for tumor cell migration and invasion in hepatocellular carcinoma (HCC) J. Winkler1, S. Roessler1, C. Sticht2, E. Drucker1, K. Holzer1, A. DiGuilio3, E. Eiteneuer1, K. Breuhahn1, N. Gretz2, P. Schirmacher1, A. Ori3, S. Singer*1, 3 Pathologisches Institut, Ruprecht-Karls-Universität, Heidelberg, Deutschland, 2Medizinische Fakultät Mannheim der Universität Heidelberg, Zentrum für Medizinische Forschung, Mannheim, Deutschland, 3European Molecular Biology Laboratory, Heidelberg, Deutschland 1

Background.  Exportins and importins represent an integral part of the nucleocytoplasmic transport system with fundamental importance for eukaryotic cell function. A variety of malignancies including hepatocellular carcinoma (HCC) show deregulation of nuclear transport factors such as overexpression of the exportin Cellular Apoptosis Susceptibility (CAS). The functional implications of CAS in hepatocarcinogenesis remain, however, poorly understood. Here, we integrated proteomics and functional assays with patient data to further characterize the role of CAS in HCC. Methods.  To identify potential “downstream” targets of CAS in HCC HLE cells were treated with two different CAS-specific siRNAs and cell extracts Der Pathologe Suppl 1 · 2016

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Abstracts were subjected to shotgun proteomics. Since the top hit of the aforementioned approach was integrin-β1 (a key factor in cell motility), the role of CAS in tumor cell migration and invasion was analyzed by using ‘scratch’ and invasion chamber assays, respectively. The latter experiments were repeated upon siRNA-mediated depletion of integrin-β1 to confirm its pivotal role in these scenarios. The prognostic relevance of the acquired experimental results was tested by interrogating a survival data set of a large (n = 247) HCC patient cohort. Results.  By analyzing ~1700 proteins using quantitative mass spectrometry in HCC cells we found that CAS depletion by RNAi led to deregulation of integrins, particularly downregulation of integrin-β1. Consistent with this finding CAS knockdown resulted in substantially reduced migration and invasion of HCC cells. This phenotype could be recapitulated by direct knockdown of integrin-β1 indicating the requirement of CAS for tumor cell motility by maintaining integrin-β1 expression. Supporting the potential in vivo relevance of these findings, we determined that high expression levels of CAS in HCC tissue samples were associated with macroangioinvasion and poorer patient outcome. Conclusions.  Our data demonstrate a previously unanticipated link between CAS and integrin signaling which correlates with an aggressive HCC phenotype.

AG01.05 The oncogenic functions of MDM4 are supported by Serum Response Factor (SRF) and ETS domain transcription factors in hepatocellular carcinoma R. Pellegrino*1, D. F. Calvisi2, A. Thavamani3, A. Eberhart1, R. Geffers4, O. Neumann1, M. Evert5, F. Dombrowski2, P. Schirmacher6, A. Nordheim3, T. Longerich1 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, Institut für Pathologie, Universitätsmedizin, Greifswald, Deutschland, 3 Interfakultäres Institut für Zellbiologie, Universität Tübingen, Tübingen, Deutschland, 4Genomanalytik, Helmholtz-Zentrum für Infektionsforschung, Braunschweig, Deutschland, 5Institut für Pathologie, Universitätsklinikum Regensburg, Regensburg, Deutschland, 6Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland 1

2

Background.  The Mouse Double Minute 4 protein (MDM4) represents a negative regulator of p53 transcriptional activity. As we previously demonstrated, MDM4 is upregulated in about 50 % of human hepatocellular carcinomas (HCC) and exerts protumorigenic functions. We hypothesized that aberrant transcriptional activation could be involved in oncogenic MDM4 upregulation. Methods.  To identify potential transcription factors (TFs) binding sites in the MDM4 promoter, we took advantage of the MAPPER platform. Existing gene expression profiling data of human HCCs were used to assess a potential association between MDM4, SRF, and co-acting factors. Gene-specific siRNAs were used to knockdown SRF and its co-factors in HCC cell lines. Luciferase assay allowed us to analyze the impact of SRF inhibition on the activity of the MDM4 promoter in vitro. Additionally, ChIP analyses were performed to investigate the binding of SRF and/or ELK1/4 at the MDM4 promoter. Furthermore, MDM4 expression was analyzed in SRF-VP16 and Ras-AKT overexpressing mice. Results.  In silico analysis of the MDM4 promoter region identified SRF as a putative transcription factor. Overexpression of SRF mRNA was observed in a collection of human HCCs; furthermore, a strong positive correlation between MDM4 and SRF expression was found in these samples, which was associated with overexpression of MKL1-2, ELK1 and ELK4, known transcriptional co-activators acting in complex with SRF. More intriguingly, ELK4 and MDM4 genes mapped at the same chromosome locus 1q32.1, which was found amplified in human HCC, prompting us to speculate for an interesting interplay between them. MDM4 mRNA and protein levels decreased in HCC cell lines upon knockdown of SRF by gene-specific siRNAs that resulted in reduction of cell viability. Similar results were observed when ELK1 and ELK4 were silenced by siRNAs. More importantly, ChIP

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analyses revealed that both SRF and ELK1 were able to bind to the MDM4 core promoter and SRF depletion decreased its activity in a MDM4 promoter reporter assay. Moreover SRF was found in complex with ELK4 in HCC cells. Finally, upregulation of MDM4 mRNA and protein levels was observed in HCCs that developed in SRF-VP16 transgenic mice; a similar increase of MDM4 was detected in Ras-AKT-induced murine HCCs. Conclusions.  Taken together, our data assign a crucial role to SRF and Ets transcription factor family members in the transcriptional activation of MDM4 expression, thereby supporting the oncogenic activity of MDM4 in HCC.

AG01.06 Concomitant activation of PIK3CA and Yap oncogenes promotes the development of hepatocellular and cholangiocellular tumors in mouse and human liver D. Calvisi*1, 2, L. Li3, A. Cigliano1, J. Tao3, S. Ribback1, G. Latte2, G. M. Pilo2, K. Evert4, K. Utpatel4, F. Dombrowski1, X. Chen3, M. Evert4 Institut für Pathologie, Universitätsmedizin, Greifswald, Deutschland, Abteilung für Klinische und Experimentelle Medizin, Universität Sassari, Sassari, Italy 3Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, Vereinigte Staaten von Amerika, 4 Universität Regensburg, Institut für Pathologie, Regensburg, Deutschland 1

2

Background.  Activation of the PI3K and Yes-associated protein (Yap) signaling pathways has been independently reported in human hepatocellular carcinoma (HCC). However, the oncogenic interactions between these two cascades in hepatocarcinogenesis remain undetermined. Methods.  To assess the consequences of the crosstalk between the PI3K and Yap pathways along liver carcinogenesis, we generated a mouse model characterized by combined overexpression of activated mutant forms of PIK3CA (PIK3CAH1047R) and Yap (YapS127A) in the mouse liver using hydrodynamic gene delivery (referred to as PIK3CA/Yap mouse). In addition, suppression of PI3K and Yap pathways was conducted in human HCC and cholangiocarcinoma (CCA) cell lines. Results.  We found that concomitant activation of PI3K and Yap pathways triggered rapid liver tumor development in mice. Histologically, tumors developed in PIK3CA/Yap mice were pure HCC, CCA, or mixed HCC/ CCA. At the molecular level, PIK3CA/Yap tumors were characterized by strong activation of the mTORC1/2, ERK/MAPK, and Notch pathways. Simultaneous activation of PI3K and Yap pathways frequently occurred in human liver tumor specimens, and their combined suppression was highly detrimental for the growth of HCC and CCA cell lines. Conclusions.  Our study is the first demonstration in vivo that the PI3K and Yap oncogenic pathways functionally cooperate along liver carcinogenesis, leading to the development of tumors with hepatocellular and cholangiocellular features. Thus, the PIK3CA/Yap mouse represents an important preclinical liver tumor model for the development of novel therapeutics against human HCC and CCA.

AG01.07 Novel immunhistochemical markers differentiate intrahepatic cholangiocarcinoma from benign bile duct lesions S. Bertram*1, J. Padden2, J. Kälsch3, M. Ahrens2, L. Pott1, A. Canbay3, F. Weber4, C. Fingas4, A.-C. Hoffmann5, A. Vietor1, J. Schlaak3, M. Eisenacher2, H. Reis1, B. Sitek2, H. Baba1 Universitätsklinikum Essen, Institut für Pathologie, Essen, Deutschland, Ruhr-Universität Bochum, Medizinisches Proteom-Center, Medizinisches Proteom-Center, Bochum, Deutschland, 3Universitätsklinikum Essen, Klinik für Gastroenterologie und Hepatologie, Essen, Deutschland, 4 Klinik für Allgemeinchirurgie, Viszeral- und Transplantationschirurgie, Universitätsklinikum Essen, Universität Duisburg-Essen, Essen, Deutschland, 5 Innere Klinik (Tumorforschung) am WTZ, Universitätsklinikum Essen, Universität Duisburg-Essen, Essen, Deutschland 1

2

Background.  The distinction between intrahepatic cholangiocarcinoma (ICC) and benign bile duct lesions can be challenging. Using our previously identified potential biomarkers for ICC we examined whether these are useful for the differential diagnosis of ICC, bile duct adenoma and reactive bile duct proliferations in an immunhistochemical approach and identified a diagnostic marker panel including known biomarkers. Methods.  Subjects included samples from 77 patients with ICC, 33 patients with bile duct adenoma and 47 patients with ductular reactions in liver cirrhosis. Our previously identified biomarkers (STIP1, SerpinH1, 14-3-3Sigma) were tested immunohistochemically following comparison with candidates from the literature (CD56, HSP27, HSP70, BCL2, p53, ki67). Results.  The expression of SerpinH1 and 14-3-3Sigma was significantly higher in ICC than in bile duct adenomas and ductular reactions (p < 0.05), whereas STIP1 expression was significantly higher (p < 0.05) in ICC than in ductular reactions, but the difference to the bile duct adenoma group was not significant. A panel of the biomarker SerpinH1, 14-3-3Sigma and ki67 (≥ 2 marker positive) showed a high diagnostic accuracy (sensitivity 87.8 %, specificity 95.9 %, accuracy 91.8 %) in the differential diagnosis of ICC versus non-malignant bile duct lesions. Conclusions.  This suggests that 14-3-3Sigma and SerpinH1 may be useful in the differential diagnosis of malignant, benign and reactive bile duct lesions in addition to ki67 where a cut-off of >5 % might be used for the distinction of malignant and non-malignant lesions.

AG01.08 Well differentiated neuroendocrine neoplasms with an elevated MIB1-index of pancreatic and extrapancreatic origin are mainly somatostatin receptor 2A positive and TP53 wild type B. Konukiewitz*1, A. M. Schlitter1, D. Pfister1, A. Segler1, A. Agaimy2, B. Sipos3, G. Zamboni4, I. Esposito5, N. Pfarr1, G. Klöppel1 Technische Universität München, München, Deutschland, 2Universität Erlangen, Erlangen, Deutschland, 3Universitätsklinikum Tübingen, Tübingen, Deutschland, 4Universität Verona, Verona, Italien, 5Universität Düsseldorf, Düsseldorf, Deutschland

1

Background.  Neuroendocrine neoplasms of the gastroenteropancreatic system are sub-classified into well differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinomas (NECs), based on morphological criteria and the MIB1-proliferation index. Recently, a small subset of well differentiated neuroendocrine neoplasms with an elevated MIB1-index (>20 %) have been described (NETs G3). The immunohistochemical expression of somatostatin receptors (SSTRs), especially SSTR2A, is a common finding in NETs, which is important for diagnosis, therapy and follow up. Little is known about the expression of SSTRs in NETs G3 and high proliferative NECs. The aim of this study is to investigate the expression of SSTR2A and SSTR5 in neuroendocrine neoplasms G3 of pancreatic and extrapancreatic origin and to correlate the SSTR expression pattern with p53 immunohistochemistry and the molecular TP53 status. Methods.  42 surgical resection specimens, including 33 NECs of different origin (pancreas/ampulla, colorectum, bladder, parotid gland, merkel cell carcinoma of the skin) and 9 NETs G3 (MIB1-index >20 %) of pancreatic (n = 7) and colorectal (n = 2) origin, were examined. Immunohistochemistry was performed for synaptophysin, chromogranin A, Ki-67, CD56, p53, progesterone receptors, CK20, SSTR2A and SSTR5. TP53-mutation analysis (exon 5–9, by Sanger-sequencing) was done in 40 cases (NECs, n = 31; NETs G3, n = 9). Results.  5/33 NECs and 7/9 NETs G3 were SSTR2A positive. 23/33 NECs were p53 positive, all NETs G3 were p53 negative. SSTR5 was expressed in 2/42 cases. Molecular analysis revealed TP53-mutations in 21/31 NECs. No TP53-mutations were seen in cases of NETs G3. 3/5 SSTR2A positive NECs were TP53-mutated. Conclusions.  SSTR2A expression is not restricted to well differentiated neuroendocrine tumors but also occurs in a subset of pancreatic and ex-

trapancreatic poorly differentiated neuroendocrine carcinomas. However, NETs G3 of pancreatic and colorectal origin retain their SSTR2A expression. In our small series, NETs G3 seem to be genetically different to NECs, due to their TP53-status. SSTR5 is just occasionally expressed in NECs and NETs G3. Progesterone receptors are neither expressed in pancreatic NETs G3 nor in pancreatic NECs.

AG01.09 Genome wide DNA copy number analysis in cholangiocarcinoma using high resolution molecular inversion probe single nucleotide polymorphism assay A. Arnold*1, A. Koch2 1 Institut für Pathologie, Berlin, Deutschland, 2Institut für Neuropathologie, Charité – Universitätsmedizin, Berlin, Deutschland

Background.  Cholangiocarcinoma (CCA) are highly malignant epithelial tumors of the bileduct system. Based on the anatomical localisation CCA are classified as intrahepatic (IH-CCA) or extrahepatic (EH-CCA) carcinoma. Although the complex mechanisms of CCA development is not entirely defined, new investigations using whole genome copy number analysis have shed light on the major processes involved in its tumor biology. But their results are limited by the number of samples analyzed, the ethnic diversity of the cases and the merging of different tumor subclasses. Methods.  In order to study molecular similarities and differences of IHCCA and EH-CCA, 24 FFPE tumor samples (13 IH-CCA, 11 EH-CCA) were analyzed for whole genome copy number variations (CNVs) using a new high-density Molecular Inversion Probe Single Nucleotide Polymorphism (MIP SNP) assay. For verification of copy number status Fluorescence-in-Situ Hybridazation (FISH), q-PCR and immunhistochemistry were used. Results.  Common in both tumor subtypes the most frequent losses were detected on chromosome 1p, 3p, 6q and 9 while gains were mostly seen in 1q, 8q as well as complete chromosome 17 and 20 (. Fig. 1). Applying the statistical GISTIC (Genomic Identification of Significant Targets in Cancer) tool we identified potential novel candidate tumor suppressor(DBC1, FHIT, PPP2R2A) and oncogenes (LYN, FGF19, GRB7, PTPN1) within these regions of chromosomal instability. Next to common aberrations in IH-CCA and EH-CCA, we additionally found significant differences in copy number variations on chromosome 3 and 14 (. Fig. 2). Conclusions.  In conclusion, new possible target genes within regions of high significant copy number aberrations were detected using a high-density Molecular Inversion Probe Single Nucleotide Polymorphism (MIP SNP) assay, which opens a future perspective of fast routine copy number and marker gene identification for gene targeted therapy.

AG01.10 MicroRNA screen identifies tumorsuppressive and oncogenic microRNAs in gallbladder carcinoma B. Goeppert1, M. Renner1, A. Fraas1, C. Sticht2, D. Scherer3, N. Gretz2, J. Lorenzo Bermejo3, P. Schirmacher*1, S. Roessler1 Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, Zentrum für Medizinische Forschung, Heidelberg, Deutschland, 3Institut für Medizinische Biometrie und Informatik, Heidelberg, Deutschland 1

2

Background.  Gallbladder carcinoma (GBC) is a rare orphan cancer entity with an incidence of less than 3/100.000 in Western Europe and the US. The main risk factors for GBC include female gender, age, ethnicity, obesity, cholelithiasis, chronic inflammation and exposure to certain chemicals. GBC treatment options are very limited and prognosis of unresectable GBC is poor with 2-year survival rates <10 %. Radical surgery is the only potentially curative treatment option and Gemcitabine plus a platiDer Pathologe Suppl 1 · 2016

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Abstracts

Fig.1 I AG01.09 8 Identification of copy number aberrations throughout the whole genome of 24 CCA (a) and 6 controls (b) and visualization of the Genomic Identification of Significant Targets In Cancer (GISTIC) algorithm (c)

Fig. 2 I AG01.09 8 Identification of differences in copy number aberrations between intra- and extrahepatic Cholangiocarcinoma (CCA) applying “Comparison” function of Nexus Copy Number

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num-based agent (e. g. cisplatin or oxaliplatin) currently seems to be the most effective chemotherapy. Therefore, there is a great need for the development of new treatment options, including targeted therapy for GBC. Methods.  To dissect the epigenetic regulation during GBC development, we performed global miRNA profiling of 40 GBC and 8 normal gallbladder tissues. MiRNAs that are associated with survival were functionally analyzed by cell proliferation and colony formation assays in two different cholangiocellular cell lines, TFK-1 and EGI-1. In addition, we performed Affymetrix gene expression microarray analysis of cell lines transfected with miRNA mimics or control. Results.  Comparison of miRNA profiles of 40 GBC and 8 normal gallbladder tissues exhibited clear differences and 992 out of 2006 miRNAs were differentially expressed (FDR <0.001). In addition, the GBC cohort showed high heterogeneity and specific survival related subgroups were identified. To select key survival associated miRNA genes, we split our cohort of 40 GBC into two groups based on median survival (18 months). This revealed 8 miRNAs to be down and 16 to be up regulated in the poor outcome group (p < 0.05). The most down regulated miRNA was miR-145-5p and the top up regulated miRNA miR-575. Next, we sought to characterize the proor anti-tumorigenic function of the most differentially expressed miRNAs. Overexpression of miR-145 led to a significant reduction of cell proliferation and colony formation. In contrary, ectopic expression of miR-575 in GBC cells resulted in an increased proliferation rate and colony formation capacity. Gene expression profiling of cell lines overexpressing miR-145 or miR-575 revealed key affected oncogenic pathways. Conclusions.  MiRNA profiling of a well-characterized German GBC cohort identified pro- and anti-tumorigenic miRNAs. Functional validation confirmed the tumor suppressive function of miR-145 and the oncogenic properties of miR-575.

AG01.13 Depletion of the extracellular matrix molecules Tenascin C or Periostin results in impaired regeneration after cerulein-induced pancreatitis I. Regel*1, S. Hausmann2, S. C. Haneder3, N. Wagner1, K. Steiger3, M. Erkan4, J. Kleeff5, 6, I. Esposito1 Heinrich-Heine Universität, Institut für Pathologie, Düsseldorf, Deutschland, Klinikum rechts der Isar, Technische Universität, Chirurgische Klinik, München, Deutschland, 3Klinikum rechts der Isar, Technische Universität, Institut für Pathologie, München, Deutschland, 4Koc School of Medicine, Chirurgische Klinik, Istanbul, Türkei, 5Heinrich-Heine Universität, Chirurgische Klinik, Düsseldorf, Deutschland, 6The Royal Liverpool and Broadgreen University Hospitals, Chirurgische Klinik, Liverpool, Vereinigtes Königreich

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2

Background.  Extracellular matrix molecules, such as Tenascin C and Periostin are secreted by activated pancreatic stellate cells, which are thought to be mainly responsible for the fibrotic response during pancreatic injury and following regeneration processes. Both molecules are also highly expressed in liver fibrosis contributing to the severity of the fibrotic response. However, the functional role of various secreted extracellular matrix molecules on regenerative pancreatic or liver epithelial cells has not been addressed so far. Here, we analyzed the function of Tenascin C and Periostin in pancreatic exocrine regeneration after the induction of a severe acute pancreatitis. Methods.  Tenascin C and Periostin-deficient mice and wildtype control animals received repetitive cerulein injections, and a detailed histological analysis of pancreatic tissues was performed. Results.  Although there was no difference in pancreatitis severity in the acute inflammatory phase, the recovery of the exocrine pancreas was massively impaired in Tenascin C and Periostin deficient mice. Depletion of Tenascin C results in prolonged acinar-to ductal metaplasia and loss of Periostin expression was accompanied by strong pancreatic atrophy and acinar-to-adipocyte differentiation, which was also reflected in gene expression patterns. Conclusions.  Our data suggest that extracellular matrix molecules, such as Tenascin C and Periostin are crucial factors for proper exocrine lineage-specific regeneration after severe acute pancreatitis.

AG01.14 High throughput analysis in pancreatic cytology H. Jütte*, S. Liffers, B. Verdoodt, A. Tannapfel Institut für Pathologie Bochum, Bochum, Deutschland Background.  Pancreatic ductal adenocarcinomas (PDAC) may be difficult to differentiate from chronic pancreatitis in cytological specimens. We wanted to examine, if multiple parameter analysing, using Next-Generation-Sequencing (NGS) may be of benefit. Methods.  For this study, we examined 26 pancreatic cytology specimens of patients with known PDAC with NGS. DNA isolated from cytology smears was prepared by multiplex PCR for cancer-relevant genes using the GeneRead system (Qiagen), followed by ligation of barcode adaptors (BIOO) to the amplicons. The resulting libraries were sequenced on a MiSeq System (Illumina); data were analysed using the NextGene v.2.4.1 (SoftGenetics LLC) software. The cytologies were split into two groups: The first group (n = 7) had negative cytology for PDAC, although it was later found in the surgical specimen. The second group (n = 19) showed positive cytology for PDAC, and this was later confirmed with histology. NGS was performed on these cytologies, afterwards we examined the readout for mutations of the most commonly mutated “driver” genes in PDAC: KRAS, TP53, SMAD4 and CDKN2A. Results.  In the group with benign cytology (and a subsequent carcinoma diagnosed in the resection specimen), three patients (43 %) had at least one mutation in the studied genes (KRAS, TP53, SMAD4, CDKN2A).

We could find altogether six mutations in this group. Two mutations were found in KRAS, TP53 and SMAD4, respectively. KRAS showed two substitution missense mutations. TP53 and SMAD4 had multiple missense mutations. The other four patients displayed wild type of the examined genes. The group with positive cytology revealed thirteen patients (68 %) with at least one mutated gene. A total of 22 mutations could be detected in this group. Ten missense mutations were found in the KRAS gene. We found nine missense mutations in the TP53 gene. SMAD4 was altered in two cases, showing one deletion/insertion and one missense mutation. CDKN2A was altered in two cases, showing one missense and one nonsense mutation. In six patients with positive cytology no mutation could be detected. Conclusions.  Our results indicate that multiple parallel analysis of several driver genes for pancreatic cancer is not suitable to differentiate PDAC from chronic pancreatitis in cytology, since we identified mutations in three of seven patients with benign cytology. Even in the samples of the patients with positive cytology, mutations could not be shown in six of nineteen cases.

AG01.15 Desmogleins as prognostic biomarkers in resected pancreatic ductal adenocarcinoma S. Ormanns*1, A. Altendorf-Hofmann2, R. Jackstadt3, D. Horst1, G. Assmann1, Y. Zhao4, C. Bruns4, T. Kirchner1, T. Knösel1 1 Ludwig-Maximilians-Universität München, Pathologisches Institut der Universität München, München, Deutschland, 2Klinik für Allgemein-, Viszeral- und Gefäßchirurgie, Friedrich-Schiller-Universität, Jena, Deutschland, 3Beatson Institute for Cancer Research, Universität Glasgow, Glasgow, Vereinigtes Königreich, 4Universitätsklinikum Magdeburg, Universitätsklinik für Allgemein-, Viszeral- und Gefäßchirurgie, Magdeburg, Deutschland

Background.  Frequent disease relapse and a lack of effective therapies, result in a very poor outcome of pancreatic ductal adenocarcinoma (PDAC) patients. Thus, identification of prognostic biomarkers and possible therapeutic targets is essential. Besides their function in cell-cell adhesion, desmogleins may play a role in tumor progression and invasion, which has not been investigated in PDAC to date. This study evaluated desmoglein expression as biomarker in PDAC. Methods.  Using immunohistochemistry, we examined desmoglein 1 (DSG1), desmoglein 2 (DSG2) and desmoglein 3 (DSG3) expression in the tumor tissue of 165 resected PDAC cases. Expression levels were correlated to the patients’ clinicopathological parameters and post-operative survival times. We confirmed these results in two independent gene expression datasets. Results.  36 % of the tumors showed high DSG3 expression, which correlated significantly with shorter patient survival (p = 0.011) and poor tumor differentiation (p < 0.001) whereas no such association was detected for DSG1 or DSG2. In RNA-Seq data and in microarray expression data, high DSG3 expression correlated significantly with poor survival (p = 0.000356 and p = 0.00499). Conclusions.  We identify DSG3 as a negative prognostic biomarker in resected PDAC, as high DSG3 expression is associated with poor overall survival and poor tumor specific survival. These findings suggest DSG3 and its downstream signaling pathways as possible therapeutic targets in DSG3 expressing PDAC.

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Abstracts AG01.16 Pregnancy-specific beta-1-glycoprotein 1 in Pancreatic Adenocarcinoma H. Füllgraf*1, S. Küsters2, M. Werner1, 3, 4, M. Kuehs1, U. F. Wellner5, J. Shahinian6, O. Schilling7, P. Bronsert1, 3, 4 1 Institut für Klinische Pathologie, Department für Pathologie, Universitätsklinikum, Freiburg, Deutschland, 2Klinik für Allgemeinund Viszeralchirurgie, Universitätsklinikum, Freiburg, Deutschland, 3 Tumorzentrum Freiburg, Universitätsklinikum, Freiburg, Deutschland, 4 German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Deutschland, 5Klinik für Chirurgie (UKSH), Campus Lübeck, Lübeck, Deutschland, 6Herzchirurgie, Universitätsspital, Basel-CH, Schweiz, 7Institut für Molekulare Medizin und Zellforschung, AlbertLudwigs-Universität, Freiburg, Deutschland

Background.  Pancreatic adenocarcinoma (PDAC) is the fourth most common gastrointestinal cancer in Europe with an annual incidence of over 200,000 pancreatic cancer cases worldwide. Pregnancy-specific beta-1-glycoprotein (PSG-1) is mostly renowned for its abundant placental expression during pregnancy. A limited number of reports suggest PSG-1 expression in adenocarcinomas as a putative onco-fetal biomarker. Here, we present first comprehensive immunohistochemical PSG-1 analyses in a PDAC cohort. Methods.  Our study cohort comprises 105 PDAC patients. Surgical and Pathological diagnostics were performed at the University Medical Center, Freiburg. After histological revalidation tumor slides were stained immunohistochemically for PSG-1. Staining intensity (0, 1, 2 and 3), percentage of positive tumor cells and PSG-1 localization (cytoplasmatic or cytoplasmatic/luminal) were evaluated. Survival analyses were performed via Kaplan-Meier-Logrank test. Multivariate analyses was performed using Cox proportional hazards model and involved clinico-pathological predictors reaching at least trend levels (p < 0.15). All statistical tests were performed two-sided. Results.  Median patient age was 66 years. According to the current UICC-Classification most patient were classified T3 (84.6 %). 32 (30.8 %) of all tumors were exclusively intracellular PSG-1 positive, 72 (69.2 %) tumors showed a PSG-1 luminal and intracellular positivity. Univariate analyses revealed lymph node ratio (p = 0.014) and PSG-1 localization intracellular vs luminal/intracellular PSG-1 (p < 0.001) as significant prognosticators. For the multivariate analysis univariate significant correlations and trends (p < 0.15) analysis were included. Hereby, only intracellular vs luminal/intracellular PSG-1 expression (p = 0.001) revealed as an independent predictor for overall survival. Conclusions.  The poor outcome of PDAC necessitates a better understanding of protein determinants particularly because of its malignant behavior. We demonstrate abundant PSG expression in PDAC as an independent prognostic factor. Examination of published reports on cancer-associated PSG expression supports our finding. Our results further highlight that co-occurrence of luminal and intracellular PSG localization correlates with a worse prognosis as compared to an exclusively luminal localization. These results highlight a new aspect of PSG biology within the context of PDAC pathology and warrant further investigation.

AG01.17 Non-invasive detection of tumor cell content as a negative predictor of survival in PDAC K. Steiger*1, I. Heid2, M. Trajkovic-Arsic3, M. Settles2, A. Steingoetter2, M. Schwaiger4, C. Jäger5, J. Kleeff5, J. Siveke3, R. Braren2, I. Esposito6 Institut für allgemeine Pathologie und pathologische Anatomie, TU München, München, Deutschland, 2Institut für diagnostische und interventionelle Radiologie, Klinikum recht der Isar, München, Deutschland, 3 II. Medizinische Klinik, Klinikum rechts der Isar, München, Deutschland, 4 Nuklearmedizinische Klinik, Klinikum rechts der Isar, München, 1

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Deutschland, 5Chirurgische Klinik und Poliklinik, Klinikum Rechts der Isar der Technischen Universität München, München, Deutschland, 6Institut für Pathologie, Universitätsklinikum Düsseldorf, Düsseldorf, Deutschland Background.  Molecular and morphological heterogeneity are key factors for prognosis, therapy response and resistance in pancreatic ductal adenocarcinoma (PDAC). Non-invasive biological imaging methods detecting PDAC growth patterns are not available to date but might allow a better prognostic or therapeutic stratification of non-resectable patients. The aim of this study was to define prognostic relevant parameters for PDAC heterogeneity by non-invasive biological imaging methods. Methods.  Murine (mPDAC; N = 141) and human (hPDAC; N = 94) PDAC were analyzed histologically and immunohistochemically. According to their growth pattern, tumors were classified by tumor-cell content using three categories: poor (PDAClow), medium (PDACmed) and rich (PDAChigh). In hPDAC, tumors were classified according to the area with the highest tumor-cell content covering at least 1mm2. The results were compared to the data obtained by diffusion weighted- and dynamic contrast enhanced-MRI (DW-MRI, DCE-MRI). Results.  In mPDAC, the tumor cell content showed excellent correlation with the DW-MRI derived ADC (apparent diffusion coefficient) parameter. When applied to hPDAC (hPDAClow, N = 55; hPDACmed, N = 27). Tumors with low cellularity exhibited a significantly better prognosis (19.8 versus 13.0 months, Log rank, p < 0.002). In analogy to the murine model, the ADC parameter allowed to define tumor cellularity in hPDAC pre-operatively. Conclusions.  This study identifies tumor cellularity as a negative predictor of prognosis in PDAC. Furthermore, the ADC DW-MRI imaging parameter is a promising biomarker for non-invasive classification of PDAC that may be used for evaluation of subtype-directed therapeutic approaches.

AG01.18 Impact of SPARC expression level on outcome in patients with advanced pancreatic cancer not receiving nab-paclitaxel: A pooled analysis from prospective clinical and translational trials S. Bächmann1, S. Ormanns*1, M. Haas2, V. Heinemann2, T. Kirchner1, S. Böck2 1 Ludwig-Maximilians-Universität München, Pathologisches Institut der Universität München, München, Deutschland, 2Ludwig-MaximiliansUniversität, Medizinische Klinik und Poliklinik III und Comprehensive Cancer Center, Klinikum Großhadern, München, Deutschland

Background.  Conflicting results on the role of secreted protein acidic and rich in cysteins (SPARC) expression have been reported in resected pancreatic ductal adenocarcinoma (PDAC) and its prognostic and/or predictive role in advanced PDAC (aPDAC) has not been extensively investigated yet. This study was designed to evaluate SPARC expression as a biomarker in aPDAC patients (pts) not receiving nab-paclitaxel. Methods.  Using immunohistochemistry, we examined the stromal as well as the tumoral (i. e. cytoplasmic) SPARC expression in tumor tissue (primary tumors and metastases) of 134 aPDAC pts participating in completed prospective clinical and biomarker trials. SPARC expression levels were correlated to the patients’ clinicopathological parameters and survival times. Results.  Sixty-seven percent of the analyzed tumors showed high stromal SPARC expression, which was not associated with survival (median 9.1 vs. 7.6 months, p = 0.316). A positive cytoplasmic SPARC expression was detected in 55 % of the tumors and correlated significantly with inferior survival (7.8 vs. 8.4 months, p = 0.032). This association was strongest for pts where primary tumor tissue was examined (7.9 vs. 11.9 months, p = 0.030), whereas no significant correlation was detected for pts where only metastatic tissue was available (7.0 vs. 7.8 months, p = 0.452). Cytoplasmic (positive vs. negative: 7.3 vs. 9.9 months, p = 0.012) but not stromal (high vs. low: 9.9 vs. 7.6 months, p = 0.180) SPARC expression was associated with survival in pts receiving palliative gemcitabine-based chemother-

apy, whereas no such association was detected for fluoropyrimidine-based chemotherapy. Conclusions.  We identified tumoral/cytoplasmic SPARC expression in the primary tumor as a biomarker associated with inferior survival in aPDAC. Cytoplasmic SPARC expression may furthermore act as a negative predictive biomarker in pts treated with gemcitabine-based chemotherapy.

AG01.19 Genetic Complexity of the Tumor Microenvironment in Pancreatic Cancer featured by Genetic and Epigenetic changes in Stromal Cells Regulating Tumor Progression M. Wartenberg*, S. Haemmig, E. Vassella, A. Perren, E. Karamitopoulou Institut für Pathologie – Universität Bern, Bern, Schweiz Background.  In Pancreatic ductal adenocarcinoma (PDAC), a highly lethal malignancy with a rich stromal component, tumor-stroma interactions have been pointed out to be a crucial factor for neoplastic progression. In this regard the process of epithelial mesenchymal transition (EMT), thought to be reflected by the presence of dissociative growing cancer cells, coined “tumor budding cells”, has drawn attention, as tumor budding is an independent prognostic factor in PDAC. Methods.  mRNA-in-situ-Hybridization and immunostaining have been applied on stroma cells of 120 well-characterized PDACs using multipunch tissue-microarrays. Levels of EMT factors (SNAIL1, TWIST, ZEB1, ZEB2) and common tumor suppressors (SMAD proteins and PTEN) were assessed on the protein and mRNA level. Additionally, Laser Capture Microdissection, RNA extraction and subsequent qRT-PCR were pursued to analyze specific microRNA-sequences from juxta-tumoral and tumor-remote stroma. Special focus was set on stromal cells surrounding EMT-like cancer cells at the invasive front. Results.  Stromal cells surrounding EMT-like cancer cells, overexpressed EMT-associated markers like SNAIL1, TWIST, ZEB1 and ZEB2 at protein and mRNA level among the tumor microenvironment of aggressive PDAC cases. For a subset of cases, a concomitant loss of SMAD proteins and PTEN in tumor and stroma cells was associated with aggressive features like distant metastasis (p = 0.0045), larger tumor size (p = 0.0112) and worse overall-survival (p = 0.021). Interestingly, stroma cells with PTEN loss showed frequent chromosome-10 deletion and miR-21 overexpression. Comparing juxta-tumoral and tumor-remote stroma, different expression patterns for miR-21, miR-210, miR-200b and miR-203 could be detected. Moreover, microRNA expression in stromal cells correlated with the expression of target proteins like ZEB1 (for miR-200b) and PTEN (for miR-21). Conclusions.  In PDAC, epigenetic changes involving microRNA expression among stromal cells of the tumor microenvironment seem to be crucially involved in the regulation of tumor progression, especially at the invasive front. Our findings emphasize a potential role of novel therapies targeting stromal cells and thus altering the tumor microenvironment in pancreatic cancer in a potential favorable manner.

AG01.20 SMAD4-, CDKN2A- and TP53- mutations indicate a differential therapeutic response to Erlotinib/Gemcitabine and Folfirinox-therapy in pancreatic ductal adenocarcinoma A. Muckenhuber*1, A. Berger2, N. Pfarr1, M. Jesinghaus1, A. Trumpp3, M. Sprick3, C. Springfeld2, W. Weichert1 1 Institut für Allgemeine Pathologie und Pathologische Anatomie der Technischen Universität München, München, Deutschland, 2Nationales Centrum für Tumorerkrankungen (NCT), Medizinische Onkologie, Heidelberg, Deutschland, 3Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Deutschland

Background.  Primary chemotherapy is the therapy of choice in non-resectable and metastasized pancreatic ductal adenocarcinoma (PDAC). Despite recent advances like the introduction of Erlotinib-augmented Gemcitabine therapy or Folfirinox-regimes as more efficacious yet more physically demanding and side-effect prone therapeutic alternatives outcomes remain poor and the survival benefit limited. Therefor pre-therapeutic patient stratification seems mandatory to limit the negative impact of inefficacious therapies. Methods.  A retrospective cohorte of 132 PDAC patients that received primary chemotherapy after histologic validation of diagnosis was created. 21 patients received Gemcitabine-monotherapy, 43 Erlotinib+Gemcitabine and 68 Folfirinox. The material of the tumor biopsies was analyzed by targeted next generation sequencing using a custom built pancreatic cancer primer panel (40 genes, 165 exons, 280 amplicons). Mutational data of 89 patients could be obtained. Results.  A wide range of mutations was found, KRAS being the most frequently mutated gene (85 samples) followed by TP53 (66), CDKN2A (31) and SMAD4 (14). Genetic alterations at lower frequencies were detected in 18 additional genes. Patients with CDKN2A-tumor mutations showed better survival in the Erlotinib+Gemcitabine therapy group (p-value 0.035) while performing poorer than CDKN2A-wildtyp tumors under Gemcitabine monotherapy (p-value 0.06) or Folfirinox. If tumors showed TP53-mutations better survival was observed under Erlotinib+Gemcitabine therapy while no survival differences could be seen in the other treatment groups. The positive survival effect of CDKN2A and TP53 mutations under Erlotinib+Gemcitabine therapy was additive (p-value 0.008). Patients with SMAD4 tumor mutations performed significantly worse under Gemcitabine based chemotherapies (p-value 0.002) while showing slightly better survival in the Folfirinox treatment group. Conclusions.  Our data indicate that mutational analyses of CDKN2A, TP53 and SMAD4 might be used as predictive biomarkers for patient stratification as patients with CDKN2A and TP53 mutations seem to have a higher benefit from Erlotinib/Gemcitabine combination therapy while patients with SMAD4 mutations show better survival under Folfirinox-therapy while performing exceptionally poor under Gemcitabine-based therapies.

AG01.21 Characterization Crohn’s disease-associated cancers of the intestine F. Lasitschka*1, O. Drukarczyk2, M. Kadmon3, S. Schiessling2 Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, Klinik für Viszeral- und Transplantationschirurgie, Ruprecht-KarlsUniversität, Heidelberg, Deutschland, 3Abteilung Medizinische Ausbildung und Ausbildungsforschung, Carl von Ossietzky Universität Oldenburg, Oldenburg, Deutschland 1

2

Background.  Whether the cancer risk is increased in patients with Crohn’s disease compared to the general population is controversial. According to a meta-analysis in 2005 there seems to be an increased risk by a factor of 2.5 for colorectal cancer (CRC). Immunohistochemical and genetic differences between the two cancer entities have not yet been adequately studied. Methods.  The data of all Crohn’s disease patients operated at the Surgical University Clinic Heidelberg since 2002 were evaluated. Currently 484 patients are documented with their individual course of the disease since 1990, including 22 patients with Crohn’s associated colon neoplasia. The data of these patients were set in comparison regarding clinical, pathological and genetic findings (using next-generation sequencing (NGS) and tissue microarrays (TMA)) and were analyzed and compared with data of sporadic CRC. Results.  22 patients (4.5 %) were diagnosed with a colon carcinoma. 15 of the carcinomas (68.2 %) were at an advanced stage (T3, T4). In four patients (18.2 %) the carcinoma was newly diagnosed after pathological work up. 21 cases (95.5 %) had a CRC one case (4.5 %)was carcinoma of the small intestine. The most common tumor location was the rectum (11, 50 %), followed by the cecum (4, 18.18 %). The average age at the time Der Pathologe Suppl 1 · 2016

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Abstracts of tumor diagnosis was 46.3 years. The timespan from the initial diagnosis of Crohn’s Disease to tumor diagnosis was on average 18.4 years, for females, this period was an average of six years longer than in men (p <0.0001). The median survival time from primary resection onwards was 61.2 months. The 10-year survival rate was 26.8 %. Only seven patients remained free of tumor over an observation period of an average of 7.52 years (25–217 months). In 14 patients an immunohistochemical analysis (TMA) was carried out and in 12 of 14 patients a mutational analysis using NGS could be done. Conclusions.  Colorectal cancer in Crohn’s disease seems more likely to be located in the area of ​​the cecum and ascending colon and patients are affected much earlier than patients with sporadic CRC. The time interval between initial diagnosis of Crohn’s disease and cancer diagnosis is significantly longer in men than in women. Crohn’s associated CRC appear to have a worse prognosis than sporadic CRC. The 10-year survival for disease-associated carcinomas was statistically significantly worse.

highly significant (p = 0.0001) after treatment. The amount of CD8+ and CD8+/GrB+ T cells in the tumor epithelium of pretheraputic biopsies corresponded to pT-stage (p = 0.022) and to tumor regression grade (TRG). The proportion of GrB+ relative to CD8+ cells was significantly higher in patients with better treatment response than in those with less tumor regression (p = 0.003). Conclusions.  The amount of CD8+ and CD8+/GrB+ T cells in pretherapeutic biopsies of rectal cancer patients corresponded to tumor stage and TRG after resection. Neoadjuvant RCTx seems to modify the CD8+/ GrB+ ratio; moreover, not only the number but in particular the ratio of CD8+GrB+ cells was associated with TRG after neoadjuvant RCTx and might be a predictive marker for favorable response to neoadjuvant RCTx and improved clinical outcome.

AG01.22 The ratio of CD8+/GranzymeB+ lymphocytes in rectal cancer patients is modified by neoadjuvant radiochemotherapy and predictive for tumor regression

A. Ahadova*1, M. von Knebel Doeberitz1, H. Bläker2, M. Kloor1

U. Sommer*1, A. Jarosch1, A. Bogner2, C. Reißfelder2, J. Weitz2, D. E. Aust1, G. B. Baretton1 Institut für Pathologie, Universitätsklinikum Carl Gustav Carus, Dresden, Deutschland, 2Chirurgische Klinik, Universitätsklinikum Carl Gustav Carus, Dresden, Deutschland

1

Background.  It has been proven, that the immunological tumor microenvironment with its various cell components is of paramount importance to tumor prognosis. Lymphocytes in general and in particular CD8+/GranzymeB+ cells (CD8+GrB+) as cytotoxic effector T-cells are major players in tumor immunity and increased numbers of tumor-infiltrating and peritumoral CD8+/GrB+ T-cells are associated with improved survival. We analyzed the immune cell infiltrate in rectal cancer patients before and after neoadjuvant treatment as well as in primarily resected patients to evaluate the association of CD3+/ki67 and CD8+/GrB+ T cells to tumor regression and the neoadjuvant treatment related modulation of the immune cell infiltrate. Methods.  A total of 139 resection specimens from rectal cancer patients, who underwent neoadjuvant radio-/radiochemotherapy (RCTx) were studied; from 24 of these cases also pretherapeutic tumor biopsies were available. In comparison a cohort of 31 primarily resected patients with matched TNM-stage and sex, was analyzed. FFPE sections were immunostained in double reactions for CD3/Ki67 and CD8/GrB. Slides were digitized and evaluated for the number of intraepithelial and peritumoral immune cells. Results.  The number of CD3+ cells in the peritumoral stroma was significantly lower in patients with neoadjuvant RCTx than in unpretreated patients (p = 0.0001), whereas the remaining cells after neoadjuvant RCTx seem to proliferate in a slightly higher manner. Whereas neoadjuvant RCTx lead to a not significant increase of CD8+/GrB+ cells in the peritumoral stroma, the proportion of CD8+ to GrB+ cells increased

AG01.23 Non-polypous colorectal cancers in Lynch syndrome 1 Pathologisches Institut, Abteilung für Angewandte Tumorbiologie, Heidelberg, Deutschland, 2Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland

Background.  Lynch syndrome is the most frequent inherited cancer Syndrome. Mutation carriers have a 50 % lifetime risk of developing colorectal cancer (CRC). Cancers in Lynch syndrome patients show high-level microsatellite instability (MSI-H), resulting from DNA mismatch repair (MMR) deficiency. Regular colonoscopy programs directed to identification and removal of colorectal adenomas as cancer precursors have significantly decreased CRC incidence and mortality in Lynch syndrome. However, a significant part of Lynch syndrome mutation carriers still develops CRCs despite regular colonoscopy. One possible reason of such interval cancers could be non-polypous precursor lesions such as the recently described MMR-deficient crypt foci. Methods.  We analyzed the histological appearance of 46 Lynch syndrome-associated CRCs and compared them to 34 sporadic microsatellite unstable cancers. Colon cancer-associated genes were analyzed for mutations, fragment length analysis and Sanger sequencing. Beta-catenin expression was monitored by immunohistochemistry. Results.  25/40 (62.5 %) Lynch syndrome-associated carcinomas lacked evidence of polypous growth, compared to 17/34 (50 %) sporadic MSI-H cancers. A phenotype suggesting immediate invasive growth was strongly associated with CTNNB1 mutations and nuclear beta-catening staining, which were present in 8/46 (17.4 %) Lynch syndrome-associated cancers, but absent in 34 sporadic MSI-H cancers (p = 0.01) (. Fig. 1). Conclusions. Interval cancers in Lynch syndrome may result from non-polypous precursor lesions, such as MMR-deficient crypt foci, and therefore represent a distinct subgroup of CRC. CTNNB1 mutations may drive the development of non-polypous Lynch syndrome-associated cancers (. Fig. 2). Active, primary preventive measures should be considered in Lynch syndrome mutation carriers to tackle non-polypous precursors, which cannot be detected by colonoscopy.

Fig. 1 I AG01.23 9 Nuclear beta-catenin staining in invasively growing cancer cells

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Fig. 2 I AG01.23 7 Colorectal cancer pathogenesis in Lynch syndrome

AG01.24 VEGFR2 signaling blocks cancer cell senescence to promote colorectal tumorigenesis in mice irrespective of angiogenesis S. Foersch*1, 2, T. Sperka3, C. Lindner2, A. Taut2, K. L. Rudolph3, G. Breier4, F. Boxberger2, T. Rau5, A. Hartmann5, M. Stürzl6, N. Wittkopf2, L. Haep2, S. Wirtz2, M. F. Neurath2, M. Waldner2 Institut für Pathologie, Universitätsmedizin, Mainz, Deutschland, 2FAU Erlangen-Nürnberg, Medizinische Klinik 1, Erlangen, Deutschland, 3LeibnizInstitut für Alternsforschung, Fritz-Lipmann-Institut, Jena, Deutschland, 4 Universitätsklinikum Carl Gustav Carus, Institut für Pathologie, Dresden, Deutschland, 5FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland, 6Chirurgische Klinik, Universität Erlangen-Nürnberg, Erlangen, Deutschland 1

Background.  Cellular Senescence is a potential barrier against malignant transformation[1]. We investigated whether the VEGF/VEGFR2 signaling pathway regulates proliferation and senescence of tumor cells in mice with colorectal cancer (CRC). Methods.  Colitis-associated colorectal cancer was chemically (AOM/ DSS) induced in VEGFR2ΔIEC mice, which do not express VEGFR2 in the intestinal epithelium, and VEGFR2fl/fl mice (controls). Inflammation and tumor development were evaluated by endoscopy. Intestinal tissues were collected for histological, immunohistochemical, immunoblot and quantitative polymerase chain reaction analyses. Findings from the in vivo experiments were confirmed in human HCT116 colorectal cancer cells. Furthermore we analyzed colorectal tumor samples from patients before and after treatment with Bevacizumab. Results. VEGFR2ΔIEC mice developed significantly fewer and better differentiated tumors than control mice. A greater number of the tumor cells from VEGFR2ΔIEC mice were in in a senescent state than tumor cells from control mice. We found VEGFR2 to activate phosphatidylinositol-4,5-bisphosphate-3-kinase and AKT, resulting in inactivation of p21 in HCT116 cells. Inhibitors of VEGFR2 and AKT induced senescence in HCT116 cells. Furthermore, cellular senescence promoted an anti-tumor immune response led by CD8+ T cells in mice. Patients whose tumor samples showed an increase senescent cells and a higher number of CD8+ T cells after treatment with Bevacizumab had longer progression-free survival than patients in which the proportion of senescent tumor cells did not change before and after treatment. Conclusions.  Our results highlight the role of VEGF/VEGFR2 signaling as an independent tumor growth factor completely irrespective of angiogenesis. Inhibition of VEGFR2 signaling leads to senescence of human and mouse colorectal cancer cells. VEGFR2 interacts with phosphatidy-

linositol-4,5-bisphosphate-3-kinase and AKT to inactivate p21. Colorectal tumor senescence and p21 level correlate with patient survival during treatment with Bevacizumab[2]. References 1. Collado M et al (2007) Cellular Senescence in Cancer and Aging, Cell, 130(2): 223–233, 2. Foersch S et al (2015) VEGFR2 Signaling Prevents Colorectal Cancer Cell Senescence to Promote Tumorigenesis in Mice With Colitis, Gastroenterology, 149(1): 177–189,

AG01.25 Genetic characterization of mixed adeno-neuroendocrine carcinomas (MANECs) of the colorectum by Next generation sequencing C. Woischke*1, C. Schaaf1, H. Yang2, D. Schaeffer3, M. Vieth4, L. Veits4, H. Geddert5, P. Greif6, S. Vosberg6, A. Jung1, T. Kirchner1, D. Horst1 Pathologisches Institut, Ludwig-Maximilians-Universität, München, Deutschland, 2Columbia University, Pathology, New York, Vereinigte Staaten von Amerika, 3University of British Columbia, Pathology, Vancouver, Kanada, 4 Institut für Pathologie, Bayreuth, Deutschland, 5St. Vincentius-Kliniken, Institut für Pathologie, Karlsruhe, Deutschland, 6Medizinische Klinik III, Ludwig-Maximilians-Universität, München, Deutschland 1

Background.  Mixed adenoneuroendocrine carcinomas (MANECs) of the colorectum are rare but highly aggressive neoplasms, and are characterized by an admixture of glandular and neuroendocrine components. Since little is known about the genetic basis of these tumors, we here comparatively evaluated the spectrum of genetic mutations in both components of MANECs. Methods.  In a collection of 15 MANECs of the colorectum, CD56, synaptophysin and NSE stainings were used to identify and distinguish neuroendocrine from glandular components. For 10 cases, these two components then were microdissected, their DNA isolated, and subjected to next generation sequencing (NGS), covering a panel of 50 cancer related genes. A subset of 3 cases was further investigated by whole exome sequencing analysis. Results.  As expected, neuroendocrine components of all MANECs expressed at least one neuroendocrine marker and showed significantly higher proliferation rates compared to respective glandular components. Panel sequencing revealed shared mutations of the two components in each case, with TP53, KRAS, APC and PIK3CA being most frequent. Additionally,

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Abstracts a subset of cases showed mutations in NOTCH1 and RB1, some of which were exclusive to the neuroendocrine component. Whole exome sequencing revealed partially overlapping copy number alterations. Conclusions.  Here we show that glandular and neuroendocrine components in MANECs share several key mutations, suggesting a clonal origin. Similar to other neuroendocrine carcinomas, TP53, NOTCH1 and RB1 were commonly found. Whole exome sequencing indicated early separation of both tumor components in MANECs.

AG01.26 Mixed adenoneuroendocrine carcinomas and adenocarcinomas of the colorectum differ in their genetic profiles: an analysis of 12 cases M. Jesinghaus1, B. Konukiewitz*1, K. Steiger1, A. Stenzinger2, W. Weichert1, N. Pfarr1, G. Klöppel1 Technische Universität München, München, Deutschland, Universitätsklinikum Heidelberg, Heidelberg, Deutschland

1 2

Background.  Mixed adenoneuroendocrine carcinomas (MANECs) of the colorectum represent a rare entity of colorectal neoplasms characterized by a gland-forming and a neuroendocrine component. These neoplasms have been intensively studied regarding their MSI-status, but information on their genetic makeup in comparison to that of colorectal carcinomas is still scarce. Methods.  MANECs of the colorectum (n = 12) were characterized by immunohistochemistry using a marker panel including synaptophysin, chromograninA, Ki-67, somatostatin receptor 2A, p53, MUC1, MUC2, CK7, CK20 and CD56. Formalin fixed tumor tissue was microdissected and analysed by Ion Torrent semiconductor-based next-generation sequencing of hotspot regions of 29 genes commonly found in colorectal carcinomas and 3 genes typically linked to neuroendocrine neoplasms, including TP53, RB1 and RET. Coverage analysis for detection of amplifications and deletions was also performed. Results.  In total, we found 20 mutations and one deletion in the investigated genes in our cohort. 6/12 MANECs harbored TP53-mutations. KRAS and BRAF were each mutated in 3/12 cases. Two cases showed SOX9 and FBXW7 mutations, respectively. MSH3, PTEN, ERBB2 and APC mutations were found in one case each, while PIK3CA mutations were absent. RB1 was deleted in one case, while point mutations were lacking. No mutations were detected in two cases. Conclusions.  Mutations typical for colorectal adenocarcinoma, such as APC and PIK3CA, or genes, linked to neuroendocrine tumor differentiation, e. g. RB1 and RET, were infrequent or even absent in our series of colorectal MANECs. This indicates that the examined colorectal MANECs are genetically distinct from both conventional colorectal carcinomas and pure neuroendocrine neoplasms, suggesting a distinct molecular pathway.

AG01.27 PD-L1 expression in malignant tumors of the right colon correlates with microsatellite instability and mutations in RAS pathway genes I. S. Feder *1, M. Karp *1, B. Verdoodt1, M. Vogt1, S.-T. Liffers1, A. Reinacher-Schick2, A. Tannapfel1 1 Institut für Pathologie, Ruhr-Universität, Bochum, Deutschland, 2St. JosefHospital, Ruhr-Universität, Bochum, Deutschland

Background.  Accumulated genetic and epigenetic alterations of tumor cells potentially allow the immune system to distinguish tumor cells from healthy cells. Especially mismatch repair deficient tumors accumulate a larger number of mutations, and may therefore become more immunogenic. However most tumors are able to escape the immune response. PD1, an immune-inhibitor, and its ligands PD-L1 and PD-L2 play a crucial role in this immune-suppressive mechanism.

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PD-L1 inhibitors are new immuno-therapeutic antibody based drugs for treatment of cancer. Blocking PD-L1 leads to enhanced anticancer immunity. These drugs are most effective when the tumor, especially the tumor infiltrating immune cells, express high levels of PD-L1. On the other hand, tumors with defects in the mismatch repair system leading to microsatellite instability (MSI) also show clinical benefit of PD-L1 blockade. The aim of this project was to investigate the PD-L1 expression of cancer of the right colon, and its correlation to MSI and to common mutations found in colon cancer. Methods.  50 FFPE samples of cancer of the right colon with UICC stage I to III resected between 2009 and 2011 were immuno-histologically stained with anti-PD-L1 antibody (abcam). The percentage of positive tumor cells and tumor-infiltrating immune cells were counted separately. For the same samples, MSI-status was determined, and mutations in genes relevant for colorectal cancer development were detected by next generation sequencing. Statistical significance between groups was determined by the Mann-Whitney-U test. Results.  Staining for PD-L1 was membranous and occurred in distinct foci of cells. Fifteen tumor samples showed staining in at least 1 % of neoplastic cells. MSI was more frequently observed in tumors with higher expression of PD-L1 (p = 0.0405). PD-L1 expression also correlated with the presence of any mutations in the RAS-pathway (KRAS, NRAS, BRAF, and MAP2K1; p = 0.01143). BRAF-mutant samples were typically MSI-high as well. No associations were observed with clinicopathologic parameters nor with mutations in other genes that are often mutated in colorectal cancer like PIK3CA, PTEN, CDKN2A, CTNNB1, MET, SMAD4, or TP53. Conclusions.  The correlation between PD-L1 expression and MSI as shown in this work may explain the prognostic importance of the MSI status for the response to treatment with a PD-L1 inhibitor. * I. S. Feder and M. Karp contributed equally to this work

AG01.28 SWI/SNF Complex expression in unselected colorectal cancer C.-I. Geppert*1, K. Erlenbach-Wünsch1, S. Merkel2, A. Hartmann1, A. Agaimy1 FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland, Chirurgische Klinik, Universität Erlangen-Nürnberg, Erlangen, Deutschland

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Background.  The switch/sucrose-non-fermenting (SWI/SNF) complex is a highly preserved group of interdependent proteins involved in the chromatin remodelling and thus in the regulation of vital cellular processes such as proliferation and differentiation. Current knowledge suggests a tumor suppressor function for the SWI/SNF complex subunits. Antibodies directed against several components of the SWI/SNF complex are still emerging with promising results. In the gastrointestinal (GI) tract, frequent loss of different SWI/SNF components has been recently shown in undifferentiated GI carcinomas with frequently rhabdoid morphology. However, SWI/SNF expression in unselected colorectal cancer (CRC) cohorts has not been studied before. Methods.  We analyzed 100 consecutive CRC cases for expression of six SWI/SNF subunits by immunohistochemistry on tissue microarrays using antibodies against SMARCB1 (INI1), SMARCA2, SMARCA4, SMARCC1, SMARCC2 and ARID1A. Results.  Complete loss of expression restricted to the tumor cells was seen in 6 cases (6 %) and included ARID1A loss (n = 3), and loss of SMARCC1 (n = 2) and SMARCA4 (n = 1). Other markers showed intact nuclear expression. The stromal cells and normal glands in the background showed retained expression. However, variable reduction in expression intensity of the different markers was seen in several cases, but the biological relevance of this is not clear yet. None of the SWI/SNF-deficient cases showed rhabdoid cell morphology and, instead, most displayed variable gland formation. Conclusions. SWI/SNF deficiency may be encountered in otherwise gland-forming (conventional) CRC cases. The relationship of this finding to the mismatch repair (MMR) status and MSI as well as the prognostic impact remains to be clarified.

AG01.29 Changes in DNA Methylation of PITX2 and the non-coding RNA PITX2-lncRNA during Transition from Normal to Malignant Colorectal Tissue B. Uhl*, A. van Ellen, S. Meller, D. Dietrich, G. Kristiansen Institut für Pathologie, Universitätsklinikum, Bonn, Deutschland Background.  The DNA methylation of PITX2 is known to be associated with the patient’s outcome in prostate, breast and lung cancer. PITX2 is also involved in the regulation of androgen receptor, MYC oncogene and Wnt/ß-catenin pathway. The latter one is an important pathway in the pathogenesis of colorectal cancer (CRC). The non-coding RNA PITX2-lncRNA is located 2 kb downstream of the PITX2 locus. In previous studies its DNA methylation revealed a sharp distinction between non-small cell lung cancer subtypes. This study investigated a possible correlation between the state of colorectal carcinogenesis and the DNA methylation of PITX2 and PITX2-lncRNA. Methods.  Colorectal tissue samples were obtained from patients who underwent surgery at the University Hospital Bonn. 176 FFPET specimens were analyzed including normal adjacent tissue (NAT) and inflammation (n = 75), subtypes of adenomas (n = 75) and cancerous colorectal tissue (n = 26). The DNA methylation levels in bisulfite-converted DNA were analyzed using quantitative, methylation-specific qPCR assays. Results.  The DNA methylation of PITX2 and PITX2-lncRNA in CRC is significantly lower as compared to NAT & inflammation or adenoma tissues (p < 0.0001). No differences were found between NAT & inflammation and adenomas. Conclusions.  Reduction of aberrant DNA methylation of PITX2 and PITX2-lncRNA seems to be a late epigenetic event in carcinogenic progression from adenoma to CRC. Further investigations are necessary to clarify and characterize the role of PITX2 and PITX2-lncRNA in colorectal cancer in more detail.

AG01.30 Targeting NOTCH signaling in colon cancer E. M. Schmidt, C. Blaj, S. Lamprecht, A. Küchler, H. Hermeking, A. Jung, T. Kirchner, D. Horst* Pathologisches Institut, Ludwig-Maximilians-Universität, München, Deutschland Background.  WNT and MAPK are two core signaling pathways in colon cancer initiation and progression. Much less is known about the role of NOTCH signaling in these tumors, another key pathway indispensable for cell fate decisions in normal intestinal epithelium. Here, we explored the role of NOTCH signaling in colon cancer and assessed strategies for potential therapeutic intervention. Methods.  To determine the distribution of NOTCH activity in colon cancer, we used immunofluorescence stainings of primary tumors, and lentiviral NOTCH reporters in colon cancer xenografts. We manipulated NOTCH activity in vitro by inducible lentiviral expression systems. For repression of NOTCH and MAPK signaling in vivo, we used inhibitors of both pathways in xenograft models. Results.  Unexpectedly, we found that high NOTCH activity marked distinct tumor cell subpopulations with low MAPK activity in colon cancer. In vitro data suggested that NOTCH repressed MAPK signaling in colon cancer, explaining this observation. In xenografts, inhibition of NOTCH or MAPK revealed high phenotypic plasticity of these two subpopulations in vivo, a potential mechanism for treatment resistance. Combined targeting of both pathways overcame treatment resistance and showed superior therapeutic effects in slowing xenograft growth. Conclusions.  Our findings demonstrate that the existence of phenotypically distinct tumor cell subpopulations with high NOTCH activity in colon cancer can be exploited for effective therapeutic targeting.

AG Urologische Pathologie AG02.04 Proteomic Heterogeneity in Tumorous and Non-tumorous Tissues of Prostate Cancer Patients T. Guo1, L. Li2, N. J. Rupp*3, Q. Zhong3, C. Fritz3, U. Wagner3, W. Jochum4, M. Umbehr4, L. Bachmann4, C. Poyet5, P. J. Wild3, R. Aebersold1, 6, A. Beyer2 Department Biologie, Institut für Molekulare Systembiologie, ETH, Zürich, Schweiz, 2CECAD, Universität Köln, Köln, Deutschland, 3Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz, 4Institut für Pathologie, Kantonsspital, St. Gallen, Schweiz, 5Klinik für Urologie, UniversitätsSpital, Zürich, Schweiz, 6Mathematisch-naturwissenschaftliche Fakultät, Universität Zürich, Zürich, Schweiz

1

Background.  Protein biomarkers are the major clinical delivery of mass spectrometry (MS)-based proteomic research. Technical issues for quantitative proteomic analysis of biopsy-level tissues, such as quantitative accuracy and sample throughput, have been alleviated by the recent integration of pressure cycling technology and SWATH-MS. What remains unclear is the impact of intra-tumor heterogeneity of protein expression on protein biomarker discovery. Here we designed and procured a set of prostate tissue samples, which enabled partitioning of multiple layers of experimental protein variations, and particularly quantification of intra-tumor heterogeneity at proteomic level. Methods.  Multiple (at least three) biopsy-level benign and cancerous prostate tissue punches of three prostate cancer patients were obtained to investigate intra- and inter- tissue variation. The quantitative proteome of each sample was analyzed in duplicates by PCT-SWATH to address technical variation. Proteins found to be expressed variable or stable in the tumorous and non-tumorous tissue compartment were investigated in duplicates by immunohistochemistry using tissue microarray technology. Results.  Our data showed that the degree of intra-tumor heterogeneity of a protein is dependent on its own properties and its context. While the majority of the thus measured proteins demonstrated a low degree of intra-tumor heterogeneity, some proteins and pathways were heterogeneously expressed in various prostate tissues. Therefore, they are unlikely robust biomarkers or therapeutic targets. Our predictions are supported by meta-analyses and clinical studies. Conclusions.  In conclusion, this study uncovered and characterized independent criteria for prioritizing future protein biomarkers and therapeutic targets for patients with prostate cancer.

AG02.05 Detection of Androgen-Receptor Splice Variant 7 Messenger RNA (AR-V7) by Branch Chain RNA In-Situ Hybridization (ISH) in Castration-Resistant Prostate Cancer J. H. Rüschoff*1, Q. Zhong1, C. Fankhauser2, U. Wagner1, N. J. Rupp1, M. Robinson3, M. Koletou1, C. Poyet2, T. Hermanns2, K. S. Arora4, V. Deshpande4, P. J. Wild1 1 Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz, 2Klinik für Urologie, UniversitätsSpital, Zürich, Schweiz, 3Institut für Molekulare Biologie, Universität Zürich, Zürich, Schweiz, 4Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, Vereinigte Staaten von Amerika

Background.  Castration-resistant prostate cancer (CRPC) is not androgen-independent and continues to rely on androgen signaling. Abiraterone and enzalutamide have recently emerged for the treatment of CRPC, showing improved survival in CRPC patients. These agents either suppress the synthesis of extragonadal androgens or target the androgen receptor directly. Nevertheless, 20–40 % of patients do not respond to abiraterone or enzalutamide. Detection of an androgen-receptor isoform encoded Der Pathologe Suppl 1 · 2016

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Abstracts by splice variant 7 (AR-V7) in circulating tumor cells from patients with CRPC was shown to be associated with resistance to enzalutamide and abiraterone. Methods.  We evaluated an RNA in situ hybridization (ISH) method using branch chain technology to detect AR-V7 mRNA (Affymetrix, Santa Clara, CA) in CRPC samples using a tissue microarray (TMA). Clinico-pathological details were reviewed for a cohort of 76 patients with CRPC (64 palliative TUR-P specimens, 10 bone metastases). RNA-ISH assays were performed using an automated platform (Bond RX, Leica Biosystems) and ViewRNATM technology (Affymetrix, Santa Clara, CA) detecting mRNA of AR-V7, AR and a cocktail probe for GAPDH, PPIB, and ACTB as housekeeping genes. Expression of AR-V7 mRNA was evaluated semi-quantitatively by a three-step scoring system (negative, weak and strong) and correlated with treatment history. Results were confirmed by RNA sequencing. Results.  The majority of CRPC cases (87.8 %, n = 65) were interpretable by the AR-V7 mRNA ISH assay. In total, AR-V7 mRNA expression was found in 32.3 % of analyzable cases. Palliative TUR-P specimens had weak (24.1 %) or strong (3.4 %) detectable AR-V7. In contrast, five of seven (71.4 %) analyzable bone metastases were only weakly positive. In the subgroup of CRPC patients with palliative TUR-P, detection of AR-V7 was significantly increased in case of previous orchiectomy (p < 0.001), status post chemotherapy (p = 0.007), and no history of treatment with LHRH agonists (p = 0.005). No association with previous anti-androgen treatment or radiation therapy was found. Conclusions.  The branch chain AR-V7 RNA-ISH assay is a robust technique to detect AR-V7 as a tissue biomarker for patients with CRPC. The predictive power of the ISH assay has to be shown in prospective clinical trials. The AR-V7 splice variant is significantly associated with the specific drug history.

SCiLS lab software was applied to discriminate the different groups from each other. Results.  A number of interesting candidate proteins, which allow the differentiation between the aggressive and non-aggressive tumor form were identified using liquid chromatography and mass spectrometry (LC-MS). For the validation of the biomarker candidates three different techniques were used: immunohistochemistry (IHC) with TMAs, western blotting with tissue and urine samples and a mass spectrometry based technique, the parallel reaction monitoring (PRM) for absolute quantification of proteins in urine. Using MALDI imaging it was possible to discriminate TMA samples into PCa subgroups. Using a linear discriminant analysis model, classification accuracy between 66 % and 91 % was achieved. Peptides responsible for the subgroup discrimination of PCa were identified using LC-MALDI. The established classification model is validated with tissue sections of PCa patients. Conclusions.  The determination of aggressive and non-aggressive prostate cancer tumors was successfully achieved using LC-MS and MALDI imaging. The validation of the identified biomarker candidates is currently performed on independent patient cohorts.

AG02.07 Pan-cancer analysis of the Mediator complex transcriptome identifies involvement of subunits CDK19 and CDK8 in prostate cancer progression N. Klümper*1, 2, J. Brägelmann1, A. Offermann1, 2, A. von Mässenhausen1, 2, D. Böhm1, 2, M. Deng1, 2, A. Queisser1, 2, C. Sanders1, 2, I. Syring1, 3, A. S. Merseburger4, W. Vogel2, J. Carlsson5, O. Andrén5, P. Brossart1, S. Duensing6, M. A. Svensson7, Z. Shaikhibrahim2, J. Kirfel8, S. Perner2 Sektion für Prostatakarzinomforschung, Institut für Pathologie, Universitätsklinikum, Bonn, Deutschland, 2Institut für Pathologie, Universitätsklinikum Lübeck und Leibniz Forschungszentrum Borstel, Lübeck und Borstel, Deutschland, 3Klinik und Poliklinik für Urologie und Kinderurologie, Universitätsklinikum, Bonn, Deutschland, 4Klinik für Urologie, Universitätsklinikum Schleswig-Holstein, Lübeck, Deutschland, 5 Klinik für Urologie, Universitätsklinikum Örebro, Örebro, Schweden, 6 Molekulare Uroonkologie, Klinik für Urologie, Universitätsklinikum Heidelberg, Heidelberg, Deutschland, 7Abeilung für Forschung und Erziehung, Universitätsklinikum Örebro, Örebro, Schweden, 8Institut für Pathologie, Universitätsklinikum, Bonn, Deutschland 1

AG02.06 Aggressive Prostate Cancer Prediction by using Innovative Proteomic Tools B. Beine1, D. Dietrich2, N. Gaisa3, D. Trede4, G. Kristiansen2, K. Steinestel5, D. Pfister6, A. Heidenreich6, R. Knüchel3, B. Sitek7, H. E. Meyer8, C. Henkel*8 Leibniz-Institut für Analytische Wissenschaften – ISAS – e. V., Biomedizinische Forschung, Dortmund, Deutschland, 2Institut für Pathologie, Universitätsklinikum, Bonn, Deutschland, 3Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, 4SCiLS GmbH, Bremen, Deutschland, 5University Hospital Münster (UKM), GerhardDomagk-Institute of Pathology, Münster, Deutschland, 6Department für Urologie, Universitätsklinikum RWTH, Aachen, Deutschland, 7RuhrUniversität Bochum, Medizinisches Proteom-Center, Bochum, Deutschland, 8 Leibniz-Institut für Analytische Wissenschaften – ISAS – e. V., Dortmund, Deutschland 1

Background.  Prostate cancer (PCa) is today’s third most common cancer diagnosed in Europe. One major problem in PCa management is the assessment of cancer progression risk and thereby the decision for a suitable therapy. There is an urgent need for markers which allow an early prognosis and personalized therapy for each patient. The aim of our work was to identify protein markers which help to predict tumor progression in PCa. Therefore, different state of the art techniques were combined to gain an overall reflection of the proteome of the aggressive and non-aggressive PCa to identify new targets for risk assessment in PCa management. Methods.  The discovery phase was performed using microdissected fresh frozen tissue from aggressive (n = 8) and non-aggressive (n = 6) tumors and the corresponding tumor free tissues. Samples were analyzed in technical triplicates and evaluated by the Progenesis software. Additionally, MALDI imaging experiments of peptides were performed on formalin fixed paraffin embedded (FFPE) material. A tissue microarray (TMA) was used comprising 177 patients (33 aggressive and 144 non-aggressive tumors).

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Background.  The multi-protein Mediator complex is essential for the transcription of protein-coding genes and serves as a hub for diverse signaling pathways and transcription factors. It has been linked to human malignancies, but a comprehensive analysis across tumor types is lacking. Methods.  Mediator subunit transcriptomes in 23 cancer entities (n = 8468 patients) were analyzed by RNA-Seq gene expression data from The Cancer Genome Atlas (TCGA). Findings regarding CDK19 and its paralog CDK8 in prostate cancer (PCa) were validated in publicly available PCa microarray data and with immunohistochemical staining (IHC) of two large PCa cohorts containing a total of 622 patient samples (benign n = 102, primary PCa (pPCa) n = 410, lymph-node metastasis (LNPC) n = 76, castration-resistant PCa (CRPC) n = 34). In vitro, effects of CDK19 and CDK8 siRNA knock-down and treatment of PCa cell lines with selective small-molecule CDK8/CDK19 inhibitor Senexin A were analyzed. Results.  Transcriptome analysis of the Mediator complex subunits segregated tumor entities in an unsupervised cluster analysis. The pPCa cluster was marked by high CDK19 expression. Concordantly in mRNA microarray and protein expression analysis CDK19 showed a significant increase during the progression of PCa to CRPC. In contrast, the paralog CDK8 exhibited a stable protein expression in pPCa and was increased in about 50 % of CRPC samples. In pPCa CDK19 expression was significantly associated with increased tumor stage, presence of an extracapsular extension, higher Gleason score, ERG rearrangement and greater Ki67 proliferation index. Moreover CDK19 overexpression was a predictor of disease

free survival of pPCa patients even after adjustment for age and Gleason score (HR 2.7, p = 0.02). In vitro, CDK19 and CDK8 knock-down did not affect proliferation in LnCap and PC-3 cells, but strongly decreased the migratory potential of PC-3 cells. Comparably, CDK8/19-inhibitor treatment significantly impaired migration and invasion of PC-3 cells dose-dependently. Conclusions.  In conclusion the in silico transcriptome analysis revealed distinct transcriptional expression profiles of the Mediator complex across cancer entities, highlighting differential modes of transcriptional regulation. CDK19 was identified to be specifically overexpressed in pPCa and its implication in the further progression was validated in two large cohorts of clinical specimens. Additionally, our in vitro data indicates the therapeutic potential of CDK19 and CDK8 as novel targets in PCa.

AG02.08 The role of testosterone metabolites in castration resistant prostate cancer F. Bremmer*1, H. Jarry2, V. Unterkircher1, J. Gehrig3, S. Kaulfuss3, P. Burfeind3, H. J. Radzun1, P. Ströbel1, P. Thelen4 Institut für Pathologie, Universitätsmedizin, Göttingen, Deutschland, Experimentelle Endokrinologie, Göttingen, Deutschland, 3Institut für Humangenetik, Göttingen, Deutschland, 4Abteilung Urologie, Universitätsmedizin (UMG), Göttingen, Deutschland 1

2

Background.  Prostate cancer (PCa) is the 2nd leading cause of cancer death in Europe. Local PCa patients can be cured by radical prostatectomy or radiotherapies and/or androgen deprivation therapy. In case of castration resistant PCa (CRPC) the prognosis is usually incurable. Novel treatments such as abiraterone acetate (AA) or enzalutamide effectively target the androgen pathway to arrest aberrant signalling. Moreover it become evident that testosterone (T) is able to eliminate tumour cell growth in CRPC. Metabolites of T are 3-β-Androstendiol (3βA) and 3-α-Androstendiol (3αA). However, their effects on PCa cells have not been investigated in detail. In the prostate the estrogen receptor β (ERβ) has features of a tumour suppressor. It is widely accepted that ERβ adopts a regulatory role in estrogen signalling, mediating antiproliferative effects. In this study we investigated the effect of T-metabolites with regard to proliferation and role in estrogen signalling. Methods.  We used the human prostate cancer cell lines LNCaP, BPH-1, PC-3, revCRPC, CRPC, CRPCAA, hiPLNCaP, and hiPLNCaPAA. VCaP (CRPC) was treated with either 1 nmol/​L T over 20 passages yielding the androgen-sensitive cell line (revCRPC). LNCaP was grown over 100 passages yielding a high passage (LNCaP) cell line (hiPLNCaP). VCaP and hiPLNCaP cell line was treated with 5 µmol/​L AA over 20 passages generating the AA-tolerant subtype CRPCAA and hiPLNCAPAA. The cell lines were treated with T, dihydrotestosterone (DHT) and R1881 as well as the T-derivatives 3βA and 3αA. Tumour cell proliferation was evaluated with BrdU-ELISA. AR, ER-β and ER-α protein expressions were investigated by Western Blot analysis. Results.  1 µmol/​L 3βA or 3αA lead to a significantly reduced proliferation in LNCaP, revCRPC, CRPC, CRPCAA, hiPLNCaP, and LNCaPAA cell lines. BPH-1 and PC-3 showed no changes of proliferation after 3βA or 3αA. In addition stimulation with 1 µmol/​L 3βA or 3αA elicits markedly reduced AR and ERα, whereas the expression of ERβ was increased in LNCaP, revCRPC, CRPC, CRPCAA, hiPLNCaP, and LNCaPAA. In BPH-1 and PC-3 cell lines no significant changes in protein expression occurred. Conclusions.  (I) 3βA or 3αA as well as DHT and R1881 significantly reduce tumour cell growth in several CRPC cell lines. Thus these substances may yield new potential therapeutic approaches in CRPC (II) Furthermore, our data solidify the tumour suppressor properties of ERβ in CRPC.

AG02.09 Prognostic Relevance of CXCL12 Promoter Methylation within the Cross-relation of ERG and Androgen Receptor Expression in Prostate Cancer D. Goltz*1, E. E. Holmes1, V. Sailer2, 3, M. Jung1, M. Röhler1, S. Meller1, J. Ellinger4, G. Kristiansen1, D. Dietrich1 1 Institut für Pathologie, Universitätsklinikum, Bonn, Deutschland, 2Weill Cornell Universität für Medizin, Abteilung für Pathologie und Labormedizin, New York, Vereinigte Staaten von Amerika, 3Weill Cornell Universität für Medizin, Englander Institut für Präzisionsmedizin, New York, Vereinigte Staaten von Amerika, 4Klinik und Poliklinik für Urologie und Kinderurologie, Universitätsklinikum, Bonn, Deutschland

Background.  The CXCR4/CXCL12 axis plays a central role in systemic metastasis of prostate carcinoma (PCa) thereby representing a promising target for future therapies. We evaluated the prognostic value of epigenetic CXCL12 gene silencing by DNA hypermethylation with respect to ERG, preoperative serum PSA, and androgen receptor (AR) expression. Methods.  CXCL12 methylation was detected by means of a methylation specific quantitative PCR analysis in a test-series of 66 paired specimens of normal, hyperplastic and carcinomatous prostate tissues and a radical prostatectomy patient cohort (n = 268) operated for primary prostate cancer at the University Hospital Bonn between 1998 and 2008. In the latter, CXCL12 methylation results were correlated to clinico-pathological parameters including biochemical recurrence-free survival. Results.  Carcinomatous prostate tissue showed hypermethylation of the CXCL12 gene locus compared to normal adjacent and benign hyperplastic tissues in the test series. CXCL12 methylation showed a significant correlation with Gleason score (p = 0.001) and nodal status (pN, p = 0.029). Univariate analyses on biochemical recurrence-free survival demonstrated a significant prognostic value for CXCL12 methylation (Hazard Ratio: HR = 1.87 [95 %CI: 1.02–3.44], p = 0.043). Of importance, in patient subgroups, stratified according to ERG- and AR-expression, as well as serum PSA, the prognostic power of CXCL12 methylation is markedly enhanced in ERG negative (HR = 2.63 [95 %CI: 1.07-6.50], p = 0.036) low-level AR expressing PCa (HR = 4.89 [95 %CI: 1.09-21.90], p = 0.038), and PCa presenting with low serum PSA levels (HR = 4.59 [95 %CI: 1.5313.73], p = 0.006), respectively. Conclusions.  CXCL12 methylation is a powerful novel prognostic biomarker for biochemical recurrence in prostate cancer patients after radical prostatectomy. Further studies are need to ascertain, if CXCL12 methylation may aid in planning active surveillance strategies or can even be a predictive biomarker for future therapies targeting the CXCR4/ CXCL12 axis.

AG02.10 Inhibition of the Amyloid precursor protein (APP) induces Ferroptosis in prostate cancer – A new cell death mechanism linking p38MAPK signaling with DNA damage V. Venkataramani*1, S. Küffer2, T. Rumkamp1, S. Hakroush2, A. Frosch2, L. Trümper1, P. Ströbel2, G. G. Wulf1 1 Abteilung Hämatologie und Onkologie, Universitätsmedizin, Göttingen, Deutschland, 2Abteilung für Pathologie, Universitätsmedizin, Göttingen, Deutschland

Background.  Ferroptosis is a new mode of non-apoptotic cell death involving the production of iron-dependent reactive oxygen species (ROS). This cell death pathway is clearly distinct from classical death pathways, such as apoptosis, autophagy and necrosis. However, the deleterious downstream signalling networks and molecular consequences of ferroptosis remain to be elucidated. We previously demonstrated that the amyloid precursor protein (APP), an androgen-receptor (AR) target gene is overexpressed in prostate cancer and is significantly associated with an inferior

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Abstracts clinical survival. Here we demonstrate for the first time that loss-of-APP either by genetic depletion or pharmacologic by the novel APP blocker JTR-009 results in iron-mediated accumulation of ROS suggesting similarities to ferroptosis. Methods.  Stable APP-knockdown cell lines were established by shRNA approach. The ferroptosis inducer Erastin and L-BSO as well as the inhibitor ferrostatin-1 were uesed. Rescue experiments were performed using NAC and the p38 inhibitor SB20358. siRNA experiments confirmed the essential impact of p38 α. The establish APP as druggable target gene, we evaluated the novel APP inhibitor JTR-009 in mimicking ferroptosis. Functional assays such as cell proliferation, colony forming assays, RNA seq, and western blot as well as xenograft analysis via CAM-Assay were performed. Results.  Beside the relevant AR regulation of APP expressing determining survival of prostate cancer cells, we demonstrate that APP enables prostate cancer stem cells to cope with redox imbalance associated with tumor progression and initiation. Elevated APP in CSCs is paralleled with lower ROS. APP silencing elevated ROS, activating the p38MAPK finally resulting in a DNA damage response without induction of other cell death pathways. Depletion of APP, either by genetic ablation or by JTR-009 treatment resulted in similar activation of molecular hallmark events as ferroptotic cell death and both could be reverted by ferrostatin-1 and iron chelation treatment. Newly, we established that p38MAPK is critical for excerting detriminal DNA damage since both SB203580 treatment and siRNA-mediated ablation of p38 α rescued the loss-of-APP phenotype. Conclusions.  Beside known factors of ferroptosis, such as depletion of glutathione or direct inhibition of glutathione peroxidase 4, we now establish APP as a new key player that directly regulates iron-meditaed cell death. The druggability of APP opens new treatment oppurtunities against prostate cancer.

AG02.11 ITIH5 loss is associated with basal-type bladder cancer while ITIH5 re-expression impairs invasive characteristics of basal-like SCaBER tumor cells in vitro M. Rose*1, J. Kistermann1, N. T. Gaisa1, T. Heide1, A. Heidenreich2, R. Knüchel1, E. Dahl1 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, Department für Urologie, Universitätsklinikum RWTH, Aachen, Deutschland

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Background.  Recently, we revealed that ITIH5 is a novel putative tumor suppressor gene in urothelial cancer. Epigenetic silencing of ITIH5 was associated with unfavorable patient’s outcome indicating a potential impact of ITIH5 protein expression as novel prognostic biomarker for high grade bladder tumors. In the current study we aim to decipher the clinical and functional impact of ITIH5 in basal-type bladder cancer that is known to be associated with high risk for poor prognosis. Methods.  In a dataset of The Cancer Genome Atlas (TCGA) of bladder tumor samples (n = 195), we analyzed ITIH5 mRNA expression in dependence of known basal- and luminal-type markers. To functionally elucidate the role of ITIH5 in basal-type bladder cancer, we stably transfected SCaBER tumor cells with an ITIH5 cDNA pBK-CMV expression vector or empty vector alone. Subsequently, functional analyses were performed using proliferation, colony formation, apoptosis and cell adhesion assays. Statistical data acquisition was done using R Statistical Software version 3.2.0. Results.  A prevalent loss of ITIH5 expression was observed in basal type bladder tumors, i. e. a close association of ITIH5 loss with the basal-type markers in bladder cancer was revealed while strong ITIH5 expression tightly clustered with the expression of luminal markers such as UPK1 and KRT20. Upon stable transfection of basal-type SCaBER cells lacking endogenous ITIH5 expression, SCaBER-ITIH5 clones significantly showed both reduced cell and colony growth compared to mock clones in vitro.

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Furthermore, cell-matrix adhesion on hyaluronic acid was significantly enhanced by ITIH5 expression in comparison to control cells. Conclusions.  Our results propose a significant impact of ITIH5 on suppression of basal-type and squamous-like bladder cancer. Future studies focusing on the role of ITIH5 in the clinical important patients’ subgroup should be performed to determine whether ITIH5 may represent a prognostic biomarker especially helping to stratify those aggressive bladder cancers.

AG02.12 Differential expression of markers of basal and luminal differentiation in nested type of urothelial carcinoma S. Bertz*1, R. Weisser1, N. Gaisa2, E. Comperat3, G. Kristiansen4, M. Otto5, R. Stöhr1, T. Brabletz6, J. Giedl1, B. Keck7, B. Wullich7, A. Hartmann1 FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland, Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, 3 Hospital Pitié Salpetriere, UPMC Paric VI, Institut für Pathologie, Paris, Frankreich, 4Institut für Pathologie, Universitätsklinikum, Bonn, Deutschland, 5 MVZ für Histologie, Zytologie und Molekulare Diagnostik, Trier, Deutschland, 6Universität Erlangen-Nürnberg, Lehrstuhl für Experimentelle Medizin I, Erlangen, Deutschland, 7Universitätsklinikum Erlangen, Klinik für Urologie, Erlangen, Deutschland 1

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Background.  Nested variant of urothelial carcinoma (NVUC) belongs to the rare variants of urothelial carcinoma (UC) with poor prognosis and poor chemotherapy response despite its mostly bland cytology. Recent studies reported luminal and basal phenotypes in bladder cancer with significant prognostic differences. Aim of this study was to describe the histopathological spectrum and investigate immunohistochemically the expression profile of basal and luminal markers and also proliferation associated antigens in NVUC. Furthermore we performed TERT promoter analysis to validate previous molecular studies demonstrating TERT mutations as useful markers in the differential diagnosis of benign mimickers of NVUC. Methods.  FFPE tumor tissue of NVUCs of 65 patients (mean age 66.1 years, range 38.89) was obtained from several pathology centres. Histomorphological reevaluation according to the current WHO and TNM classifications was performed by two uropathologists (A. H., S. B.). After TMA construction immunohistochemistry for luminal and basal markers (CD44, CK5, CK7, CK20, EGFR, GATA3, p63, FOXA1, CD24, CK14) and cell cycle associated antigens (TP53, Ki-67) was performed. After manual microdissection DNA was isolated and TERT promoter mutation analysis was performed by Sanger sequencing or SNaPshot-Assay. Results.  TERT promoter mutations were found in 63.5 % of tumours. Concomitant urothelial carcinoma in situ (CIS) and dysplasia were found in 19 and 2 cases respectively. Pure NVUC was found in 21 cases, all other cases were associated with conventional UC or other subtypes e. g. micropapillary, plasmacytoid and sarcomatoid UC. Pure low-grade cytological atypia was found in 14 cases, all other cases revealed at least a focal highgrade component. Evaluation of basal/luminal pattern was performed in 56 cases and showed predominance of the luminal phenotype. Most cases showed low proliferative activity assessed by Ki-67 immunohistochemistry but frequent p53 overexpression. Conclusions.  NVUC shows differential expression of luminal and basal markers with predominance of the luminal phenotype. The pattern of expression of cell cycle proteins support studies reporting increased chemoresistance in NVUC. Furthermore TERT promoter mutations distinguishing NVUC from its benign mimickers were found in a frequency according to previous studies. Thus, telomerase may represent a useful diagnostic marker and a potential therapeutic target in this rare variant of UC.

AG02.13 Detection of new pathways/biomarkers in cisplatin resistant GCT cell lines F. Bremmer*1, H. Bohnenberger1, H. Jarry2, P. Thelen3, H. J. Radzun1, P. Ströbel1, F. Honecker4, S. Balabanov5, S. Küffer1, C. L. Behnes1 Institut für Pathologie, Universitätsmedizin, Göttingen, Deutschland, Experimentelle Endokrinilogie, Göttingen, Deutschland, 3Abteilung Urologie, Universitätsmedizin (UMG), Göttingen, Deutschland, 4Tumorund Brustzentrum ZeTuP, St. Gallen, Schweiz, 5Abteilung für Hämatologie, UniversitätsSpital, Zürich, Schweiz 1

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Background.  Germ cell tumors (GCTs) are the most common malignancies in young men. Most patients with GCT can be cured with cisplatin-based combination chemotherapy, even in metastatic disease. However in case of therapy resistance, the prognosis is usually poor. In addition new biomarkers may help to understand pathophysiological processes in these tumours. In addition, new treatment options for refractory GCTs are needed. Methods.  We used the human GCT-cell lines NCCIT, NTERA-2 and the both cisplatin-resistant sublines NCCIT-R and NTERA-2R. For quantitative proteomic studies we used high-resolution mass spectrometry in combination with the SILAC method to compare cisplatin-sensitive and resistant cells. Gene ontology and network analysis has been performed using either the Metacore software or the R package Clusterprofiler. In addition tyrosine kinase PamChip arrays were performed comparing NCCIT-/R and NTERA-2/​R GCT cell lines. Tyrosine kinase substrate spots with a differential phosphorylation were filtered and taken as specific cisplatin reactivity pattern and upstream kinases were predicted using Bionavigator. Results.  The quantitative proteomic investigations revealed a total number of 69 differential expressed proteins in NCCIT-R and 53 differential expressed proteins in NTERA-2R compared to cisplatin-sensitive variants. Differential expressed proteins were loaded into Metacore software and significantly enriched biological processes, molecular functions and pathway networks were extracted for significantly regulated proteins. Network analyses were performed for enrichment of shortest pathways inside the group of differentially regulated proteins. The shortest path algorithm links the original proteins identified in the SILAC approach with additional objects from the Metacore database along a directed path. In tyrosin kinase arrays a total number of eight (NCCIT/-R) and seven (NTERA-2/R) significant activated upstream kinases could be detected. Conclusions.  (I) High-resolution mass spectrometry in combination with the SILAC method is suitable to detect differential expressed proteins and signaling pathways. (II) Kinase inhibition profiles have been shown to be useful tools to predict treatment response in tissue. Our results show a distinct pattern for a potential resistant phospho-pattern to cisplatin in GCT cell lines. (III) Finally the two presented methods could be combined to obtain the phosphorylation status of differential expressed proteins.

AG02.14 ErbB-2 protein expression and HER2 gene amplification in upper urinary tract urothelial carcinoma A. Zimpfer1, M. Mosig1, H. Zettl*2, I. Petersen1, M. Maruschke3, O. W. Hakenberg4, A. Erbersdobler5 Institut für Pathologie, Universitätsklinikum Jena, Jena, Deutschland, Klinisches Krebsregister, Universität Rostock, Rostock, Deutschland, 3Helios Hanseklinikum Stralsund, Urologische Klinik, Stralsund, Deutschland, 4 Abteilung Urologie, Universität Rostock, Rostock, Deutschland, 5Institut für Pathologie, Universitätsmedizin Rostock, Rostock, Deutschland 1

2

Background.  Upper urinary tract urothelial carcinomas (UUT UC) occur in about 5–10 % of all UCs and are frequently discovered at a high-stage

disease. We aimed to evaluate Her2 protein expression and HER2 gene amplification in a series of 130 UUT UC patients with nephroureterectomy or rarely UUT biopsy. Methods.  A total of 130 UUT UCs were analyzed immunohistochemically (Her2-score 0/1+/2+/3+) and with fluorescence in situ hybridization applying a tissue microarray technique. HER2 gene gains or amplification was defined by HER2/CEN17 ratio 1.8–2.2 or >2.2, respectively. Grade, TNM stage, lymphovascular invasion, surgical margins, morphologic variants were reviewed in all tumours. Results.  Her2 immunoreactivity was seen in 68/120 tumours. 20 cases were 2+ score and 12 cases were 3+ score. HER2 gene amplification or gains (enrichment) in HER2 gene was observed in 4/114 (3.5 %) or 8/114 (7.0 %) evaluable cases. 7/8 HER2-enriched/amplified cases showed high grade morphology and were stage pT3 or pT4. A strong correlation between Her2 expression and amplification was noted (p = 0.001). Pearson’s χ2 test demonstrated significant relations between Her2-score and tumour grade (p = 0.002). Survival data revealed a trend towards prolonged overall survival in Her2-immunonegative patients (p = 0.221), but a significant shortened progression-free survival in UUT UCs with Her2-score 2+ or 3+ (p = 0.003). A significant number of invasive UUT UCs showed polysomy of chromosome 17. These cases were significantly related with a non-inverted tumour growth (p = 0.007). Also, significant relations between polysomy 17 and grading, muscle or non-muscle-invasive tumours, nodal status, resection status (p = 0.029, p < 0.029, p = 0.005, p = 0.012) were demonstrated. Conclusions.  The results suggest that c-erbB2/Her2 positivity is a biomarker for progression of UUT UCs. As previously shown, HER2 gene amplification is infrequent. But, this small number of patients might benefit from HER2-targeted cancer therapy. Furthermore, polysomy of chromosome 17 is an important molecular marker correlated with higher tumour stage and grade categories.

AG02.15 Rhabdoid Phenotype in Renal Cell Carcinoma: Analysis of 25 Cases Indicating a Common Final Pathway of Dedifferentiation Frequently Associated with SWI/SNF Complex Deficiency A. Agaimy1, L. Cheng2, L. Egevad3, O. Hes4, S. Sioletic5, A. Hartmann*6 FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland, 2Molecular Diagnostics and Molecular Pathology Laboratory, Indiana University School of Medicine, Indianapolis, Vereinigte Staaten von Amerika, 3 Department of Pathology, Karolinska University Hospital, Stockholm, Schweden, 4Department of Pathology, Charles University, Medical Faculty and Charles University Hospital Plzen, Plzen, Tschechische Republik, 5 Azienda ospedaliera di udine Früher Pennine Acute Hospitals NHS Trust, Udine, Italien, 6Pathologisches Institut, Universitätsklinikum, Erlangen, Deutschland

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Background.  Rhabdoid features are increasingly recognized as adverse prognostic findings in renal cell carcinoma (RCC). The molecular pathogenesis of this aggressive phenotype in RCC has not been studied sufficiently before. Recently, several studies identified alterations involving one or more components of the SWI/SNF chromatin remodeling complex in the pathogenesis of dedifferentiated carcinomas in different organs such as gastrointestinal tract, pancreas and endometrium where the SWI/SNF deficiency was frequently associated with rhabdoid dedifferentiation. Methods.  In this study, we have analyzed the histomorphological and immunohistochemical features of 24 RCC having in common either an undifferentiated (anaplastic) phenotype or prominent rhabdoid features. All cases were stained with 6 members of the SWI/SNF pathway (SMARCB1, SMARCA2, SMARCA4, ARID1A, SMARCC1 and SMARCC2) in addition to conventional RCC markers. Results.  Patients were 15 males and 10 females aged 40–78 years. Most tumors presented with advanced stage (pT3 or more). A differentiated component varying from microscopic foci or intermingled cells to major

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Abstracts component was detected in 17 cases (15 of clear cell type and of 2 chromophobe type). The dedifferentiated component varied from pure rhabdoid dyscohesive cells to large compact epithelioid sarcoma-like to small monotonous anaplastic cells. Variable loss of at least one subunit of the SEI/SNF complex was noted in 19/25 cases. Loss was characteristically seen in the undifferentiated component compared to intact or reduced expression in the differentiated component. However, a few cases showed loss of expression of SWI/SNF components in the clear cell but not in the sarcomatoid component. Conclusions.  This is the first study exploring the role of SWI/SNF deficiency in the dedifferentiation process of RCC. Our results highlight the close link between rhabdoid phenotype in RCC and the SWI/SNF complex, consistent with studies on similar neoplasms in other organs.

AG Hämatopathologie AG03.01 A novel approach to detect resistance mechanisms reveals FGR as a factor mediating resistance to the HDAC inhibitor SAHA in B-cell lymphoma M. Joosten*1, S. Ginzel2, 3, C. Blex4, D. Schmidt4, M. Gombert2, C. Chen2, R. M. Linka2, O. Gräbner4, A. Hain5, B. Hirsch1, A. Sommerfeld1, A. Seegebarth1, U. Gruber1, C. Maneck4, L. Zhang2, 6, K. Stenin4, H. Dieks4, M. Sefkow4, C. Münk5, C. D. Baldus7, R. Thiele3, A. Borkhardt2, M. Hummel1, H. Köster4, U. Fischer2, M. Dreger4, V. Seitz1 Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland, Heinrich Heine Universität, Klinik für Kinder-Onkologie, -Hämatologie und klinische Immunologie, Düsseldorf, Deutschland, 3Hochschule Bonn-RheinSieg, Institut für Angewandte Informatik, Sankt Augustin, Deutschland, 4 Caprotec bioanalytics GmbH, Berlin, Deutschland, 5Heinrich Heine Universität, Klinik für Gasteroenterologie, Hepatologie und Infektiologie, Düsseldorf, Deutschland, 6Medizinische Universität Fujian, Klinik für Hämatologie, Fuzhou, China, Volksrepublik, 7Institut für Hämatologie und Onkologie, Charité – Universitätsmedizin, Berlin, Deutschland 1

AG02.16 RBBP8/CtIP promoter methylation: potential usage as specific biomarker for early bladder cancer detection and its putative prognostic impact T. Heide*1, N. T. Gaisa1, E. Burghardt1, S. Gostek1, T. C. de Ruijter2, A. Heidenreich3, R. Knüchel1, J. Veeck1, M. Rose1 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, Medizinische Onkologie, Universitätsklinikum Maastricht, Maastricht, Niederlande, 3Department für Urologie, Universitätsklinikum RWTH, Aachen, Deutschland 1

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Background.  Aims: Recently, we identified RBBP8 as almost exclusively hypermethylated in bladder cancers among 27 tumor entities based on TCGA data sets. In the current study we aim to decipher the clinical significance of RBBP8 methylation in bladder cancer and its potential biomarker impact for early cancer detection. Methods.  Methods: RBBP8 methylation was determined based on TCGA data and validated by methylation-specific PCR (MSP) in cancer cell lines and primary urothelial tissues as well as in urines derived from bladder cancer patients (n = 25). Age-matched healthy donors and patients with urologic diseases such as prostate cancer were included as controls (overall n = 27) to improve specificity. Statistical data acquisition was done by R Statistical Software version 3.2.0 and survival analyses (univariate Kaplan-Meier and multivariate Cox regression) were performed using SPSS 17.0. Results.  Results: Based on TCGA data, RBBP8 was most exclusively and frequently methylated in primary bladder cancer. Univariate Kaplan-Meier analysis revealed that RBBP8 promoter hypermethylation was significantly (p = 0.011) associated with longer OS (mean OS: 3,391 days) when compared to low RBBP8 methylation levels (mean OS: 1,782). The calculated Cox regression model confirmed the potential prognostic impact: Bladder cancer patients with increased RBBP8 methylation have an estimated twofold decreased risk for death (multivariate hazard ratio: 0.536, p = 0.022). In vitro MSP analysis showed RBBP8 methylated in 2 out of 4 bladder cancer cell lines while not in any of 37 cancer cell lines from six other tumor entities. In normal urothelium tissue RBBP8 was unmethylated but methylated in 42 % of primary bladder tumors. Finally, RBBP8 methylation was detectable in 11 out of 25 urine samples of bladder cancer patients while none of age-matched controls including urines from prostate cancer patients exhibited methylation signals: Overall RBBP8 promoter methylation currently gains a sensitivity of 44 % combined with a specificity of 100 %. Conclusions.  Conclusions: Applying RPPP8 could improve specificity for early bladder cancer detection or monitoring of high risk patients with invasive subtypes. Of clinical importance, RBBP8 methylation may also provide a prognostic impact, but the biological causative of the statistical correlation has to be addressed in future studies. Owing to the proven role of RBBP8 in homologous recombination-mediated DNA double-strand break repair, a predictive impact of RBBP8 may be given.

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Background.  Histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA) are able to achieve partial or complete remissions in a proportion of relapsed or refractory B-cell lymphoma patients. The understanding of the underlying molecular mechanisms leading to SAHA-resistance could lead to stratification of patients eligible for this promising treatment option. Methods.  We designed an integrative approach combining drug efficacy testing with exome and captured target analysis (DETECT). In brief, we tested SAHA-sensitivity in 26 B-cell lymphoma cell lines using flow cytometry (Annexin V/propidium iodide staining (PI)). SAHA-interacting proteins were determined in SAHA-resistant and SAHA-sensitive cell lines employing capture compound mass spectrometry (CCMS). In addition, we performed whole exome sequencing to assess the impact of mutations in SAHA-interacting proteins. Candidates were validated by expression analysis and knock-out experiments. Results.  Our integrated network analysis revealed that the Src tyrosine kinase Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog (FGR) is associated with SAHA-resistance. FGR was specifically captured by the SAHA-CC in resistant cells. In line with this observation, we found that FGR expression was significantly higher in SAHA-resistant cell lines. As functional proof, CRISPR/Cas9 mediated FGR knock-out in SAHA-resistant cells increased SAHA-sensitivity. In silico analysis of B-cell lymphoma samples (n = 1200) showed a wide range of FGR expression indicating that FGR expression might help to stratify patients who clinically benefit from SAHA therapy. Conclusions.  The comprehensive analysis of SAHA-interacting proteins highlights FGR as a factor mediating SAHA-resistance in B-cell lymphoma. Our DETECT approach is a new and promising way to study drug resistance mechanisms and to identify predictive biomarkers.

AG03.02 SIP-F1: A new gray zone lymphoma cell line? B. Rengstl*, M.-L. Hansmann, S. Newrzela Dr. Senckenbergisches Institut für Pathologie, Universitätsklinikum, GoetheUniversität, Frankfurt am Main, Deutschland Background.  Lymphoid cancers are mainly classified according to their clinical presentation, morphology, immunology and molecular genetic features. Diagnosis of Hodgkin’s lymphoma (HL) is mainly based on pathognomonic Hodgkin and Reed/Sternberg cells (HRS). However, in the group of non-Hodgkin lymphomas (NHL), diffuse large B-cell lym-

phoma (DLBCL) can resemble typical features of HL. In some cases, a diagnostic pitfall exists at the interface of HL and DLBCL, so that, morphological overlaps and missing clear-cut diagnostic criteria complicate classification of these so-called gray zone lymphomas (GZL). Methods.  Here, we analyzed a newly established GZL cell line (SIP-F1) derived from a lymph node biopsy of a male 20-year-old lymphoma patient. Histological sections of the primary GZL were characterized by striking pleomorphism of in part multinuclear blast infiltrates and areas of necrosis. Immunohistochemistry revealed tumor cell positivity for the markers CD15, CD20 and CD30. SIP-F1 presented clonal Ig gene rearrangement and flow cytometry analysis showed expression of B-cell markers as well as 50–60 % positivity for HL marker CD30. This new GZL cell line was further characterized by a xenograft mouse model, gene expression profiling (GEP) and co-culture studies with T cells from the same patient. Results.  Transplanting SIP-F1 cells into immunodeficient mice, we observed tumor growth morphologically resembling the primary tumor with lacunar HRS-like cells growing in sheets. For closer characterization, we performed gene expression profiling (GEP) and compared SIP-F1 to several B-cell lymphoma cell lines. As expected, the GEP of SIP-F1 did not cluster with any of the compared entities. Interestingly, but yet quite surprisingly, cytogenetic analysis revealed a normal karyotype. As SIP-F1 is positive for EBV, the cell line might display a transformed B-cell clone rather than a GZL cell line. The patient, SIP-F1 is derived from, showed fast tumor rejection after initialization of chemotherapy. We were able to receive T cells for a co-culture study and could show that his immune system regained anti-tumor strength. Conclusions.  Considering the paucity in prospective differentiation of GZL cases, their distinct clinical behavior and treatment, we think that SIP-F1 could be a helpful tool to study morphological and molecular features of GZL and to better understand the biology of these unusual cases, although we are still tackling the question, what is the real tumor cell clone.

AG03.03 Cell-of-Origin-Classification in Diffuse Large B-cell Lymphoma using the Lymph2Cx Predictor H. Heike*1, A. M. Staiger1, M. Ziepert2, 3, T. F. E. Barth4, W. Bernd5, A. C. Feller5, W. Klapper6, M. Hummel7, H. Stein8, D. Lenze7, M.-L. Hansmann9, S. Hartmann9, P. Möller4, S. Cogliatti10, M. Pfreundschuh11, L. Trümper12, M. Löffler2, N. Schmitz13, A. Rosenwald14, G. Ott15 1 Dr. Margarete Fischer-Bosch-Institut für Klinische Pharmakologie, Stuttgart, Deutschland, 2IMISE, Universitätsklinikum, Leipzig, Deutschland, 3Institute for Medical Informatics, Statistics and Epidemiology, Universität Leipzig, Leipzig, Deutschland, 4Institut für Pathologie, Universität Ulm, Ulm, Deutschland, 5Institut für Pathologie, Universitätsklinikum SchleswigHolstein, Campus Lübeck, Lübeck, Deutschland, 6Institut für Pathologie, Universitätsklinikum Schleswig-Holstein, Campus Kiel, Kiel, Deutschland, 7 Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland, 8 Pathodiagnostik Berlin, Berlin, Deutschland, 9Institut für Pathologie, Goethe Universität, Frankfurt, Deutschland, 10Institut für Pathologie, Kantonsspital St. Gallen, St. Gallen, Schweiz, 11Medizinische Klinik I, Saarland University Medical School, Homburg/Saar, Deutschland, 12Abteilung für Hämatologie und Onkologie, Georg-August-Universität, Göttingen, Deutschland, 13Abteilung für Hämatologie, Asklepios-Klinik St. Georg, Hamburg, Deutschland, 14Pathologisches Institut, Universität Würzburg, Würzburg, Deutschland, 15Abteilung für Klinische Pathologie, Robert-BoschKrankenhaus, Stuttgart, Deutschland

Background.  Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous lymphoid tumor with respect to morphology and site of origin, clinical outcome and genetic parameters. The cell-of-origin (COO) classification, subdividing patient samples into prognostically relevant germinal center B-cell- (GCB) and activated B-cell- (ABC)-derived DLBCL is of

pivotal interest for risk stratification at diagnosis. With the introduction of nCounter®-based targeted gene expression analysis, it is possible to reliable classify also DLBCL patient samples originating from formalin-fixed paraffin-embedded (FFPE) tissue, investigating merely a subset of 20 genes (Scott et al. 2014)1. Methods.  To analyze the prognostic power of this 20-gene-COO-predictor (Lymph2Cx) in a large DLBCL cohort, patient samples from two prospectively randomized clinical trials (Ricover-60: n = 422 samples and R-MegaCHOEP: n = 114 samples) of the German High Grade Lymphoma Study Group (DSHNHL) were analyzed performing targeted gene expression profiling with the nCounter®-platform. Results.  In total, COO-classification was possible in 383/422 (91 %) and in 108/114 (95 %) samples. In the Ricover-60 trial, including patients aged over 60 years, a GCB-subtype was detected in 177/422 (42 %), while 153/422 (36 %) DLBCL samples were classified as ABC. An unclassified subtype was observed in 53/422 (13 %). In contrast, in the R-MegaCHOEP-trial enrolling patients aged between 18 and 60 years, patients were classified as GCB in 66/114 (58 %), as ABC in 28/114 (25 %) and as unclassified in 14/114 (12 %). These results were discrepant to the immunohistochemistry-based Hans-classifier in 14 %, respectively. In the Ricover-60-trial, overall survival (OS) was significantly shorter in patients classified as ABC-DLBCL, when compared to GCB-DLBCL (p = 0.013), when the patients were treated with CHOP. However, the COO-classification yielded no prognostic information, when patients were treated with R-CHOP (p = 0.189). Conclusions.  Although DLBCL patients within DSHNHL trials could be reliable classified according to their COO type, its impact on clinical outcome, as described by Scott and colleagues [1,2] could not be verified in R-CHOP treated patients in the Ricover-60 trial. Statistical analysis concerning patients in the R-Mega-CHOEP trial is ongoing and will be presented. References 1. Scott DW et al (2014) Blood 123:1214-7 2. Scott DW et al (2015) J Clin Oncol 33:2848-56

AG03.04 Highly recurrent mutations of SGK1, DUSP2 and JUNB in nodular lymphocyte predominant Hodgkin lymphoma S. Hartmann*1, B. Schuhmacher2, T. Rausch3, L. Fuller2, C. Döring2, M. Weniger4, A. Lollies4, C. Weiser2, L. Thurner5, B. Rengstl2, U. Brunnberg6, M. Vornanen7, M. Pfreundschuh5, V. Benes3, R. Küppers4, S. Newrzela2, M.-L. Hansmann2 Universitätsklinikum Frankfurt, Institut für Pathologie, Frankfurt/Main, Deutschland, 2Institut für Pathologie, Goethe Universität, Frankfurt, Deutschland, 3EMBL, Heidelberg, Deutschland, 4Institut für Zellbiologie, Universitätsklinikum, Universität Duisburg-Essen, Essen, Deutschland, 5José Carreras Center für Immun- und Gentherapie, Homburg, Deutschland, 6 Klinik für Innere Medizin, Hämatologie/Onkologie, Goethe-Universität, Frankfurt, Deutschland, 7Pathologie Fimlab, Tampere, Finnland 1

Background.  Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) – a subtype of Hodgkin lymphoma (HL) – is characterized by a low content of tumor cells, the LP cells. Transformation into diffuse large B cell lymphoma (DLBCL) occurs in about 10 % of patients and usually shows an aggressive behavior. Methods.  We performed whole genome mutation analysis of the DLBCL components from two composite lymphomas consisting of clonally related NLPHL and DLBCL as a means to identify candidate tumor suppressor genes and oncogenes in NLPHL. Selected genes were further analyzed by ultra deep targeted sequencing of additional primary NLPHL cases with the usual low tumor cell content of 1–5 %. Results.  Analysis of the clonal related NLPHL and DLBCL composite lymphoma components revealed that most mutations identified in the

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Abstracts DLBCL are also present in the LP cells, indicating a close relationship between the two components. Analysis of 62 selected genes in NLPHL by targeted ultradeep sequencing revealed three novel highly recurrently mutated genes (each mutated in about 50 % of cases), i. e. DUSP2, SGK1 and JUNB. SGK1 was expressed in LP cells of primary NLPHL cases and in the NLPHL cell line DEV. Administration of an SGK1 inhibitor induced apoptosis in the NLPHL cell line DEV and the DLBCL cell line Farage, suggesting a pathogenetic role of SGK1 in LP and DLBCL cells. Conclusions.  In summary, the present study identifies SGK1, DUSP2, and JUNB as novel key players in the pathogenesis of NLPHL.

AG03.05 The novel PTP1B variant PTP1BΔ6 controls JAK-STAT signaling in classical Hodgkin’s lymphoma M. Zahn*, T. F. Barth, I. Melzner, J. Heinrich, R. Marienfeld, P. Möller Institut für Pathologie, Universitätsklinikum, Ulm, Deutschland Background.  Constitutively activated JAK-STAT signaling is crucial for the survival and proliferation of Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of the classical Hodgkin’s lymphoma (cHL). Given the essential role of PTP1B and SOCS1 for the constitutive JAK-STAT signaling in cHL, the identification and characterization of mechanisms controlling the activity of these negative JAK-STAT regulators is crucial for the understanding of their role for the pathogenesis of cHL. Here, we describe the expression of a shorter PTP1B isoform as a novel control mechanism for the JAK-STAT pathway in cHL. Methods.  PTP1B mRNA of cHL cases and cell lines was Sanger sequenced. PTP1B protein was analyzed by western blot and immunohistochemistry. PTP1B mRNA variants were analyzed by qPCR. L428 cell clones expressing either PTP1BWT, PTP1BC215S, or PTP1BΔ6 were generated by lentiviral transduction. Functions of the novel PTP1BΔ6 variant was determined by western blot, luciferase reporter analysis and EMSA. Results.  To determine the molecular basis of the diminished PTP1B expression in selected cHL cases, we sequenced the PTP1B mRNA. In contrast to the reported inactivating point mutations in the PTP1B gene, we obtained a set of shorter PTP1B variants. Since shorter PTP1B protein variants were also detectable in cHL cell lines, we determined the molecular functions of the most prominently expressed PTP1B variant, PTP1BΔ6. PTP1BΔ6 expressed in HEK293 cells augmented STAT1 and STAT6 activity induced by IFN γ or IL-4. Moreover, while ectopic overexpression of PTP1BWT in L428 cells attenuated STAT phosphorylation, STAT DNA binding activity and STAT transcriptional activity as measured by luciferase reporter assays, epression of the PTP1BΔ6 variant augmented the basal STAT activation similar to the ectopic expression of a dominant negative PTP1BC215S mutant. Moreover, PTP1B as well as PTP1BC215S increased L428 cell proliferation, whereas PTP1BWT had a negative impact on L428 cell growth. Of note, PTP1BΔ6 effects were not limited to the JAK-STAT pathway, but also affected the NF-κB signaling system. L428 cells overexpressing PTP1BWT, for instance, display a diminished expression of the NFκB transcription factors RelA and RelB, which was found to be increased in PTP1BΔ6 expressing L428 cells. Conclusions.  Together, the data presented here suggest that JAK-STAT signaling in cHL is regulated by shorter PTP1B variants like the novel PTP1BΔ6 variant which act as dominant negative regulators for PTP1B.

AG03.06 Application of next generation sequencing for IGH clonality testing in malignant lymphoproliferations P. Raisch*1, I. Bonzheim1, M. Schulze2, M. Sturm2, J. Steinhilber1, P. Bauer2, L. Quintanilla-Martinez1, F. Fend1 Institut für Pathologie, Universitätsklinikum, Tübingen, Deutschland, Institut für Medizinische Genetik und Angewandte Genomik, Universitätsklinikum, Tübingen, Deutschland

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Background.  Established methods for clonality testing like multiplex PCR based GeneScan analyses are often problematic for small extranodal biopsies, because the limited lymphocyte content results in a restricted repertoire of IGH-rearrangements within the DNA sample, which in turn may lead to pseudoclonal amplification products and false positive results. This study aimed to establish next generation sequencing (NGS) of PCR-amplified IGH-rearrangements for clonality testing in B-cell populations and thereby achieve an improvement in molecular diagnosis of lymphomas. Methods.  NGS of IGH rearrangements was conducted using BIOMED-2 FR1 and FR2 primer sets in a two-step PCR approach to add Fluidigm Access Array Barcode Library adapters for Illumina MiSeq sequencing and products were sequenced on the Illumina MiSeq platform. First, optimal PCR conditions and the detection limit of NGS were determined based on lymphoma cell line dilutions. Then well characterized primary B-NHL FFPE samples were analyzed to test the method’s suitability based on samples with a high content of malignant cells as well as on samples taken from patients over the course of disease. Results.  In dilutions of lymphoma DNA a cluster of the detected IGH-rearrangements which decreased with lower concentrations of lymphoma DNA in the sample could be assigned to that of the lymphoma cell line. It was detectable in dilutions up to 1:1000. Individual IGH-rearrangements of the reactive background ranged around or below 1 % of total detected rearrangements. In patient samples, IGH-rearrangements of a malignant clone that had previously been determined by Sanger sequencing, could be detected via NGS. Even in samples in which Sanger sequencing had failed, a prominent clonal population of individual IGH-rearrangements could be detected among a background of rearrangements deriving from reactive B-cells. In samples taken over the course of disease, individual lymphocyte populations could be detected and monitored based on their IGH-rearrangement. Conclusions.  Analysis of amplified IGH-rearrangements via NGS can provide information on the presence of clonality. Furthermore, this method allows the detection and monitoring of individual lymphocyte populations. By providing a detailed characterization of the IGH-repertoire, NGS can help interpreting problematic samples with low lymphocyte content. The preliminary data of this study promise an improvement of clonality detection in samples with low tumor cell content and in monitoring disease course.

AG03.08 Comprehensive characterization of targeted sequencing platforms to elucidate AML-specific mutational landscapes U. Wagner*1, C. Fritz1, N. Valtcheva1, M. Rechsteiner1, S. Balabanov2, P. Wild1 Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz, Abteilung für Hämatologie, UniversitätsSpital, Zürich, Schweiz

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Background.  Routine molecular diagnostics in acute myeloid leukemia (AML) is presently based mainly on the concurrent screening of several genes for mutations of therapeutic and prognostic significance. Using a multiple single-gene assay approach is challenging, as it requires large amounts of DNA and is work and time-intensive. Next-generation sequencing-based platforms are now being tested for and introduced to highly parallelized detection of mutations in a clinical setting. However, a consensus has not yet been reached on which platform is the most suita-

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ble for a given situation. Here, we present a comprehensive comparison of three different NGS platforms for AML diagnostics. Methods.  We carried out targeted sequencing of 38 DNA samples from 26 patients with AML and three replicate samples of Acrometrix Oncology Hotspot Control (AOHC) DNA each using the Ion Ampliseq Comprehensive Cancer panel (CCP, Thermo Fisher) on Ion Torrent Proton, the Ion Ampliseq AML Panel (AMLP, Thermo Fisher) on Ion Torrent Personal Genome Machine, and the TruSight Myeloid Panel on MiSeq (MMP, Illumina). Data analysis was carried out using CLC Genomic Workbench, Ion Reporter Software (CCP and AMLP data), Somatic Variant Caller as implemented in Illumina BaseSpace (MMP data) and Genome Analysis Toolkit (GATK). Selected individual results were verified by conventional single-gene assays. Results.  We were able to determine a wide range of performance characteristics of all three platforms. We show the extent of inter- and intra-platform variability for coverages, quality measures, variant calls, allele frequencies and other parameters. The AOHC samples allowed us to determine sensitivity and specificity of variant calls thus enabling us to fine-tune data analysis settings. We demonstrate that choosing the right data analysis software with settings adapted to the respective platform strongly enhanced the performance of all platforms. Furthermore, we were able single out regions covered by the essays that performed in a suboptimal manner. Conclusions.  Here we present the results of a comprehensive comparison of the properties of three major targeted sequencing platforms for AML. Although all three platforms can be used to elucidate the AML-specific mutational landscape of clinical samples, MMP and CCP can also be used in a wider context and thus have different conceptual properties. The information given here should support individuals in making informed decisions on which platform to use given a specific purpose.

AG03.09 Comprehensive mutation profiling of bone marrow trephines using next generation sequencing in the routine diagnostics of myeloid neoplasms: technical validation, reliability, and clinical usefulness S. Bartels*, B. Hasemeier, E. Schipper, G. Büsche, J. Schlue, H. H. Kreipe, U. Lehmann-Mühlenhoff Institut für Pathologie, Medizinische Hochschule Hannover, Hannover, Deutschland Background.  In order to enable comprehensive mutation profiling of bone marrow trephines suspicious for a myeloid neoplasm a customized next generation sequencing gene panel comprising 78 exons from 23 genes frequently mutated in myeloid neoplasms was developed, validated, and tested under routine conditions. Methods.  A list of 23 genes most frequently mutated in myelodysplastic syndrome (MDS) and myeloproliferative neoplasia (MPN) was compiled after a comprehensive literature search. Sequencing libraries for the Ion Torrent/PGM were synthesized from 30 ng of genomic DNA extracted from routinely processed bone marrow trephines. For technical validation 24 samples with known mutations were tested. Quality performance parameters were: number of total reads, percentage of reads on target, uniformity, mean read depth, mean read length, and quality score for sequence variants. Afterwards 200 samples from the daily routine were analysed. Data were evaluated using Ion Torrent Variant caller, Cartagenia Bench Lab NGS software version 4.0.2, and the IGV browser. Selected sequence variants were independently analysed suing pyrosequencing or Sanger sequencing. Results.  With a mean of 627,853 reads per sample (Range 627,853– 1,460,412) and a mean coverage of 2674 (Range 707–6327) a sufficient count of reads sequencing depth for mutation profiling could be achieved. Only five samples failed (2.5 %). In 200 samples we found in total 284 pathogenic variants (Mean 1.4 variants per patient, Range 0-5), whereby 139 patients exhibit at least one pathogenic mutation (69.5 %). The variants show allele frequencies ranging from 6.7 % up to 88.7 %. Only five genes

(BRAF, FLT3, KIT, NRAS and WT1) are mutated in less than 1 % of the patients. The most frequently mutated genes were TET2 (24.8 %), SRSF2 (18.6 %), TP53 (10.6 %), and ASXL1 (9.7 %). Compared to serial sequence analysis of individual genes this approach is much faster and more cost-effective. In numerous cases the mutation profiling confirmed a suspected diagnosis or even determined the final diagnostic evaluation. Conclusions.  Comprehensive mutation profiling using next generation sequencing is feasible and cost-effective under routine conditions in the work up of bone marrow trephines and improves the diagnostic reliability.

AG03.10 Activation of the innate immune system is linked to the pathogenesis of anemia in myelodysplastic sydrome R. Schneider-Kramann*1, G. Büsche2, U. Germing3, U. Platzbecker4, T. H. Brümmendorf5, R. Knüchel1, B. L. Ebert6 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, Institut für Pathologie und Molekularpathologie, Medizinische Hochschule Hannover, Hannover, Deutschland, 3Klinik für Hämatologie und Onkologie, Heinrich-Heine Universitätsklinikum, Düsseldorf, Deutschland, 4 Medizinische Klinik und Poliklinik 1, Universitätsklinikum Carl Gustav Carus, Dresden, Deutschland, 5Abteilung für Hämatologie und Onkologie, RWTH Universität, Aachen, Deutschland, 6Department of Hematology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Vereinigte Staaten von Amerika 1

2

Background.  Heterozygous deletion of RPS14 occurs in del(5q) MDS and has been linked to impaired erythropoiesis, characteristic of this disease subtype. Methods.  We generated a murine model with conditional inactivation of Rps14 in hematopoietic stem and progenitor cells. Results.  We demonstrated a p53-dependent erythroid differentiation defect resulting in age-dependent progressive anemia, megakaryocyte dysplasia, and loss of hematopoietic stem cell (HSC) quiescence Rps14 haploinsufficient mice. When hematopoietic cells were cultured in vitro, without in vivo compensatory mechanisms, Rps14 haploinsufficient hematopoietic stem and progenitor cells (HSPC) failed to undergo terminal differentiation into hemoglobinized CD71-Ter119+ cells in vitro. Rps14 haploinsufficient mice were not able to recover from hemolytic stress and died from severe anemia associated with a delayed reticulocyte response. We next asked whether Rps14 haploinsufficiency causes reduced protein synthesis, thereby contributing to the erythroid differentiation defect. We applied a fluorogenic assay using O-Propargyl-puromycin (OP-Puro), which incorporates into nascent polypeptides and enables quantitation of protein synthesis in individual cells in vivo. Protein synthesis was significantly reduced in Rps14 haploinsufficient hematopoietic stem cells and myelo-erythroid progenitor cells relative to wild-type cells. We explored the mechanistic basis for the anemia using unbiased, quantitative mass spectrometry in erythroid progenitor cells. We found powerful induction of proteins involved in innate immune signaling, particularly the danger associated molecular pattern (DAMP) heterodimeric S100A8/S100A9 proteins. We functionally validated that S100A8 is necessary and sufficient for the erythroid differentiation defect due to Rps14 haploinsufficiency. Moreover, we validated the association between ribosomal haploinsufficiency, induction of S100A8 and a severe erythroid phenotype in bone marrow samples of patients with del(5q) MDS. We rescued the erythroid differentiation defect in Rps14 haploinsufficient HSCs by genetic inactivation of S100a8 expression. Conclusions.  Our data link Rps14 haploinsufficiency to activation of the innate immune system via induction of S100A8/A9 and the p53-dependant erythroid differentiation defect in del(5q) MDS. These findings have direct implications for the biology and therapy of del(5q) MDS, as well as for other disorders of ribosomal protein haploinsufficiency.

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Abstracts AG03.11 Correlation of genetic mutations and clinical progression in pediatric Langerhans Cell Histiocytosis and their long-term consequences P. Schneckenburger*1, D. Nann2, J. Steinhilber2, G. Metzler3, R. Beschorner2, C. P. Schwarze1, P. Lang1, R. Handgretinger1, F. Fend2, I. Bonzheim2, M. Ebinger1 Klinik für Kinder- und Jugendmedizin, Kinderheilkunde I – Allgemeine Pädiatrie, Hämatologie und Onkologie, Tübingen, Deutschland, 2Institut für Pathologie, Universitätsklinikum, Tübingen, Deutschland, 3Klinik für Dermatologie, Universitätsklinikum, Tübingen, Deutschland 1

Background.  Langerhans Cell Histiocytosis (LCH) is a histiocytic disorder characterized by CD207+ dendritic cells which are surrounded by an inflammatory infiltrate. Although recent studies identified mutations in BRAF or MAP2K1 genes as molecular characteristics of LCH, data on clinical progression and long-term consequences in correlation with genetic features are sparse. The aim of the study was to analyze the BRAF and MAP2K1 mutation status and compare it to clinical parameters and follow-up data in a series of pediatric LCH. Methods.  BRAF codon 600, as well as MAP2K1 hotspot regions in exons 2 and 3 were analyzed in paraffin-embedded specimens of 37 pediatric LCH patients by mutation-specific immunostaining using VE1 antibody, allele-specific PCR, and Sanger sequencing, respectively and correlated with clinical progression and long-term consequences, which were retrospectively collected by contacting the patients. Results.  Seventeen of 37 (46 %) cases showed the BRAF p.V600E mutation, and in 11 (30 %) of the cases with wild-type BRAF a MAP2K1 mutation was detected. Additionally, we found one case with a BRAF p.V600D mutation with bone involvement. Patients with BRAF mutations more frequently showed multisystemic progression in comparison to wild-type cases. In contrast, the majority of the MAP2K1 mutated cases had unisystemic progression. Involvement of skin and internal organs were also more frequently associated with mutations; specifically, eight of nine cases with skin affection and all Hashimoto-Pritzker cases (n = 3) were BRAF p.V600E mutated. Furthermore, all cases considered clinically at risk due to involvement of internal organs had either a BRAF or MAP2K1 mutation. Patients with BRAF mutations showed a two year relapse-free survival of 72 %, in contrast to 100 % without mutation. Including also LCH patients without available molecular data (46 patients, follow-up range: 4.2 to 33.7 years, follow-up median: 13.0 years), long-term consequences caused by LCH were present in 41 %, in particular orthopedic abnormalities, hormonal disorders, loss of teeth and others. Conclusions.  The distribution of BRAF and MAP2K1 mutations in our study of pediatric LCH is similar to reported data. Skin involvement, multisystemic progressive disease and relapses were associated with a BRAF mutation. In long-term follow-up, pediatric LCH revealed an overall high frequency of long-term disease-related medical consequences.

AG03.12 Oncogenic signaling in multiple myeloma: correlation of RAS/RAF mutations and MAPK pathway activation in primary myeloma biopsies J. Xu*1, 2, 3, N. Pfarr3, 4, V. Endris3, E. K. Mai2, N. H. Md Hanafiah1, N. Lehners1, 2, R. Penzel3, W. Weichert3, 4, A. D. Ho2, P. Schirmacher3, H. Goldschmidt2, 5, M.-S. Raab1, 2, M. Andrulis3, 6 Max-Eder-Nachwuchsgruppe Experimentelle Therapien hämatologischer Neoplasien, Innere Medizin V, Universitätsklinikum Heidelberg und Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Deutschland, 2 Medizinische Klinik, Innere Medizin V, Universitätsklinikum Heidelberg, Heidelberg, Deutschland, 3Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, 4Technische Universität München, Pathologie, München, Deutschland, 5Nationales Centrum für Tumorerkrankungen (NCT), Heidelberg, Deutschland, 6Universitätsklinikum Ulm, Institut für Pathologie, Ulm, Deutschland

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Background.  Multiple myeloma (MM) is a plasma cell malignancy characterized by high molecular heterogeneity. Most frequent mutations are found in RAS/RAF genes, pointing to the MAPK signaling pathway (RAF/ MEK/ERK) as a therapeutic target. However, recently reported RAS/RAF mutant MM patients (pts) treated with the MEK inhibitor- trametinib, led to heterogeneous response rates, suggesting a non-linear dependency of MAPK signaling in RAS/RAF mutant MM. The aim of the study was to explore this question by correlating RAS/RAF mutations with activation of MEK/ERK signaling pathway in primary MM biopsies. Methods.  Bone marrow or soft tissue biopsies from 102 pts with newly diagnosed myeloma (NDMM) and 77 pts with relapsed/refractory myeloma (rrMM) were included in the study. Immunohistochemical staining with primary antibody against phosphorylated-ERK (pERK) was used to detect activated RAF/MEK/ERK cascade. Mutation analysis was performed by targeted sequencing using the Ion Torrent NGS technology. Results.  Mutation analysis revealed frequent KRAS (25 %), NRAS (24 %), and BRAF (8 %) mutations as recently reported. Overall, RAS/RAF mutations were significantly more frequent in rrMM compared to NDMM cases (p = 0.01). The nine most frequent recurrent mutations were KRAS Q61H (n = 11), NRAS Q61R (n = 11), NRAS Q61K (n = 10), KRAS G12D/ G12V (n = 6 each), BRAF V600E (n = 6), NRAS G13D (n = 5) and NRAS G13R/Q61H (n = 4 each). KRAS but not NRAS mutations were significantly associated with EKR activation as compared to RAS/RAF-wild type (wt) cases (p = 0.003). When each recurrent RAS/RAF mutation was tested against all RAS/RAF-wt samples only KRAS G12D and BRAF V600E were consistently associated with ERK activation (p < 0.001 each). This indicates that ERK activation is dependent on the type of mutation. Conclusions.  In summary, we demonstrated for the first time that RAS/ RAF mutations are not generally associated with RAF/MEK/ERK pathway activation in MM. Specifically, only KRAS G12D and BRAF V600E are consistently associated with MAPK pathway activation in MM. These findings provide a plausible explanation for heterogeneous responses to MAPK inhibition in MM and indicate that a confirmation of MAPK activation on protein-level should be considered for optimal patient stratification. Furthermore, our data imply that immunohistochemistry for pERK is a cost-effective and reliable method to complement the mutation analysis. The prospective validation in future clinical trials for targeting the MAPK pathway is warranted.

AG03.13 Rare SNPs in receptor tyrosine kinases are negative outcome predictors in multiple myeloma S. Keppler1, S. Weißbach1, C. Langer2, S. Knop1, J. Pischimarov1, M. Kull2, T. Stühmer1, T. Steinbrunn1, R. Bargou1, H. Einsele1, A. Rosenwald1, E. Leich*1 1 Universität Würzburg, Würzburg, Deutschland, 2Universitätsklinikum Ulm, Ulm, Deutschland

Background.  Multiple myeloma (MM) is a malignancy of finally differentiated B-cells that is characterized by a great genetic heterogeneity. Recent investigations by whole exome sequencing revealed an accumulation of tumor-associated mutations in receptor tyrosine kinases (RTKs) which may also trigger the activation of survival pathways in MM. Methods.  To investigate the clinical impact of RTK-mutations in MM, we sequenced the coding DNA-sequence of EGFR, EPHA2, ERBB3, IGF1R, NTRK1 and NTRK2 which were found to be mutated in MM in a previous study by amplicon sequencing, in 75 uniformly treated MM patients of the “Deutsche Studiengruppe Multiples Myelom”. Finally, the detected mutations were correlated with common cytogenetic alterations and clinical parameters. Results. We identified 11 novel non-synonymous SNVs or rare patient-specific SNPs that were not listed in the SNP databases 1000 genomes and dbSNP in 10 MM patients. The mutations accumulated in the tyrosine-kinase and ligand-binding domains. No correlation with cytogenetic parameters was found. Interestingly, however, the occurence of RTK-mu-

tations was associated with a worse survival. Specifically patients with rare patient-specific SNPs, showed a significant lower overall survival, eventfree survival and progression-free survival. Conclusions.  This indicates that RTK-mutations, specifically rare patient-associated RTK SNPs are of prognostic relevance in MM. Thus, MM patients with RTK-mutations may profit from a treatment with RTK-inhibitors.

AG Thoraxpathologie AG04.01 Histomorphological and molecular characterization of human plexiform vasculopathy and its Sugen (SU 5416)/hypoxia induced rat model L. Mägel*1, B. Ludewig1, P. Borchert1, O. Rafikova2, M. Kellner2, M. Kühnel3, D. Jonigk1 Institut für Pathologie, Medizinische Hochschule Hannover, Hannover, Deutschland, 2University of Arizona, College of Medicine, Center for Lung Vascular Pathobiology, Tucson, Arizona, Vereinigte Staaten von Amerika, 3 Institut für Funktionelle und Angewandte Anatomie, Medizinische Hochschule, Hannover, Deutschland

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Background.  Pulmonary Hypertension (PH) is characterized by progressive and irreversible remodeling of the pulmonary vasculature and is associated with a poor prognosis. Due to limited availability of human lung tissue samples, research in human PH is restricted to postmortem autopsies or analysis of endstage lung explants. Therefore, in order to study the development of the disease, a animal model, which mimics the pathophysiology of human PH, is required. It has been put forward that a “2-hit” model based on subcutaneous application of SU 5416 in rats combined with chronic hypoxia represents the best model for PH, since the animals develop histomorphological changes which resemble human plexiform vasculopathy. However, until now it is not known whether these histopathological changes mimics the human disease on a genetic level or not. In the present study, we aimed to address this question by examining histomorphologic and gene expression profiles of plexiform vasculopathy in human lung explants and SU 5416/hypoxia exposed rats. Methods.  We selected 25 human bilateral lung explants with prominent plexiform vasculopathy from patients with PH. Downsizing lung tissue of donor lungs (n = 18) served as human reference. Furthermore, a total of 28 Sprague– Dawley rats (female, n = 15, male, n = 13) were used. PH was induced via a high dose of SU 5416 (200 mg/kg, sc) followed by 3 weeks exposure to hypoxia (n = 14) as described by Voelkel and Tuder. Compartment-specific (normal and neighboring arteries, concentric and plexiform lesions) mRNA expression analysis of angiogenesis related genes in rat and human samples were performed by using lasermicrodissection and microarray analysis. Results.  We could confirm that SU 5416/hypoxia induced rat model develop histomorphlogical changes resembling human plexiform vasculopathy. On a molecular level the rat model revealed significant differences between male and female rats. It turned out that the gene expression profile of male rats was consistent with the human genetic profiles of plexiform/ concentric lesions as well as lung arteries compared to controls regarding the angiogenic genes (Cav2, FGF2, HIF1a, KDR, Notch4, VCAM1 and VEGFC), irrespective of sex. Conclusions.  Histomorphological and molecular changes of SU 5416/hypoxia model in male rats seems to resemble human situation in PH end stage lungs. Therefor, this rat model seems to be a promising approach to study the time-dependent development of the disease.

AG04.02 Prospective Molecular Profiling in Human Lung Allografts F. Länger*1, N. Izykowski1, J. Rische1, P. Braubach1, J. Gottlieb2, T. Welte2, A. Haverich3, G. Warnecke3, H.-H. Kreipe1, M. Kühnel4, D. Jonigk1 1 Institut für Pathologie, Medizinische Hochschule Hannover, Hannover, Deutschland, 2Klinik für Pneumologie, Medizinische Hochschule, Hannover, Deutschland, 3Klinik für Herz, Thorax-, Transplantations- und Gefäßchirurgie, Medizinische Hochschule, Hannover, Deutschland, 4Institut für Funktionelle und Angewandte Anatomie, Medizinische Hochschule, Hannover, Deutschland

Background.  Chronic lung allograft dysfunction (CLAD) is the main reason for the poor long-term outcome of lung transplanted patients, with bronchiolitis obliterans representing the predominant histopathological feature. It is morphologically defined by a progressive fibrous obliteration of the small airways, thought to be triggered by a combination of non-immune bronchial injury and allo-/autoimmune mechanisms. As biopsies are too insensitive to reliably detect bronchiolitis obliterans and a decline in lung function tests, which is clinically used to define chronic lung allograft dysfunction, is rather unspecific and does not detect early stages, there is need for alternative approaches for early diagnosis. Methods.  Here, we present the analysis of the cellular composition and differential expression of 45 tissue remodelling-associated genes in transbronchial lung biopsies from two cohorts with 18 patients each: patients, who did not develop chronic lung allograft dysfunction within 3 years after transplantation (48 biopsies) and patients rapidly developing chronic lung allograft dysfunction within the first three postoperative years (57 biopsies). For expression analysis of fibrosis associated genes whole sections from biopsies without BO lesions were taken and following RNA extraction low-density/high throughput (RT-PCR) array-based analyses was performed. Results were confirmed by appropriate in-situ methods (immunohistochemistry) and correlated with morphological parameters and clinical outcome. This approach has recently been expanded to all biopsies from the surveillance biopsy program of our transplantation service. Results.  We could identify a subset of genes overexpressed in morphologically inconspicuous biopsies from patients either prior to or after the clinical diagnosis of CLAD. Integrating the mRNA expression levels of the five most significantly dysregulated genes from the TGF-β-axis (BMP4, IL6, MMP1, SMAD1 and THBS1) into a score, patient groups could be confidently separated and the clinical outcome predicted (p < 0.001). Additionally the first results from the analyses of the surveillance biopsy programme are presented. Conclusions.  We conclude that expression analyses of fibrosis-associated genes may be valuable as tissue-based molecular biomarker to more accurately diagnose or predict chronic lung allograft dysfunction. This hypothesis is currently tested in our surveillance biopsy programme of lung transplant patients.

AG04.03 Remodelling-related molecular profiles in different subforms of chronic lung allograft dysfunction D. Jonigk*1, B. Rath1, L. Mägel1, H. Golpon2, N. Izykowski1, T. Welte2, S. Janciauskiene2, J. Gottlieb2, G. Warnecke3, A. Haverich3, H.-H. Kreipe1, M. Kühnel4, F. Länger1 Institut für Pathologie, Medizinische Hochschule Hannover, Hannover, Deutschland, 2Klinik für Pneumologie, Medizinische Hochschule, Hannover, Deutschland, 3Klinik für Herz, Thorax-, Transplantations- und Gefäßchirurgie, Medizinische Hochschule, Hannover, Deutschland, 4Institut für Funktionelle und Angewandte Anatomie, Medizinische Hochschule, Hannover, Deutschland 1

Background.  Chronic lung allograft dysfunction (CLAD) is the major obstacle to long term survival following lung transplantation (LuTX). Bronchiolitis obliterans represents the most frequent and well characterized histological correlate of CLAD. However,recently a divergent clinical manDer Pathologe Suppl 1 · 2016

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Abstracts ifestation of CLAD showing restrictive changes in lung physiology has been introduced as restrictive allograft syndrome (RAS). Morphologically RAS is characterized by a pattern reminiscent to pleuroparenchymal fibroelastosis (PPFE) often in combination with acute pattern injuries. A differential analysis of the morphology and governing molecular fibrosis pathways of RAS in comparison to BO is warranted to elucidate the pathophysiology and better predict the development of CLAD. Methods.  We performed compartment-specific analyses using laser microdissection, RT-PCR based microarray techniques and immunohistochemistry combined with the novel imaging technique two photon scanning laser optical tomography (2P-SLOT) in explanted lung allografts from patients with BO or RAS. In addition we performed a comparative analysis with BO triggered by stem cell transplantation (BO-GvHD). Results.  Low-density/high throughput arrays proved to be a reliable tool for analysing gene expression in microdissected formalin fixed paraffin embedded (FFPE) samples of lung transplanted patients with predominant BO and RAS characteristics, respectively. Likewise, the complementary clearing resin (CRISTAL) embedded 2P-SLOT samples allowed for assessment of complementary morphological changes. Particularly genes belonging to the TGF-β superfamily showed a compartment-specific alteration of expression in BO and RAS phenotypes of CLAD, respectively, while BO-GvHD was identical to BO after LuTX. Among the differentially expressed genes 5 members of the MMP family were found to be exclusively expressed in BO, but not in RAS. Conclusions.  Airway lesions after LuTX and BO-GvHD show a similar regulation of fibrosis-associated pathways. They especially share relevant similarities in activated pathways involved in tissue-remodelling, but at the same time have fundamental differences in MMP expression profiles. These MMPs are therefore promising candidates as diagnostic markers of RAS, as these gene products are soluble and located in the alveolar spaces and therefore detectable in lavage fluids as well as transbronchial biopsies.

AG04.04 COPD-like inflammation drives the progression of K-ras induced early cancerous lesions C. Jungnickel*1, R. Bals1, R. M. Bohle2, C. Zaharia2, C. Beisswenger1, P. A. Schnabel2 Universität des Saarlandes, Innere Medizin V, Homburg Saar, Deutschland, Universität des Saarlandes, Institut für Allgemeine und Spezielle Pathologie, Homburg Saar, Deutschland

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Background.  Chronic obstructive pulmonary disease (COPD) is a risk factor for lung cancer (LC). COPD is characterized by chronic inflammation of the airways and lung infections. Adenocarcinomas are frequently associated with an activating mutation in the K-ras gene. The aim of this study was to determine the effect of COPD-like inflammation on the progression of K-ras induced cancerous lesions. Methods.  Mice (n ≥ 7 per group) were exposed to cigarette smoke (CS) 5 days per week. CS- or air exposed mice were exposed to a clinical isolate of heat inactivated non-typeable Haemophilus influenzae (NTHi) 3 days per week. Histologic analysis were performed on formalin fixed and paraffin embedded sections. Pre-cancerous lesions were divided into alveolar hyperplasia, alveolar adenomatous hyperplasia (AAH), and adenocarcinoma. Alveolar Hyperplasia were defined by an increase of cells along the interalveolar septa in the alveolar space. Alveolar Hyperplasia developed from single-layered lesions into alveolar adenomatous hyperplasia, characterized by a further increase in cellularity. The structure of the alveoli was still recognizable, without any invasive growth. Cells within adenocarcinoma were charcterized by a round shape, nuclear atypia and less differentiation compared to hyperplasia. [1] Results.  Mice with an oncogenic K-ras allele in lung epithelium were exposed to CS, NTHi, and the combination of CS and NTHi for 12 weeks. Exposure of mice to NTHi resulted in an increased total burden and in increased numbers of AAH lesions, whereas exposure to CS alone did not

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affect tumor growth. Combined exposure to CS and NTHi led to larger pre-cancerous lesions and an increased adenocarcinoma formation. Conclusions.  CS and NTHi promote the progression of K-ras induced early cancerous lesions resulting in a more severe phenotype. References 1. Sutherland KD, Song JY, Kwon MC, Proost N, Zevenhoven J, Berns A (2014) Multiple cells-of-origin of mutant K-Ras-induced mouse lung adenocarcinoma, Proc Natl Acad Sci U S A

AG04.05 PTEN is upregulated in the matricellular stroma of non-small cell lung carcinoma (NSCLC) K.-F. Deml*1, H.-U. Schildhaus2, U. Rolle3, I. Schmitt-Opitz4, R. Casanova3, A. Soltermann3 UniversitätsSpital Zürich, Institut für Klinische Pathologie, Zürich, Schweiz, Institut für Pathologie, Universitätsmedizin, Göttingen, Deutschland, 3 Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz, 4Klinik für Thoraxchirurgie, UniversitätsSpital, Zürich, Schweiz 1

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Background.  Phosphatase and tensin homolog (PTEN) is an important tumor suppressor in malignant tumor epithelia but few is known about its function in the surrounding stroma. Recently, Trimboli et al. demonstrated that PTEN inactivation in stromal fibroblasts of mouse mammary glands accelerates malignant transformation of mammary epithelial tumors and leads to massive remodeling of extracellular matrix (ECM). We investigated the protein expression of PTEN in the immediate peritumoral stroma, and the corresponding tumor epithelia of non-small cell lung carcinoma (NSCLC) together with relevant extracellular matrix proteins. Methods.  Expression of PTEN and ECM proteins including collagen, versican, vimentin and periostin were immunohistochemically determined on a tumor tissue microarray of 456 patients with NSCLC of all pT stages. The H-score was correlated with histological subtype, pTNM, metastasis and survival. Results.  Our study included 233 squamous cell carcinomas (SCC) and 223 adenocarcinomas (ADC). For all NSCLC, PTEN expression in both cytoplasm and nucleus was higher in the matricellular cancer-associated fibroblasts (CAFs) than in the carcinoma epithelia. A complete loss of stromal PTEN was infrequent (<0.5 % of tumors). High PTEN in both stroma and tumor was associated with lower pT and smaller tumor size. Stratification for histology indicated that ADC displayed higher nuclear PTEN than SCC. Further, complete loss (H-score 0) of epithelial PTEN occurred more frequently in SCC compared to ADC. Among SCC, high PTEN in both cytoplasm and nucleus of CAFs as well as in the cytoplasm of tumor epithelia correlated again with smaller tumor size. No correlation was observed with lymph node or distant metastasis. High vimentin, versican and collagen but not periostin expression was positively correlated with PTEN in both tumor and stroma. Conclusions.  PTEN loss in both cytoplasm and nucleus occurs more frequently in lung squamous cell than adenocarcinoma. Peritumoral cancer-associated fibroblasts have a higher PTEN protein expression than the adjacent malignant epithelia. However, protein synthesis decreases with increasing tumor size, probably via alteration of the extracellular matrix composition.

AG04.08 Prognostic significance of the autophagy markers p62 and LC3B in stage I/II NSCLC A. Schläfli1, O. Adams1, 2, J. A. Galván1, L. Bubendorf3, S. Savic-Prince3, M. Gugger1, 4, R. A. Schmid5, M. P. Tschan1, R. Langer1, S. Berezowska*1 Institut für Pathologie, Universität Bern, Bern, Schweiz, 2Graduate School for Cellular and Biomedical Sciences, Universität Bern, Bern, Schweiz, 3 Institut für Pathologie, Universitätsspital, Basel, Schweiz, 4Promed SA Laboratoire Medical, Fribourg, Schweiz, 5Universitätsspital Bern, Klinik für Thoraxchirurgie, Bern, Schweiz

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Background.  Autophagy plays a divergent role in cancer, as it may support tumor cell survival or promote cell death. Protein light chain 3B (LC3B) and p62 are proteins associated with autophagosomal membranes that engulf cytoplasmic content during autophagy, which is subsequently degraded. We studied the expression of these two markers in early stage non-small cell lung cancer (NSCLC). Methods.  Immunohistochemistry for LC3B and p62 was performed on a tissue microarray with 400 NSCLC: 175 Adenocarcinomas (AC), 198 squamous cell carcinomas (SCC), 27 large cell carcinomas (LCC). Dot-like cytoplasmic expression of the two markers as well as cytoplasmic and nuclear staining for p62 was determined using an established protocol. Results were compared with clinical and pathological parameters. Results.  LC3B expression showed a positive correlation with p62 dotlike (p < 0.001) and nuclear staining (p = 0.007). There was no significant intratumoral heterogeneity. The percentage of LC3B high expressing tumors was higher in AC (p = 0.001) whereas SCC showed more frequent nuclear p62 expression (p = 0.009). Apart from a higher percentage of high p62 expression (dot like) among stage IA tumors (p = 0.035), there was no association with stage, age or gender. Survival analysis revealed a small group of patients with high LC3B/low p62 expressing tumors with a better outcome compared to the remaining cases (p = 0.103). This marker combination has been suggested to reflect intact activated autophagy. However, the best prognostic value was observed for the identification of “triple low p62” expressing tumors (consisting of dot-like, cytoplasmic and nuclear), that showed a significantly better outcome compared to tumors with any high p62 staining in univariate analysis (p = 0.026) and in multivariate analysis (p = 0.043; HR = 1.64) in addition to tumor stage (p = 0.01; HR = 1.90). Conclusions.  The expression of the autophagy markers LC3B and p62 can be detected in NSCLC, supporting the biologically significant role of autophagy in these tumors. High LC3B levels seem to be linked to lower tumor aggressiveness, while high p62 expression was significantly associated with aggressive tumor behavior. Further investigations are needed to link expression data to conclusions about functional autophagy states. Moreover, the role of p62 may not be restricted to autophagy and it needs to be shown if other factors may contribute to our findings.

AG04.09 Activation of ABC transporter and inhibition of apoptosis as the main driver of the Etoposide and Cisplatin resistance mechanism in the thymic carcinoma cell line 1889c S. Hesse*1, S. Küffer1, F. Bensch2, V. Venkataramani3, H. Bohnenberger1, P. Ströbel1 Institut für Pathologie, Univeristätsmedizin, Göttingen, Deutschland, University Medical Center Groningen, Department of Medical Oncology, Groningen, Niederlande, 3Abteilung Hämatologie und Onkologie, Universitätsmedizin, Göttingen, Deutschland

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Background.  Thymomas (TH) and thymic carcinomas (TC) are rare but the most common primary mediastinal tumours. They tend to be aggressive and have a high frequency for local therapy failures. Chemotherapy

is used in patients with unresectable or recurrent disease, however a optimal treatment has not yet been established and tumours often become resistant. In order to better understand the resistance mechanisms against chemotherapeutics in TC we investigated two TC cell lines resistant against Cisplatin and Etoposide. Using stable isotope labelling by/with amino acids in cell culture (SILAC) we characterized the specific resistance mechanisms of Cisplatin and Etoposide and reveal possible problems and opportunities in the long term treatment of TH and TCs. Methods.  The TC cell line 1889c was adapted to increasing levels of Etoposide and Cisplatin over 18 month. Resistance was regularly tested via cell viability assay MTT test. Quantification of proteins by mass spectrometry was perfomed in treated and control cell after feeding cells with normal and heavy isotope labelled amino acids. Apoptosis and Cell Cycle were detected with the Muse system 24 hours after Cisplatin (0,5 µg/ml) and Etoposide (2 µg/ml) treatment. DNA damage response (DDR) was recorded with an antibody against p-H2AX by western blot. Reactive oxygen species (ROS) levels were detected by FACS analysis using a fluorescence antibody against 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA). Rescue experiments were either done be specific gene knock down using siRNAs or specific chemical inhibitors. Results.  Viability assays showed that the established resistance is stable over a large range of increasing concentrations. The Etoposide resistance showed an increase cellular export by activation of ABC-transporters and the Cisplatin resistant 1889c showed an increased DDR, inhibition of apoptosis and cell cycle arrest. Both cell lines showed a up to 100-fold level of reactive oxygen species (ROS) than parental cell lines. Conclusions.  Cisplatin resistance in 1889c is preferentially established by an apoptosis inhibition and a possible G2/M arrest, whereas the Etoposide resistance is mainly caused by increasing a specific export via ABC transporter. However this does not exclude that either mechanism is active in both resistances. In addition, resistant cells showed a lower level of ROS and lost their resistance over time when not treated. This study provides possible resistance mechanisms of TH and TC.

AG04.10 Tracking the Clonal Origin and Genetic Heterogeneity of Malignant Pleural Mesothelioma K. Oehl*1, B. Vrugt1, U. Wagner1, A. J. Christiansen1, M. Rechsteiner1, Q. Zhong1, M. Meerang2, M. B. Kirschner2, W. Weder2, I. Opitz2, P. J. Wild1 UniversitätsSpital Zürich, Institut für Klinische Pathologie, Zürich, Schweiz, UniversitätsSpital Zürich, Klinik für Thoraxchirurgie, Zürich, Schweiz

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Background.  Malignant Pleural Mesothelioma (MPM) is a rare but aggressive neoplasm with a median life expectancy is around 12 months, despite widely used multimodal treatment efforts including chemotherapy followed by surgery. In addition, no effective second-line treatment could be established so far for patients with MPM. To investigate the underlying mechanisms of MPM development leading to treatment resistance, the objective of this study was to track the clonal origin and to investigate the intra-tumor heterogeneity of MPM at different time points during treatment. Methods.  A cohort of MPM patients undergoing treatment with Cisplatin and Pemetrexed followed by surgery were chosen for analysis. DNA was isolated from FFPE samples which were taken at the initial treatment-naïve biopsy (referred to as phase 1), at extrapleural pneumonectomy after combined induction chemotherapy (phase 2) and at relapse (phase 3), followed by ultra-deep targeted amplicon sequencing. For this purpose, based on a systematic literature review, a custom-designed MPM-specific sequencing panel was established, targeting the coding sequence of the 30 most frequently mutated genes in MPM. Analysis was conducted following internally established guidelines for diagnostic sequencing.

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Abstracts Results.  Sequencing revealed high intra-tumor heterogeneity between and even within the three different phases. In some samples, an accumulation of private mutations occurred in phase 2. Only very few mutations could be found in every phase analyzed. One of them is a possible driver mutation in exon 8 of NF2 that leads to an amino acid change as well as to a truncation of the protein. Conclusions.  Our results indicate a very high heterogeneity within the same tumor tissue and between the samples taken at different time points during the course of disease. This is especially evident in the surgical samples taken after chemotherapy, which could be explained by the high selective pressure in favor of chemo-resistant tumor cell populations. Altogether the high heterogeneity could explain the relatively poor response rates to chemotherapy in MPM patients and probably limits the efficacy of future targeted therapies. However, targeting the possible driver mutations, for example in NF2, could be a promising approach for the personalized treatment of MPM.

AG04.11 T790M mutation testing from tissue and plasma: results and conclusions of the first german round robin testing J. Fassunke, M. Ihle, S. Merkelbach-Bruse* Institut für Pathologie, Universitätsklinikum, Köln, Deutschland Background.  Targeted therapy using Osimertinib (Tagrissoâ) has obtained conditional approval by the European Medicines Agency (EMA) in February 2016 for the treatment of adult patients with locally advanced or metastatic epidermal growth factor receptor (EGFR) T790M mutation-positive non-small cell lung cancer (NSCLC). Osimertinib is indicated for patients with T790M mutation-positive NSCLC, irrespective of previous treatment with an EGFR tyrosine kinase inhibitor (TKI). Osimertinib targets both the EGFR mutation that triggers cancer development and T790M, a mutation that makes tumours resistant to existing treatment with EGFR-TKIs. Nearly two out of three patients with NSCLC whose disease progresses after treatment with an EGFR inhibitor develop the T790M mutation, for which treatment options are limited. A small number of patients (approximately 3–5 %) have the T790M mutation at NSCLC diagnosis. Eligibility for treatment with osimertinib will be dependent on mutation status, to be determined through a validated diagnostic test based on a tumour tissue sample or plasma. In order to initiate and supervise large-scale and nationwide quality controlled testing for EGFR mutations in NSCLC in Germany a steering panel consisting of six institutions was implemented. T790M was tested on tissue as well as on blood samples. The blood samples were prepared from healthy donors and spiked with different amounts of cell line DNA (MCF7 and NCI-HT1875). For better preservation, blood samples were shipped in Cell-Free DNA BCT tubes with a formaldehyde-free preservative stabilizing nucleated blood cells. Methods for plasma preparation, DNA extraction and mutation analysis could be chosen freely in each institute. Based on the results and experiences from the internal trial an open round robin trial was initiated in March 2016.

AG04.12 Molecular profiling of lung adenosquamous carcinoma: hybrid or genuine type? E. Vassella*1, S. Schäfer2 Universität Bern, Institut für Pathologie, Bern, Schweiz, 2Uniklinik Köln/ Institut für Pathologie, Köln, Deutschland 1

Background.  Lung adenosquamous carcinoma is a particular subtype of non-small cell lung carcinoma that is defined by the coexistence of adenocarcinoma and squamous cell carcinoma components. The aim of this

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study was to assess the mutational profile in each component of 16 adenosquamous carcinoma samples from a Caucasian population. Methods.  The mutational profile was assessed by a combination of next generation sequencing using the cancer hotspot panel as well as the colon and lung cancer panel and FISH. Identified mutations were confirmed by Sanger sequencing of DNA from cancer cells of each component collected by Laser Capture microdissection. Expression of adenocarcinoma and squamous cell carcinoma-specific markers was assessed by immunohistochemistry. Expression of classifier miR-205 was assessed by real-time PCR. Results.  Mutations typical for adenocarcinoma as well as squamous cell carcinoma were identified. Driver mutations were predominantly in the trunk suggesting a monoclonal origin of adenosquamous carcinoma. Most remarkably, EGFR mutations and mutations in the PI3K signaling pathway, which accounted for 30 % and 25 % of tumors respectively, were more prevalent while KRAS mutations were less prevalent than expected for a Caucasian population. Surprisingly, expression of classifier miR-205 was intermediate between that of classical adenocarcinoma and squamous cell carcinoma suggesting that adenosquamous carcinoma is a transitional stage between these tumor types. Conclusions.  Our findings that both components share the same driver mutations and express intermediate levels of classifier miR-205 suggest that adenosquamous carcinoma is not a hybrid cancer but rather constitute an own entity. Owing to the high prevalence of therapy relevant driver mutations, a higher proportion of patients may benefit from targeted therapy than expected for a Caucasian population.

AG04.13 Detection of genetic rearrangements in lung adenocarcinomas by targeted RNA-sequencing N. P. Velizheva*1, M. P. Rechsteiner1, C. E. Wong1, Q. Zhong1, M. Rössle2, B. Bode1, H. Moch1, A. Soltermann1, P. J. Wild1, V. Tischler1 1 UniversitätsSpital Zürich, Zürich, Schweiz, 2Kantonsspital Graubünden, Chur, Schweiz

Background.  Molecular testing of lung adenocarcinomas (ADCAs) is crucial for therapy selection. Besides mutations (EGFR, ERBB2, BRAF, KRAS) and amplifications (MET), gene rearrangements (ALK, ROS1, RET) are tested to guide treatment in patients with advanced lung ADCA. Because of limited tissue and low incidence of predictive rearrangements, parallel testing by targeted RNA-sequencing is preferred. We aimed to establish the simultaneous detection of therapeutically relevant gene rearrangements by next-generation sequencing (NGS). Methods.  A comprehensive cohort of advanced lung ADCAs (n = 112) was tested in this study. Sixteen lung ADCAs (13 FFPE tissue blocks, 3 cytological FFPE cell blocks, and 8 cytological smears matching for 8 out of all 16 cases) were selected as a first training cohort, which were previously molecularly characterized by Sanger sequencing and FISH. Additionally, 96 FFPE tissue samples were selected for independent targeted RNA-sequencing as validation set. ALK, ROS1 and RET status was pre-detected by FISH. RNA from all ADCAs was isolated using the Maxwell 16 LEV RNA FFPE Purification Kit. The obtained RNA was quantified with Qubit 2.0. The quality was assessed by the Agilent 2100 Bioanalyzer. For NGS library amplification, the Ion AmpliSeq RNA Fusion Lung Cancer Research panel and Oncomine Panel for Fusion Transcripts were applied with subsequent sequencing on the Ion Torrent Personal Genome Machine (PGM). Results.  All FFPE samples and cytological smears yielded sufficient RNA for library amplification and PGM sequencing. Fifteen of 16 FFPE samples as well as seven of eight cytological smears from the training cohort produced acceptable sequencing parameters (total valid mapped reads >20,000, minimum 3 of 5 internal reference controls expressed). NGS failed in one of 16 ADCAs by not reaching enough mapped reads for sensitive detection of potential translocations. Importantly, all previously de-

tected ALK and ROS1 gene rearrangements were confirmed, resulting in 94 % performance, 100 % sensitivity, and 100 % specificity. All but one sample from the validation set (n = 96) reached appropriate quality metrics and were successfully sequenced (overall performance 99 %). Conclusions.  NGS-based targeted RNA-sequencing was established in a retrospective clinical setting. This assay allows the input of different types of specimens with limited material, proved to be accurate for therapeutically significant gene rearrangements, and will hopefully reduce time until oncological treatment decision.

AG04.14 Critical aspects of the KIF5B-RET fusion variant detection in NSCLC R. Penzel*, A.-L. Volckmar, M. Kriegsmann, F. Lasitschka, P. Schirmacher, A. Stenzinger, V. Endris Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland Background.  Beyond established targeted therapies for lung adenocarcinoma patients targeting EGFR or ALK, recurrent gene fusions involving protein kinases impose new diagnostic opportunities. Genetic rearrangements of the RET (Rearranged during Transfection gene) locus are in the focus as the first RET inhibitor has been approved by FDA and EMA for Medullary Thyroid Cancer. About 1–2 % of lung adenocarcinoma patients are ‘triple negative’ for EGFR KRAS mutations and ALK rearrangements, but display a RET fusion thereby probably profiting from RET targeted therapy. Methods.  As consecutive analysis of EGFR mutations and ALK-, ROS1and RET fusions is time consuming, taking about 9 working days, we implemented a combined NGS analysis for DNA mutations and RNA-based fusion analysis, The NSCLC fusion panel covers ALK-, ROS-, RET- and NTRK1-fusions in the same assay. In addition ALK immunohistochemistry is performed in parallel. Positive ROS1 and RET rearrangements are cross validated by FISH. Results.  Besides ALK and ROS1 fusions, we currently identified RET positive cases in 5/539 (1 %) analysed samples. The RET fusion positive cases detected by routinely performed NGS fusion panel screening were cross validated using FISH analysis (ZytoLight SPEC RET Dual Color Break Apart Probe). All but one harboured a KIF5B (kinesin family 5B gene)RET fusion as a consequence of a small inversion of chromosome 10. Two of the variants (KIF5B Ex 15-RET Ex 12: 11.08 mbp; KIF5B Ex 24-RET Ex 11: 11.1 mbp) are characterised by even smaller split distances than EML4-ALK fusions (12 mbp). Therefore, half of the KIF5B-RET positive cases detected by NGS-screening could not be identified by blinded FISH validation. To validate the RET fusions, we designed primers for conventional RT-PCR and Sanger sequencing and verified the fusion transcripts in all cases the. FISH analysis was only successful for the identification of the K15R12 inversions. For the K24R11 types FISH failed to unambiguously identify inversions. Conclusions.  For diagnostic screening of small inversions like KIF5B-RET, FISH break apart may be critical. We propose conventional RT-PCR with subsequent Sanger sequencing as a time and cost efficient confirmatory method.

AG04.15 Mutational profiling of non-small cell lung carcinoma (NSCLC) brain metastases D. Förbs*1, M. Klier-Richter1, R. Rodriguez1, M. Früh2, W. Jochum1 Institut für Pathologie, Kantonsspital St.Gallen, St.Gallen, Schweiz, 2Klinik für Onkologie/Hämatologie, Kantonsspital St.Gallen, St.Gallen, Schweiz 1

Background.  Brain metastases are frequent in patients with non-small cell lung carcinoma (NSCLC) and are associated with poor prognosis. Systemic treatment with EGFR or ALK inhibitors may be effective in NSCLC with

EGFR mutations or ALK translocation, respectively. Limited information is available on the mutational profile of NSCLC brain metastases. The objective of our study was to compare the mutational profile of NSCLC brain metastases with paired primary tumors. Methods.  Thirteen patients with NSCLC (adenocarcinoma, poorly differentiated carcinoma) were identified for whom brain metastasis and paired primary tumor tissues were available. Sequence analysis was performed using the Ion Torrent PGM platform and the Ion AmpliSeq Colon and Lung Cancer Panel v2, which allows for the parallel analysis of 22 genes including established NSCLC oncogenic drivers. Results.  Primary carcinomas of 78 % (10/13) of patients harbored at least one mutated panel gene, including the EGFR, KRAS, PIK3CA, PTEN and TP53 genes. The frequencies for these mutations were: EGFR 15 % (2/13), KRAS 23 % (3/13), PIK3CA 8 % (1/13), PTEN 8 % (1/13) and TP53 46 % (6/13). Three primary carcinoma samples (23 %) showed concurrent mutations in two or three different genes. Mutations in the EGFR, KRAS, PIK3CA and PTEN genes were mutually exclusive. Twelve out of 13 mutations (92 %) detected in primary carcinomas were also found in the matched brain metastases. In 15 % (2/13) of patients, the brain metastasis harbored additional mutations compared to the matched primary carcinoma. Conclusions.  In NSCLC brain metastases additional mutations may occur that are potentially actionable.

AG04.16 Subtyping and molecular characterization of NSCLCs correlated with miRNAs as „new kids on the genomic block“ M.-C. Demes* Gemeinschaftspraxis für Pathologie, Molekularpathologie, Wiesbaden, Deutschland Background.  The dissertation tackled the current and problematic situation in differential lung cancer diagnosis which is paving the way in therapy and prognosis. The aim of the study was also to determine the diagnostic value of specific miRNAs miR-20b, miR-21, miR-93, miR-99b, miR-155, miR-205 and miR-221 of NSCLC histologies carrying varied mutations. Methods.  Distinct variables of interest were assessed in 139 NSCLC patients with single tumour lesions and with multiple tumour lesions. A total of 186 neoplastic lesions were analysed The mutation status of EGFR, KRAS and ALK-EML4 genes was investigated through fluorescence in-situ hybridization, sequencing, SNaPshot and fragment analysis, respectively. Quantitative real-time PCR was the method of choice for miRNA analysis, which was also tested as a reliable method for routine diagnostics by assessing the impact of the fixation time and heterogeneity on the final expression. Results.  In addition to histological and immunohistochemical assessment, molecular markers such as EGFR, KRAS and ALK-EML4 were analysed in two distinct sets of patients suffering from primary tumours and multiple tumour lesions respectively. EGFR mutations were found in 14.7 % of ADCs and 5 % of SQCs. KRAS mutations were only found in 24 % of ADCs and 3 % of SQCs. 5 % of ADCs show an ALK-EML4 rearrangements. The current study has demonstrated no evidence of intratumoral heterogeneity. The fixation time of 6, 12 and 24 hours has no influence on expression data. The data presented show no significant differences in miRNA expression within a tumour lesion. The dissertation suggests a correlation of the miR-93 expression with the presence of intranodal metastases (p = 0.051). miR-205 (p = 2.43 × 10-22), miR-221 (p = 6.9 × 10-5) and miR-21 (p = 3.9 × 10-5) are found to be differently expressed in adenocarcinomas and squamous cell carcinomas.

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Abstracts Conclusions.  1. One can conclude that EGFR-positive SQCs are also potential candidates for TKIs. 2. The results suggest that it is not necessary to test multiple areas within a single tumour. 3. The differentiation of a synchronous tumour growth from intrapulmonary metastases determines the tumour staging and ultimately the treatment strategies. 4. miR-205 and miR-221 seem to be an adjunctive differential tool for supporting diagnosis and defining new therapeutic approaches in adenocarcinoma as well as squamous cell carcinoma histologies carrying varied mutations.

AG Gynäko- und Mammapathologie AG05.01 Reliability of histological grade in breast cancer core needle biopsies depends on biopsy size: a comparative study with subsequent surgical excisions C. Focke*1, T. Decker1, P. van Diest2 Dietrich Bonhoeffer Klinikum, Institut für Pathologie, Neubrandenburg, Deutschland, 2Universitair Medisch Centrum Utrecht, Pathology, Utrecht, Niederlande

1

Background.  In breast cancer patients undergoing neoadjuvant chemotherapy or heat ablation therapy, histologic grading needs to be done on core needle biopsies that may not be representative of the whole tumour when they are small. Our aim was to study the influence of biopsy sizes on agreement rates of histological grade between core needle biopsies (CNB) and subsequent surgical excision biopsies (SEB). Methods.  Histological grading was performed by one observer in CNB and subsequent SEB of 283 early stage invasive breast cancers. Percentage agreement and Cohen’s κ were calculated for grade and mitotic scores in CNB vs SEB. The number of cores, total core length, total tumour length and tumour: tissue ratio were assessed for each CNB, and tumour size was measured in the SEB. Agreement rates for grade and mitotic scores were calculated for different classes of core numbers and tumour: tissue ratio, and for total core lengths and tumour lengths per biopsy set in 5 to 15 mm intervals. Results.  Agreement of grade between CNB and SEB was 71 % (κ 0.55). The main cause for discordance were changes in the mitotic score (Agreement 66 %, κ 0.47) with underestimation of grade in CNB in 25 % and overestimation in 4 % of cases. The total core length was significantly higher in grade concordant cases (p = 0.03) and the total core length and the total tumour length were significantly higher in cases with concordant mitotic scores (p = 0.001, and p = 0.001, respectively). Likewise, there was significantly higher concordance between CNB and SEB at ≥50 vs <50 mm total core length (grade: 81 % vs 67 % agreement, p = 0.017; mitotic score: 81 % vs 60 % agreement, p = 0.001), and ≥15 vs <15 mm tumour length in the biopsy (grade: 77 % vs 65 % agreement, p = 0.04; mitotic score: 75 % vs 57 % agreement, p = 0.001). The number of cores, the tumour: tissue ratio, and pathological tumour size were not statistically different between grade concordant and discordant cases. Conclusions.  Agreement rates of histological grade and mitotic score in CNB vs SEB improve with increasing biopsy sample size.

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AG05.02 Magnetic Resonance Imaging of Ductal Carcinoma in situ: Influence of Histology and Molecular Parameters on the Accuracy of Preoperative Lesion Sizing A. Staebler1, J. Pawlowski*1, P. Schulze Temming-Hanhoff2, G. Helms3, B. Wiesinger2, U. Vogel1, H. Nassar Warzecha1, M. Hahn3, C. Roehm3, F. Fend1, B. Wietek2 Universität Tübingen, Institut für Pathologie und Neuropathologie, Tübingen, Deutschland, 2Universitätsklinikum Tübingen, Abteilung für diagnostische und interventionelle Radiologie, Tübingen, Deutschland, 3 Universität Tübingen, Universitätsfrauenklinik, Tübingen, Deutschland 1

Background.  For patients with ductal carcinoma in situ (DCIS) precise evaluation by imaging is of utmost importance for operative planning and outcome. Our study addresses the influence of histological and molecular parameters on the accuracy of magnetic resonance imaging (MRI) in pure DCIS. Methods.  100 consecutive cases of DCIS with available MRI were reviewed retrospectively by two radiologists and a consensus size was determined. The histological size was prospectively evaluated by a systematic and segmental evaluation of the specimens (mean 39.6 mm, median 32.5 mm, range 1.5 mm to 130 mm, 53 segmentectomies and 47 mastectomies). In a second pathology review the cases were evaluated for subgross distribution, histological type, nuclear grade, comedo-necrosis, microcalcifications, infiltrating lymphocytes, ER, PR, HER2 and molecular subtype. For MRI accuracy was considered sufficient for a close correlation if the size differed less than 10 mm from the histological finding. Results.  Sufficient accuracy was obtained with MRI for 87 % of cases, 7 % were overestimated and 6 % were underestimated which was significantly improved from mammography. The correlation of MRI and histology was positively influenced by segmental resection vs. mastectomy (96.2 % and 76.6 % close correlation, p = 0.008 Chi square) and subgross distribution in patchy vs. diffuse growth pattern (100 % vs, 74.4 % p = 0.017). There was no significant effect of other histological or molecular subtypes on the accuracy of MRI. Conclusions.  Our study demonstrates a close correlation with histology and MRI in 96.2 % of cases, if a segmental and systematic pathology workup of breast resections is performed.

AG05.03 Oncotype DX recurrence score and Immunohistochemistry in intermediate risk breast cancer P. Sinn*, M. Kriegsmann Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland Background.  Multigene expression profiling assays, such as the Oncotype DX assay, were developed in order to more objectively evaluate the prognosis and to predict therapy results in early-stage breast cancer. Clinically, this is most useful in luminal type HER2-negative breast cancer of intermediate risk and may help in the selection of patients for adjuvant treatment and may help to avoid unnecessary cytotoxic treatment. Here we report the results of the Oncotype DX assay in a comparison with routine Immunohistochemistry. Methods.  In this unicentric study a consecutive series of early stage-breast cancer (UICC stage IA – IIA) that had received primary surgical treatment in 2014 or 2015 at the University of Heidelberg were analyzed. Inclusion criteria were positive estrogen hormone receptor status by immunohistochemistry (> 1 % positive tumor nuclei), negative HER2 status, and Ki-67 proliferation rate of > 10 % and less or equal of 30 % of tumor nuclei. In 113 patients that fulfilled these criteria an Oncotype DX assay had been ordered to better define individual patient risk. Results.  All cases were confirmed ER positive and HER2 negative by ODX test results. ODX recurrence scores (RS) were reported as low risk in 61

cases (54 %), followed by intermediate risk (43 cases, 38 %), and high risk (9 cases, 8 %). The median RS for the whole group was 19. However, when revised thresholds for the ODX recurrence score as applied in the prospective TAYLERx trial, 62 % were classified as intermediate risk. RS was calculated as 17 for G2 tumors, as compared with 22 for G3 tumors, but this association was statistically not significant because of the low number of G3 tumors. RS for Luminal-A vs. Luminal-B tumors were 15 vs. 20 (n. s.). 24 cases (21 %) were negative for PgR by ODX assay. This compared to only 5 % of negativity by IHC, but all except one case negative for PgR by IHC were also correctly identified as PgR negative by ODX. Risk categories by ODX were high (RS > = 31) in 11 cases with negative PgR status by ODX, but only 2 of these high risk cases were negative for PgR by IHC. Median Ki-67 values were 25 for PgR negative cases by ODX and also 25 for PgR negativity by IHC. Conclusions.  In luminal type breast cancers, the evaluation of PgR status by IHC may underestimate the proportion of PgR negative cases and did not identify most cases of tumors with a high risk of recurrence. This may be caused by an overly high sensitivity of PgR status in routine automated immunohistochemistry.

AG05.04 Standardized evaluation of tumor-infiltrating lymphocytes in breast cancer – results of the ring studies and development of a standardized analysis system

of different ICCs was evaluated in an in-silico ring study in 580 tumors from the GeparSixto study by mathematical modeling. Results.  In RS1 the prespecified endpoint was not reached (ICC 0.70; 95 %CI 0.62–0.78, 32 pathologists). Based on an analysis of sources of variation, we developed a more advanced digital image evaluation system including reference images for RS2, which improved the ICC to 0.89 (95 %CI 0.85–0.92, 28 pathologists). The Fleiss’ kappa value for <60 % vs. ≥60 % TILs improved from 0.45 (RS1) to 0.63 in RS2 and the mean concordance improved from 88 to 92 %. The in-silico ring study showed that the clinical results for prediction of response to neoadjuvant therapy by different TIL levels are stable across a wide range of different ICCs between 0.4 and 0.9. Conclusions.  Tumor-infiltrating lymphocytes are a promising parameter for monitor the immunogenicity of breast cancer. This large international standardization project shows that reproducible evaluation of TILs is feasible in breast cancer. In particular the software-guided image evaluation approach used in ring study 2 may be of value as a tool for TIL evaluation in clinical trials and diagnostic practice.

AG05.05 Molecular alterations and prognostic factors in myoepithelial breast cancer F. Leo1, S. Bartels1, L. Maegel1, T. Framke2, G. Büsche1, D. Jonigk1, M. Christgen1, U. Lehmann1, H. H. Kreipe*1 Institut für Pathologie, Medizinische Hochschule Hannover, Hannover, Deutschland, 2Institut für Biometrie, Medizinische Hochschule Hannover, Hannover, Deutschland

1

C. Denkert*1, S. Wienert1, A. Poterie2, S. Loibl3, J. Budczies1, Z. Bago-Horvath4, M. Christgen5, B. Ingold Heppner1, H. Kreipe5, B. Sinn1, P. Sinn6, G. von Minckwitz3, F. Klauschen1, M. Untch7, P. A. Fasching8, T. Reimer9, S. Michiels10, S. Loi11, R. Salgado12 Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland, Institute Gustave Roussy, Villejuif, Frankreich, 3German Breast Group, Neu-Isenburg, Deutschland, 4Institut für klinische Pathologie, Medizinische Universität, Wien, Österreich, 5Institut für Pathologie und Molekularpathologie, Medizinische Hochschule Hannover, Hannover, Deutschland, 6Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, 7Klinik für Gynäkologie und Geburtshilfe, Helios Klinikum Berlin-Buch, Berlin, Deutschland, 8University Hospital Erlangen, Department of Obstetrics and Gynecology, Erlangen, Deutschland, 9 Universitätsfrauenklinik Rostock, Rostock, Deutschland, 10Institut Gustave Roussy, Département d’Epidémiologie et de Biostatistiques, Villejuif, Frankreich, 11Division of Research and Clinical Medicine, Peter MacCallum Cancer Center, Melbourne, Australien, 12Breast Cancer Translational Research Laboratory, Institut Jules Bordet, Brüssel, Belgien 1

2

Background.  Accumulating evidence from several studies indicates that tumor-infiltrating lymphocytes (TILs) are predictive for response to neoadjuvant therapy and prognostic after adjuvant chemotherapy. This suggests that monitoring of tumor-infiltrating lymphocytes (TILs) might have potential relevance for response to immune checkpoint inhibitor therapy. While many groups are currently evaluating TILs, there is no standardized system for diagnostic applications. This study is presented on behalf of the International Immuno-oncology Biomarker Working Group (“TIL working group”), which was established in 2012 with the goal of creating an internationally standardized approach for TIL evaluation in breast cancer and which has published a guideline document as the first step. This study reports the results of the TIL ring studies (RS) that were conducted by the International Working Group on Immuno-oncology biomarkers for futher standardization. Methods.  A total of 32 pathologists from 27 Institution in nine countries participated first ring trial, 28 paticipated in the second ring trial, as well. Digital slides with a web-based system in RS1 and a more standardized software-system in RS2, with the pre-defined aim of an intraclass correlation coefficient (ICC) above 0.7 (lower limit of 95CI). The clinical relevance

Background.  Whereas prospective studies have identified prognostic factors in invasive breast carcinoma of no special type, knowledge is limited in the rare special types. Methods.  A retrospective study on myoepithelial breast cancer (n = 42, median follow-up time 43 months)was performed. Disease free survival (DFS), overall survival (OS) and types of treatment were assessed. Molecular studies were done on DNA sequences of mutation hot spots in AKT1, ALK, APC, BRAF, CDH1, CTNNB1, EGFR, ERBB2, FBXW7, FGFR2, FOXL2, GNAQ, GNAS, KIT, KRAS, MAP2K1, MET, MSH6, NRAS, PDGFRA, PIK3CA, PTEN, SF3B1, SMAD4, SRC, SRSF2, STK11, TP53, and U2A. Furthermore, copy numbers for EGFR, c-myc, FGFR, PLAG, c-met) were investigated. Results.  None of the patients had axillary lymph node involvement. In 11 cases, local recurrence developed after surgery (26 %). Distant metastasis occurred in 7 patients (17 %; 5 after local recurrence) (. Fig. 1, 2). The most frequent genetic alteration was PIK3CA mutation (50 % of cases). None of the pathological parameters (size, grade, stage, Ki-67 labelling

Fig. 1 I AG05.05 8 Kaplan Meier plots of diseae free survival in a cohort of 41 patients with spindle cell myoepithelial carcinomas Der Pathologe Suppl 1 · 2016

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Abstracts (TP53, NCOR1, NF1, PTPRD and RB1) were significantly associated with high loads of mutated genes. Multivariate analysis of overall survival revealed a worse survival for patients with high numbers (≥ 22) of mutated genes (hazard ratio = 4.6, 95 % CI: 1.0 – 20.0, p = 0.044). Conclusions.  In summary, we show that specific levels of the mutational load underlie different morphological and biological phenotypes, which collectively constitute the current basis of pathological diagnosis. Our study is a step towards genomics-informed breast pathology and provides a basis for future studies in this field bridging the gap between morphology, tumor biology and medical oncology.

AG05.08 SPAG6, NKX2-6 and PER1 as novel methylation specific biomarkers for liquid biopsy based early breast cancer detection Fig. 2 I AG05.05 8 Kaplan Meier plots of survival probability

J. Mijnes*1, J. Tiedemann1, D. Bauerschlag2, N. Maass2, S. von Serenyi1, R. Knüchel1, V. Kloten1, E. Dahl1 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, Klinik für Gynäkologie und Geburtshilfe, Universitätsklinikum SchleswigHolstein, Campus Kiel, Kiel, Deutschland

1

2

index) were significantly associated with disease-free or overall survival (DFS, OAS). PIK3CA mutation, especially the H1047R type, tended to adversely affect OAS. Mode of resection (mastectomy versus breast -conserving therapy, width of margins) or adjuvant radiotherapy had no influence on DFS or OAS, whereas in the group treated with radio-/chemotherapy, no local recurrence or metastasis and no death occurred. Conclusions.  We conclude that the spindle cell type of MBC with myoepithelial features exhibits a higher frequency of PIK3CA mutation than other types of metaplastic or basal-like breast cancer and may benefit from combined radio-/chemotherapy.

AG05.06 Classical pathology and mutational load of breast cancer – integration of two worlds J. Budczies*1, M. Bockmayr1, C. Denkert1, F. Klauschen1, J. Lennerz2, B. Györffy3, M. Dietel1, S. Loibl4, W. Weichert5, A. Stenzinger6 Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland, Harvard Medical School, Massachusetts General Hospital, Boston, Vereinigte Staaten von Amerika, 32nd Department of Pathology, Semmelweis University Budapest, Budapest, Ungarn, 4German Breast Group, Neu-Isenburg, Deutschland, 5Institut für Allgemeine Pathologie und Pathologische Anatomie der Technischen Universität München, München, Deutschland, 6Pathologisches Institut, Ruprecht-Karls-Universität, Heidelberg, Deutschland 1

2

Background.  Breast cancer is a complex molecular disease comprising several biological and clinical subtypes. However, daily routine diagnosis is still based on a small set of well-characterized clinicopathological and molecular variables. Methods. We evaluated the number of mutated genes with somatic non-silent mutations in different subgroups of breast cancer based on clinicopathological and molecular tumor characteristics. The analysis was performed in a cohort of 687 primary breast cancer patients with mutational profiling, gene expression and clinico-pathological data available from The Cancer Genome Atlas (TCGA) project. Results.  The number of mutated genes was strongly associated with tumor grade (positive association, p = 1.4e–14) and with the molecular subtypes of breast cancer (p = 1.4e–10 for immunohistochemistry-based subtypes, p = 4.3e–10 for PAM50-based subtypes). Mutational load and expression levels of genes related to proliferation were significantly associated in breast cancer and in hormone receptor-positive breast cancer, but not in hormone receptor-negative breast cancer. Mutations in specific cancer genes

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Background.  Breast cancer remains the most frequently diagnosed cancer and leading cause of cancer deaths amongst women. Early breast cancer detection is key to favourable outcome. Mammography and magnetic resonance imaging are employed for early breast cancer detection. Nevertheless both technologies show important limitations. Blood-based approaches provide minimally invasive screening tools to characterize epigenetic alterations of tumor suppressor genes. Detection of cell-free DNA (cfDNA) hypermethylation could serve as a liquid biopsy, complementing current image-driven technologies. Previously, we identified DKK3 and ITIH5 as highly specific hypermethylation biomarkers, however sensitivity was limited to 40 %. Therefore, we aimed to identify additional biomarkers, to create a clinically reliable and sensitive biomarker panel for early breast cancer detection. Methods.  Potential biomarkers were identified from the Cancer Genome Atlas (TCGA), using HumanMethylation450 BeadChip data. We focussed on distinct CpG dinucleotides in promoter regions of candidates, discriminating significantly between breast cancer and normal tissue. Promoter methylation status was examined qualitatively and quantitatively in luminal- and basal-like breast cancer cell lines. Furthermore, methylation frequency of potential biomarkers was investigated in a tissue and serum collective. Results.  Based on TCGA we identified SPAG6, NKX2-6 and PER1 as candidates, showing high promoter CpG dinucleotide methylation in breast cancer versus normal tissue. Examination of our tissue collective confirmed abundant biomarker methylation in tumor compared to normal tissue. Importantly, analysis of serum cfDNA showed a significant higher methylation frequency of biomarkers in cfDNA from breast cancer patients compared to healthy individuals. SPAG6 proved the most sensitive single marker, reaching 44 % sensitivity for DCIS (AUC: 0.65, p = 0.02) and 30 % sensitivity for tumor (AUC: 0.66, p = 0.007) detection, at 94 % specificity. Yet, a three-gene panel increased sensitivity for DCIS detection to 51 % (AUC: 0.81, p < 0.0001) and tumor detection to 49 % (AUC: 0.74, p < 0.0001), at a specificity of 92 %. Conclusions.  In conclusion, tumor-specific hypermethylation of SPAG6, NKX2-6 and PER1 (SNP panel) provides a promising tool for blood-based breast cancer detection. Next, validation will be performed in a larger and independent serum collective, in combination with DKK3 and ITIH5, to develop a highly sensitive system for early breast cancer detection.

AG05.09 Predictive role of HER2/neu, topoisomerase-II-alpha, and tissue inhibitor of metalloproteinases (TIMP-1) for response to adjuvant taxane-based chemotherapy in patients with intermediate-risk breast cancer: results from the WSG-AGO EC-Doc trial

AG05.10 Identification of 19q12 (CCNE1/URI) amplification in endometrial cancer

R. Erber*1, O. Gluz2, 3, N. Brünner4, H. H. Kreipe5, E. Pelz6, R. Kates2, A. Bartels4, J. Huober7, 8, S. Mohrmann9, Z. Moustafa2, C. Liedtke10, V. Möbus11, D. Augustin12, C. Thomssen13, F. Jänicke14, M. Kiechle15, W. Kuhn16, U. Nitz2, 3, 9, N. Harbeck17, A. Hartmann1

Universitätsspital Zürich, Zürich, Schweiz

Universitätsklinikum Erlangen, Pathologisches Institut, Erlangen, Deutschland, 2West German Study Group, Mönchengladbach, Deutschland, 3 Evangelisches Krankenhaus Bethesda Mönchengladbach GmbH, Brustzentrum Niederrrhein, Mönchengladbach, Deutschland, 4Section of Molecular Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Dänemark, 5Institut für Pathologie, Medizinische Hochschule Hannover, Hannover, Deutschland, 6 Institut für Pathologie, Viersen, Deutschland, 7Department of Obstetrics and Gynecology, University Clinics Tuebingen, Tübingen, Deutschland, 8 Department of Obstetrics and Gynecology, University Clinics Ulm, Ulm, Deutschland, 9Department of Obstetrics and Gynecology, University Clinics Düsseldorf, Düsseldorf, Deutschland, 10Department of Obstetrics and Gynecology, University Clinics Schleswig-Holstein, Lübeck, Deutschland, 11 Department of Obstetrics and Gynecology, Städtisches Klinikum, Frankfurt, Deutschland, 12Clinics Deggendorf Mammacenter Ostbayern, Deggendorf, Deutschland, 13Department of Gynecology, Martin-Luther-University Halle-Wittenberg, Halle/Saale, Deutschland, 14Department of Gynecology, University Medical Center Hamburg-Eppendorf, Hamburg, Deutschland, 15 Department of Obstetrics and Gynecology, TUM, München, Deutschland, 16 Department of Obstetrics and Gynecology, University Clinics, Bonn, Deutschland, 17Breast Center, Ludwig Maximilian University of Munich, München, Deutschland 1

Background. Taxane-anthracycline-based adjuvant chemotherapy is standard of care in patients with node-positive breast cancer (BC) but is also associated with severe side effects and significant costs. It is yet unclear, which biomarkers would predict benefit from taxanes and/or general chemoresistance. In this study, we investigate a large cohort of patients with intermediate-risk BC treated within the WSG EC-DOC Trial for the predictive impact of topoisomerase-II-alpha, HER2/neu, and TIMP-1. Methods.  Tumor tissue was available in a representative cohort of 772 cases of the WSG EC-DOC Trial collective which compared 4xEC-4xDoc versus 6xCEF/CMF. In addition to hormone receptor status and Ki-67, HER2/neu+ and topoisomerase-II-alpha status using fluorescence in situ hybridisation (FISH) and immunohistochemistry, TIMP-1 using immunohistochemistry, and aneuploidy of chromosome 17 using FISH were evaluated and correlated with outcome and taxane benefit. Results.  There was significant superiority of EC-Doc over CEF regarding 5-year DFS (90 vs. 80 %, respectively, p = 0.006) particularly in patient subgroups defined by HR+, HER2/neu+, high proliferation (i. e., Ki-67 ≥ 20 %), patient age > 50 years old and normal chromosome 17 status, high TIMP-1 and low topoisomerase-II-alpha protein expression. Significant prognostic factors in multivariate analysis were EC-Doc therapy (HR = 0.61; 95 %CI 0.38-0.986), age ≤ 50 years old (HR = 1.682; 95 %CI 1.025–2.579), centrally assessed grade 3 (HR = 4.657; 95 %CI 1.809–11.989), and high Ki-67 (HR = 2.232; 95 %CI 1.209–4.121). Interestingly, we observed a significant interaction between treatment arm (EC-Doc vs. CEF) and high topoisomerase-II-alpha protein expression (HR = 0.427; 95 %CI 0.203–0.900) in multivariate interaction analysis. Conclusions.  Despite of univariate predictive effect of HER2/neu status among other factors only topoisomerase-II-alpha protein expression was associated with significant benefit from EC-Doc compared to CEF by multivariate interaction analysis.

A. Noske*, S. Brandt, N. Valtcheva, U. Wagner, Q. Zhong, D. Fink, H. Moch, P. Wild Background.  One of The Cancer Genome Atlas (TCGA) genomic subgroups of endometrial cancer (EC) is characterized by extensive copy number alterations. CCNE1 located at 19q12 is frequently amplified in this tumor type and a target for anti-cancer therapy. The relevance of URI which is also part of the 19q12 locus is unknown. To gain more insight on the prevalence of 19q12 (CCNE1/URI) status in EC, we investigated different histologic types by chromogenic in situ hybridization (ISH) techniques. Methods.  We applied a dual-color 19q12 ISH assay for detection of CCNE1/URI copy numbers and Chromosome 19 as a surrogate on EC samples (n = 270) using a VENTANA BenchMark XT Platform and conventional bright field microscopy. The 19q12 status was validated with the OncoScan assay in a subset of ECs (n = 21) and compared to protein expression of Cyclin E1, URI, p53, and pathological parameters. Results.  Amplification of 19q12 was found in 10.4 % (28/270) of EC samples and could be confirmed by the OncoScan assay, showing copy number gain of CCNE1 and URI. 19q12 amplification was significantly associated with type II (high grade and non-endometrioid) EC (p < 0.0001), advanced FIGO stage (p = 0.001), Cyclin E1 expression (p = 0.008), and aberrant p53 expression (p = 0.04). Conclusions.  Amplification of 19q12 including CCNE1 and URI occur predominantly in type II ECs. The 19q12 ISH assay is a feasible tool for the guidance of targeted tumor therapy in patients with EC. Whether the combination of 19q12 amplification status and Cyclin E1/URI protein expression is a useful predictive test for chemotherapy and/or CDK inhibitors has to be further assessed in prospective clinical trials.

AG05.11 Fumarate hydratase (FH)-deficient uterine smooth muscle neoplasms can be identified by characteristic histological features and confirmed by FH immunohistochemistry R. Erber*1, D. L. Wachter1, M. W. Beckmann2, S. Burghaus2, F. Haller1, A. Hartmann1, A. Agaimy1 Universitätsklinikum Erlangen, Pathologisches Institut, Erlangen, Deutschland, 2Universitätsklinikum Erlangen, Frauenklinik, Erlangen, Deutschland 1

Background.  HLRCC is a rare autosomal dominant disease caused by germline mutations in the FH gene mapping to 1q42-43. Affected individuals develop cutaneous leiomyomas (~80 %), uterine leiomyomas (~77 %) and aggressive RCC (~1/3). In the absence of cutaneous manifestations, uterine leiomyomas and RCC can be easily overlooked if one is not familiar with their distinctive morphology. To date, only very few publications described the characteristic histology of FH-deficient uterine smooth muscle tumors. Methods.  We reviewed 9 presumed FH-deficient uterine leiomyomas collected prospectively over a period of 2 years from our routine and consultation files. FH IHC was performed using a monoclonal antibody (clone J-13, 1:50, Santa Cruz). In addition, tissue microarray slides containing 23 extra-uterine leiomyosarcomas, 8 uterine leiomyosarcomas, 6 atypical uterine smooth muscle neoplasms and 8 uterine leiomyomas were screened for FH loss. Results.  All 9 FH-deficient cases were initially diagnosed on routine H&E sections and then confirmed by IHC. Patients’ ages ranged from 25 to 70 years (median: 35). Eight patients had multiple nodules (2–14) measuring up to 9 cm. The oldest patient had a solitary nodule in a hysterectomy spec-

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Abstracts imen for endometrial cancer. Histologically, all tumors showed consistent and reproducible features: moderate cellularity, remarkable cytoplasmic eosinophilia with fibrillary quality, focal neuropil-like cytoplasmic material surrounded by nuclear palisades reminiscent of neuroblastic/schwannian neoplasms, scattered enlarged or bizarre nuclei with pseudoinclusions, variable perinucleolar halos, hyaline globular bodies and prominent vasculature. Rhabdoid cells were seen in one tumor. All tumors strongly expressed smooth muscle actin, desmin and h-caldesmon with complete loss of FH by IHC. Screening of the TMA cohorts showed only one case with FH loss (previous enucleation from one of the 9 cases). Conclusions.  FH-deficient uterine leiomyomas are probably more frequent than expected. They can be recognized by their distinctive histological features. FH IHC is an excellent confirmatory marker that is superior to 2SC as it is commercially available and it is not complicated by interpretative difficulties known for 2SC immunostaining. The relationship of FH-deficient uterine smooth muscle tumors to the HLRCC syndrome needs further clarification. Genetic counselling and molecular FH analysis are warranted because of the extremely poor prognosis of renal cell carcinomas in the HLRCC syndrome.

AG05.12 Systematic analysis of Programmed Cell Death-1 (PD-1) and PD-Ligand 1 (PD-L1) Expression in Cancer Cells and Tumor-Infiltrating Lymphocytes of High-Grade Serous Ovarian Carcinoma C. A. Kunze*1, S. Wienert2, I. Braicu3, 4, J. Budczies1, M. Bockmayr1, H. Kulbe4, J. Lindner1, J. Sehouli3, 4, M. Dietel1, C. Denkert1, K. Jöhrens1, S. Darb-Esfahani1, 4 Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland, VM Scope GmbH, Berlin, Deutschland, 3Frauenklinik Charité VirchowKlinikum, Charité – Universitätsmedizin, Berlin, Deutschland, 4Tumorbank Ovarian Cancer Network (TOC), Berlin, Deutschland 1

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Background. High-grade serous ovarian carcinoma (HGSOC) is a poor-prognosis cancer, however high levels of tumor-infiltrating-lymphocytes (TILs) are features of patients with a better survival, strongly suggesting that the anti-tumoral immune response could be exploited as a therapeutic option. Antibodies targeting the checkpoint molecules programmed cell death 1 (PD-1) and its ligand PD-L1 are emerging cancer therapeutics. In this study, we systematically investigated PD-1 and PD-L1 expression patterns in primary HGSOC. Methods.  PD-1 and PD-L1 protein expression was determined by IHC on TMAs from 215 primary cancers both in cancer cells and in TILs. mRNA expression was measured by qRT-PCR. The Cancer Genome Atlas (TCGA) gene expression datasets were used for in silico validation. Results.  PD-1 and PD-L1 protein expression in cancer cells, as well as CD3+, PD-1+, and PD-L1+ TILs densities were positive prognostic factors for progression-free (PFS) and overall survival (OS). Similarly, high PD-1 and PD-L1 mRNA levels were significantly linked to better survival. All factors were significant for PFS (p < 0.035 each), and most were significant for OS. Multivariate analysis including age, stage, and residual tumor showed independent significance for most markers. Moreover, high PD1+ TILs as well as PD-L1+ TILs densities added prognostic value to CD3+ TILs (PD-1+: p = 0.002,; PD-L1+: p = 0.002). Our findings could be validated on the mRNA level in the TCGA gene expression datasets (p = 0.02 and p < 0.0001, respectively). Conclusions.  Surprisingly and despite their reported immune-modulatory function, high PD-1 and PD-L1 levels are indicators of a good prognosis in HGSOC. Our data indicate that PD-1 and PD-L1 molecules are biologically relevant regulators of the immune response in HGSOC, which is an argument for the evaluation of immune checkpoint inhibiting drugs in this aggressive tumor entity.

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AG05.13 Dynamics of the immune infiltrate during progression of high-grade serous ovarian carcinoma M. Stanske*1, S. Wienert2, D. Cacsire Castillo-Tong3, C. Kreuzinger3, I. Vergote4, S. Lambrechts4, H. Gabra5, C. Gourley6, J. Sehouli7, 8, C. Denkert1, H. Kulbe8, W. Schmitt1, M. Dietel1, K. Jöhrens1, I. Braicu7, 8, S. Darb-Esfahani1, 8 Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland, VM Scope GmbH, Berlin, Deutschland, 3Medizinische Universität Wien, Klinik für Gynäkologie und Geburtshilfe, Comprehensive Cancer Center, Wien, Österreich, 4Leuven Cancer Institute, Klinik für Gynäkologie und Geburtshilfe, Leuven, Belgien, 5Imperial College London, Medizinische Fakultät, Klinik für Chirurgie und Krebsmedizin, London, Vereinigtes Königreich, 6Edinburgh Cancer Research Center, Edinburgh, Vereinigtes Königreich, 7Frauenklinik Charité Virchow-Klinikum, Charité – Universitätsmedizin, Berlin, Deutschland, 8Tumorbank Ovarian Cancer Network (TOC), Berlin, Deutschland 1

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Background.  High-grade serous ovarian carcinoma (HGSOC) in general is a poor-prognosis tumor. However, the immune microenvironment has been shown to significantly influence the clinical course of the disease as high numbers of tumor-infiltrating lymphocytes (TILs), particularly intraepithelial TILs, are robust positive prognostic factors in the primary setting. No data exist to date on the dynamics of the immune infiltrate during ovarian cancer progression. Methods.  We determined T cell subpopulations as well as MHC1 and MHC2 expression in tumor cells by immunohistochemistry in the TOC (Charité) cohort of paired primary/recurrent HGSOC (n = 61 pairs) from the FP7 EU project OCTIPS (http://www.octips.eu/). TILs quantification was performed manually, with software-support (VM Slide Explorer 2.2, VM Scope), as well as automatically by image analysis (Cognition Master Professional Suite, VM Scope). Results.  Informative data were available for automatic and manual evaluation of intraepithelial and total (intraepithelial and stromal) CD3+ TILs (n = 59 pairs) and CD4+ TILs (n = 54 pairs), and automatic evaluation of total CD8+ TILs (n = 54 pairs). Automatic and manual evaluation of intraepithelial CD3+ and CD4+ TILs were highly correlated with each other (Spearman rho >0.8, p < 0.0001 each). Intraepithelial CD3 and CD4 TILs showed a moderate, however significant correlation between primary and recurrent tumors (Spearman rho >0.27, p < 0.03 each), whereas pairwise comparison showed no evidence of a directed change (Wilcoxon n. s. each). Total TILs (CD3, CD4, CD8) as determined by automatic evaluation were not significantly correlated in primary/recurrent HGSOC and not different in pairwise comparison (n. s. each). MHC1 and MHC2 expression in tumor cells strongly correlated with each other (Spearman rho >0.4, p < 0.0001 each) as well as with TILs number in primary and recurrent tumors (Mann-Whitney p < 0.005 each). MHC1, not MHC2 expression furthermore showed a significant increase in recurrent tumors in pairwise comparison (Wilcoxon p = 0.009). Conclusions.  Our data suggest that recurrent HGSOC reconstitute their immune micromillieu according to intraepithelial CD3 and CD4 TILs. T cell numbers might be regulated by MHC1 and MHC2 expression in tumor cells. An expansion of our analyses to the total OCTIPS FFPE cohort including samples from three other partners in the consortium (n > 100 pairs) is ongoing.

AG05.14 APOBEC3B on protein and gene expression is associated with survival in histologically defined subtypes of ovarian cancer U. Unger*1, C. Denkert1, C. A. Kunze1, J. Lindner1, K. Jöhrens1, H. Kulbe2, 3, J. Sehouli2, 3, M. Dietel1, E. I. Braicu2, 3, S. Darb-Esfahani1, 2 Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland, Tumorbank Ovarian Cancer Network (TOC), Berlin, Deutschland, 3 Frauenklinik Charité Virchow-Klinikum, Charité – Universitätsmedizin, Berlin, Deutschland

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Background.  Not only in virally caused tumors but also in cancers not directly associated with virus infections the immune system and its players become more interesting. APOBEC3-enzymes are part of the innate immune system and important in retroviral defense through their function as RNA-editing enzymes. With increasing levels of APOBEC3B mRNA the number of mutations grows in ovarian cancer. It could be shown that APOBEC3B mRNA is upregulated in both ovarian cancer cell lines and in ovarian cancer tissue compared to normal ovarian tissue. We are the first having evaluated APOBEC3B in histologically defined subtypes of ovarian cancer to identify its influence on overall (OS) and progression-free survival (PFS). Methods.  Tissue microarrays from 221 patients with high-grade (HGSC), 58 with low-grade (LGSC), 35 with endometrioid (EC) and 48 with clear cell (CC) ovarian carcinoma were stained using an antibody against APOBEC3B. The slides were digitalized and evaluated using the immunoreactivity score to assess intensity and number of stained tumor cells. Real-time quantitative PCR was performed to detect APOBEC3B mRNA levels in 310 cases of HGSC, in 34 cases of EC and in 29 cases of CC. Tumor-infiltrating lymphocytes (TILs) had been evaluated in a previous project. Results.  APOBEC3B staining was cytoplasmic as well as nuclear and both were positively correlated (p < 0.001). The distribution of nuclear staining differed in ovarian cancer with predominately negative staining in HGSC and CC, with positive staining in LGSC and with an equilibrium of positive and negative stained tumor cells in EC (p < 0.001). In HGSC only a trend was detectable for positive cytoplasmic staining as favorable regarding to OS (p = 0.206) and PFS (p = 0.162). High levels of APOBEC3B mRNA correlated with prolonged PFS in HGSC in univariate analyses (p = 0.034) as well as in multivariate analyses (HR 0.503; 95 %CI 0.312– 0.811; p = 0.005). A combined positive staining in nucleus und cytoplasm was predictive for prolonged PFS in LGSC (p = 0.014) and for prolonged OS in CC (p = 0.03). In exploratory analyses APOBEC3B was correlated with TILs such as CD3+ (p = 0.003) and CD8+ (p = 0.005). Conclusions.  APOBEC3B expression is a positive prognostic factor in ovarian cancer and is related to an active immune infiltrate. An extension of the cohorts, particularly of special histological type is ongoing.

AG05.15 Prognostic significance of Wilms Tumor Protein 1 (WT1) in high-grade serous ovarian carcinoma – alone and in combination with ER-α E. T. Taube*1, C. Denkert1, J. Sehouli2, 3, C. A. Kunze1, M. Dietel1, I. Braicu2, 3, A. Letsch4, S. Darb-Esfahani1, 3 Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland, Frauenklinik Charité Virchow-Klinikum, Charité – Universitätsmedizin, Berlin, Deutschland, 3Tumorbank Ovarian Cancer Network (TOC), Berlin, Deutschland, 4Klinik für Onkologie und Hämatologie, Charité Universitätsmedizin, Berlin, Deutschland 1

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Background.  Wilms tumor protein 1 (WT1) was first discovered as a tumor suppressor gene. Its expression is used in gynecological pathology as a diagnostic marker of serous differentiation, and is frequently co-expressed with ER-α. Because WT1 is not widely expressed in adult tissue it

is a promising target for immunotherapy and early phase studies on WT1 vaccine in gynecological cancers are ongoing. In this study we aimed to determine the prognostic value of WT1 alone and in combination with ER-αin serous high-grade ovarian carcinoma. Methods.  Immunohistochemical expression of WT1protein and ER-α receptor were determined by immunohistochemistry in a cohort of 207 primary high-grade serous ovarian carcinomas. Additionally WT1 mRNA expression was evaluated in a cohort of 1137 ovarian carcinomas from publically available gene expression datasets. Results.  High WT1 expression was a significant positive prognostic factor in primary high-grade serous ovarian carcinoma regarding overall survival (OS, p = 0.008) and progression free survival (PFS, p = 0.015) and remained an independent prognostic marker in multivariate analysis(OS: p = 0.024, PFS: p = 0.047), independent of age, stage, and residual tumor. On the mRNA level the prognostic effect was validated in silico in publically available gene expression datasets including TCGA data (OS: p = 0.002, PFS: p = 0.011).In our cohort WT1 expression was significantly associated with ER-α expression (p = 0.001), and tumors that co-expressed both markers (WT1+/ER-α+) had a longer survival time than tumors of all other marker combinations (OS: p = 0.002, PFS: p = 0.013). Conclusions.  Our study demonstrates that WT1 is an independent prognostic factor for primary high-grade serous ovarian carcinoma, a finding which could be validated in silico in a series of publically available gene expression datasets. Most interestingly, tumors that co-expressed WT1 and ER-α had a particularly good prognosis.Our results should be kept in mind when evaluating clinic trials for immunotherapy against WT1 in high-grade serous ovarian carcinoma to prevent mixing a positive therapy effect with an intrinsic better survival for WT1 positive high-grade serous ovarian cancer patients.

AG05.16 Tubal intraepithelial lesions have distinct places of origin and can be detected by LEF1 expression E. Schmoeckel*1, A. Odai-Afotey2, C. Lee Pelletier2, M. Rottman3, T. Kirchner1, A. Y. Nikitin2, D. Mayr1 1 Pathologisches Institut, Ludwig-Maximilians-Universität, München, Deutschland, 2Department of Biomedical Sciences and Cornell Stem Cell Program, Cornell University, Ithaca, Vereinigte Staaten von Amerika, 3Tumorregister München, Institut für medizinische Informationsverarbeitung, Biometrie und Epidemiologie, München, Deutschland

Background.  Several tubal intraepithelial lesions, such as serous tubal intraepithelial carcinoma (STIC), P53-signature and secretory cell outgrowth (SCOUT) are currently being considered as putative precursors of many high-grade serous ovarian carcinomas (HGSOCs). However, it remains uncertain if these lesions represent a continuum in HGSOC pathogenesis. Recently it has been reported that STICs are located in the vicinity of tubal-peritoneal junction (TPJ), consistent with the cancer-prone features of many epithelial transition regions. To test if P53-signatures and SCOUTs also localize to TPJs, we compared distances from the TPJs to STICs, P53-signatures and SCOUTs in the fallopian tubes of patients undergoing salpingo-oophorectomy due to sporadic HGSOCs or as a prophylactic procedure for carriers of familial BRCA1 or 2 mutations. Methods.  All slides were digitalized in the laboratory of Prof. Nikitin (Cornell Universitiy) using Aperio Scanscope technology. Measurement of size and distance to the TPJ of tubal intraepithelial lesions and evaluation of immunohistochemical stains were analyzed by using the ImageJ software. Results.  In both groups the majority of STICs and P53-signatures were detected in the tubal fimbria (85.7 % for STICs and 62.5 % for P53-signatures), while all SCOUTs were located in non-fimbriated regions of the fallopian tube. STICs were located closest to the TPJs with an average distance of 1.31 mm. The average distance for TP53-signatures was 1.96 mm.

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Abstracts Since HGSOC frequently show WNT pathway dysregulation, we have also determined the expression of its markers LEF1 and ß-catenin in the tubal intraepithelial lesions. LEF1 was consistently expressed in all lesions, independent of P53 status. ß-catenin was mostly expressed cytoplasmic in STICs and TP53-signatures, whereas all SCOUTs presented a marked nuclear expression Conclusions.  Taken together, our findings suggest that tubal intraepithelial lesions have distinct pathogenesis and are unlikely to represent continuous stages of neoplastic progression towards HGSOC. LEF1 expression may serve as a useful auxiliary marker for the detection of tubal intraepithelial lesions.

AG05.17 Serous tubal intraepithelial carcinoma (STIC) – reduced tumor infiltrating lymphocytes (TIL) are suggestive of immunosuppressive mechanism in early tumor progression S. Wenz*1, M. Nieser1, H. Bösmüller1, K. Greif1, H. Schuster2, J. Peper2, P. Wagner3, J. Sperveslage4, F. Fend1, A. Staebler1 Universität Tübingen, Institut für Pathologie und Neuropathologie, Tübingen, Deutschland, 2Universität Tübingen, Institut für Immunologie, Tübingen, Deutschland, 3Universität Tübingen, Universitätsfrauenklinik, Tübingen, Deutschland, 4Universitätsklinikum Münster, Gerhard Domagk Institut für Pathologie, Münster, Deutschland 1

Background.  Tumour infiltrating lymphocytes (TILs) in high-grade serous ovarian carcinomas (HGSC) are of significant prognostic relevance. However, little is known about the role of the immune response and the tumor microenvironment for the development and progression of serous tubal intraepithelial carcinoma (STIC), the putative precursor of HGSC in the fallopian tube. Methods.  147 cases of high-grade serous carcinomas were reviewed for the presence of STIC. 26 lesions were identified by HE morphology and confirmed by overexpression of p53 and Ki67 labeling. Tumor infiltrating lymphocytes (TILs) were detected by immunohistochemistry for CD3, CD8 and CD103 and quantified using image analysis (Definiens) in normal tube, STIC and corresponding carcinoma. Results.  Paired analysis showed a stepwise increase of T-lymphocytes in surrounding stromal lymphocytes from normal tubal epithelium to STIC and from STIC to invasive carcinoma (CD3: average 56 vs. 378 vs. 920 per mm2, CD8: 29 vs. 182 vs. 660 per mm2, CD103: 111 vs. 121 vs. 184 per mm2, for all pairs p < 0.001). However, intraepithelial lymphocytes did not increase from normal tube to STIC, there was even a noticeable decrease (not significant) followed by an increase in the carcinomas (CD3: 329 vs. 225 vs. 522, CD8: 295 vs. 208 vs. 257, CD 103: 9 vs. 44 vs. 206, all per mm2). Conclusions.  Our study demonstrates the slow and stepwise increase of tumor-infiltrating lymphocytes from normal tubal epithelium to STIC and HGSC with a significantly lower TIL number in STIC as compared to the corresponding carcinomas. This finding is suggestive of a reduced cellular immune response in STIC as compared with the corresponding carcinomas, possibly the result of focal immune suppression or slow onset of immune response during the evolution from precursor to carcinoma.

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AG Dermatopathologie AG06.01 From pruritic symptoms to bullous diseases H. Mittag*, M. Hertl Klinik für Dermatologie und Allergologie, Marburg, Deutschland Background.  Bullous autoimmune diseases are heterogenous in clinical and histological aspects. Methods.  Morphological methods, including immunopathology, and immune serologic investigations are considered. Results. 55Certain histological feature in the development of blisters demonstrate the advance of a prebullous state to bullous disease, for instance in bullous pemphigoid. 55Morphological and immunopathological methods are useful tools in establishing differential diagnoses among bullous diseases. 55Furthermore, immune serologic investigations support establishing new entities within the bullous autoimmune diseases, for instance „pruritic diseases of the elderly“. Conclusions.  Modern diagnostic pathways are useful in clarifying nosologic and systematic aspects within the heterogenous spectrum of bullous autoimmune diseases.

AG06.02 Malignant Skin Adnexal Tumours – Criteria for Malignancy T. Brenn* Western General Hospital, Pathologie, Edinburgh, Vereinigtes Königreich Background.  The histological spectrum of skin adnexal tumours is wide and includes tumours with differentiation towards the hair follicle, the sebaceous glands and the sweat glands/ducts. According to their behaviour they are traditionally separated into benign, low-grade malignant and high-grade malignant. The precise classification of these tumours is often challenging. Particular problems include the rarity of the majority of these tumours, especially the malignant examples, and the inconsistent use of the terminology. The criteria for malignancy are often poorly defined und entity specific, and little is known about the true biological behaviour of many of the skin adnexal carcinomas. Methods.  This talk will provide an overview of malignant skin adnexal tumours with emphasis on high-grade malignant sweat gland neoplasms. It will focus on entity specific criteria for malignancy, behaviour and outcome, recent advances and differential diagnosis. Results.  High-grade malignant sweat gland tumours are characterised by potential for local and distant recurrence and associated mortality. The main entities in this tumour group are eccrine porocarcinoma, hidradenocarcinoma, spiradeno- and cyindrocarcinoma, apocrine carcinoma and aggressive digital papillary adenocarcioma. Their diagnosis is made particular challenging due to the deceptively bland histological appearances of some of these tumours. Aggressive digital papillary adenocarcinoma, in particular, is often mistaken for a benign disease. In contrast, features traditionally associated with malignancy, such as perineural or lymphovascular invasion, tumour necrosis or involvement of locoregional lymph nodes have recently been demonstrated in tumours with indolent clinical behaviour. Other diagnostic pitfalls are reliable separation from cutaneous metastases of visceral primaries, which applies especially but not exclusively to apocrine carcinoma and hidradenocarcinoma. Furthermore, more recent comprehensive studies have provided further insights into the behaviour of these tumours and have identified histological features as a predictor of outcome in some ­instances.

Conclusions.  Over recent years, our understanding of the biology of malignant skin adnexal tumours has changed significantly. They remain a diagnostic challenge but confident and accurate diagnosis is possible in most cases using a combination of careful interpretation of the histological features and sampling, awareness of the many pitfalls and judicious use of immunohistchemical markers.

AG06.04 Correlation of mutational spectrum and PD-1/PD-L1 expression between primary tumour and metastasis in melanoma M. Ihle*1, B. Bühler2, M. Schlaak2, A. Scheel1, N. Friedrichs1, N. Brenner2, C. Mauch2, R. Büttner1, S. Merkelbach-Bruse1 Universitätsklinikum Köln, Institut für Pathologie, Köln, Deutschland, Universitätsklinikum Köln, Klinik und Poliklinik für Dermatologie und Venerologie, Hauttumorzentrum, Köln, Deutschland

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AG06.03 Detection of BRAF V600E mutations in benign nevi and Melanocytic Tumors of Uncertain Malignant Potential (MELTUMPs) R. Seitz*1, J. Villacorta Hidalgo1, C. Kreutz2, C. Kayser1, A. von Deimling3, K. Technau-Hafsi4, C. Diaz5, P. Fisch1 Institut für Klinische Pathologie, Department für Pathologie, Universitätsklinikum, Freiburg, Deutschland, 2Albert-Ludwigs-Universität Freiburg, Physikalisches Institut, Freiburg, Deutschland, 3Abteilung für Neuropathologie, University Hospital Heidelberg, Heidelberg, Deutschland, 4 Klinik für Dermatologie und Venerologie, Universitätsklinikum, Freiburg, Deutschland, 5Zentrum für Dermatopathologie, Freiburg, Deutschland 1

Background.  A subgroup of melanocytic lesions referred to as Melanocytic Tumors of Uncertain Malignant Potential (MELTUMPs) cannot easily be classified as benign or malignant on the basis of histopathological analysis. BRAF (V600E) mutations are generally a poor prognostic factor in patients with cancer and therapies targeting BRAF mutations have become standard in the care for advanced melanomas. Thus, we examined the BRAF V600E mutation frequency in MELTUMPs and benign nevi using three distinct detection methods. Methods.  85 formalin-fixed and paraffin-embedded (FFPE) tissue samples (40 benign melanocytic nevi and 45 MELTUMPs) were tested for their BRAF V600E mutational status using immunohistochemistry (IHC) with the anti-BRAF V600E-specific monoclonal antibody (mAb) VE1 followed by evaluation with a modified immunoreactive score (IRS). DNA isolated from the FFPE material was examined by a semi-nested BRAF exon 15 PCR followed by Sanger sequencing as well as a sensitive V600E allele specific PCR (AS-PCR) and fragment analysis. Results.  IHC with the BRAF V600E VE1 mAb and AS-PCR for the V600E mutation were almost equally sensitive (60 % vs. 56 %) in detecting the V600E mutations while Sanger sequencing was significantly less sensitive (27 %). All IHC V600E negative cases were also negative by AS-PCR. Consequently, we used IHC as a reference to determine the frequency of BRAF V600E mutations in benign nevi and MELTUMPs. The histological samples were independently evaluated by two expert pathologists. In the samples initially classified by pathologist A as “benign nevi”, we found 70 % BRAF V600E positive cases, while there were 51 % positive cases in MELTUMPs. In the samples diagnosed by pathologist B as “benign nevi” 63 % were BRAF V600E positive. In BRAF positive MELTUMPs where more than 90 % of cells stained positive in IHC a final benign diagnosis was significantly more common than that of melanoma. BRAF V600E mutations were significantly more frequent in tissue samples taken from the trunk as compared to all other locations. In the samples taken from the legs BRAF V600E mutations were significantly less frequent. Conclusions.  The V600E mutations appear to be more frequent in benign nevi than in MELTUMPs. Thus, MELTUMPs do not seem to preferentially develop from V600E positive nevi. Although BRAF positive melanomas are clinically more aggressive, the BRAF V600E mutation is not more frequent in malignant than in benign melanocytic lesions.

Background.  In recent years, the growing molecular understanding of melanoma led up to the development of targeted therapies. However, most patients progress under treatment. One major clinical issue in the context of resistance is the early detection of resistant subclones. Therefore we aimed at evaluating the mutational spectrum and expression of 19 different markers in primary melanoma and the corresponding metastasis to evaluate the intertumour heterogeneity in melanoma and to detect early resistance mechanisms. Methods.  A cohort of 61 samples consisting of 26 pairs of primary melanoma and metastases was analysed by targeted multiplex PCR based parallel sequencing. A customized melanoma panel of 17 different genes or gene regions including clinical relevant ones such as BRAF, NRAS, KIT, GNAQ and GNA11 was designed by Qiagen. Amplification and library preparation were performed by the GeneReadTM DNAseq targeted Panels V2 kit (Qiagen). 12 pM of each library were analysed on an Illumina MiSeq sequencing platform. In addition, PD-1 and PD-L1 expression were determined by immunohistochemistry (antibodies were from Cell Signalling, clone E1L3N, 1:1000 and Abcam, clone NAT105, 1:100, respectively). Results.  Concordant PD-L1 immunohistochemistry was detected in eight of 16 melanoma pairs (negative staining in the primary tumour as well as in the metastasis). In eight pairs discordant results were detected. Concordant PD-1 expression was likewise detected in eight pairs. Nine pairs showed discordant PD-1 expression. 59 of 61 samples showed sufficient tumour content for parallel sequencing analysis. Mutations were called with an allele frequency of >5 % mutated alleles in a background of wildtype alleles having a coverage of at least >200x. Using these quality criteria, 36.2 % NRAS, 24.1 BRAF mutations, 12.1 % TP53 and 0 % KIT mutations were detected. Additionally, 8.6 % CDKN2A, 5.2 % PTEN and 5.2 % RAC1 mutations were found. Sequencing data were checked bioinformatically for gene amplifications. Mutational intertumour heterogeneity between primary tumour and corresponding metastasis was observed in two pairs. Conclusions.  Immunohistochemical PD-L1/PD-1 intertumour heterogeneity was detected in about 50 % of the analysed pairs. Mutational intertumour heterogeneity was detected in 8.7 %. Based on these results it seems to be insufficient to analyse only one specimen to enable the best practical care for patients. No minor clones leading to resistance were detected.

AG06.05 Basal cell carcinoma triggered by piercing in a young patient L. Morawietz*1, C. Weise2, L. Landeck2 Diagnostik Ernst von Bergmann GmbH, Institut für Pathologie, Potsdam, Deutschland, 2Klinikum Ernst von Bergmann gGmbH, Klinik für Dermatologie, Venerologie und Allergologie, Potsdam, Deutschland 1

Background.  Basall cell carcinoma is a common skin tumor, usually observed in elderly patients due to lifelong accumulation of sun damage. Methods.  This is a case report of basal cell carcinoma on the left alar wing of the nose in an unusually young patient (39 years). Anamnestic exploration revealed that she had been wearing a piercing for several years at the site of the tumor. The extralesional skin showed no signs of overly sun damage or premature aging. Micrographic excision was performed and the specimen was completely worked up for histopathology using standard methods. Der Pathologe Suppl 1 · 2016

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Abstracts Results.  Histologic examination demonstrated an invasive epithelial neoplasia, consisting of basaloid cells. The architecture was heterogeneous, some parts of the tumor presenting a nodular configuration, while other areas had a more diffuse growth pattern and extensive desmoplastic stroma reaction. The diagnosis of basal cell carcinoma with nodular and sclerodermiform segments was established. The immediate center of the lesion was spared, here the skin appendages were replaced by connective tissue, resempling the scar at the site of the former piercing. Furthermore, the connective tissue in the upper dermis showed the typical basophilic degeneration of solar elastosis. Conclusions.  Due to the immediate co-localization it seems undoubted that the tumor was induced by the piercing. The jewelry the patient had been wearing contained a small gemstone with a shape reminiscent of an optical lense. Therefore we assume that this stone focused the sunlight in such a way that it caused damage directly to the adjacent skin and finally led to the development of basal cell carcinoma. This theory might be supported by the fact that the skin also showed extensive solar elastosis and that the direct piercing site was spared by the tumor. We conclude that basal cell carinoma can occur in younger patients and that it might be evoked by jewelry, especially by gemstones with shapes similar to optical lenses.

AG06.06 Biphasic Sarcomatoid Transdifferentiation in a Case of Metastatic Melanoma Identified by Next Generation Sequencing N. J. Rupp*1, M. Rechsteiner1, D. Lenggenhager1, P. K. Bode1, U. Camenisch1, E. Marques Maggio1, M. Urosevic2, D. Mihic-Probst1 Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz, Dermatologische Klinik, UniversitätsSpital, Zürich, Schweiz

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Background.  Metastatic melanomas, mimicking many different tumors, are a morphological challenge for pathologists. Experiments with melanoma cell lines have shown the capacity of tumor cells to transdifferentiate into different lineages and to even have the plasticity to contribute to neovascularization. However, comprehensive melanoma tumor tissue transdifferentiation into another lineage is a little-known phenomenon in diagnostic practice and only few cases have been published so far. Here, we describe a highly unusual case of a metastatic melanoma manifested as groin lymph node metastasis in a 61 years old female with two distinct concomitant subcutaneous sarcomatoid lesions on the same leg, whereas no evidence for a conventional primary tumor was found. Methods.  All three tumorous lesions were formalin-fixed paraffin embedded and examined histologically on Hematoxylin & Eosin staining. Further characterization encompassed extensive immunohistochemical staining, in particular for known melanocytic markers as well as investigation of BRAF (Exon 15) and NRAS (Exons 2–4) genes by Sanger sequencing. Additionally, next generation sequencing was performed using the Ion AmpliSeq™ Colon and Lung Cancer Panel (22 genes) and Comprehensive Cancer Panel (409 genes). Results.  The lymph node metastasis showed obvious morphological and immunohistochemical differentiation of melanoma. The two subcutaneous lesions were conventionally consistent with high-grade myxofibrosarcoma and primarily soft tissue mixed tumor, respectively. All three lesions showed BRAF wildtype and harbored a NRAS p.Q61R mutation. Next generation sequencing data could confirm these findings and reveal further concordant mutations in 22 genes. Conclusions.  Although there was no morphological or immunohistochemical conclusive evidence of melanoma in the mesenchymal lesions, the concordant genetic profile of BRAF and NRAS as well as the other concordant mutations in 22 genes indicate biphasic sarcomatoid transdifferentiation of melanoma in this case. Particular in patients with previously known melanoma, unusual presentation of metastasis, even without conventional evidence for melanoma, should raise the possibility of transdif-

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ferentiation. Next generation sequencing comparing mutational profiles may therefore help to establish this potentially difficult diagnosis.

AG Knochen-, Gelenk- und Weichgewebspathologie AG07.01 Ewing sarcoma cells own a sub-population with stem cell properties – a next generation sequencing (NGS) study N. V. Mallela1, E. Korsching*1, J. Seggewiß2, M. Hotfilder3 Universität Münster, UKM, Institut für Bioinformatik, Münster, Deutschland, Universität Münster, UKM, Institut für Humangenetik, Münster, Deutschland, 3Universität Münster, UKM, Pädiatrische Hämatologie und Onkologie, Münster, Deutschland 1

2

Background.  Ewing sarcoma, a bone tumor, belongs to the group of rare diseases. The genomic situation is characterized by some few genomic alterations with low explanatory power and one prominent fusion gene based on an EWS portion (including a transcription activating domain) and a second portion from FLI or ERG (to name the two prominent fusions [1]) retaining their DNA binding activity. Apart from that the regulatory network and the distinct molecular details of this pathophysiological process remain nebulous. Therefore diagnostic as well as therapeutic options can not be targeted systematically. Here we present a NGS based study where we want to characterize a sub-population of Ewing sarcoma cells of the model cell line CADO-ES1 (EWS-ERG fusion), and compare these properties with primary cell cultures of Ewing cells, MSC stem cells and normal fibroblasts as a reference. Methods.  The CADO-ES1 cells were FACS sorted using a dye-efflux-assay. The characterization of the side population (SP) cells showed stem cell like properties in proliferation, differentiation and tumor forming assays. The color-space (CS) reads of the sequencing were handled in four different ways according to transcriptomic or genomic origin and according to different analysis procedures. A huge effort was invested to customize the raw reads to get a high coverage with high quality reads. The NGS pipelines mainly based on established tools (e. g.TopHat, CuffLinks, Bowtie, edgeR, deFuse) were enriched by own developments (Cpp based CS to base-space converter) and several scripts. Results.  Some first results show, that we have a remarkable number of differential genes between the Ewing SP cells and the primary cell culture of Ewing cells adjusted by the technical controls (912). CHN2 (down in the side population) owns alterations on the DNA and RNA level and is already described as a highly significant factor by Hu-Lieskovan et al. [2] (a differentiation study) consistently with our observations. Conclusions.  The regulatory networks of the cell and the resulting visible phenotypes can be often successfully decoded by applying systems biology approaches on high dimensional data snap-shots. In this first report we focused on the NGS analysis strategy and the developed tools to accomplish this challenging analysis process to decipher the properties of the Ewing SP cells presumably driving tumor progression. References 1. Potratz J, Jurgens H, Craft A, and Dirksen U. (2012) Ewing Sarcoma: Biology-Based Therapeutic Perspectives., Pediatr Hematol Oncol, 29:12–27 2. Hu-Lieskovan S, Zhang J, Wu L, Shimada H, Schofield DE, Triche TJ (2005) EWS-FLI1 fusion protein up-regulates critical genes in neural crest development and is responsible for the observed phenotype of Ewing’s family of tumors., Cancer Res, 65(11):4633–44

AG07.02 EWSR1-FLI1-mediated suppression of the evolutionary conserved RAS-antagonist Sprouty 1 (SPRY1) confers aggressiveness to Ewing sarcoma F. Cidre-Aranaz1, T. G. P. Grünewald*2, L. García-García1, D. Surdez3, J. Carlos Lázaro1, A. Sastre4, P. García-Miguel4, S. Álvarez1, S. Monzón1, O. Delattre3, J. Alonso1 Instituto de Investigación de Enfermedades Raras, Instituto de Salud Carlos III, Madrid, Spanien, 2Pathologisches Institut der LMU München, Labor für Pädiatrische Sarkombiologie, München, Deutschland, 3Institut Curie Research Center, INSERM U830 Genetics and Biology of Cancers, Paris, Frankreich, 4Hospital Infantil Universitario La Paz, Unidad hemato-oncología pediátrica, Madrid, Spanien 1

Background.  Ewing sarcoma is a highly aggressive bone-associated cancer mostly affecting adolescents and young adults. It is characterized by chromosomal translocations fusing the EWSR1 gene with various members of the ETS family of transcription factors – most commonly FLI1. EWSR1-FLI1 drives tumorigenesis of Ewing sarcoma by either transcriptionally inducing or suppressing specific target genes. Recent data showed that basic fibroblast growth factor (bFGF) promotes proliferation of Ewing sarcoma cells and that FGF receptors (FGFRs) are hyper-phosphorylated in primary tumors, although activating FGFR mutations are exceedingly rare. Methods.  We analyzed the effect of Sprouty 1 (SPRY1), which is an evolutionary conserved negative feedback inhibitor of FGFRs and other RAS-activating receptors, on the aggressive phenotype of Ewing sarcoma cells with an inducible SPRY1 re-expression system. In addition, we performed several in silico and in situ experiments. Results.  We show that SPRY1 is strongly suppressed by EWSR1-FLI1 and that EWSR1-FLI1 knockdown specifically and dramatically increases SPRY1 re-expression while other Sprouty family members remain unaffected. Indeed, SPRY1 is not expressed in many Ewing sarcoma cell lines derived from very aggressive primary tumors or metastases, suggesting that its suppression may confer a growth advantage. In agreement, SPRY1 re-expression functionally impaired proliferation, clonogenic growth as well as migration and invasion in three different Ewing sarcoma cell lines. This was accompanied by reduced ERK/MAPK signaling upon stimulation with serum or bFGF. Consistently, treatment of Ewing sarcoma cells with the potent FGFR inhibitor PD-173074 reduced bFGF-induced proliferation and colony formation, thus mimicking SPRY1 activity in Ewing sarcoma cells. Interestingly, SPRY1 was variably expressed in primary Ewing sarcoma tumors and very low expression levels were significantly associated with worse outcome in a large patient cohort. Conclusions.  Taken together, our data indicate that EWSR1-FLI1-mediated suppression of SPRY1 leads to unrestrained FGFR-signaling and bFGF-induced cell proliferation, and suggest that targeting the FGFR/MAPK pathway may constitute a promising therapeutic approach. Our data support that EWSR1-FLI1-mediated suppression of SPRY1 may explain the observed hyper-phosphorylation of FGFRs in primary tumors and that the SPRY1 expression level may serve as a prognostic biomarker to guide individualized therapy of Ewing sarcoma patients.

AG07.03 CD274/PD-L1 Gene Amplification and PD-L1 Protein Expression in high grade bone and soft tissue sarcomas M. Straub*1, H. Rechl2, R. von Eisenhart-Rothe2, K. Specht1 Technische Universität München, Institut für Allgemeine Pathologie und Pathologische Anatomie, München, Deutschland, 2Technische Universität München, Klinik und Poliklinik für Orthopädie und Sportorthopädie, München, Deutschland 1

Background.  Sarcomas are a heterogeneous group of rare tumors. According to their genotype, they can be grouped into sarcomas with com-

plex genotypes and sarcomas with recurrent genetic abnormalities. They display relative insensitivity to conventional chemotherapy, and for most subtypes, targeted therapeutics are missing. Immune checkpoint blockade targeting PD-1 receptor and its ligand PD-L1 has shown promising results in clinical trials in several solid malignancies. In this context, PD-L1 expression might be a potentially valuable predictive marker. Methods.  The study cohort comprised 102 cases of undifferentiated pleomorphic sarcomas, 45 synovial sarcomas, 38 angiosarcomas, 12 epithelioid sarcomas, 10 alveolar soft part sarcomas (ASPS), 11 clear cell sarcoma, 39 CTX-naive osteosarcomas and 30 high-grade chondrosarcomas. PDL1 protein expression was examined by immunohistochemistry; positive PD-L1 staining was defined as >5 % membrane staining of any intensity. PD-L1 copy number status was analyzed by fluorescence in situ hybridization. PD-L1 amplification was defined as (1) percentage of tumor cells containing ≥5 PD-L1 signals ≥50 %,(2) PD-L1/CEP9 ratio ≥2.0 (3) average number of PD-L1 signals per nucleus ≥6, (4) percentage of tumor cells containing ≥15 PD-L1 signals ≥10 %. (1) was defined as low level, (2)–(4) as high level amplification. Results.  In a preliminary analysis, PD-L1 amplification was seen in 22 % of UPS and 4 % of osteosarcomas. No amplification was detected in ASPS and chondrosarcomas. PD-L1 protein expression was present in 14 % of UPS, 31 % of ASPS, 6 % of epithelioid sarcomas, 46 % of chondrosarcomas, 50 % of angiosarcomas and 56 % of clear cell sarcomas. Staining was not concordant between primary tumor and metastasis in several cases. Conclusions.  PD-L1 amplification is a frequent event in STS with complex genotypes, whereas in sarcomas with simple genotypes and bone sarcomas, genetic alterations of the PD-L1 gene locus are rare. PD-L1 protein expression is seen in all subtypes of bone and soft tissue sarcomas with variable frequency. Expression is not concordant in primary tumor and metastasis in several cases, possibly as a result of inducible protein expression or tumor heterogeneity. Especially in sarcoma cases with PD-L1 amplification, PD-1 blockage could be a therapeutic option, with PD-L1 amplification status as predictive biomarker.

AG07.04 Genome abstraction analyses identify deficiencies in homologous recombination repair as a common trait in osteosarcoma M. Kovac*1, C. Blattmann2, J. Smida3, S. Ribi1, N. Mueller3, F. Engert4, F. Castro-Giner5, S. Fulda4, S. Bielack2, G. Jundt1, I. Tomlinson5, J. Korbel6, M. Nathrath3, D. Baunhoer1 Universitätsspital Basel, Pathologie, Basel, Schweiz, 2Klinikum Stuttgart – Olgahospital, Stuttgart, Deutschland, 3Helmholtz Zentrum München, Neuherberg, Deutschland, 4Goethe-Universität, Frankfurt, Deutschland, 5The Wellcome Trust Centre for Human Genetics, Oxford, Vereinigtes Königreich, 6 European Molecular Biology Laboratory, Heidelberg, Deutschland 1

Background.  Osteosarcomas are aggressive bone tumors with a high degree of genetic heterogeneity which complicates the identification of driver genes and the development of more individualized treatment approaches. Methods.  We inferred signatures of the underlying mutation processes in 31 osteosarcoma exomes and validated these findings by SNP array and re-sequencing data in a set of 92 tumors. Since the vast majority of tumors revealed mutations in the BRCA pathway of homologous recombination repair, we carried out in vitro cell viability tests using the phase-3 PARP inhibitor Talazoparib and the osteosarcoma cell line MNNG/HOS. Results.  We identified 14 distinct driver genes in our set of tumors, of which five were formerly unknown in the context of osteosarcoma. None of the drivers was clearly responsible for the majority of tumors and even TP53 mutations were frequently mapped into subclones. However, >80 % of osteosarcomas exhibited a specific combination of single base substitutions, LOH, or large-scale genome instability signatures characteristic for BRCA1/2-deficient tumors that are known to respond to treatment with PARP inhibitors. Since osteosarcoma cell lines with BRCA1/2 deficiency

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Abstracts do not exist, we carried out testing of MNNG/HOS osteosarcoma cells carrying ATM and PTEN mutations, which inactivate the BRCA pathway upstream from BRCA1. Indeed, the in vitro tests showed good evidence of cell viability reduction with standalone Talazoparib treatment and an even increased response in combination with the alkylating agent Temozolomide and the topoisomerase I inhibitor SN-38. Conclusions.  Our findings imply that multiple oncogenic pathways can drive chromosomal instability during osteosarcoma evolution and result in the acquisition of BRCA-like traits in the majority of tumors, which could be therapeutically exploited.

AG07.06 Clinical and morphological characterization of pediatric and adult sarcomas harboring a novel gene fusion involving the kinase NTRK1

Methods.  Tissue microarrays (TMA) of 240 soft tissue tumors were analyzed for the expression and correlated with clinicopathological parameters including long term survival. Results.  Expression of PDL1 was observed in 81 undifferentiated pleomorphic sarcomas (UPS) in 22 %, 48 leiomyosarcomas in 13 %, 28 synovial sarcomas in 4 %, 45 dedifferentiated liposarcomas in 13 %, 5 angiosarcomas in 60 %, 10 MPNSTs in 0 % and 23 mixed sarcomas in 23 %. Expression of PDL1 showed a significant correlation with shorter patients five-year survival (p < 0.05), higher grading (G2 vs G3, p < 0.03) and PD1 positive tumor infiltrating lymphocytes (TILs, p < 0.001). Furthermore high PD1 in TILs (more than 3) also showed a significant correlation with shorter patients survival in high grade tumors. In UPS high PD1 expression in TILs was also correlated with shorter patients survival. Conclusions.  PDL1 expression is significantly associated with shorter patients survival, grading and PD1 positive TILs. PDL1 expression might guide the use of PDL1 inhibitors in immunotherapy in soft tissue tumors.

A. Agaimy*, J. Knopf, E. A. Moskalev, A. Hartmann, F. Haller FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland Background.  Using whole-genome and RNA-sequencing in a case of infantile hemangiopericytoma, we observed a novel gene fusion involving the kinase NTRK1/TrkA. Similar gene fusions were recently described in rare cases of lung cancer, glioblastoma, melanoma and soft tissue sarcoma. Notably, dramatic responses in patients with advanced disease were reported using specific TrkA inhibitors. For the current study, we aimed to identify specific clinical and morphologic features that might be of potential value to recognize cases of pediatric and adult sarcomas harboring NTRK1 gene fusions. Methods.  The index case was analysed by whole-genome and RNA-sequencing using Illumina Hiseq sequencers. NTRK1 gene fusions were analysed by fluorescence in situ hybridization employing a dual color break apart probe. Other cases were identified based on specific morphological features as the index case irrespective of age. Results.  Four patients were identified (two children aged 11 months and 2 years and two adults aged 51 years and 80 years). While adults tumors were strikingly myopericytoma-like but with clear-cut atypical features, pediatric cases were more akin to infantile myofibromatosis/hemangiopericytoma. All cases contained numerous thick-walled dysplastic-like vessels with segmental or diffuse nodular myxohyaline myointimal proliferations of smooth muscle actin-positive cells occasionally associated with frequent thrombosis. Immunohistochemistry showed variable expression of smooth muscle actin and CD34, but other mesenchymal markers including STAT6 were negative. Conclusions.  Our observation is the first clue which pediatric and adult soft tissue sarcomas need to be evaluated for the presence of therapeutically relevant NTRK1 gene fusions. Future studies are necessary to delineate the full morphological spectrum of this subset of sarcomas.

AG07.07 PDL1 expression is significantly associated with shorter patients survival and grading in soft tissue tumors E. Kampmann1, A. Altendorf-Hofmann2, V. Buecklein1, L. Lindner1, R. Issels1, T. Kirchner3, T. Knösel*3 Onkologische Klinik der LMU, München, Deutschland, 2Klinik für Allgemein-, Viszeral- und Gefäßchirurgie, Friedrich-Schiller-Universität, Jena, Deutschland, 3Pathologisches Institut, Ludwig-Maximilians-Universität, München, Deutschland

1

Background.  The cell surface receptor programmed death-1 (PD1) and its ligand (PDL1) have been detected in various cancer types. However, the expression has never been studied in soft tissue tumors in a large cohort. In this study, we investigated, whether PDL1 and PD1 expression have any impact on the prognosis or clinicopathological parameters of soft tissue tumors.

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AG07.08 Functional characterization of the novel gene fusion LMNA-NTRK1 J. Knopf*, E. A. Moskalev, A. Hartmann, A. Agaimy, F. Haller FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland Background.  The tropomyosin-receptor kinase A (TrkA) encoded by the gene locus NTRK1 is involved in the regulation of cell proliferation, differentiation and apoptosis. NTRK1 gene fusions recently caused increasing interest through recurrent reports of these fusions in lung cancer, glioblastoma, melanoma and soft tissue sarcoma cases. Unfortunately, cell lines with NTRK1 gene fusions are limited. Therefore, we aimed to develop a cell culture model that stably expresses the LMNA-NTRK1 gene fusion to enable functional characterization with subsequent tyrosine kinase inhibitor testing. Methods.  The fusion gene LMNA-NTRK1 was cloned into the lentiviral pCDH vector and transduced into human skin fibroblasts (CCD-1137Sk). ShRNA-mediated knockdown of p16INK4A was additionally achieved by lentiviral transduction to reconstruct the in vivo setting as sarcomas harboring the LMNA-NTRK1 gene fusion frequently exhibit an additional disruption of the CDKN2A locus. The resulting transduced fibroblast model was further characterized using Western blotting, cell proliferation and cell viability assays. Responsiveness to different TrkA kinase inhibitors Lestaurtinib, AZ-23, Dovitinib and Crizotinib was analysed in comparison to a kinase dead control. Results.  Activation of PLCγ1 as a direct target of TrkA was observed using Western blotting. Additional knockdown of p16INK4A led to higher proliferation rates and better viability compared to transduction of the LMNA-NTRK1 fusion alone. The responsiveness to the different kinase inhibitors was highest for AZ-23 compared to the kinase dead control. However, Lestaurtinib showed inhibitory effects at the lowest concentrations (~1 nM). Conclusions.  The successful establishment of a LMNA-NTRK1 cell culture model demonstrates the usefulness of synthetic gene fusions and lentiviral transduction to study rare gene fusion events if no correlating cell line exists. We showed that the LMNA-NTRK1 gene fusion activates cell proliferation through PLCγ1, and we provided first clues on in vitro treatment options comparing different TrkA inhibitors.

AG07.09 Functional characterization of two distinct NAB2-STAT6 gene fusion variants A. Ackermann*, E. A. Moskalev, A. Hartmann, A. Agaimy, F. Haller FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland Background.  Recently, a recurrent gene fusion involving NGFI-A-binding protein 2 (NAB2) and the transcription factor STAT6 was observed in tumors of the solitary fibrous tumor (SFT)/hemangiopericytoma (HPC) family. In a previous study, we reported on distinct clinical and morphological features of tumors with the canonical NAB2-STAT6 fusion variants 4-2 and 6-16, correlating to classical fibrous pleuro-pulmonary SFT and deep-seated HPC, respectively. For the current study, we aimed to generate cell culture models stably expressing the two canonical NAB2-STAT6 fusion variants to characterize potential differential effects on gene expression and cell proliferation. Methods.  The two canonical NAB2-STAT6 fusion variants with breakpoints at exons 4-2 and exons 6-16, respectively, were cloned to rwpLX305 lentiviral vector allowing stable transduction of CCD-1137Sk human skin fibroblasts. Canonical NAB2-STAT6 expressing cell populations were selected using blasticidin. Efficiency of ectopic expression was controlled by NAB2STAT6 fusion-specific TaqMan multiplex qPCR and Western blotting using an N-terminal NAB2 antibody. Differential gene expression was explored via Microarray-based gene expression analysis (Affymetrix Whole-Transcript Expression Analysis & Profiling Human Transcriptome Array (HTA) 2.0). Results.  Stable expression of the NAB2-STAT6 fusion variants resulted in nuclear localization of the chimeric protein as determined by STAT6 immunofluorescence, whereas wildtype STAT6 was expressed in the cytoplasm. Differential gene expression analysis was evaluated using the Affymetrix Expression Analysis Console Software, and differentially expressed genes were mapped to specific signaling pathways. Additionally, publicly available gene expression data sets of SFT patient samples were analysed for differentially expressed candidate genes. Four promising candidate target genes were validated by SYBR Green qPCR. Conclusions.  In the current study, an in vitro CCD-1137Sk human skin fibroblast cell model with stable constitutive expression of the two canonical NAB2-STAT6 fusion variants 4-2 and 6-16 was established. Using gene expression analysis, target candidate genes were identified which potentially contribute to the distinct clinical and morphological phenotype of SFTs/ HPCs harboring these gene fusions.

AG07.10 SWI/SNF-deficiency as a novel pathway of tumor initiation and/or dedifferentiation in diverse neoplasms in adults and the elderly A. Agaimy*

Conclusions.  Alterations of the SWI/SNF complex represent a novel pathway of dedifferentiation in diverse neoplasms from different organs. There is a close link between the SWI/SNF pathway and the aggressive rhabdoid phenotype. The complexity of the molecular profiles of these aggressive tumors and the short survival periods after diagnosis underscores the urgent need for prompt and thoruough molecular testing on an individualized level to highlight potential therapeutic targets.

AG07.11 H3F3A mutations in giant cell tumors (GCT) of bone and GCT cell lines are associated with decreased detection of H3K36me3 protein J. Schreiber*, J. Lüke, R. Marienfeld, J. Nell, P. Möller, K. Mellert, T. F. Barth Institut für Pathologie, Ulm, Deutschland Background.  The giant cell tumor of the bone is a rare tumor, usually developing in the epiphysis of long bones. GCT is a heterogenous tumor consisting of two cellular components: the neoplastic stromal cells, derived from mononuclear cells of the bone marrow, and the multinuclear giant cells, which are recruited by the stromal cells. The giant cells are responsible for the local destruction of bone substance, which can lead to pathological fractures. The stromal cells are characterized by a point mutation in the H3F3A gene, which is regarded as a driver mutation in this tumor. H3F3A encodes the histone variant H3.3. The mutation causes an amino acid change at position 34 (G34 W). Due to this mutation the methyltransferase SETD2 cannot trimethylate the neighboring Lysine 36 (H3K36) (Lewis et al, Science 2013,17;857–861). Methods.  We established two GCT cell lines consisting of a monomorphous stromal cell-like tumor cell population. Both lines carry the mutated H3F3A gene as shown by PCR based mutation analyses. We compared the two lines to eleven non-mutated tumor cell lines using the Line-1 assay to measure DNA-methylation. Western Blot and immunocytochemistry were used to detect H3K36me3 and SETD2. Furthermore we performed immunohistochemistry on 11 H3F3A-mutated giant cell tumors as well as seven non-mutated giant cell containing bone lesions, using an H3K36me3-specific monoclonal antibody. Results.  We could not find DNA-methylation patterns specific to the giant cell tumor cell lines in the Line-1 assay. However, in Western blot analyses H3K36me3 is not expressed in the H3F3A-mutated GCT cell lines, while SETD2 protein is present. By immunohistochemical staining for anti-H3K36me3 we detected a lower expression in the eleven H3F3A mutated giant cell tumors of the bone as compared to the seven other H3F3A non-mutated bone lesions containing giant cells. Conclusions.  In H3F3A-mutated GCT cell lines in vitro and GCT in situ the mutations seems to be linked to a lower detection of H3K36me3; therefore lower expression of H3K36me3 might serve as a surrogate marker of a H3F3A mutation.

FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland Background.  The switch/sucrose-non-fermenting (SWI/SNF) complex is a highly preserved group of interdependent proteins involved in the chromatin remodelling and thus in the regulation of vital cellular processes such as proliferation and differentiation. At least 8 genes encoding core subunits of the SWI/SNF complex have been identified to date. Recent studies uncovered a surprisingly high frequency of mutated SWI/SNF subunits in a variety of neoplastic as well as developmental diseases in human. Methods.  A review of common an rare alterations involving the SWI/ SNF pathway from previous studies and our own previous publication on this topic with a focus on organ-specific phenotype-genotype correlations. Results.  A variety of neoplasms in adults and the elderly may show alterations of one or more of the core subunits of the SWI/SNF complex, frequently associated with rhabdoid dedifferentiation and highly aggressive course. The morphologic spectrum of these entities varies greatly from small cell basaloid to large cell frankly rhabdoid phenotypes with organ-specific marker profile.

AG07.12 Perilipin 1 helps differentiate adipocytic tumors from other soft tissue tumors B. K. Straub*, L. Pawella, E. Eiteneuer, M. Hashani, M. Renner, P. Schirmacher, G. Mechtersheimer Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland Background.  The formation of fat droplets is a hallmark of adipogenesis, and a morphologic characteristic of adipose tissue and adipocytic tumours including liposarcoma. Lipid droplet-associated structural proteins of the perilipin-/PAT-family account for the most frequent and characteristic proteins during adipogenesis and consist of 5 members in mammalians: perilipin 1-5 (formerly known as perilipin, adipophilin, TIP47, S3-12, MLDP). Whereas perilipin 1 is restricted to mature lipid droplets

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Abstracts in adipocytes, sebaceocytes and steatotic hepatocytes, perilipins 2-5 are widely expressed. As perilipin 1 helps differentiate hepatocellular from other epithelial tumors, the value of perilipin immunohistochemistry for the differential diagnosis of liposarcoma should be tested. Methods.  We therefore comprehensively investigated perilipins in 478 human soft tissue tumours as well as 60 respective normal tissues and derived cultured cells with immunohistochemistry, immunofluorescence microscopy, immunoblot and rtPCR analysis. Results.  Whereas perilipin 1 is positive at large lipid droplets in normal adipose tissue (22/22), all benign adipocytic tumors (13/13), in all well differentiated liposarcoma (24/24) and the vast majority of myxoid liposarcoma (43/47 = 91 %) and pleomorphic liposarcoma (10/14 = 72 %), perilipin 1 only marks the well differentiated part of dedifferentiated liposarcoma (50/50) and is absent from the dedifferentiated part and absent from all other soft tissue sarcoma (0/356) including Ewing sarcoma, myxofibrosarcoma, fibromatoses, GIST, leiomyosarcoma, rhabdomyosarcoma, MPNST, neuroblastoma, synovial sarcoma and undifferentiated pleomorphic sarcoma. Only in some high grade sarcoma, faint nuclear staining with perilipin 1 antibodies was noted, but no lipid droplet staining pattern. In contrast, perilipin 2 was absent or only faintly positive in lipoma and well differentiated liposarcoma, but demonstrated increased numbers of lipid droplets in pleomorphic liposarcoma and other high grade sarcoma. Cultured cells of the pleomorphic liposarcoma line LiSa-2 treated with adipogenic differentiation medium also showed differential perilipin-expression. Upon siRNA downregulation of perilipins 2 or 3, cell viability was mildly reduced. Conclusions.  Immunohistochemistry for the lipid droplet protein perilipin 1 helps differentiate liposarcomas from other soft tissue sarcoma. In contrast, other perilipins such as perilipin 2 are upregulated in high grade tumours and may represent a potential therapeutic target.

AG07.13 Medial-Shelf syndrom – first results of a systematic histopathological characterization C. Brochhausen*1, 2, C. Meyer-Scholten2, J. Grifka3, K. Matzinger1, A. Mamilos1, D. Grevenstein4, M. Evert5 REPAIR-Lab, Institut für Pathologie, Universität Regensburg, Regensburg, Deutschland, 2Zentrum für Rheumapathologie, Universitätsmedizin Mainz, Mainz, Deutschland, 3Orthopädische Uniklinik, Regensburg, Deutschland, 4 Hochtaunusklinik, Abteilung für Orthopädie und Unfallchirurgie, Bad Homburg, Deutschland, 5Institut für Pathologie, Universität Regensburg, Regensburg, Deutschland

1

Background.  Synovial plicae represent normal anatomical structures, which are thougth tob e embryological remnants of septical structures during joint development. There are three described plicae from which especially one, namely the plica mediopatellaris could become symptomatic in adolescence and young adults. The proper diagnose oft eh so called plica syndrome is difficult and there are no histo-pathological description within this entity. Methods.  In the present retrospective study plicae resections from 50 patients were analysed histologically and histochemically. After arthroscopic resection the specimens were fixed over night in buffered formalin (4 %) and embedded in paraffin according to standard methods. Afterwards 5 µm thick sections were stained with Haematoxylin & Eosin and Elastica van Gieson according to standardised protocols. The specimens were analysed by three independent pathologists. Results.  All analysed specimens showed an increase of the subsynovial fibrous tissue. In a number of specimens oedema could be observed, which also were evident in pre-interventional magnetic resonance tomography. Furthermore, circumscribed hypertrophy of the synovial cell layer and a slight lymphatic cell infiltrate were evident. Occasionally scattered granulocytic infiltrates were seen.

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Conclusions.  We present the first systematically description of plicae resections. Beside a slide oedema, which was also described in the magnetic resonance tomography, hyperplasia of the synovial cells as well as infiltrates of lymphocytes and granulocytes as a morphological correlate for inflammatory changes were observed. These histo-pathological changes underline the pathological value of isolated plica-changes, named plica syndrome. Our results represent an interesting base for further histo-pathological analyses for the development of a potential histo-pathological scoring system of plica changes.

AG07.14 Is the diagnosis of meniscus degeneration and trauma dependent on the technique of the pathological preparation a prerequisite for successful material based meniscus implantation? C. Brochhausen*1, 2, A.-K. Desch3, D. Grevenstein3, A. Mamilos1, V. Schmitt3, P. Drees4, M. Evert5, C. J. Kirkpatrick3 REPAIR-Lab, Institut für Pathologie, Universität Regensburg, Regensburg, Deutschland, 2Zentrum für Rheumapathologie, Universitätsmedizin Mainz, Mainz, Deutschland, 3REPAIR-Lab, Institut für Pathologie, Universitätsmedizin, Mainz, Deutschland, 4Zentrum für Orthopädie und Unfallchirurgie, Universitätsmedizin, Mainz, Deutschland, 5Institut für Pathologie, Universität Regensburg, Regensburg, Deutschland 1

Background. It is well known, that lesions of the menisci represent risk-factor for the development of osteoarthritis. Therefore, it would be necessary, to be able to regenerate the menisci. At the moment, we do have implants, which do not completely fulfill all the high demands they would have to. One very important requirement for the improvement of these implants, would be a reliable histopathological scoring-system, to be able to classify the lesions. Such a score does not exist yet. The menisci can be subdivided in three zones: the inner avascular “white zone”, the intermediate “red-white zone” and the vascularized outer “red zone”. Up to now, it is unknown, if the diagnosed grade of degeneration is dependent on the direction of the histopathological cut. Methods.  In this retrospective study, five partly resected menisci were cut in tangential and five in triangular technique. We performed a HE-, Alcianblue and Safranin-O staining, after that the specimen were blinded and evaluated by an experienced pathologist. Results.  The triangular horizontal cuts allowed to identify the three typical zones. Fibrillar areas at the surface and lacerations in the avascular zone could be detected without fail. Dehiscences of the fibers and mucoid matrix degeneration was mostly localized in the border-zone. In the tangential cuts, the possibility to evaluate the surface or the avascular zone was very limited. These specimen consisted mostly of the border-zone, which lead to an overdiagnosis of the grade of degeneration. Conclusions.  The whole transverse section should be used to develop a histopathological scoring-system, because it allows to evaluate the entire meniscus, which is an important prerequisite to decide wether an scaffold implantation could be successful or not.

AG07.15 Clival and sacral chordomas and the cell lines derived therefrom show differences in HOX gene expression but are both defective in the p16 pathway D. Jäger*1, A. von Witzleben1, R. Marienfeld1, H. Barth2, A. Lechel3, M. Kornmann4, R. Mayer-Steinacker5, A. von Baer6, M. Schultheiß6, A. M. Flanagan7, P. Möller1, S. Brüderlein1, K. Mellert1, T. F. Barth1 Universität Ulm, Institut für Pathologie, Ulm, Deutschland, 2Universität Ulm, Institut für Pharmakologie und Toxikologie, Ulm, Deutschland, 3Universität Ulm, Klinik für Innere Medizin I, Ulm, Deutschland, 4Universität Ulm, Klinik für Allgemein- und Viszeralchirurgie, Ulm, Deutschland, 5Universität Ulm, 1

Klinik für Innere Medizin III, Ulm, Deutschland, 6Universität Ulm, Klinik für Unfallchirurgie, Ulm, Deutschland, 7University College London, Cancer Institute, London, Deutschland Background.  Chordoma is a rare type of bone tumour arising along the vertebrate body axis, predominantly at the sacral and clival region. There is an ongoing discussion whether chordomas at different positions of the spine share the same clinical and biological characteristics. Methods.  We analysed our chordoma tissue bank of 43 patients with regard to possible differences between clival and sacral chordomas. We established a novel cell line, U-CH14, derived from a clivus chordoma and performed DNA-microarray-based gene expression analysis of U-CH14 in comparison with 8 chordoma cell lines derived from the sacrum region. In one further clivus chordoma cell line we performed immuncytochemical analysis of several genes from HOXA-Cluster, namely HOXA3, HOXA5, HOXA7 and HOXA11, a group of transcription factors known to play a critical role in the formation of the anterior-posterior body axis. We performed analysis of the p16/CDK4-pathway using immunohistology (IHC) and growth inhibition experiments. Results.  Analysis of our chordoma cohort (n = 43, among 5 with clival chordoma and 24 with sacral chordoma) revealed that clivus chordoma patients tend to be younger than patients with sacrum chordoma (median age: 49 years versus 70 years). Metastasis occurred in 0/5 clival and 8/24 sacral chordomas, recurrent disease was seen in 0/5 clival and 13/24 sacral chordoma patients. Clivus chordomas had lower Ki-67 indices and a smaller tumour size. R0 resection was possible in 0/5 clival and 4/24 sacral chordoma patients. On genomic, mRNA and protein expression levels, U-CH14 is a true chordoma cell line from the clivus. Gene expression analysis shows several genes to be significantly different regulated in U-CH14 in comparison to sacral chordoma, including genes of the HOX-gene family. In IHC analyses 4/10 sacral chordoma cell lines and 1/2 clival cell lines were positive for HOXA3, HOXA5 was positive in 1/2 clivus cell lines. HOXA11 was positive in 2/10 sacrum and 1/2 clivus chordoma cell lines. Surprisingly, all chordoma cell lines and their parental tumours were positive for HOXA7. As seen in sacral chordomas, U-CH14 and UM-Chor1 showed loss of p16 and activation of the CDK4-pathway and responded to inhibition by palbociclib. Conclusions.  Chordomas of the clivus have different clinical parameters than sacral chordomas and express a different set of HOX genes, while inhibition of the defective p16 pathway is a common feature of both types of chordomas.

AG Informatik, innovative Bildgebung und Biobanking AG08.01 Audio-Video-Podcasts as innovative teaching module B. Müller*1, K. Hetzel1, A. Hartmann2, M. Scheib1 1 Universität Erlangen-Nürnberg, Medizinische Fakultät, Erlangen, Deutschland, 2FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland

Hintergrund.  In einem durch die Medizinische Fakultät der Friedrich-Alexander-Universität Erlangen geförderten Projektes soll evaluiert werden, inwieweit Audio-Videopodcasts den Studierenden einen Mehrwert für ihr Studium im Hinblick auf Prüfungsvorbereitung und Wiederholung der Vorlesungsinhalte bieten. Seit 2013 erstellen Studierende im Rahmen des Medcastprojekts, Audio- und Videopodcasts in in unterschiedlichen Fachdisziplinen wie Pathologie, Histologie, Innere Medizin etc, die eine zusätzliche Lernunterstützung für ihre Kommilitonen bieten sollen.

Methoden.  Insgesamt wurden 53 Audio-Video-Podcasts erstellt, davon 8 im Fach Pathologie. Die einzelnen Medcastler widmen sich unterschiedlichen Aufgabengebieten wie: Texte erstellen, vertonen, Videos erstellen, Public Relation, sowie Bereitstellung auf den Social Media Portalen. Alle Beiträge werden in enger Kooperation mit dem jeweiligen fachvertreter erstellt. Um die Reliabilität zu überprüfen finden jeweils zu Semesterende, Paper/Pencil-Umfragen unter den Studierenden statt. Zudem werden die Zugriffszahlen seit Beginn der Bereitstellung auf den unterschiedlichen Portalen erhoben. Ergebnisse.  Bei der letzten Paper/Pencil-Umfrage im Februar 2015 haben 159 Studierende teilgenommen. Die große Mehrheit aller Studierenden (93 %), die Medcast nutzen, attestiertem dem Angebot einen Mehrwert und 91 % würden ihren Kommilitonen zu einer Nutzung raten. Das Angebot erfreut sich einer stetigen Beliebtheit sowohl auf dem FAU eigenen Videoportal (2379 Zugriffe), als auch auf Youtube (15.884 Zugriffe). Ein Audio-Videopodcast aus der Pathologie wird vorgestellt. Schlussfolgerung.  Durch den Einsatz von Medcast können sich Studierende besser auf Klausuren und Prüfungen vorbereiten, Medcast bieten eine zusätzliche Lernunterstützung, da sie von Kommilitonen aus höheren Semestern bereitgestellt werden, die sich intensiv mit der Thematik auseinander gesetzt haben.

AG08.02 An unsupervised computational approach to detect PTEN loss in prostate cancer using an in situ hybridization (ISH) assay F. Yousefi*1, Z. Dai1, Q. Zhong2, P. J. Wild2, C. H. Ek3, N. Lawrence1 University of Sheffield, Department of Computer Science, Sheffield, Vereinigtes Königreich, 2Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz, 3KTH, Stockholm, Schweden

1

Background.  In prostate cancer (PC), loss of the phosphatase and tensin homolog (PTEN) gene has been shown to be associated with tumor progression and is used as a prognostic factor for overall survival in PC. Detecting PTEN loss is usually performed by visual inspection of trained pathologists, which is time-consuming and prone to inter-observer variability. In this work, we aimed to automatically detect PTEN loss in a dual-color chromogenic and silver in situ hybridization (DISH) assay by an unsupervised computational approach. Methods.  Comprehensive prostate cancer tissue microarray (TMA) images that were stained with a PTEN DISH assay showing black and red signals, indicating the PTEN genes and the corresponding centromeric probe (CEP10) in individual cells. The loss of PTEN can be quantified by the ratio of PTEN to CEP10 in designated regions. We developed an unsupervised approach for detecting the loss of PTEN, using a set of computational methods, such as Fourier transformation, non-parametric Gaussian Process Latent Variable Model (GP-LVM), and Bayesian optimization. Results.  We used 71 TMA images (2000 × 2000 pixels) labeled as PTEN deletion or non-deletion by a trained pathologist, using fluorescence in situ hybridization (FISH) and copy number arrays as ground truth. We first cut individual TMA images into small overlapping patches, containing roughly a single signal. The total number (4,468,936) of image patches was then grouped by K-means into 1000 clusters. We then applied two-dimensional Fourier transformation to each color channel of cluster centers to obtain shift-invariant image patches. Further, we learned a low-dimensional latent space of the cluster centers with GP-LVM, in which PTEN and CEP10 signals formed separate clusters. We next identified PTEN and CEP10 by two separate bounding boxes in the latent space. The PTEN to CEP10 ratio could be estimated as the ratio of the number of image patches in the corresponding bounding boxes. We finally used Bayesian optimization to search for the bounding boxes configuration providing the highest accuracy, which allowed us to explore the configuration space efficiently.

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Abstracts Conclusions.  Our initial result showed that our unsupervised latent variable approach was comparable with supervised techniques. The proposed method is flexible. Currently, we are applying the same method to detect genetic gains or losses in other tumor tissue types, e. g. HER2 amplification in breast cancer.

AG08.03 A computational framework for systems pathology of prostate cancer M. Koletou*1, 2, 3, M. Gabrani2, T. Guo3, Q. Zhong1, U. Wagner1, R. Aebersold3, P. Wild1, M. Rodriguez Martinez2 Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz, 2IBM Zurich Research Laboratory, Zürich, Schweiz, 3Institut für Molekulare Systembiologie, ETH Zürich, Zürich, Schweiz 1

Background.  Prostate cancer is the second most frequent cancer type in men but it is not always possible to accurately estimate the prognosis for the survival of the patient. This is mainly resulting from the lack of biomarkers that could be prognostic of a more aggressive phenotype. In this project we aim to search for prostate cancer specific genomic alterations and study how they could improve the stratification of prostate cancer in two classes, significant and insignificant disease. Methods.  We are developing a novel computational framework based on pattern detection via dictionary learning that has been successfully applied in signal processing. The method is able to detect patterns of genomic alterations coming from relatively very small number of samples, representing rare or infrequent variants, but also epistatic interactions. We intent to use the method in the TCGA Prostate Adenocarcinoma datasets and then validate it using an independent Zurich cohort, the Zurich Prostate Cancer Outcome Cohort study. Additionally, it is possible to integrate different types of genomic alterations, such as single nucleotide variations, copy number alterations and mRNA expression, and infer patterns across different omics datasets. Results.  Here we will show some preliminary results of the method and present an overview of further improvements in the computational framework. More specifically, we want to demonstrate how the method can find groups of genomic alterations that are specific to Prostate Cancer. Additionally, we want to present a possible stratification of the TCGA cohort into samples with significant and insignificant disease and compare our results with the available clinical data. Conclusions.  Our computational framework offers a novel perspective into analysing genomic data from relatively very small number of samples and can integrate multiple omics datasets. This is an interdisciplinary PhD project funded from SystemsX.ch and it aims to impact the capacity building in Systems Biology not only in the methodology and algorithmic level but also on the clinical level supporting precise, predictive and personalized medicine (3P-medicine).

AG08.04 10-year Digital and Computational Pathology at the University Hospital Zurich: Past, Present and Future Q. Zhong*1, N. Wey1, M. Bieri1, A. Wethmar1, T. J. Fuchs2, P. J. Schüffler3, J. M. Buhmann3, H. Moch1, R. Barnert4, P. J. Wild1 Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz, Memorial Sloan-Kettering Cancer Center, New York, Vereinigte Staaten von Amerika, 3Lehrstuhl Informatik, ETH Zürich, Zürich, Schweiz, 4think beyond AG, Bellmund, Schweiz 1

2

Background.  Recent advances of virtual microscopy, image-based machine learning algorithms, and computer hardware have enabled a rapid progress in the development and application of digital techniques and computational methods in pathology. In the past decade, the Institute of

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Surgical Pathology at the University Hospital Zurich (USZ) has transformed its research and clinical practice from analog investigation of tissue slides into a computer-aided framework, using advanced data analytics for knowledge discovery. Here, we describe the evolution of digital and computational pathology at our institute, depict current developments of a slide management system, and address future challenges Methods.  We used virtual microscopy technology from Bacus Laboratories and slide scanners from Hamamatsu, Carl Zeiss and Ventana to digitize tissue microarrays and whole glass slides. A novel virtual slide management system has been introduced based on Microsoft DirectShow that interconnects slide handling robots, scanners, software packages, and reporting systems. Results.  The first online histopathology class in 2006, using virtual microscopy technology, marked the debut of digital pathology at the USZ. A year later, we started to scan glass slides as few as 10 assignments per year. In 2015, the number of monthly scanning jobs reached 1000 on average. Several pathologists are routinely digitizing more than 50 glass slides on a monthly basis. In 2008, we coined the term computational pathology with our ETH Zurich collaborators. Since then, we have developed a series of software and algorithms. The accomplishment of these projects has led to a generic, integrative, and open-source software framework TMARKER for image-based data analysis, including the ISHProfiler plugin for evaluating copy number variations and quantifying tumor heterogeneity at multiple levels. In 2016, the Oracle Health Sciences platform will be tested for a cohort of 40 patients with prostate cancer with the goal of developing a comprehensive clinical computer system that integrates clinical, imaging (radiology, pathology), serological and proteogenomic data into a single standardized data model for cost-effective, secure and unbiased outcome prediction. Conclusions.  Based on the intrinsic multi-disciplinary nature of digital and computational pathology at the USZ, we are convinced that our phenomics approach along with virtual microscopy will play a significant role in achieving personalized precision medicine in the future.

AG08.05 Mapping of the lymphocyte distribution in the bone marrow: comparison of neoplastic and non-neoplastic changes C.-A. Weis*1, F. G. Zölner2, A. Marx1 Pathologisches Institut, Universitätsmedizin Mannheim, Mannheim, Deutschland, 2Universitätsklinikum Mannheim, Medizinische Fakultät Mannheim der Universität Heidelberg, Computergestützte Klinische Medizin, Mannheim, Deutschland

1

Background.  Bone marrow is a complex tissue composed of multiple different cell populations (e. g. myeloid cells, lymphocytes). In the course of different non-neoplastic (e. g. reactive bone marrow changes) and (pre-)neoplastic processes (e. g. myelodysplastic syndromes, MDS) lymphocytic infiltrations can occur, which could be considered as “tumor infiltrating lymphocytes”. To analyze whether the spatial distribution of the lymphoid components in reactive and (pre-)neoplastic BM trephines is different, we implemented a complex image analysis framework. Methods. The frameworks comprises: 1) Immunohistochemical (IHC)-staining: Sections of trephines of non-neoplastic (reactive) bone marrow changes and MDS-cases are sequentially stained for a subset lymphocytic markers (e. g. CD3, CD20). 2) Whole slide image (WSI): Slides are completely digitalized using an Aperio ScanScope and uploaded on an OMERO-server. 3) Lymphocyte-segmentation: By tile-wise download (MATLAB-OMERO-toolbox) and a multistep segmentation stained and non-stained cells within one complete WSI are segmented (MATLAB). 4) Slide registration: Based on thumbnail registration, transformation values (translation, rotation) are applied to all object coordinates (MATLAB). 5) Cell- and cluster-description: Continuous density maps are calculated on basis of scalar functions (MATLAB). Depending on the field shape dif-

Fig. 1 I AG08.05 8 Spatial description of lymphocytes: a) shows in red all CD20(+) and in blue the remaining CD20(-) cells. b) and c) show the density map for all (b) and for the CD20(+) cells (c) in one WSI ferent modes of spatial interaction (e. g. direct cellular interaction) could be mapped. Results.  All cells within one trephine could be detected and identified as “positive” (+) or “negative” (-) for the applied IHC-staining (. Fig. 1). Furthermore, due to sequential staining and data registration, the composition of the lymphatic infiltrate (e. g. CD3(+) T-cells, FoxP3(+) regulatory T-cells) could be visualized. Conclusions.  By this high-throughput and big data producing approach, we are able to objectively detect and describe subsets lymphocytes and in the next step we seek to allocate them to certain compartments (e. g. peritrabecular niche).

AG08.06 Image Quality – Requirements for clinical and research applications N. Zerbe*1, 2, 3, D. Heim1, M. Domhardt2, T. Wetzel1, S. Wienert3, 4, A. Alekseychuk5, K. Schlüns1, S. Lohmann1, 3, P. Hufnagl1, 2, 3 Institut für Pathologie, AG Digitale Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland, 2Institut für Pathologie, ZeBanC, Charité – Universitätsmedizin, Berlin, Deutschland, 3Hochschule für Technik und Wirtschaft – HTW Berlin, Fachbereich Angewandte Informatik, Berlin, Deutschland, 4VMscope GmbH, Berlin, Deutschland, 5Vision in X industrial imaging GmbH, Berlin, Deutschland

1

Background.  The digitalization of slides and subsequent utilization of whole slide images has a highly visible penetration within digital pathology workflows. Not only research applications moving towards daily use, but also clinical pathology applies digital images increasingly. The use of virtual microscopy, with all of their advantages and benefits, requires additional efforts to assure sufficient image quality. This consist of multiple aspects to be fulfilled, such as sharpness, tissue completeness, color fastness primary and additional secondary properties like existence of artifacts, compression, availability of digitalization metadata and others. Methods.  Each dedicated requirement towards image quality has to be investigated separately, due to the fact that they differ in their origin. Distortions may be introduced through slide preparation, calibration of scanning device or even scanner software parameter. Therefore, we developed multiple algorithms and software tools to calculate a quality measure for each aspect automatically. Recently we are focusing on image sharpness, color fastness and completeness of tissue. Sharpness is measured by a no-reference focus algorithm per slide. Color fastness is calculated based on CIEDE2000 for a reference IT8.7/1 color target mounted onto a glass slide per scanner. Completeness is automatically analyzed based on registration and comparison of whole slide overview image and preview camera data. Secondary quality parameters were not part of this investigation.

Results.  We established a standard operation procedure to automatically apply slide based tests directly after digitalization. This enables scanning personal to execute quality inspection at a glance and schedule insufficient whole slide images for a rescan. Moreover, we made some of these tools available as a web-based service including a based web-frontend with user management. This enables everyone within the digital pathology community to validate their slides, scanning devices and scanning parameter. Conclusions.  Scanning devices use multiple different technologies such as brightfield, laser or confocal. Moreover, they partly divide into patch and line scanning concepts. But not only hardware is constantly changing due to upgrades or further developments but also software algorithms, e. g. focus and stitching, are modified. This requires a standardized measurement tools and procedures to assure an appropriate quality for relevant aspects, depending on dedicated use-cases.

AG08.07 German biobank node: establishing a national biobanking research infrastructure tightly connected to the european network BBMRI-ERIC M. Hummel*, C. Rufenach Charité – Universitätsmedizin Berlin, German Biobank Node, Institut für Pathologie, Berlin, Deutschland Background.  The German Biobank Node (GBN/bbmri.de) aims to establish an excellent biobanking infrastructure in Germany and to increase cooperation and harmonization between biobanks. These are prerequisites to facilitate access and to foster the use of biological samples and data for academic and industrial research. Biologic samples and data collected in biobanks are valuable resources for innovations in personalized medicine, development of biomarkers for diagnostics and therapeutics as well as for prevention of diseases. Methods.  The main goal of GBN is to set up a focal point for national biobanks and simultaneously act as the national hub for the European biobanking research infrastructure BBMRI-ERIC (Biobanking and Biomolecular Resources Research Infrastructure). GBN will thus comprise the representation of Germany in BBMRI-ERIC by the national coordinator who takes part in the BBMRI negotiations and instigate Germany’s role in BBMRI-ERIC. Being the central point of contact and exchange for the German biobank community GBN aims: 1. to facilitate exchange of experiences between biobanks 2. to develop standards for quality assurance 3. to develop IT concept for the sample and data exchange 4. to inform the public about biobank activities

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Abstracts Results.  The core of GBN is the central office, which is at the Charité in Berlin. An annual national biobanking symposium serves as the central scientific meeting for all German biobanks and enables a continuous exchange of GBN with all national biobanks and federated networks that operate in the biobank community. Most important, working groups for IT, quality management and public engagement are actively developing concepts and strategies to reach the above mentioned goals. These concepts are in very close alliance with the activities pursued in BBMRI-ERIC. Especially for IT GBN has taken a major role in the common service IT of BBMRI-ERIC and will contribute significantly to the progress of the European IT infrastructure. One of the tasks is the development of a sample locator which is already in a pilot phase will be developed further in the European network. Conclusions.  With a new funding period starting in summer 2016 the Federal Ministry of Research will fund GBN together with a German Biobank Alliance in order to establish important infrastructures based on the current concepts and to connect the German biobanks seamlessly into all BBMRI-ERIC activities. References Hummel M, Rufenach C (2015) Biomaterialbanken als Grundlage für die Entwicklung genetisch basierter Präventionskonzepte, Bundesgesundheitsblatt – Gesundheitsforschung – Gesundheitsschutz, Vol. 58, 127–130

AG08.08 A web-based application for antibody management F. Roßner*, I. Anagnostopoulos, M. Dietel Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland Background.  Diagnostic immunohistochemistry of tumours is becoming increasingly complicated due to the constantly growing number of available antibodies. Promising approaches to build comprehensive compendiums to assist the pathologist in his daily routine exist in vade mecum (www.e-immunohistochemistry.info/) and Immunoquery (www.immunoquery.com). Here we present an unique, electronic research and management system for pathology departments involved in routine diagnostics and research. Methods.  We conducted a comprehensive analysis of the antibody stocks in our institute and simultaneously built a database via Text Mining in

Fig. 1 I AG08.08 8 Consolidated antibody panel

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standard textbooks of pathology (. Fig. 1). Furthermore, we developed a user-friendly front end. Results.  The database currently contains about 11,000 records ofthe WHO classification of malignant tumours, antigen expression profiles and currently offers detailed information about more than 400 antibodies. This results in at least 2300 antigen expression combinations, which are possible as of today. Conclusions.  The short-term goal is a Charité wide access to the database with comprehensive, up-to-date information about antibody stocks of all routine and research laboratories of our Institute. Further goals are a more broad availability and multilingual support. In comparison to “Vade Mecum” our approach offers a direct correlation with the WHO classification and is more flexible to use, and in contrast to “Immunoquery” it is free to use.

AG08.09 TissueMAPS: Web-based, cluster-supported visualization and analysis of large scale microscopy image datasets M. D. Herrmann*, R. Hafen, L. Pelkmans Institute of Molecular Life Sciences, Zürich, Schweiz Background.  Microscopy image datasets acquired within high-throughput screens or whole slide scans often exceed the storage and processing capacity of local computers and necessitate remote solutions, where images are stored and processed within a cluster or cloud environment. Yet, most image-processing software is designed for desktop use with poor support of batch processing. Methods.  TissueMAPS, in contrast, has a client-server architecture and is primarily designed for cluster computing. Its server runs on a virtual machine within a cloud, which provides access to large storage and compute resources. The user interacts with the program via the Internet and a standard web-browser. Results.  TissueMAPS provides automated workflows to process images acquired in brightfield as well as fluorescence mode and is compatible with most microscope file formats. The web interface supports browsing entire image datasets in real-time, featuring interactive zoom, dynamic intensity rescaling, channel toggling, and additive blending to virtually combine channels to color images. In ad-

dition, segmented objects can be visualized on the map and dynamically colorized according to extracted feature values. For example, each cell can be color-coded for measured biomarker intensities. Using state of the art web technologies ensures efficient caching and fast rendering on the client side and results in a smooth zooming experience across different resolution levels. The major strength of TissueMAPS, however, is based on its interactive, explanatory data analysis and machine learning tools. The user can for example select individual objects in the map to query information about them or label phenotypically distinct objects to perform supervised classification. The generated models can then be used to determine characteristic features of the sample or make predictions for other samples. Thereby, users can apply computer vision to supplement classical histological analysis with quantitative readouts and quickly test statistical hypothesis in a visually assisted way. Conclusions.  TissueMAPS is available online at www.tissuemaps.org. The software is extensively documented and well tested. It is easily extendible, integrates with open-source software such as OpenSlide, and can be setup on various cloud infrastructures. In conclusion, we envision TissueMAPS to be an asset for image-based systems biology and digital pathology.

AG08.10 Computationally extracting unique protein signatures from IHC assays per grade and protein for personalized treatment E. Zerhouni1, B. Prisacari2, Q. Zhong3, P. Wild3, M. Gabrani*2 1 Zurich Laboratory, IBM Research-Zurich, Cognitive Computing and Industry Solutions, Rueschlikon, Schweiz, 2Zurich Laboratory, IBM Research-Zurich, Rueschlikon, Schweiz, 3Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz

Background.  Immunohistochemistry (IHC) assays are used to identify patients most likely to respond to targeted therapy. Currently, IHC image analysis focuses on the staining intensity performed mostly in a manual way. Emerging computational techniques use metrics like the H-score, or the Aperio metric. Recent studies, however, show that to tailor a patient’s treatment and to monitor treatment progression, the staining intensity needs to be correlated to the grade of disease; that is, to the morphological and cellular architecture. To address the correlation between staining intensity, morphology, and topology we propose a novel computational framework that combines all three types of information. We quantitatively compare the proposed framework with existing techniques and demonstrate the significance of each type of information in the grading of IHCstained images for different proteins. Methods.  We have collected a cohort of 39 prostate cancer patients with different degrees of tumor grade evaluated by Gleason score, and constructed a tissue microarray (TMA), followed by IHC staining of several prostate-related proteins. We developed a computational workflow where we explored the use of deep learning to extract features that capture both morphology and color, at image patches around identified based on color stained cells. To represent tumor heterogeneity, we captured spatial layout information using a commute time matrices approach. Results.  We used the commute time matrices to define unique signatures per grade and protein. We compared our results to the Aperio metric. Secondly, we used as features vector the normalized 3 bins histogram of the staining intensity. Finally, we computed the commute time matrix-based signature, using color intensity instead of the morphological features. We quantitatively compared the grading accuracy per protein. The results showed that while the importance of the staining intensity varied per protein, both morphology and topology contributed significantly in the accuracy, which increased by 20 to 40 %. Combining protein signatures increased also the accuracy implying the complementarily of the various proteins. Conclusions.  Our computational analysis of IHC assays shows that besides staining intensity both morphology and topology are significant contributors for disease grading but also for identifying the significance of each

protein per grade, contributing to the development of personalized therapy for patients.

AG08.11 A New High Throughput Staining Device H. Merz*, K. van der Wolk Hämatopathologie Lübeck, Lübeck, Deutschland Background.  Immunohistochemistry (IH) is used in almost all pathology laboratories. Routine diagnostics was further improved with automation of IH staining’s. IH load increases continuously. It is convenient to use a panel of up to 10 or even more IH tests for characterizing breast cancer, CUP, lymphoma, or leukemia. Typical staining platforms used today allow for the automated work flow of IH; 30 slides can be run simultaneously, up to 90 slides can be processed during a day- and overnight. Current systems normally allow for the analysis of one antigen per tissue slide with a reagent volume from 100–150 µl. Increasing demands in IH, rising costs for automation and staining reagents were opposed by constraints to economize the health care system. Methods.  The aim of our project was to design a special slide for parallel analysis, a tissue arrayer to assemble the slides and to construct a sophisticated high throughput automated staining device (asd) (. Fig. 1). Results.  The new slides were segmented in a variable number of equally dimensioned reaction zones with hydrophobic borders for a variable number of tissue slices (1, 2, 3, 6, 18) from a given sample (e. g. tumor), ideally, each with the same region of interest. Slides with 3, 6 or 18 reaction zones may mount tissue slices up to 14×19 mm, 6×19 mm and 6×6 mm, respectively. Up to 77 glass slides with varying number of tissue slices will then be placed in the asd. The asd will be available in two different formats. One with a cavro valve assisted 6-channel head for simultaneous liquid handling (up to 6 fluids). The other one will be a very fast and complex system with a 66 channel head with pressure assisted liquid dosing (1 to 18 fluids, eg prim. antib. at once). Conclusions.  77 slides, irrespective of the slide format (1, 2, 3, 6, 18) may be processed in one run. The turnaround time will be 210 min when fully loaded: reliable staining of up to 77 slides/patient samples with 77, 144, up to 1368 specimen for up to 66 different antigens. Reagent cost will be reduced as will be costs for slides, physical and digital storage. The platform may support affiliation of path-labs to face the necessities of QM, the increase in work load and the economic pressure.

AG08.12 Digital analysis and epigenetic regulation of the signature of rejection in colorectal cancer L. Sokol*1, V. Kölzer2, 3, S. Zahnd1, L. Christe1, I. Zlobec1, A. Lugli3 1 Universität Bern, Institut für Pathologie, Bern, Schweiz, 2Kantonspital Baselland, Liestal, Schweiz, 3Institut für Pathologie, Universität Bern, Bern, Schweiz

Background.  The immune system plays a crucial role in the development and progression of colorectal cancer (CRC). Tumor immune rejection has been previously linked to activation of the interferon stimulated genes pathway including STAT1, IRF5 and IRF1. We tested the associations of these proteins with clinicopathological factors, cytotoxic T cell (CTL) infiltration, granzymeB and perforin in the tumor microenvironment, and tumor expression of intercellular adhesion molecule 1 (ICAM-1). Further, we investigated the poorly understood regulation of STAT1 and IRF1 by microRNAs in CRC. Methods.  Resection specimens from 244 CRC patients with full clinicopathological data were included in this study. Next generation tissue mi-

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Fig. 1 I AG08.11 8 Slides for parallel analysis, tissue arrayer and staining device croarray slides containing 6 tumor punches per case were immunostained for the proteins of interest. miRNA expression was visualized on consecutive slides by ISH. Precise correlation analysis of miRNA and target proteins expression was performed using digital image analysis. Results. STAT1, IRF1 and IRF5 expression in the tumor microenvironment negatively correlated with the presence of distant metastasis (p = 0.013, OR = 0.98, 95 %CI = 0.96–0.99; p = 0.014, OR = 0.96, 95 %CI = 0.94–0.99; p = 0.009, OR = 0.96, HR = 0.93–0.99). High expression of miR-34a and miR-93 corresponded to a strong decrease of their target proteins, STAT1 and IRF1 (p = 0.006, OR = 0.39, 95 %CI = 0.20–0.77; p = 0.058, OR: 0.48 95 %CI = 0.23–1.02). A combined score, generated from all three protein markers, was used to represent an immune activated and a “quiescent” phenotype. The activated phenotype was associated with elevated CD8+ CTL infiltration (p = 0.007, HR = 2.07, 95 %CI = 1.22–3.52), an increase in granzymeB (p < 0.001, HR = 3.22, 95 %CI = 1.70–6.08), perforin (p = 0.020, OR = 1.90, 95 %CI = 1.10–3.26) and ICAM-1 expression (p = 0.006, OR = 0.2.17, 95 %CI = 1.26–3.85). Patients with this phenotype had a considerably reduced risk of developing distant metastases (p = 0.001, OR = 0.034, 95 %CI = 0.006–0.183). Conclusions.  Here, we describe a combined score that can help to identify patients with an activated immune phenotype of CRC. The score is based on a quantitative assessment of STAT1, IRF1 and IRF5 expression. Increased tumor infiltrating CTLs, granzymeB, perforin and tumor ICAM-1 expression, as well as a lower risk of metastases characterize these patients. Additionally, targeting miR 34a and miR-93 may be a promising strategy to activate the anti-tumoral immune response and will be validated in future experimental studies.

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AG08.13 Anti-CCL2 treatment to induce vascular normalization and improve image-guided drug delivery to breast tumors J. Ehling*1, 2, D. Möckel2, M. Bartneck3, R. Knuechel1, F. Kiessling2, F. Tacke3, T. Lammers2 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, 2Institut für Experimentelle Molekulare Bildgebung, RWTH Universitätsklinikum, Aachen, Deutschland, 3Klinik für Gastroenterologie und Hepatologie, RWTH Universitätsklinikum, Aachen, Deutschland 1

Background.  CCL2-dependent bone marrow-derived inflammatory macrophages are known to be important mediators of pathological angiogenesis. In this regard, we recently showed that in fibrosis-associated angiogenesis in chronically injured livers, anti-CCL2 treatment can prevent vascular denormalization (Ehling et al, Gut 2014). We now set out to evaluate if anti-CCL2 treatment can also induce vascular normalization and thus assist in improving image-guided drug delivery to tumors. Methods.  In total, n = 30 mice bearing orthotopic 4T1 mammary carcinoma tumors received anti-CCL2 treatment at two different dosing levels (5 and 20 mg/kg; thrice weekly). Contrast-enhanced micro-CT (µCT) imaging was performed to monitor the relative blood volume (rBV). Vascular normalization and its impact on drug delivery were evaluated by injecting fluorescent liposomes or polymers (n = 15 mice per drug carrier). The accumulation of liposomal and polymeric nanomedicines was in vivo imaged using hybrid computed tomography – fluorescence molecular tomography (CT-FMT). In vivo findings were validated by conventional molecular methodologies including FACS, immunohistochemistry and 2-photon microscopy. Results.  The accumulation of ~100 nm sized liposomes in orthotopic 4T1 tumors ranged from 1–3 % of the injected dose (%ID) for 20 mg/ kg anti-CCL2-treatment, from 3–5 %ID for 5 mg/kg, and from 5–7 %ID for vehicle-treated control mice. Significant higher accumulation levels were found for ~10 nm sized polymers: 15–18 %ID for 20 mg/kg anti-CCL2-treatment, 12–15 % for 5 mg/kg, and 7–10 % for control mice. Drug delivery to tumors was compromised by a dose-dependent anti-an-

giogenic effect of anti-CCL2 treatment, reflected by decreased rBV values. FACS analyses revealed a shift in macrophage polarization from the pro-tumorigenic M2 to the anti-tumorigenic M1 phenotype. Histological analyses further revealed that this shift in macrophage polarization was parallel by induced vascular normalization (increased vessel maturity and functionality) upon anti-CCL2 treatment. Conclusions.  We demonstrate that anti-CCL2 treatment induces vascular normalization in breast cancer by changing the phenotype of tumor-associated macrophages. Strikingly, this resulted in improved delivery of smallsized polymeric but not of large-sized liposomal nanomedicines. Our data indicate that the pharmacological modulation of the CCL2-CCR2 chemokine axis might be a promising strategy to improve image-guided drug delivery to breast cancer.

AG08.14 The third dimension of applied Pathology A. Brobeil*, A. Bräuninger, S. Gattenlöhner Pathologie, Justus-Liebig Universität Gießen, Gießen, Deutschland Background.  Up to now, functional in situ diagnostic tools are rare in the daily pathological routines. For mutational and chromosomal analyses there are robust methods, e. g. next generation sequencing (NGS) or fluoresence in situ hybridization (FISH). These methods provide useful prognostic and therapeutical informations. Yet, these investigations do not allow any morphological or functional data for understanding tumorigensis or progression of diseases, e. g. vascular invasion. Moreover, little is known about normal or altered cellular signaling networks in diseased tissue, regardless benigne or malignant. Methods.  In this study, functional in situ assays are combined with 3D confocal microscopy. Displaying protein-protein interaction the DuoLink proximity ligation is used according to the manufacturer’s standard protocol. Furthermore, based on the proximity ligation assay the protocol is adapted in regard to the target detection method for visualization of protein-DNA interactions. Mutational in situ data are generated using padlock probes, e. g. for detecting KRAS mutation. The assays are carried out on formalin fixed parrafin embedded tissue with 500 µm thickness. The sections are visualized by a ZEISS LSM 800 confocal laser scanning microscope. Results.  The obtained data gives new insights in histomorphological distribution of distinct receptors, e. g. the epidermal growth factor receptor in normal lung and in diseased lungs with fibrosis or adenocarcinoma. Moreover,the coupled downstream signaling pathways are displayed in regard to their localization within normal lung structures or the tumor. This holds also true for the investigated gene mutations (KRAS, EGFR). Conclusions.  The in situ assays combined with newest 3D confocal microscopy are a powerful new tool getting new insights in normal and diseased tissue, which can help to develop new strategies in disease and cancer treatment. It opens the third dimension for pathological assessment with new possibilities in the diagnostic process.

AG08.15 The SFB850 translational core project Z1 – 6 year experience L. Lutz* , N. Bittermann , M. Werner 1

1

, S. Laßmann

1, 2, 3

1, 2, 3, 4

Universitätsklinikum, Institut für Klinische Pathologie, Freiburg, Deutschland, 2Universitätsklinikum, Comprehensive Cancer Center Freiburg, Freiburg, Deutschland, 3Deutsches Konsortium für Translationale Krebsforschung (DKTK) und Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Deutschland, 4Albert-Ludwigs-Universität, BIOSS Centre for Biological Signalling Studies, Freiburg, Deutschland 1

Background.  Validation and translation of basic research findings is possible by integration of histological and molecular information derived from

human tissue specimens. Here, an overview of collaborative work of the SFB850 Z1 project is given. Methods.  So far, 30 project requests (6x/2009, 1x/2010, 6x/2011, 5x/2012, 4x/2013, 3x/2014, 5x/2015) were filed, with 7/30 projects published, 9/30 projects awaiting publication by collaborators and 11/30 ongoing. Technical support was provided in 3/30 requests for laser-capture microdissection. The focus is on tissue specimens analyses of colorectal, pancreatic, lung, breast and ovarian cancers (primary, metastatic). Hence, selection of appropriate cohorts for the scientific question is crucial and involves diagnostic report review, tissues retrieval and re-classification. Generally, validation of 2–5 proteins per project are requested, which involves IHC staining, data interpretation, visualization and statistical evaluation. Since most proteins are novel candidates, prior establishment and validation of antibodies is necessary. Results.  In total, the approach was successful for 32/36 antibodies and yielded about n = 2800 stainings. Increasing requests are seen for analysis of mouse xenografts. Xenograft tissues undergo SOP-driven macroscopic grossing, tissue processing and paraffin embedding. So far, histologic evaluation (n = 170 mice; each 1–2 organs/xenografts) and immunohistochemistry (mean 3–4 proteins per xenograft) were performed. One key topic addressed is oncogenic signaling regarding the RASRAF-MAPK signaling pathway. In two collaborative projects promising results were revealed: First, morphologically and immunohistochemically characterized epithelial differentiation was shown in BRAF-mutant colorectal cancer cells treated with B-Raf inhibitors. Second, the so far uncharacterized kinase RioK1 was shown to be differentially expressed in non-small cell lung cancers dependent on the histological subtype. Interestingly RioK1 expression appears to be less frequent in adenocarcinomas, which harbor more often a RAS mutation compared to squamous cell carcinomas. Conclusions.  This project illustrates the volume, workflows and necessities of a translational core project. Importantly, it underlines its key contribution to validate basic research findings in a clinico-pathological setting, which is essential for further translation, transfer and potential application. *funded by DFG SFB850 Z1 Project.

AG Herz-, Gefäß-, Nieren- und Transplantationspathologie AG09.03 Constitutive activation of PDGFR-β in renal mesenchymal cells drives renal fibrosis E. M. Buhl*1, 2, S. Djudjaj1, B. M. Klinkhammer1, E. Borkham-Kamphorst3, R. Weiskirchen3, L. E. Olson4, J. Floege2, P. Boor1, 2 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, Klinik für Nieren und Hochdruckkrankheiten, Rheumatologische und Immunologische Erkrankungen, Uniklinik RWTH, Aachen, Deutschland, 3 Institut für Molekulare Pathobiochemie, Experimentelle Gentherapie und Klinische Chemie, Uniklinik RWTH, Aachen, Deutschland, 4Programm für kardiovaskuläre Forschung, Oklahoma Stiftung für medizinische Forschung, Oklahoma City, Vereinigte Staaten von Amerika 1

2

Background.  Platelet-derived Growth Factor Receptor β (PDGFR-β) is involved in organ fibrosis, yet only few studies suggested its involvement in renal fibrosis. PDGFR-β is upregulated in fibrosis and specifically expressed on the mesenchymal cells centrally involved in renal fibrosis, i. e. glomerular mesangial cells, interstitial fibroblasts and pericytes. Methods.  Here we analyzed the role of activation of PDGFR-β specifically in renal mesenchymal cells, using a transgenic FoxD1-driven activation of a constitutively active PDGFR-β mutant. Results.  Compared to controls, mutant mice developed glomerular mesangial hypercellularity and sclerosis as well as expansion of interstitial (myo)fibroblasts and interstitial fibrosis. This was progressive in time and neither associated with a prominent inflammatory response nor hyperDer Pathologe Suppl 1 · 2016

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Abstracts tension. The mutant mice also developed renal anemia in the absence of prominent uremia as well as secondary tubular injury in later time-points. Compared to controls, mutant mice developed a more severe fibrosis after ischemia-reperfusion injury. Conclusions.  In conclusion, activation of PDGFR-β specifically in renal mesenchymal cells is sufficient to induce and drive progressive renal fibrosis independently of inflammation. Our model might be a valuable tool to study renal fibrosis. Importantly, our data suggest that fibrosis is not simply a consequence or renal injury but per se leads to progression of renal disease evidenced by tubular damage and reduced renal function.

AG09.04 Amloid load in cardiac biopsies is a prognostic and predictive biomarker A. Kristen1, E. Brokbals1, F. aus dem Siepen1, R. Bauer1, S. Hein1, M. Aurich1, J. Riffel1, H.-M. Behrens2, S. Krüger2, P. Schirmacher3, H. Katus1, C. Röcken*2 Klinik für Kardiologie, Angiologie und Pneumologie, Universitätsklinikum, Heidelberg, Deutschland, 2Institut für Pathologie, Christian-AlbrechtsUniversität, Kiel, Deutschland, 3Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland 1

Background.  Heart failure is unfavourable in AL and ATTR amyloidosis. It increases the risk for cardiac decompensation and/or sudden cardiac death. Only limited studies are available comparing cardiac histology with clinical data. In this study we filled this gap of information and carried out a retrospective study testing the following hypotheses: 1) AL and ATTR amyloid show unique distribution patterns in the heart; 2) AL and ATTR amyloid show distinct clinical presentations; 3) Amyloid load in tissue specimens correlates with clinico-pathological patient characteristics and severity of (cardiac) disease; 4) Amyloid load is a prognostic and/ or predictive biomarker. Methods.  216 patients with histologically confirmed cardiac amyloidosis were included. ECG, echocardiography, and laboratory testings were assessed. The percentage of the amyloid area of endomyocardial biopsies was evaluated by counting immunohistochemically stained and non-stained pixels. Univariate and multivariate analyses were carried out. Results.  We found amyloid-type specific differences, putative age-dependent differences and differences probably related to both. Median amyloid load was 30.5 % without any amyloid load above 70 %. During follow-up (median 19.1 months; range 2.5–145.7 months) 112 patients died. Chemotherapy had a significant effect on overall survival in AL amyloidosis. Patients with <20 % AL amyloid load responding to chemotherapy showed significantly better survival than non-responders. No difference was found if amyloid load exceeded 20 %. Univariate analysis revealed gender, Karnofsky index, NYHA class, diastolic blood pressure, eGFR, NT-proBNP, MR antagonists, low voltage, left atrial diameter, ineligibility for chemotherapy, response to chemotherapy, and amyloid load as predictors of survival in AL amyloidosis. Multivariate analysis revealed eGFR, NT-proBNP, and amyloid load as independent predictors of mortality. In ATTR amyloidosis survival correlated with Karnofsky index, NYHA, TnT, diastolic blood pressure, use of diuretics, QRS, LV ejection fraction, and impaired RV function. Following Cox regression analysis, NYHA remained the only independent predictor of patient survival in ATTR amyloidosis. Conclusions.  In AL amyloidosis, endomyocardial biopsies appear to be of major importance for prediction of survival at diagnosis as only patients with amyloid load below 20 % benefit from future chemotherapy.

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AG09.05 A recurrent activating PLCG1 mutation in cardiac angiosarcomas increases apoptosis resistance and invasiveness of endothelial cells* K. Kunze*1, T. Spieker2, U. Gamerdinger1, K. Nau1, J. Berger1, T. Dreyer1, J. R. Sindermann3, A. Hoffmeier3, S. Gattenlöhner1, A. Bräuninger1 Pathologie, Justus-Liebig-Universität, Gießen, Deutschland, 2Pathologie, St. Franziskus-Hospital, Münster, Deutschland, 3Thorax-, Herz- und Gefäßchirurgie, Westfälische Wilhelms-Universität, Münster, Deutschland

1

Background.  Primary cardiac angiosarcomas are very rare tumors originating from endothelial cells. They are characterized by a high proliferation rate and a marked propensity to metastasize. The prognosis is extremely adverse due to the inoperable localization and a distinct insensitivity towards chemo- and radiotherapy. Knowledge regarding the pathogenesis of cardiac sarcomas is so far very limited. To gain insight into the pathogenesis of primary cardiac angiosarcomas, a collection of cases was investigated by targeted next generation sequencing (tNGS). Methods.  The IonTorrent AmpliSeq Comprehensive Cancer Panel was used for the detection of somatic mutations in 409 frequently mutated oncogenes and tumor suppressor genes. To analyze the consequences of the recurrent PLCG1 mutation, HUVECs were transiently transfected for apoptosis detection as well as migration and invasion assay. Whole cell lysates were prepared for western blot analyses to investigate the PLCγ1 specific signaling pathways. Results.  Repeatedly mutated genes identified by tNGS were KDR with different nonsynonymous mutations, MLL2 with different nonsense mutations and PLCG1 with a recurrent nonsynonymous mutation (R707Q) in the highly conserved autoinhibitory SH2 domain in three of 10 cases. PLCγ1 is usually activated by Y783 phosphorylation and activates PKC and Ca2+-dependent second messengers with effects on cellular proliferation, migration and invasiveness. Ectopic expression of the PLCγ1-R707Q mutant in endothelial cells revealed reduced PLCγ1-Y783 phosphorylation with concomitant increased c-RAF/MEK/ERK1/2 phosphorylation, increased IP3 amounts and increased Ca2+-dependent calcineurin activation compared to ectopically expressed PLCγ1-wildtype. Furthermore, cofilin, whose activation is associated with actin skeleton reorganization, showed decreased phosphorylation and thus activation after expression of PLCγ1-R707Q compared to PLCγ1-wildtype. At the cellular level expression of PLCγ1-R707Q in endothelial cells had no influence on proliferation rate but increased apoptosis resistance and migration and invasiveness in in-vitro assays. Conclusions.  Together, these findings indicate that the PLCγ1-R707Q mutation causes constitutive activation of PLCγ1 and may therefore represent an alternative way of activation of KDR/PLCγ1 signaling beside KDR activation in angiosarcomas with implications for VEGF/KDR targeted therapies. *published in Cancer Research, 2014, 74:6173–6183

AG09.06 Recombinant spider silk proteins for cardiac tissue engineering J. Petzold1, T. Aigner2, T. Scheibel2, F. Engel*1 Universitätsklinikum Erlangen, Nephropathologie, Erlangen, Deutschland, Universität Bayreuth, Lehrstuhl Biomaterialien, Bayreuth, Deutschland

1 2

Background.  The human heart cannot regenerate after an injury. Lost cardiomyocytes are replaced by scar tissue resulting in reduced cardiac function causing high morbidity and mortality. One possible solution to this problem is cardiac tissue engineering. Amongst natural biomaterials available, silk proteins possess unique mechanical strength, biocompatibility and relative ease in fabrication into diverse morphologies. In addition, recombinant spider silk proteins can be biotechnologically produced in a reproducible and pure form allowing their application in patients. Thus, we have investigated the suitability of spider silk proteins for cardiac tissue engineering.

Methods.  Neonatal rat cardiomyocytes as well as non-myocytes (fibroblast, endothelial cells, smooth muscle cells) were isolated and seeded on glass coverslips coated with fibronectin or recombinant spider silk proteins. Subsequently, cell attachment (cell count based after cell-type-specific immunofluorescence staining), cell survival (LIVE/DEAD assay based on Calcein and EthD-1), cell-to-cell contact (Connexin 43 staining), contractility (live imaging), and cell behaviour to external stimuli (proliferation, hypertrophy)were tested. Results.  Our analysis revealed that cardiomyocytes as well as non-myocytes attach to spider silk films as well as to their natural matrix fibronectin. The cells show a normal appearance on spider silk protein films and cardiomyocytes form regular cell-to-cell connections and beat regularly. In addition, cardiomyocytes responded upon stimulation with FGF1/p38 inhibitor with cell cycle re-entry and upon phenylephrine, isoprotenerol, as well as fetal bovine serum with hypertrophy. Conclusions.  In conclusion, our findings demonstrate that cardiomyocytes not only attach to recombinant spider silk proteins but exhibit normal behavior. Moreover, also non-myocytes attach and thus recombinant spider silk proteins are suitable to engineer complex multicellular cardiac patches.

AG09.07 Effects of GLP-1 on metabolic kidney and heart disease B. M. Klinkhammer*1, J. Möllmann2, R. Stöhr2, C. Lebherz2, N. Marx2, M. Lehrke2, P. Boor1, 3 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, Klinik für Kardiologie, Pneumologie, Angiologie und Internistische Intensivmedizin, RWTH Universitätsklinikum, Aachen, Deutschland, 3 Klinik für Nieren und Hochdruckkrankheiten, Rheumatologische und Immunologische Erkrankungen, Uniklinik RWTH, Aachen, Deutschland

1

2

Background.  Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates glucose homeostasis by increasing insulin secretion and sensitivity. Enzymatic cleavage of the active GLP-1(7–37) generates cleavage products GLP-1(9–37) or the nonapeptide GLP-1(28–37). Both cleavage products do not activate the GLP-1 receptor, but were reported to exert receptor independent cardioprotective and immunomodulatory functions. Methods.  Here we analyzed the effect of GLP-1(7–37), its superactive mutant GLP-1(7–37Mut8) and its two cleavage products, GLP-1(9–37) and GLP-1(28–37), on renal and cardiac function and morphology in diabetic db/db mice. Using an adeno-associated viral vector system, GLP-1(7–37), GLP-1(9–37), GLP-1(28–37), GLP-1(7–37Mut8) and LacZ as a control were overexpressed in diabetic db/db mice on high fat diet (n = 10–13/ group). Blood pressure was measured using tail cuff method and heart function using millar catheter. Twenty-four hour urine was collected and kidneys were analyzed using flow cytometry, histology, immunohistochemistry and qRT-PCR. Results.  Compared to LacZ control, overexpression of the active GLP1(7–37) and the superactive GLP-1(7–37Mut8), but not of the two cleavage products led to significantly improved glucose metabolism. However, diabetes-induced proteinuria was only reduced by the superactive GLP1(7–37Mut8), but not by the active GLP-1(7–37) or its cleavage products. Diabetes-induced mortality was reduced by overexpression of all GLP-1 constructs, however neither bloodpressure nor heart function differed between the groups. Compared to LacZ controls, kidneys of all GLP-1 groups exhibited significantly reduced tubulointerstitial damage and mRNA expression of the tubular injury markers KIM-1 and NGAL. Expression of all GLP-1 constructs also significantly reduced accumulation of renal macrophages and number of infiltrating T cells compared to LacZ control. Conclusions.  In conclusion, both GLP-1(7–37) and its superactive mutant GLP-1(7–37Mut8) improved glucose metabolism in db/db mice, but only the superactive GLP-1(7–37Mut8) reduced proteinuria. Interestingly, tubular injury and inflammation were reduced in a glucose-independent manner by all GLP-1 peptides. Our data show for the first time that not only active GLP-1(7–37), but also its cleavage products are reno-protective in metabolic kidney injury.

AG09.08 Neolymphangiogenesis in experimental renal fibrosis V. Kos*1, B. M. Klinkhammer1, P. Boor1, 2 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, Medizinische Klinik II, RWTH Universität, Aachen, Deutschland

1 2

Background.  The lymphatic vasculature plays an important role in immune surveillance, lipid absorption and maintenance of tissue fluid balance. The physiological and pathological role of renal lymphatic system is not fully elucidated, although some studies showed a prominent neolymphangiogenesis during renal diseases, in particular during fibrosis and after kidney transplantation. The aim of this study was to establish specific lymphatic markers to identify and quantify lymphatic vessels in healthy and diseased murine kidneys. Methods.  First, we tested four specific lymphatic markers, i. e. Podoplanin, Prox-1 (prospero-related homeobox transcription factor 1), VEGFR-3 (vascular endothelial growth factor receptor 3) and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1), with LYVE-1 performing best in immunohistochemistry or immunofluorescence. Next, we quantified the number of lymphatic vessels in three murine models of fibrosis with distinct mechanisms of initial kidney damage, i. e. unilateral ureteral obstruction (UUO, 12 h, day 1, 2, 3, 5, 7, 9, 14, 21), unilateral ischemia/reperfusion injury (I/​R; 12 h, day 1, 3, 5, 7, 10, 14, 21, 28, 56) and Adenine-induced nephropathy (Adenine, day 1, 3, 5, 7, 14, 21, 28). Results.  In healthy murine kidneys lymphatic vessels are exclusively located in the cortex in close proximity to bigger blood vessels. In all three fibrosis models we found newly formed lymphatic vessels apart from blood vessels in the renal cortex. In the medulla they still remained absent. The total number as well as the area filled fraction of lymphatic vessels increased with disease progression. In all three models we found the maximum increase at the most advanced stage of disease (increase compared to sham: UUO d21: +54 %, I/R d56: +187 %, Adenine d28: +124 %). Accordingly, neolymphangiogenesis correlated with fibrosis progression estimated by quantification of αSMA and Collagen 3. Conclusions.  In conclusion, we have established a method for quantitative analyses of renal lymphatics showing a prominent neolymphangiogenesis in three murine models of renal fibrosis. These data and methods might serve as the basis for future analyses of the impact of angiogenic molecules on renal lymphatics.

AG Kinder- und Fetalpathologie AG10.01 SOX2 is a pro-proliferative EWSR1-FLI1 target gene and its overexpression defines a subset of Ewing sarcoma patients with poor outcome G. Sannino*1, A. Marchetto1, M. Baldauf1, M. Dallmayer1, R. Alba Rubio1, J. Musa1, M. F. Orth1, M. M. Kiran1, C. Vokuhl2, I. Leuschner2, U. Dirksen3, J. Alsonso4, T. Kirchner5, T. G. P. Grünewald1 1 Labor für Pädiatrische Sarkombiologie, Pathologisches Institut der LMU München, München, Deutschland, 2Institut für Pathologie des UKSH Kiel, Sektion Kinderpathologie, Kinder-Tumorregister, Kiel, Deutschland, 3Abteilung für pädiatrische Hämatologie und Onkologie, Universitätsklinikum Münster, Münster, Deutschland, 4Instituto de Investigación de Enfermedades Raras, Instituto de Salud Carlos III, Madrid, Spanien, 5Pathologisches Institut der LMU München, München, Deutschland

Background.  Ewing sarcoma (EwS) is the second most common pediatric bone cancer, which likely originates from mesenchymal stem cells. 85 % of EwS cases are characterized by a specific chromosomal translocation fusing the EWSR1 and the FLI1 gene. EWSR1-FLI1 encodes an aberrant Der Pathologe Suppl 1 · 2016

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Abstracts transcription factor, which induces many of its target genes through binding to enhancer-like GGAA-microsatellites, whose activity increases with the number of consecutive GGAA-repeats. Previous reports showed that ectopic EWSR1-FLI1 expression in mesenchymal stem cells induces the embryonic stem cell marker SOX2. Methods.  To characterize the role of SOX2 in EwS we employed a broad panel of techniques such as DNA microarrays, ChIP-Seq, qRT-PCR, Western blot, immunohistochemistry, RNA-interference and cell-based functional assays. Results.  Analysis of DNA microarrays revealed that SOX2 is highly expressed in about 20–30 % of EwS samples, which was confirmed on the protein level in a tissue microarray. This subset of EwS patients had a significantly worse outcome relative to those with low SOX2 levels. Gene-set enrichment analysis of SOX2 co-expressed genes in 117 primary EwS tumors indicated that high SOX2 expression is related to that of genes involved in stemness, proliferation and drug resistance. Interestingly, RNA interference-mediated knockdown of EWSR1-FLI1 in SOX2-high EwS cells was accompanied by a downregulation of SOX2. In addition, we observed strong EWSR1-FLI1 binding to an enhancer-like GGAA-microsatellite close to the SOX2 in available ChIP-Seq and epigenetic datasets generated in different EwS cell lines. In line with the fact that GGAA-microsatellites naturally underlie strong germline variation, we hypothesize that inter-individual variation in the number of GGAA-repeats at the SOX2-associated GGAA-microsatellite correlates with the variable SOX2 expression levels in primary tumors. Notably, knockdown of SOX2 specifically induced a marked decrease of proliferation in two SOX2-high EwS cell lines, whereas no effect of SOX2 knockdown was noted in a cell line with low SOX2 expression levels. In addition, knockdown of SOX2 reduced colony formation capacity of SOX2-high EwS cell lines. Conclusions.  Our results suggest that SOX2 is a pro-proliferative EWSR1-FLI1 target gene, whose overexpression defines a subset of EwS patients with poor outcome. Future experiments will determine how EWSR1-FLI1 steers SOX2 expression and the ways through which this protein exerts its pro-proliferative effects.

AG10.02 SOX6 is an EWSR1-FLI1 target gene contributing to proliferation and clonogenic growth of Ewing sarcoma A. Marchetto*1, G. Sannino1, M. Dallmayer1, R. Alba Rubio1, M. Baldauf1, J. Musa1, M. M. Kiran1, M. F. Orth1, J. Alonso2, C. Vokuhl3, I. Leuschner3, U. Dirksen4, T. Kirchner5, T. G. P. Grünewald1 Labor für Pädiatrische Sarkombiologie, Pathologisches Institut der LMU München, München, Deutschland, 2Instituto de Investigación de Enfermedades Raras, Instituto de Salud Carlos III, Madrid, Spanien, 3 Institut für Pathologie des UKSH Kiel, Sektion Kinderpathologie, KinderTumorregister, Kiel, Deutschland, 4Abteilung für pädiatrische Hämatologie und Onkologie, Universitätsklinikum Münster, Münster, Deutschland, 5 Pathologisches Institut der LMU, München, Deutschland 1

Background.  Ewing sarcoma is a highly aggressive pediatric bone cancer of likely mesenchymal origin. It is characterized by chromosomal translocations fusing the EWSR1 gene to various members of the ETS family of transcription factors – most commonly FLI1. EWSR1-FLI1 diverts GGAA-microsatellites as enhancers to regulate many of its target genes. SOX6 is an embryonic transcription factor involved in differentiation of osteochondro-progenitors. Methods.  Using DNA microarrays and ChIP-Seq data, qRT-PCR, Western blot, immunohistochemistry and RNA interference in conjunction with in vitro experiments we functionally characterized the role of SOX6 in Ewing sarcoma. Results.  Analysis of SOX6 expression in >2,700 DNA microarrays comprising 71 normal tissue types and 50 cancer entities (such as bone-, soft-tissue, embryonic- and brain cancers as well as acute leukemias) revealed that SOX6 is highly overexpressed in Ewing sarcoma relative to

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normal tissues and other cancers. These data were validated in a panel of cancer cell lines on the mRNA level and in a tissue microarray of Ewing sarcoma tumors on the protein level. We noted that SOX6 is generally highly expressed in Ewing sarcoma, albeit with substantial inter-tumor heterogeneity. Using RNA interference, we observed that knockdown of EWSR1-FLI1 in Ewing sarcoma cells is accompanied by a decrease of SOX6 expression. Conversely, ectopic EWSR1-FLI1 expression in mesenchymal stem cells increases SOX6 expression. Analysis of publicly available ChIP-Seq and epigenetic data exhibited two prominent EWSR1-FLI1 peaks within intron 1 of SOX6 that overlaid with DNase 1 hypersensitivity sites and histone marks pointing to active enhancers. Both sites map to GGAA-microsatellites, strongly suggesting that SOX6 is an EWSR1-FLI1 induced target gene. Consistent with the known role of SOX6 as a splicing and transcription factor during chondrogenic and osteogenic differentiation, gene-set enrichment analysis of SOX6 co-expressed genes in primary Ewing sarcoma indicated that SOX6 expression is correlated with that of genes involved in RNA metabolism, cell cycle and metastasis. In accordance, siRNA-mediated SOX6 knockdown in different Ewing sarcoma cell lines significantly reduced proliferation and clonogenic growth through inhibition of cell cycle progression and induction of cell death. Conclusions.  Our data support that SOX6 is an EWSR1-FLI1 target gene, which contributes to proliferation and clonogenic growth of Ewing sarcoma cells.

AG10.03 β-Catenin inhibition and overexpression in RMS tumor cells A. Seils*, F. Viehweger, A. Marx, K. Simon-Keller Med. Fakultät Mannheim der Universität Heidelberg, Institut für Pathologie, Mannheim, Deutschland Background.  Rhabdomyosarcoma (RMS) is a soft tissue malignancy mainly affecting children and young adults. Histologically, embryonal and alveolar subtypes are distinguished. Common for both subtypes is an expression of muscle differentiation markers like Desmin and Myogenin. Despite increasing cure rates for localized tumors, the prognosis for patients with advanced disease still remains poor. Therefore new therapeutic targets are urgently needed. For myogenesis and somite patterning, the Wnt-pathway with the multifunctional transcription factor β-Catenin is of high importance. Despite its function in most tumors, β-Catenin induces muscle differentiation during the development. To investigate the effect of β-Catenin on differentiation, proliferation and migration in RMS tumors two different inhibitors (FH53 and XAV939) as well as a β-Catenin specific siRNA were used to inhibit the canonical β-Catenin pathway. Wnt-3a medium was used for its stimulation. To investigate the effect of a β-Catenin specific overexpression a canonical active β-Catenin in a TetR system was used. Methods.  Three different RMS Cell lines (CRL2061, FLOH1 and RD) were treated with a) the inhibitors, b) the siRNA and c) the constitutively active β-Catenin with or without addition of Wnt-3a medium to stimulate the β-Catenin pathway. MTT-and scratch assay were used to determine cell proliferation and migration. To investigate the changes in the expression profile of differentiation muscle differentiation markers, like AChR, CKM2, Desmin, MuSK, were tested via qRT-PCR, western blot and FACS analysis. Results.  Inhibition of the canonical β-Catenin pathway by FH535 and XAV939 has no effect on proliferation and differentiation in comparison to untreated cells. In contrast stimulation with Wnt-3a medium, as well as overexpressing of constitutively active β-Catenin, leads to a reduced proliferation rate and a higher expression of muscle-specific differentiation markers. Conclusions.  This implies that the differentiation status of RMS tumor cells is influenced by the canconical β-Catenin pathway.

AG10.04 Genomic status of HOXC6 in nephroblastomas B. Gürtl-Lackner*1, V. Stiegelbauer2, I. Leuschner3, G. Höfler4, D. Gisselsson-Nord5 Institut für Pathologie, Skåne University and Regional Laboratories, Universitätshospital Lund, Lund, Schweden, 2Universitätsklinik für Innere Medizin, Medizinische Universität Graz, Klinische Abteilung für Onkologie, Graz, Österreich, 3Kindertumorregister Kiel, Sektion für Kinderpathologie, Universität Kiel, Kiel, Deutschland, 4Institut für Pathologie, Medizinische Universität Graz, Graz, Österreich, 5Department Klinische Genetik, Universität Lund, Lund, Schweden 1

Background.  The transcription factor HOXC6 plays a role in the development of the mammary gland, a pathological overexpression has been found in medulloblastomas, osteosarcomas, and carcinomas of the lung, breast and prostate. Previously we were able to demonstrate an overexpression of HOXC6 in approximately one third of the sporadic nephroblastomas investigated. Methods.  In formalin-fixed, paraffin-embedded samples of nephroblastomas the number of gene copies of HOXC6 was analysed by fluorescence in-situ hybridisation (FISH). Results.  In our study the we investigated different subtypes of nephroblastomas and separately examined the different morphological areas present. The fluorescence signals of HOXC6 and the centromere of chromosome 12 were recorded and analysed. A few single cases showed trisomy of chromosome 12, but no high amplification was identified so far. Conclusions.  Upregulation of HOXC6 is apparently not caused by an excessive amplification of the genomic copies of this gene.

AG10.05 The TGF-Beta Signalling Pathway in Nephroblastomas M. Pincus*1, P. Vesely1, V. Stiegelbauer1, I. Leuschner2, H. Gerald1, B. Gürtl1 Institut für Pathologie, Medizinische Universität Graz, Graz, Österreich, Institut für Pathologie, Christian-Albrechts-Universität, Kiel, Deutschland

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Background.  Activation of the TGF-β pathway can either promote tumorigenesis or exhibit tumorsuppressive characteristics. Micro RNAs (miRNAs) are small non-coding RNAs, regulating gene expression by post-transcriptional mechanisms. Activation of the TGF-β signalling pathway by deregulation of miRNAs has already been described in nephroblastomas. In previous experiments we were able to demonstrate a downregulation of miR-27a in HEK293 Cells and a downregulation of miR-145 and miR-193-5p in HT29 Cells. Furthermore miR-27a showed a lower Expression in some nephroblastomas. Methods.  Target genes and miRNAs of the TGF-β pathway were analysed by the DIANA–mirPath programme, which is a tool to identify molecular pathways targeted by single or multiple miRNAs. In order to investigate the predicted influence of microRNA on SMAD-Proteins we transfected the human cancer cell-lines HEK293 and HT29 with microRNA and measured the change in SMAD-Protein expression by Western Blotting. Results.  The in-silico analysis demonstrated miR-27a, miR-145 and miR193-5p to target several genes within the TGF-β signalling pathway. Conclusions.  Our results suggest that a deregulation of the TGF-β pathway might be modulated by several miRNAs.

AG10.06 Investigation of the Tumormicroenvironment of Rhabdomyosarcoma K. Simon-Keller*1, A. Seils1, C. Vokuhl2, I. Leuschner2, A. Marx1 Institut für Pathologie, Med. Fakultät Mannheim der Universität Heidelberg, Mannheim, Deutschland, 2Institut für Pathologie, Universitätsklinikum Schleswig-Holstein, Campus Kiel, Kiel, Deutschland 1

Background.  Rhabdomyosarcoma (RMS) is a soft tissue malignancy mostly affecting children and young adults. Histologically two subtypes can be differentiated: alveolar (aRMS) and embryonal RMS (eRMS). Although eRMS usually shows a favorable prognosis with 5-year survival rates up to 70 %, prognosis of highly metastatic RMS is still poor. Therefore new therapeutic options are required. We choose an adoptive immunotherapy targeting the fetal acetylcholine receptor to recognize and lyse RMS tumor cells. Although immunotherapies seem to be a highly promising option, infiltration of the tumor tissue with effector cells is an important precondition for the success of such a therapy. Methods.  To examine the infiltration of RMS tumor tissue with stimulating and/or suppressive immune cells we tested 50 RMS paraffin embedded tissues for cytotoxic T cells, Treg cells, NK cells, Makrophages (M1 and M2) and Myeloid derived suppressor cells (MDSCs) by using the following markers: CD3, CD4, CD8, CD11b, CD20, CD25, CD33, CD68, CD163, Granzym, iNOS, FoxP3, Stablin-2, PD1 and PD-L1. Furthermore we investigated the integrity of the blood vessels (using ERG, smooth muscle actin and CD34) as a precondition for immune cell infiltration. As healthy controls normal skeletal muscle tissue were used. The stainings were compared to other childhood tumors (like Ewing Sarcoma and Wilms Tumor) as well as other sarcoma types like GIST and Syovialsarcoma in contrast to highly immunogenic tumor types like melanoma and colon carcinoma. Results.  We found almost no infiltration of RMS with cytotoxic immune cells (0–5 %), no infiltration with TRegs or MDSCs and only a small number of tumor associated macorphages (0–30 %). No expression of PD-L1 was detected in RMS tumors. Staining of the capillary system of RMS tumors shows only a small number of tumor supplying blood vessels. A larger number of blood vessels were detected in the direct neighborhood of the tumor tissue. Conclusions.  It seems obviously that the reduced infiltration of RMS tumor tissue with immune cells is associated with deficiencies in the capillary system. This will probably negatively influences a potential RMS specific immune therapy. Further investigation about the nature of potential blood vessels deficiencies are needed to optimize the application of prospective immune therapeutics in RMS.

AG10.07 Expression of Cancer Testis (CT) Antigens in Pediatric and Juvenile Melanomas N. Behrendt1, T. Schultewolter2, D. Frosina3, K. Busam3, A. Jungbluth*3 Roskilde Hospital, Department of Surgical Pathology, Roskilde, Dänemark, Fachklinik Hornheide, Abteilung für Dermatologie, Münster-Hornheide, Deutschland, 3Memorial Sloan-Kettering Cancer Center, Abteilung für Pathologie, New York, Vereinigte Staaten von Amerika 1

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Background.  Cancer Testis antigens (CTAs) are named after their typical pattern of expression since they are found in various types of malignant tumors but in normal tissues solely in germ cells of the testis. Based on this tumor-associated expression pattern, CTAs such as MAGE-A, NY-ESO-1 and GAGE are regarded as potential vaccine targets but may also be useful as diagnostic markers. The diagnosis of melanocytic lesions can be cumbersome and oftentimes the distinction between benign versus malignant based on morphological criteria is equivocal. This is particularly true for childhood melanocytic lesions.

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Abstracts CTAs are highly expressed in adult primary and metastatic melanomas. Since little is known about the presence of CTAs in pediatric and juvenile melanomas, we have analyzed a series pediatric melanomas for the presence of CTAs. Methods.  To ensure presence of melanoma, only cases with biological signs of malignancy (metastatic disease and/or fatal outcome) were selected. CTA expression was analyzed by immunohistochemistry using the following monoclonal antibodies (mAbs)/to the following CTAs: mAb MA454/MAGE-A1; mAb 57B/MAGE-A4, mAb CT7-33/MAGEC1(CT7), mAb E978/NY-ESO-1 and mAb #23/GAGE. Extent of expression was graded semi-quantitatively. Results.  Based on our selection criteria, 12 melanoma cases could be retrieved from the archives of the department of dermatopathology of Fachklinik Hornheide. Patient age ranged from 2–16 years. The immunoreactivity/protein expression of CTAs was as follows: MA454/MAGE-A1: 1/12 (8 %); 57B/MAGE-A4: 2/12 (17 %); CT7/ MAGE-C1(CT7): 3/12 (25 %); E978/NY-ESO-1: 0/12; #23/GAGE: 3/12 (25 %). 4/12 (33 %) melanomas showed expression of at least one CTA. The expression pattern was exclusively heterogeneous and maximal 50 % of the tumors were CTA-positive while most cases showed immunopositivity in <5 % of the tumor. Conclusions.  Surprisingly, our study shows that CTAs are poorly expressed in pediatric melanomas. In adult melanomas, previous studies demonstrated an incidence of 60–70 % of the present panel of CTAs versus 25 % in our series. In adult melanomas, approximately 80 % of tumors were positive for at least one of our tested CTA panel versus 33 % in our series. Also, CTA expression pattern in pediatric tumors was exclusively heterogeneous versus a homogeneous pattern in most adult melanomas. Our study indicates that Cancer Testis Antigens are not useful as diagnostic markers and/or vaccine targets in pediatric melanomas.

AG10.08 Pediatric Low-grade Fibromyxoid Sarcoma: Three New Cases And Literature review A. Agaimy*1, F. Haller1, A. Hartmann1, M. Metzler2 FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland, FAU Erlangen-Nürnberg, Universitäts-Kinderklinik, Erlangen, Deutschland

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Background.  Low-grade fibromyxoid sarcoma (LGFMS) is a rare soft tissue sarcoma with deceptively benign-looking histological appearance but a paradoxically aggressive clinical course with frequent local recurrences and distant metastasis on long-term follow-up. LGFMS is notorious for being often misdiagnosed as “fibroma”, “fibromatosis” “neurofibroma” or “nodular fasciitis”. Pediatric cases of LGFMS are rare with only 39 cases reported to date, but this entity might be under-recognized in pediatric patients. Methods.  We describe the clinicopathological and molecular features of three cases of LGFMS studied by immunohistochemistry and FISH analysis for the FUS-CREB3L fusions. Results.  Patients were three boys aged 8, 8 and 10 years respectively. Site of primary tumor was deep muscle of the subscapular region, the rectus abdominis muscle and the thigh. All patients received only surgical treatment (primary excision for unsuspected sarcoma followed by re-excision with free margins). One patient developed multiple pulmonary and soft tissue metastases 6 years later and is currently alive with disease 76 mo after primary diagnosis. The second patient is alive with no evidence of disease at 21 months. Histology was desmoid-like (1 cases), pleomorphic with vague giant rosettes and sclerosing foci (1 case) and cellular angiofibroma-like (1 case). All tumors expressed vimentin and diffusely MUC4 but were negative with all other lineage-specific markers. FISH analysis confirmed the presence of a translocation involving the FUS gene locus in all three cases. Conclusions.  LGFMS might be under-recognized in the pediatric population and should be included in the differential diagnosis of all spindle

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cell neoplasms in children, particularly in bland-looking paucicellular or cellular fibrous lesions. LGFMS can mimic a variety of benign soft tisssue tumors including in particular desmoid tumors, solitary fibrous tumors and other benign entities.

AG10.09 Microarray and Methylation Studies in Stillbirths with Unexplained IUGR I. U. Nicklaus-Wollenteit*1, L. Cooper-Charles2, D. McMullan2, T. Marton3, D. Lim2, L. Brueton2, P. Cox3 1 Birmingham Children’s Hospital, Paediatric Pathology, NHS Foundation Trust, Birmingham, Vereinigtes Königreich, 2West Midlands Regional Genetics Laboratory, Birmingham Women’s Health Care NHS Trust, Birmingham, Vereinigtes Königreich, 3Pathology Department, Birmingham Women’s Health Care NHS Trust, Perinatal Pathology, Birmingham, Vereinigtes Königreich

Background.  Intrauterine death (IUD) at ≥24 weeks gestation affects ~1 in 200 pregnancies, with intrauterine growth restriction (IUGR) present in approximately 50 %. Although frequently due to placental pathology, genetic abnormalities may also underlie a significant proportion and Silver-Russell syndrome (SRS) may be implicated in some. Objective.  This is the first comprehensive pathological and genetic study to investigate non-placental IUGR, hypothesizing recurring abnormalities common to this group. There is limited experience with these techniques in IUD’s. The results of this study will advance the understanding of the genetic influence in the pathogenesis of IUGR and IUD and will inform future routine clinical diagnostic practice to ensure that valuable resources are used in a cost effective manner and may be applicable to prenatal diagnosis in cases of detected IUGR in utero. Methods.  31 IUD’s ≥24 weeks gestation with non-placental IUGR (<3rd centile), with or without congenital anomalies were selected. Following standard chromosome analysis, cases with a normal result were submitted for Comparative genomic hybridisation microarrays/aCGH on DNA extracted from a skin or placental biopsy. Array-based analysis of single nucleotide polymorphism (SNP)/methylation arrays identified abnormalities of imprinted gene expression and uniparental disomy (UPD). Results.  No clinically significant copy number imbalance (CNI): 17. 11 cases showed CNI of uncertain significance, 3 inherited and benign, others undergo further analysis. One fetus with malformations and homozygous deletion of part of the FANCA gene was consistent with Fanconi anaemia. Conclusions.  The comprehensive genetic study of a representative cohort has not shown a frequently recurring underlying genetic abnormality and suggests that causes of IUGR are complex. Further investigations of the cases with CNI of uncertain significance will determine whether this is pathogenic in this situation. Acknowledgement: This project has been grant funded by the Pathological Society of Great Britain and Ireland.

AG10.10 Congenital sodium diarrhea due to mutation in the SPINT2 gene is associated with ubiquitin C mediated loss of cell polarity C. Thoeni*1, A. Martinez2, E. Cutz3, P. Wales4, H. Huynh5, A. Lacson6, A. Muise2, Y. Avitzur2 Pathologisches Institut, Ruprecht-Karls-Universität, Heidelberg, Deutschland, 2The Hospital for Sick Children, Abteilung für Gastroenterologie, Hepatologie und Ernährung, Toronto, Kanada, 3The Hospital for Sick Children, Abteilung für Pathologie, Toronto, Kanada, 4The Hospital for Sick Children, Abteilung für Transplantationschirurgie, Toronto, Kanada, 5Stollery Children’s Hospital, Abteilung für Gastroenterologie, Hepatologie und Ernährung, Edmonton, Kanada, 6Stollery Children’s Hospital, Abteilung für Pathologie, Edmonton, Kanada 1

Background.  Congenital sodium diarrhea (CSD) caused by mutations in the SPINT2 gene leads to congenital enteropathy clinically manifesting as intractable diarrhea with high fecal sodium concentration. Furthermore, it has been shown that a specific mutation, Y163C in exon 5 of the SPINT2 gene, leads to reduced activity of this serine-protease inhibitor, but the precise molecular mechanisms, of how a non-functional serine-protease inhibitor causes structural abnormalities of the intestinal epithelium resulting in diarrhea is poorly understood. In our study we described 2 patients with a homozygous Y163C mutation in the SPINT2 gene, showing mislocalized cell polarity markers including EpCAM and Na/​K ATPase in ubiquitin C positive cytoplasmic aggregates, implicating, a non-functional SPINT2 might cause secretory diarrhea via loss of cell polarity, in particular impairment of tight junctions due to ubiquitin C mediated protein degradation. Methods.  Epithelial cell polarity markers as epithelial cell adhesion molecule (EpCAM), E-Cadherin, beta Catenin and Na/​K ATPase and their association to ubiquitin C were tested with multilabel immunofluorescence and confocal microscopy on formalin fixed and paraffin embedded (FFPE) duodenal biopsy samples from patients and controls according to our previously established protocols. Results.  We showed that in duodenal villus enterocytes from both patients with homozygous SPINT2 mutations the markers of cell polarity, in particular the tight junctional protein EpCAM as well as the ion pump Na/​K ATPase were mislocalized within ubiquitin C positive cytoplasmic aggregates. In addition, cell adhesion molecule markers, E-Cadherin and beta Catenin were partially internalized and co-localized with ubiquitin C in the cytoplasm. Conclusions.  Taken together, results of ubiquitin C accumulation in enterocytes with features of disturbed cell polarity, shown by changes in nucleus-cytoplasm relation, internalization of tight junctional and cell-cell adherens proteins, accompanied by redistribution of those cell polarity proteins, in particular EpCAM, to ubiquitin C positive cytoplasmic aggregates, implicate that a non-functional SPINT2 might cause cell polarity loss over ubiquitin C induced protein degradation within differentiated duodenal enterocytes primarily resulting in lack of tight junctions, disruption of the intestinal barrier and finally in severe CSD.

AG10.11 Esophageal biopsies in the pedatric setting B. Gürtl-Lackner* Institut für Pathologie, Skåne University and Regional Laboratories, Universitätshospital Lund, Lund, Schweden Background.  Biopsies from the esophagus are quite frequently encountered in the daily routine practice. In many cases an assessment of possible reflux disease is warranted, but additionally other differential diagnosis have to be taken into account as well. Methods.  In the vast majority of cases formalin-fixed, paraffin-embedded material is being investigated. In the usual setting a routine H&E section and PAS and Alcian stains are being performed. Results.  In the estimation of reflux disease the appraisal of the quality and the quantity of the inflammatory infiltrate at the gastro-esophageal junction is very important. Furthermore to outrule other differential diagnosis morphologial features of biopsies from different levels of the esophagus should be taken into consideration. In contrast to biopsies from adult patients, Barrett’s mucosa is an extremtly rare diagnosis. During childhood eosinophilic esophagitis is one of the most important differential diagnosis to reflux esophagitis. Conclusions.  Esophageal biopsies in the pediatric patient population show a different spectrum of diseases in comparison to adult patients. Consideration of clinical findings is a very important part of interpretation of morphological features.

AG10.12 Fetal Trisomy – New osseous soft markers T. Hager*1, A. Achter2, R. Fimmers3, U. Gembruch4, A. M. Müller5 Institut für Pathologie, Universitätsklinikum Essen, Essen, Deutschland, Universitätsklinikum Bern, Klinik für Innere Medizin, Bern, Schweiz, 3Institut für Medizinische Biometrie, Informatik und Epidemiologie, Universität Bonn, Bonn, Deutschland, 4Abteilung für Geburtshilfe und Pränatalmedizin, Universitätsklinikum, Bonn, Deutschland, 5Department für Paidopathologie, Universitätsklinikum, Bonn, Deutschland 1

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Background.  For detection of fetal trisomy so called “soft markers” (morphological variants) are used in ultrasound diagnosis. The diagnostic certainty of fetal trisomy increases by detecting a certain quantity of them. There are only few accepted osseous soft markers for trisomy so far. Potential osseous soft markers for first and second trimester ultrasound screening for trisomy 13, 18 and 21 were studied. Methods.  Postmortal x-rays (ap, lateral) of 193 fetuses (trisomy 13: n = 38, trisomy 18: n = 46; trisomy 21: n = 109) were examined and compared with those of a control group of 165 euploid fetuses with intrauterine fetal death due to placental insufficiency, chorioamnionitis or maternal medical indicated abortion. Findings were monitored for their statistical significance. Results.  For trisomy 13 – longer os nasale, elevated clavicula slope, premature sternum, delayed os sacrum ossification, delayed/premature cranium ossification, reduced number of ribs, coronal clefts, reduced os maxillare-jaw-corner distance, shortened ramus mandibulare, augmented orbita height, shortened os metacarpale V and a tendency for a shortened os metacarpale IV – could be verified. For trisomy 18 – elevated clavicula slope, reduced number of ribs, bell-shaped thorax, coronal clefts, reduced os maxillare-jaw-corner distance, shortened ramus mandibulare, shortened os metacarpale IV and V, augmented ratio between biparietal diameter and (osseus and soft-tissue) shoulder width – were observed. In trisomy 21 associated soft markers were un-timely os sternale ossification, delayed os sacrum ossification, shortened os maxillare, reduced os maxillare-jaw-corner distance, augmented orbita height, premature os calcaneus ossification, bell-shaped thorax, coronal clefts, trend to wider binocular as well as wider intraocular distances. Conclusions.  Several not yet published, but statistically significant osseous soft markers associated with trisomy 13, 18 and 21 were recognized. As an alternative to still risky invasive diagnostics as well as to not everywhere available newer non-invasive methods, these new soft markers may help to ensure aneuploidy diagnoses sonographically.

AG10.13 Fetal autopsy findings – a 10-year analysis of two University centers J. Andruszkow*1, W. Weichert2, R. Knüchel1, F. Erlmeier2 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, Institut für Pathologie, Technische Universität München, München, Deutschland

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Background.  Intrauterine death remains a severe complication during pregnancy, although the tools of prenatal diagnostics have improved in the last years. As fetal autopsy represented the gold standard distinguishing fetal from placental or maternal causes of intrauterine death in the last decades, it was the aim of our study to analyze the concordance of clinical diagnosis and autopsy results. Methods.  Fetal autopsy reports between 2005 and 2014 of two university institutes of pathology were reviewed. Gestational age and sex of the fetus were documented. In addition, clinical data and autopsy results were compared. Results.  Among the 428 fetal autopsies that were performed in both institutes, the median gestational age was 20 weeks. 243 fetuses (56.8 %) were of male gender. 127 pregnancies (29.7 %) were terminated due to prenatal diagnosis of anomalies. In 119 of 127 (93.7 %) of these terminated fetuses, the autopsy report detected macroscopically visible malformations in acDer Pathologe Suppl 1 · 2016

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Abstracts cordance with clinical data (p < 0.05). In the remaining 8 autopsy reports, one abortion was performed due to a feto-fetal transfusion syndrome during a gemini pregnancy. 7 autopsy reports (5.5 %) did not reveal indicatory findings while clinical data were missing. Also, 301 autopsies (70.3 %) were performed after stillbirth. The most frequent reason for stillbirth was chorioamnionitis (97 of 301 autopsies: 32.2 %). Within this cohort merely 24 autopsies (8.0 %) revealed macroscopically visible malformations (p < 0.05). Here, the most frequent anomalies were isolated cardiac malformations (29.2 %). Conclusions.  Although improved prenatal diagnostic enables clinicians to diagnose most fetal malformations, fetal autopsy still represents an important tool to confirm clinical diagnosis and to ensure the safety of the diagnostic process. In addition, autopsy contributes to enlighten the causes of stillbirth to the parents in order to prevent future intrauterine deaths.

AG Kopf-Hals-Pathologie AG11.01 Atmospheric pressure plasma applicated in the oral cavity of mice is well tolerated in short term experiments K. Evert*1, M. Evert1, F. Dombrowski2, A. Schindler3, T. Kocher4, L. Jablonowski4 Institut für Pathologie, Klinikum der Universität, Regensburg, Deutschland, Institut für Pathologie, Universitätsmedizin, Greifswald, Deutschland, 3 IOM Leipzig, Leipzig, Deutschland, 4Zentrum für Zahn-, Mund- und Kieferheilkunde, Universitätsmedizin, Greifswald, Deutschland

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Background.  Bacteria in the oral cavity lead to infections of dental implants resulting in inflammatory bone destruction (periimplantitis) and eventually to implant loss. So far, the treatment of the infected implant has been performed with rotating instruments, such as laser or air-powder abrasive techniques. However, the latter therapeutic approaches induce the contamination of the surface of the implant, thus preventing its reosseointegration. As a consequence, no efficient therapy for periimplantitis has been established to date. Atmospheric pressure plasma may represent a valid alternative to the previous treatments; however, no toleration studies have been performed thus far. Methods.  B6C3F1 male mice were treated with two different sources of atmospheric pressure plasma (kinpen09 or PS-MWM). Corresponding control groups received UV or no treatment. Fifteen mice per group were treated for 10 s (in analogy to the intended treatment time in humans) and 60 s under narcosis at the right oral mucosa. After the treatment, mice were monitored for 1 day or 1 week, respectively, and then sacrificed. Tissue samples of the treated and the untreated mucosa of each animal were examined in serial HE-stains and PAS-reactions and compared morphologically with the control groups. Results.  The mice tolerated the treatment and the narcosis well, not showing any problem in food intake. Indeed, no severe weight loss was observed in the treated mice. One day after the treatment, the mucosa showed histologically focal ulceration and necrosis with superficial homogenization of the underlying stroma accompanied by a mild inflammatory reaction. One week after the treatment, the damaged epithelium of the oral mucosa was replaced by normal epithelium, which was associated with remnants of granulation tissue in the stroma. Histological patterns were almost equivalent regardless of the treatment duration, whereas controls did not show any histological alterations. Conclusions.  Treatment with atmospheric pressure plasma is well tolerated in B6C3F1 male mice with only minimal differences between the two plasma sources in the short-term-experiments. Long-term-experiments are now necessary to evaluate the adverse effects more in depth. In particular, a possible co-carcinogenic effect of the treatment must be ruled out before treatment with atmospheric pressure plasma can be established as an alternative treatment for periimplantitis.

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AG11.02 MERTK is a therapeutic target in head and neck squamous cell carcinoma C. Sanders*1, 2, 3, A. von Mässenhausen1, 2, 3, B. Thewes1, 2, 3, M. Deng4, A. Queisser1, 2, 3, W. Vogel4, G. Kristiansen2, 3, A. Schröck3, 5, F. Bootz3, 5, F. Göke1, 2, 3, A. Franzen1, 2, 3, L. Heasley6, J. Brägelmann1, 3, 7, S. Perner4 Pathologie, Prostate Cancer Research, Bonn, Deutschland, 2Institut für Pathologie, Universitätsklinikum, Bonn, Deutschland, 3Zentrum für integrierte Onkologie Köln/Bonn, Universitätsklinikum, Bonn, Deutschland, 4 Institut für Pathologie (UKSH), Campus Lübeck, Lübeck, Deutschland, 5 Klinik und Poliklinik für Hals-Nasen-Ohrenheilkunde, Universitätsklinikum, Bonn, Deutschland, 6Institut für Kraniofasziale Biologie, University of Colorado at Denver, Aurora, Vereinigte Staaten von Amerika, 7Medizinische Klinik und Poliklinik III Hämatologie und Onkologie, Universitätsklinikum, Bonn, Deutschland 1

Background.  Malignant tumors of the head and neck region are the 6th most common malignancy worldwide. However there are hardly any alternatives to surgery and radio and/or chemotherapy. Because of their importance in cell differentiation, proliferation and migration receptor tyrosine kinases (RTKs) are a good option for targeted cancer therapies having fewer side effects than standard therapies. We identified c-mer proto-oncogene tyrosine kinase (MERTK), a RTK possibly being relevant for HNSCC. MERTK has already been described as a potential therapeutic target in melanoma and gastric cancer and can be inhibited by the selective small molecular inhibitor UNC 1062. The aim of our study is to analyze the role of MERTK as a potential therapeutic target in HNSCC. Methods.  Bioinformatic analysis of available data from The Cancer Genome Atlas (TCGA) was performed to identify the mRNA expression level of MERTK and mutation status in HNSCC (n = 306). Immunohistochemistry (IHC) of MERTK was done on a HNSCC cohort (n = 537). MERTK or GFP as control were overexpressed in HN cells using the vector pCSGIBAwt1. Furthermore, MERTK positive Detroit 562 and MERTK negative HN cells were treated with UNC 1062. Proliferation was assessed using MTT Assay. Migration and invasion were tested with Boyden Chamber Assays. Results.  MERTK was overexpressed in tumor tissue in our IHC cohort. Mutational analysis of MERTK in HNSCC did not show any recurrent mutations. HN cells overexpressing MERTK showed an increased proliferation, migration and invasion compared to control cells. The treatment with UNC 1062 did not show any difference on proliferation between MERTK negative HN or positive Detroit 562 cells. Migration and invasion of MERTK positive cells were significantly reduced compared to MERTK negative cells using UNC 1062. In a next step, we will correlate our IHC results to clinical data. Subsequently we will use MERTK knockdown to confirm the importance of MERTK for migration and invasion in HNSCC. Furthermore, we will analyze effects of MERTK in downstream signaling pathways in HNSCC using Western Blot. Conclusions.  Our data implicate that MERTK could be a therapeutic target in HNSCC.

AG11.03 The role of AXL in Head and Neck Squamous Cell Carcinoma H. Billig*1, 2, 3, A. von Mässenhausen1, 2, 3, J. Brägelmann1, 3, 4, B. Thewes1, 2, 3, M. Deng1, 2, 3, A. Queisser1, 2, 3, W. Vogel1, 2, 3, F. Göke1, 2, 3, A. Franzen1, 2, 3, G. Kristiansen2, 3, A. Schröck3, 5, F. Bootz3, 5, S. Perner1, 2, 3, 6 Sektion für Prostatakarzinom-Forschung, Universitätsklinikum Bonn, Bonn, Deutschland, 2Institut für Pathologie, Universitätsklinikum, Bonn, Deutschland, 3Zentrum für integrierte Onkologie, Universitätsklinikum Bonn, Köln/Bonn, Deutschland, 4Poliklinik Hämatologie und Onkologie, 1

Universitätsklinikum Bonn, Bonn, Deutschland, 5Klinik und Poliklinik für Hals-Nasen-Ohrenheilkunde/Chirurgie, Universitätsklinikum Bonn, Bonn, Deutschland, 6Institut für Pathologie des Universitätsklinikums SchleswigHolstein und Leibniz-Zentrum Borstel, Lübeck, Deutschland Background.  Although Head and neck Cancer (HNSCC) is the sixth most common tumor entity worldwide, therapy options remain mostly limited to surgery followed by radio- and/or chemotherapy leading to 5-year survival rates of only 50 %. Dysregulation of the receptor tyrosine kinase AXL is implicated in the pathogenesis of several human cancers. AXL as a novel therapeutic target may support standard therapies or even be a therapy alternative with fewer side effects. The small molecule inhibitor BGB324 targets AXL selectively and is currently tested in early-stage clinical studies. Due to this background, we aim to evaluate the role of AXL and its use as a potential therapeutic target in HNSCC. Methods.  Immunohistochemistry (IHC) of AXL was done on a large HNSCC cohort (n = 364). Subsequently, bioinformatic analysis of available data from The Cancer Genome Atlas (TCGA) was performed (n = 306). AXL or GFP as control were stably overexpressed in SCC-25 cells which originally have almost no AXL expression. The effect of AXL-knockdown was investigated in HN cells that we showed to have a high AXL expression. Furthermore, HN and SCC-25 cells were treated with BGB324. Proliferation was assessed using MTT Assay. Boyden Chamber assays were used to evaluate migration and invasion. Results.  In the IHC-study tumor tissue had a higher staining intensity for AXL than normal tissue. Our analysis of the TCGA data supports the thesis of tumors having a higher AXL-expression than normal mucosa. The SCC-25 cells (low AXL expression) overexpressing AXL showed, compared to GFP controls, no difference for proliferation but migration and invasion of AXL-overexpressing cells was increased. Interestingly, HN cells (high AXL expression) with a knockdown of AXL proliferated better than the control cells. Migration of knockdown cells was decreased, whereas invasion was not affected. Cells treated with BGB324 did not show a difference in proliferation in comparison to SCC25 cells but migration and invasion were significantly decreased. As a prospect, we intend to investigate the effects of BGB324 and AXL-knockdown on downstream signalling by performing western blots. Also, we will correlate the IHC and TCGA data with clinical information. Conclusions.  AXL is frequently overexpressed in HNSCC and may play a role in tumor progression. However, the exact role of AXL remains controversial and has to be evaluated in further experiments.

AG11.04 Targeting DDR2 in Head and Neck Squamous Cell Carcinoma with dasatinib C. Sanders*1, 2, 3, A. von Mässenhausen1, 2, 3, J. Brägelmann1, 3, 4, M. Konantz5, A. Queisser1, 2, 3, W. Vogel6, G. Kristiansen2, 3, A. Schröck3, 7, F. Bootz3, 7, F. Göke1, 2, 3, A. Franzen1, 2, 3, C. Lengerke5, S. Perner6 1 Pathologie, Prostate Cancer Research, Bonn, Deutschland, 2Institut für Pathologie, Universitätsklinikum, Bonn, Deutschland, 3Zentrum für integrierte Onkologie Köln/Bonn, Universitätsklinikum, Bonn, Deutschland, 4 Medizinische Klinik und Poliklinik III Hämatologie und Onkologie, Universitätsklinikum, Bonn, Deutschland, 5Universität Basel, Departement Biomedizin, Basel, Schweiz, 6Institut für Pathologie (UKSH), Campus Lübeck, Lübeck, Deutschland, 7Klinik und Poliklinik für Hals-Nasen-Ohrenheilkunde, Universitätsklinikum, Bonn, Deutschland

Background.  Though malignant tumors of the head and neck region (HNSCC) are a common malignancy, good alternatives to surgery and radio and/or chemotherapy are missing. Receptor tyrosine kinases (RTKs) are important molecules for targeted cancer therapies having fewer side effects

than standard therapies. The RTK discoidin domain receptor 2 (DDR2) has been implicated in several tumors. In contrast to prototypic RTKs, which are activated by soluble factors in a short time, DDR2 is slowly activated by collagen. It has been shown that DDR2 is important for migration and invasion in HNSCC. The FDA approved drug dasatinib is described as a non-selective small molecular inhibitor of DDR2. The aim of our study is the investigation of DDR2 during tumor progression and its role as a prognostic marker. Moreover, we want to analyze if DDR2 can be inhibited by dasatinib. Methods.  Immunohistochemistry (IHC) of DDR2 was done on a HNSCC cohort (n = 554). DDR2 was stably overexpressed in FaDu cells using pLenti-C-mGFP vector containing DDR2 and the empty vector was used as control. Proliferation was assessed using MTT Assay, migration as well as invasion were tested with Boyden Chamber Assays and adhesion was checked by counting adherent cells after different time points. Experiments were performed with or without Dasatinib treatment. Results.  DDR2 was significantly overexpressed in tumor tissue in our IHC cohort. FaDu cells overexpressing DDR2 proliferated significantly more than control cells on cell culture treated plates as well as on collagen I coated plates. On collagen I coated plates proliferation of DDR2 overexpression cells was inhibited by dasatinib compared to control cells. Furthermore, FaDu cells overexpressing DDR2 showed increased adhesion, migration and invasion. These increased tumorigenic behaviors could be reduced by Dasatinib treatment. To support our data we will repeat our experiments in another HNSCC cell line. Moreover, to confirm our data in an in-vivo model, zebrafish xenotransplantation will be performed. IHC results will be correlated to clinical data. Conclusions.  All in all our data suggest that the inhibition of DDR2 with Dasatinib could be a therapeutic option in HNSCC.

AG11.05 SHOX2 and SEPT9 DNA Methylation in Blood for Diagnosis, Staging, Prognosis and Monitoring of HNSCC Patients D. Dietrich*1, A. Leisse2, F. Bootz3, A. Schröck3, G. Kristiansen1 Institut für Pathologie, Universitätsklinikum, Bonn, Deutschland, Medizinische Klinik II – Innere Medizin (Kardiologie, Pneumologie), Universitätsmedizin, Bonn, Deutschland, 3Klinik und Poliklinik für HalsNasen-Ohrenheilkunde, Universitätsklinikum, Bonn, Deutschland 1

2

Background.  Head and neck squamous cell carcinomas (HNSCC) represent a heterogeneous group of cancers thereby impeding the compilation of appropriate treatment guidelines. Biomarkers in minimally invasively obtained liquid biopsies facilitating disease staging, prediction of outcome and monitoring of disease progression are especially valuable in optimizing an individualized treatment of HNSCC patients. For this purpose, methylated cell-free circulating tumor DNA in blood was evaluated in an observational prospective cohort study. Methods.  Two-hundred-eighty-four HNSCC patients and 122 patients with non-malignant diseases were recruited. DNA methylation of SHOX2 and SEPT9 in plasma was quantified accurately and sensitively prior to first-line treatment (surgery with optional adjuvant radio-chemotherapy or definitive radio-chemotherapy) and longitudinally during surveillance. Results.  Fifty-nine percent (sensitivity) of HNSCC patients were methylation-positive at 96 % specificity. Plasma methylation was strongly correlated with tumor and nodal stage (p < 0.001), therefore representing a powerful staging parameter. Patients with initially elevated SEPT9 methylation level had a higher hazard of death (HR = 5.27, p = 0.001). Furthermore, SEPT9 methylation levels were predictive for loco-regional recurrence-free (p < 0.001), distant metastases-free (p = 0.002), and progression-free (p < 0.001) survival. Fifty-eight percent of deaths showed a positive test result during surveillance 213±117 days before death. Dis-

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Abstracts ease progression was diagnosed up to 377 days earlier compared to current clinical practice. Conclusions.  SHOX2 and SEPT9 are powerful biomarkers for molecular disease staging, risk stratification, and disease monitoring. Patients at high risk to suffer from recurrence might benefit from intensified first-line treatment and surveillance. The early detection of a recurrent tumor will allow for a timely initiation of a consecutive treatment, thereby improving patients’ outcome.

AG11.06 Mapping the genomic landscape of head and neck squamous cell carcinoma for predicting the response to combined treatment with EGFR-blocking and radiation therapy N. Valtcheva*1, K. Ikenberg1, M. Rechsteiner1, S. Stieb2, F. Singer3, M. Prummer3, D. Stekhoven3, H. Moch1, M. Guckenberger2, G. Studer2, O. Riesterer2, P. Wild1 Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz, 2Klinik für Radio-Onkologie, UniversitätsSpital Zürich, Zürich, Schweiz, 3ETH Zürich, NEXUS Personalized Health Technologies Clinical Bioinformatics Unit, Zürich, Schweiz 1

Background.  Head and neck squamous cell cancer (HNSCC) is the sixth most common cancer worldwide. Despite advances in diagnostics, more than 60 % of patients present with an already advanced stage of the disease. Unfortunately, advanced cancer patients do have a high risk of metastasis and recurrence. Therefore, a combined therapy approach is the standard of care. However, prediction of response to these intense treatment modalities is still insufficient. Methods.  A defined cohort of 11 patients with primary advanced HNSCC, who responded well to a combined treatment of radiation and targeted anti-EGFR therapy (Cetuximab), was compared with 12 non-responder patients. For the latter set of patients, primary cancer specimens and matching recurrent tumors were analyzed. We used the Ion Torrent Platform and a custom-designed panel for re-sequencing of genes commonly mutated in HNSCC as reported in exome sequencing studies. Additionally, we screened the cohort for copy number variation using Affymetrix OncoScan arrays, optimized for formalin-fixed, paraffin-embedded (FFPE) samples. Results.  Tumors of these two patient groups (responder vs. non-responder) showed a differential mutational landscape. Moreover, in the non-responder group we found three groups of variants: i) variants not affected by the therapy, ii) variants found in the primary tumor but not detectable after the therapy, and iii) variants absent in the primary tumor but emerging in the relapse samples. We are especially interested in the alterations that accumulate after therapy, since they provide information on the clonal selection advantage due to treatment. A phylogenetic analysis on the aberrations identified in the primary and recurrent tumor was performed. Conclusions.  The confirmed mutations and copy number changes are potential new predictive biomarkers and might also represent novel drug targets. After validation analyses by Sanger sequencing, we will screen the confirmed genes in a larger patient cohort.

AG11.07 CD274/PD-L1 Gene Amplification and PD-L1 Protein Expression are Common Events in Squamous Cell Carcinoma of the Head and Neck M. Straub1, E. Drecoll1, N. Pfarr1, W. Weichert1, R. Langer2, A. Hapfelmeier3, C. Götz4, A. Kolk4, K. Specht*1 Technische Universität München, Institut für Allgemeine Pathologie und Pathologische Anatomie, München, Deutschland, 2Institut für Pathologie, Universität Bern, Schweiz, 3Technische Universität München, Institut für Medizinische Statistik und Epidemiologie, München, Deutschland, 4 Technische Universität München, Klinik und Poliklinik für Mund-, Kiefer-, Gesichtschirurgie, München, Deutschland 1

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Background.  Squamous cell carcinomas comprise over 95 % of malignant tumors in the head and neck area. Immunomodulatory therapies, targeting the immune checkpoint ligand-receptor complex PD-L1/PD-L1 have shown promising results in early phase clinical trials in several solid malignancies, including SCC of the head and neck (HNSCC). In this context, PD-L1 protein expression determined by immunohistochemistry has been proposed as a potentially valuable predictive marker. To date, there is only limited data with regard to the expression of PD-L1 and its receptor PD-1 in HNSCC. Methods.  Expression of PD-L1 and PD-1 was evaluated by immunohistochemistry in 88 patients with predominantly HPV-negative, stages T1-T4 HNSCC and 31 associated nodal metastasis. In addition, CD274/PD-L1 gene copy number status was assessed by fluorescence in-situ hybridization analysis. Results.  PD-L1 immunohistochemical expression with ≥5 % carcinoma cells showing PD-L1 membranous staining was detected in 37/88 (42 %) of cases. PD-L1 expression in primary tumor and corresponding nodal metastasis was concordant in only 21/31 (68 %) of cases, with 10/31 (32 %) cases displaying either loss of PD-L1 expression in the nodal metastasis or a negative staining result in the primary tumor and positive expression in the metastasis. PD-1 receptor expression was found in tumor-infiltrating lymphocytes (TILs) but not in tumor cells. 27/37 (73 %) PD-L1 positive carcinomas and 19/51 (37 %) of PD-L1 negative tumors contained PD-1 positive TILs. CD274/PD-L1 gene amplification was found in 18 % of HNSCC, with high level PD-L1 amplification present in 13/88 (15 %), and low level amplification in 3/88 (3 %) of cases. Interestingly, in only 75 % of cases, CD274/PD-L1 gene amplification was associated with positive PD-L1 immunostaining. PD-L1 copy number status was concordant in primary tumor and associated lymph node metastasis. Clinically, PDL1 positivity was associated with higher prevalence of nodal metastasis at diagnosis. Disease-free free survival was significantly poorer in PD-L1 positive patients. Conclusions.  PD-L1 amplification is present in 18 % of HNSCC, representing a genetically defined subgroup of HNSCC. Only 75 % of PDL1 amplified cases show PD-L1 expression by IHC. Based on our findings we propose to include the PD-L1 copy number status in addition to protein status in screening programs for clinical trials with immunotherapeutic strategies targeting the PD-L1/PD-1 axis in HNSCC.

AG11.08 PD-L1: a novel prognostic biomarker in head and neck squamous cell carcinoma T. Müller*1, D. Dietrich1, G. Kristiansen1, P. Brossart2, F. Bootz3 Institut für Pathologie, Universitätsklinikum, Bonn, Deutschland, Medizinische Klinik und Poliklinik III Hämatologie und Onkologie, Universitätsklinikum Bonn, Bonn, Deutschland, 3Klinik und Poliklinik für Hals-Nasen-Ohrenheilkunde, Universitätsklinikum, Bonn, Deutschland 1

2

Background.  PD-1/PD-L1 pathway seems to play a pivotal role in T cell downregulation during immune response to external antigens and self-antigens to prevent tissue damage and limit autoimmunity. Today there is no data available about PD-L1 expression in HNSCC, which prompted us to analyze PD-L1 expression in HNSCC. Methods.  Two independent cohorts of HNSCC (n1 = 98, n2 = 195) in a TMA format were analysed for PD-L1 expression by immunohistochemistry (clone EPR1161(2)) and evaluated by two observers. Immunoreactivity was scored semiquantitatively as negative vs. low vs. high. Statistical analysis was conducted with SPSS. Results.  High expression levels of PD-L1 were detected in 15.3 % of tumor cases in the training cohort and 27.7 % of tumor specimens in the test cohort. Kaplan-Meier analysis demonstrated a highly significant prognostic value of PD-L1 in both cohorts (both p < 0.004), which was confirmed in a multivariate analysis encompassing the recognized prognostic factors tumor stage, nodal status, distant metastases, lymphatic invasion, vascu-

lar invasion, grading, extracapsular expansion and surgical margin status (p = 0.02; HR = 2.926 [95 %CI = 1.183–7.235]). Moreover strong PD-L1 expression was associated with the presence of distant metastases in the test cohort (p = 0.025). Conclusions.  In summary, we identified PD-L1 as expressed in HNSCC, which may provide a rational basis for anti PD-1/PD-L1 therapy in HNSCC patients. Additionally, it may serve as a multivariate prognostic biomarker in HNSCC, which deserves further study.

AG Zytopathologie AG12.01 Complemental role of smears and cell blocks as integral parts of the FNA sampling of the thyroid nodules for auxiliary studies B. Bode-Lesniewska*, P. Bode Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz

AG11.09 Rapid analysis of resection margins of bone in head and neck squamous cell carcinoma by immunofluorescence C. Haase*1, J. Teichmann2, A. Cassataro1, R. Knüchel1, F. Hölzle2, T. Braunschweig1 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, Klinik für Mund-, Kiefer- und Gesichtschirurgie, Universitätsklinikum, Aachen, Deutschland

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Background.  The purpose of this study is to develop a rapid and handy method to analyse bone resection margins of patients with head and neck squamous cell carcinoma (SCC) in an intraoperative setting, similar to frozen section. Such a method can result in minimizing bone excision, thus better reconstruction possibilities and better rehabilitation outcome. Concerning squamous cell carcinoma resection safety margins of 0.5 cm are desirable additional to the complete excision of squamous cell carcinoma. Routinely, to ensure carcinoma-free margins, frozen sections of soft tissue or epithelium/mucosa are carried out surrounding the tumor. In case of suspicion of bone infiltration, to ensure carcinoma free bone margins, a wide dissection of tumor site related bone is standard, due to lack of frozen section possibilities in bone. Histological examination of bone tissue includes 24 hours for decalcification after fixation and is therefore suitable for postoperative analysis only. To our knowledge, none of published methods is established in intraoperative routine yet due to complex methodology. Methods.  We used specimens of porcine bone and skin (simulating SCC properties) to establish two different approaches: Direct staining and staining of membrane tissue prints of 3 mm sawed bone sections combined with skin by adapted immunofluorescence staining protocol against cytokeratins. We tested several saws (hand saws, oscillating saw, band saw, etc.), secondary antibodies with different fluorochrome labelling, three types of detection devices (Typhoon 9410 scanner, Licor Odyssey® Sa IR Scanner and fluorescent microscopy, Zeiss Axiovert 100) and different protein transfer methods for printing. Results.  By immunofluorescence using an inverse microscope, skin tissue entrapped within bone could be detected positive with low background but difficult to handle and low contrast, hard to detect by camera. Scanning techniques as listed above didn’t show good contrast of the bone/skin sample itself. After protein transfer to a nitrocellulose membrane, scanning showed specific staining of skin and less background, best in IR scan. Conclusions.  Most contrasting and fastest results were obtained by sawing with water-cooled diamond band saw, direct staining of the 3 mm section and detection with fluorescent microscopy at 488 nm. The use of tissue printing is more time consuming so far but is highly promising because of better contrast. With this method best results were obtained in the infrared spectrum with Licor scanner.

Background.  As the imaging methods refine, the number of incidentally discovered thyroid nodules increases. The management of patients with both, clinically detectable and incidental nodules strongly depends on their estimated malignant potential. Fine needle aspiration (FNA) is the most important study in the examination and triaging of the thyroid nodules, providing essential information on the content of the intrathyroidal masses. The recently introduced classification schemes (British Thy- and American Bethesda- systems)of the results of the FNAs of the thyroid nodules facilitate the communication between the cytopathologists and clinicians, however the indeterminate categories of both systems remain problematic. Various auxiliary studies may optionally be used in order to minimalize the indeterminate rate and accurate specify the etiology of the examined nodules. Methods.  FNAs of the thyroid nodules were performed under the ultrasound guidance, either in the walk-in-clinics of the Cytopathology Division of the Institute of Surgical Pathology of the University Hospital Zurich or in outside institutions. For each nodule maximally 1 to 3 direct smears per pass and a common cell block (thrombin method) is prepared as a standard sampling. If indicated by the results of the microscopical findings on the Papanicolaou stained smears and/or HE stained cuts of the cell block, additional studies (immunohistochemistry and molecular methods) are performed on the paraffin embedded material. The results of the FNAs are categorized according to the slightly modified 5 tier Thy-British system. The review of the results of the thyroid FNAs with the correlation to the histology results of the resected organs over 2 years (2012–2013) was performed. Results.  2245 nodules in 1753 patients were examined over the study period. 74 % of the nodules were diagnosed as benign (Thy2) and 7 % as highly suspicious or malignant (Thy4 and Thy5). The frequency of the undetermined category (Thy3) was low (7 %). The histological correlation was available for 17 % of the nodules with sensitivity of 100 % and specificity of 98.6 %. The application of auxiliary methods was helpful or essential for the diagnosis of 10 % of cases. Conclusions.  The standard use of both, direct smears and cell blocks for sampling of the FNAs of the thyroid nodules is well suitable for keeping the rate of the undetermined FNA results at a low level and and for the rendering of correct final specific cytopathologc diagnoses.

AG Molekularpathologie AG13.01 NGS Guidelines for diagnostic use: The Basel approach with the Cancer Hotspot Panel V2 of the Ion Torrent platform M. Bihl*, L. Quagliata, L. Terracciano, S. Höller Institut für Pathologie, Molekularpathologie, Basel, Schweiz Background.  The deep sequencing method called next generation sequencing (NGS) has known a tremendous development over the last years. This method allows a massive parallel sequencing of genes of interest by primer multiplexing amplification. Its sensitivity is higher compared to Sanger sequencing and gives gene alteration results in a quantitative manner. In the absence of gold standards, the purpose of this study is to establish diagnostic guidelines from preanalytic management to data interpretation. Der Pathologe Suppl 1 · 2016

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Fig. 1 I AG13.01 8 Definition of safety zones with different material, tumor cell content and DNA quantity Methods.  This study comprises 540 cases which were analyzed by NGS under diagnostic conditions. A detailed analysis of many technical parameters (like overall coverage, amplicon-specific coverage, uniformity and allelic ratio etc.) was done on 110 cases with the goal to define cut-offs for semi-automatic analysis of NGS data. Results.  Due to the high diversity of analysed diagnostic samples in pathology the “ad hoc” cut-off, if a given mutation is specific or not, must be adapted to the given situation (e. g. low tumor cell content and microlaser capture). Our approach is described in . Fig. 1. A filter for the selection of variants was chosen, which is geared to p-values showing also variants with low frequencies below 5 %. SNPs present in more than 10 % of the population are excluded. As a quality factor of the analysis, we also investigate the global coverage and uniformity of the coverage of generated amplicons. In case of low uniformity, there are interestingly often the same amplicons involved with a low or absent expression (underneath also exon 20 of the EGFR gene). Additionally we will present our diagnostic NGS molecular pathology report. Conclusions.  In our approach, we aimed to define quality standards in the context of NGS for routine pathology diagnostics. This is mainly based on two pillars: first, a good characterization of the sample to be analyzed (tumor cell content and type of selection of the tumor cell area)and second, a careful evaluation of all analytical parameters (i. e. coverage, uniformity, homopolymer artefacts etc.). Only a well-grounded knowledge of the parameters mentioned above will allow us in a next step their integration into a semi-automated way of data anaylsis.

AG13.02 Results from Routine Genotyping of Colorectal Cancer using Next-Generation-Sequencing at the IPH-Center of Molecular Diagnostics M. Jesinghaus*1, N. Pfarr1, V. Endris2, M. Kloor3, A.-L. Volckmar2, R. Brandt2, E. Herpel2, A. Muckenhuber1, F. Lasitschka2, P. Schirmacher2, R. Penzel2, W. Weichert1, A. Stenzinger2 TU München-Pathologisches Institut, München, Deutschland, Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, 3 Abteilung für Angewandte Tumorbiologie, Universität Heidelberg, Heidelberg, Deutschland

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Background.  Although chemotherapy is still the mainstay of oncological therapy, cancer precision medicine approaches have opened up new ave-

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nues for the treatment of colorectal cancer (CRC). To fully realize its potential, high-throughput sequencing platforms that allow genotyping beyond KRAS, need to be implemented into routine diagnostics and require performance assessment. Methods.  Here we comprehensively analyzed first-year data of 202 consecutive formalin-fixed paraffin embedded (FFPE) CRC samples for which genotyping at the Institute of Pathology (IPH) – Center of Molecular Diagnostics was requested. We performed deep targeted genotyping using a semiconductor-based sequencing platform and a self-designed panel of 30 CRC-related genes. Additionally, microsatellite status (MS) was interrogated by a PCR-based assay. Results.  Of 202 tumor samples, 97 % were suitable for deep targeted sequencing and in 87.8 % of the cases MS could be assessed. The low dropout rates of 6 cases and 24 cases respectively were due to too low amounts or heavy degradation of tumor DNA. The input amount of DNA sufficient for library preparation was as low as 6 ng. Of 557 detected non-synonymous mutations, 90 have not been described in COSMIC yet. Forty-three cases (21.9 %) had double- or triple mutations affecting a single gene. We identified genetic alterations influencing oncological therapy in 64.2 % of the cases. 7.6 % (MSI phenotype: 6.1 %; mutated POLE: 1.5 %) of the patients were eligible for treatment with immune checkpoint inhibitors as their genetic make-up suggests high mutational loads. Of 56.1 % of CRC with KRASwt alleles that were potentially amenable to anti-EGFR treatment 30 % presented with mutations in BRAF/NRAS. Mutated PIK3CA was detected in 21.4 % of the cases. Conclusions.  Summarising, we here demonstrate real-life routine diagnostics data that not only show the robustness and feasibility of deep targeted sequencing and MS-analysis of FFPE CRC resection specimens and biopsies but also highlight its ability to contribute to the molecular alterations underlying CRC. Most importantly, our approach helps to provide the best oncological treatment currently available for more than half of the patients in our cohort.

AG13.03 RNA based parallel sequencing to detect ALK, RET and ROS1 translocations in lung adenocarcinoma M. Ihle*, A. Scheel, C. Heydt, R. Büttner, S. Merkelbach-Bruse Universitätsklinikum Köln, Institut für Pathologie, Köln, Deutschland Background.  Break-apart fluorescence in situ hybridization (FISH) is currently the gold standard for the detection of translocations, e. g. ALK, RET or ROS1, in non-small cell lung cancer (NSCLC). Patients with such rearrangements may benefit from targeted therapies. However, assay interpretation is challenging especially in cases of borderline results and evaluation of FISH analysis is time consuming. Therefore, we established an RNA based parallel sequencing approach for the simultaneous detection of ALK, RET and ROS1 translocations. Methods.  Our cohort consists of 44 lung adenocarcinoma cases with ALK, RET, ROS1 and NRG1 translocations as previously tested by FISH and immunohistochemistry (for ALK). RNA was isolated from three 10 µm, formalin-fixed paraffin-embedded slides each using the Maxwell® 16 LEV RNA FFPE kit and the semiautomated Maxwell® 16 system (Promega). 500 ng of each case were reverse transcribed into cDNA using the Omniscript® Reverse Transcription kit from Qiagen with random hexamer primers. 10 ng of cDNA was amplified by a Multiplex-PCR and library was prepared with the OncomineTM Solid Fusion Transcript kit (Thermo Fisher Scientific). Sequencing was performed on the MiSeq (Illumina). Results.  Medium RNA concentration was 72.6 ng/µl from FFPE sections representing an excellent RNA yield. Medium cDNA concentration was 1119.9 ng/µl. Using the OncomineTM Solid Fusion Transcript kit, Multiplex-PCR and library preparation was easy to implement with hands on time of only one working day. Sequencing on the MiSeq System requires only one additional adenylating step compared to the manufacturers protocol. This method allows the reliable and simultaneous detection of spe-

cific translocations in ALK, RET and ROS1. In addition, the implemented 3’-5’imbalance assay facilitates the detection of so far unknown translocations. Conclusions.  This study shows that RNA based next generation sequencing is a good amendment to FISH analysis especially in the case of borderline FISH results. In addition, we provided first evidence that the OncomineTM Solid Fusion Transcript kit is applicable on the MiSeq System.

AG13.04 Diagnostic testing of ovarian/breast cancer samples for BRCA1/2 mutations: one year of experience V. Endris*1, A.-L. Volckmar1, N. Pfarr2, H. P. Sinn1, W. Weichert2, A. Stenzinger1, P. Schirmacher1, R. Penzel1 Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, Pathologisches Institut, TUM, München, Deutschland

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Background.  Since the end of 2014, a new therapeutical option using the PARP inhibitor Olaparib (Lynparza®) for the treatment of platin-sensitive high grade serous ovarian cancer is available. The European Medical Agency (EMA) approved the medication for female patients with somatic or hereditary mutations in one of the two breast cancer genes BRCA1 or BRCA2. The complex molecular screening necessary to identify BRCA mutations in FFPE tumor specimens with short turnaround times is a novel challenge for molecular pathology, requiring mandatory next-generation sequencing (NGS). Methods.  Routine ovarian and breast cancer samples were analysed for BRCA1/2 mutations using an FFPE-optimised BRCA1/2 Ampliseq panel and sequenced on an IonTorrent PGM. Mutations were called using the TorrentSuite variant caller plugin. Variants in homopolymeric regions were further exploited by a custom analysis script. Amplicon coverage analyses were used to identify exonic deletions/duplications. Results.  In our routine diagnostics, we analysed more than 240 cases of ovarian and breast cancer samples BRCA1/2 mutations in 2015. The fraction of BRCA1/2 mutated cases was 21 % (51/240 cases), with 13 % being mutated for BRCA1 and 8 % carrying mutations in BRCA2. The most common mutations were frameshift deletions (35 %), followed by stopgain (nonsense) mutations (20 %) and non-synonymous variants (18 %). In two cases, splice site mutations were identified. In addition, we identified a sample with a deletion of the last exon of BRCA2 by NGS. Further analyses using RT-PCR and Sanger sequencing confirmed the exonic deletion. Allelic imbalances indicative of an LOH of the BRCA1 or BRCA2 locus was present in a large fraction of the tumor samples. Conclusions.  The median turnaround time from sample registration to pathological report was 6.5 days. The frequency of identified mutations in BRCA1/2 corresponds to the expected range of 15–20 %. Amplicon coverage analysis can be applied to identify exonic deletions/duplications in the tumor FFPE sample.

AG13.05 Impact of Genetic Heterogeneity in Synchronous Colorectal Cancers on Genotyping Approaches and Treatment Strategies M. Jesinghaus*1, N. Pfarr1, M. Kloor2, V. Endris3, L. Tavernar3, A. Muckenhuber1, M. von Knebel-Döberitz2, R. Penzel3, W. Weichert1, A. Stenzinger3 1 TU München-Pathologisches Institut, München, Deutschland, 2Abteilung für Angewandte Tumorbiologie, Universität Heidelberg, Heidelberg, Deutschland, 3Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland

Background.  Synchronous colorectal carcinomas (sCRC) are clinically challenging variant of colorectal neoplasms. Although the epidemiological characteristics of sCRC have been described quite well in the liter-

ature, the molecular basis underlying these synchronous tumors is still poorly understood. Methods.  Therefore, comprehensive molecular profiling was performed on 23 sCRC comprising 50 primary tumors, 5 metastases and corresponding normal tissue by targeted deep sequencing using a CRC-specific panel of 30 CRC-realted genes, microsatellite analysis and MLH1-methylation analysis. Results.  Our study identified striking inter- and intratumoral heterogeneity in twenty (87 %) out of twenty-three sCRC cases, leaving only three cases with an identical genetic make-up. Genetic heterogeneity was frequent in clinically actionable genes, e. g. 44 % of cases harbored tumors of which one tumor was KRAS wildtype and the other was KRAS mutated. In addition, 48 % of the cases harbored at least double, sometimes even triple or quadruple mutations in KRAS, APC, TP53,PIK3CA and TGFBR2. Furthermore, microsatellite instability (MSI) was present in four cases (17 %), with one case presenting with one MSI- and one distinct microsatellite carcinoma. Conclusions.  Here, we demonstrate a striking genetic heterogeneity not only between different sCRC of a single case but also within a single tumor. These results contribute to the biological understanding of sCRC and directly impact genotyping strategies and oncological decision making. In the sCRC setting, testing one tumor or a single metastasis may not be sufficient, as clinically relevant and tumor-specific genetic information may remain undetected, compromising optimal oncological therapy.

AG13.06 Comparative analysis of primary and metastatic colorectal cancer M. Hühns*1, M. Linnebacher2, D. Koczan3, H. Murua Escobar4, B. Schneider1, F. Prall1 Universitätsmedizin Rostock, Institut für Pathologie, Rostock, Deutschland, Universitätsmedizin Rostock, Molekulare Onkologie und Immuntherapie, Rostock, Deutschland, 3Universitätsmedizin Rostock, Institut für Immunologie, Rostock, Deutschland, 4Universitätsmedizin Rostock, Klinik III, Hämatologie, Onkologie und palliative Medizin, Rostock, Deutschland

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Background.  Genetic heterogeneity between carcinomas and their metastases may reflect tumour progression or diversity of the primary tumour. In this study, we investigated the genetic heterogeneity between primary colorectal cancer (CRC) and their liver metastases. Methods.  Next generation sequencing (NGS) of 50 genes included in the Ion AmpliSeq Cancer Hotspot panel v2 (Ion Torrent) was done with 9 primary CRCs and their matched liver metastases, 8 microsatellite stable and one microsatellite instable. Results. As expected, microsatellite analyses (Bat25, Bat26, Cat25, 17p13.1, D9S942, D9S1748, D5S346; D5S1385) and methylation specific PCRs (MLH1, CDKN2A, NEUROG1, CRABP1, CACNA1G, MGMT, IGF2, SOCS2, RUNX3) did not reveal frequent differences, although differences in microsatellite stability (up to 2 markers) was observed in 4 cases and variations in methylation pattern (up to 3 methylation marker) occurred also in 4 cases. NGS identified mutations in 20 of the genes interrogated by the panel. These were frequent in the common candidate cancer genes (“mountains” of the genomic landscapes), such as TP53 (92 %), KRAS (71 %) and APC (64 %). Identical mutation patterns were observed in 6 cases. However, in 3 cases additional mutations in these genes were found in the primary tumour. Furthermore, diversity between primary CRC and metastasis occurred in less frequently mutated cancer genes (“hills”) mutations affecting IDH1, KIT, HRAS, ERBB4, AKT1, FBXW7, SMO and STK11 in the primary CRC and SMARCB1 and RB1 in metastases. Conclusions.  Our data indicate genetic heterogeneity between matched primary and metastatic colorectal cancer pairs. Patterns observed, on one hand, are consistent with tumour progression; however in a surprisingly high fraction a pattern more consistent with genetic diversity within the primaries was seen. More extensive analyses are necessary and underway.

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Abstracts AG13.08 Comparative evaluation of ccfDNA in CRC and NSCLC A.-L. Volckmar*1, R. Penzel1, V. Endris1, M. Singh2, S. Dietz3, C. Springfeld2, H. Sültmann3, P. Schirmacher1 Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, Nationales Centrum für Tumorerkrankungen (NCT), Heidelberg, Deutschland, 3Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Deutschland 1

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Background.  Circulating cell-free (ccf) DNA is released into the blood stream by both normal tissue and tumor cells after cellular necrosis or apoptosis. Hence it can potentially be a source for mutational analysis in the context of targeted therapies. This is of high interest for those patients that are not suitable for tissue biopsies. Although the suitability for ccfDNA for mutation detection has been proven under laboratory (ideal) conditions, real life data is still lacking. Therefore we are currently examining different approaches of ccfDNA in comparison to tissue based mutational testing workflows under real life clinical diagnostic conditions. Methods.  Blood of CRC and NSCLC patients with known EGFR/RAS mutation status was collected with minimal cell shearing in tubes with preservatives for ccfDNA (Streck, Amoy DX). The patients had late stage disease and were thus screened for mutations indicating targeted therapies. The ccfDNA was extracted from serum using the PME free-circulating DNA Extraction Kit (Analytik Jena), the Amoy DX Circulating DNA Kit (Amoy DX), or the QIAamp Circulating Nucleic Acid Kit (Qiagen). The ccfDNA was analyzed by using the qPCR-based KRAS mutation detection kit (Amoy DX), targeted Next-Generation Sequencing (IonTorrent PGM, CHPv2) and by digital droplet PCR (QuantStudio 3D Digital PCR System). Results.  The amount of ccfDNA obtained from identical blood serum samples varied strongly between the different extraction kits (2.5 ml serum/50 µl elution: PME free-circulating DNA Extraction: 4–4.3 ng/µl, Amoy DX Circulating DNA: 0.2–0.3 ng/µl, QIAamp Circulating Nucleic Acid: 0.7–1.3 ng/µl). For two extraction methods (PME free-circulating DNA Extraction, Amoy DX Circulating DNA), library amplification for NGS (AmpliSeq) could not be obtained. In ccfDNA of CRC and NSCLC samples obtained with the QIAamp Circulating Nucleic Acid Kit, the allelic mutation frequencies determined by NGS were extremely low (0.3–0.5 %, coverage >1000 reads) only slightly above the background level for wildtype controls. Variant analysis with a 0.3 % MAF threshold resulted in a high number of variants which were previously not detected in the same sample (NGS of tumor biopsy material). Conclusions.  For the reliable and precise detection of mutations using the currently available techniques (NGS and digital droplet PCR), the amount of ccfDNA and especially of the tumor ccfDNA in blood is critical. Hence, a marker for tumor derived ccfDNA in the sample is highly warranted.

AG13.09 Next-generation sequencing of circulating cell-free DNA from oesophageal carcinoma patients in primary staging H. Pasternack*1, J. Fassunke2, J. Weiss3, S. Chon4, S. Perner1, T. Zander3, A. Quaas2, H. Alakus4 Universitätsklinikum Schleswig-Holstein, Campus Lübeck und LeibnizForschungszentrum Borstel, Pathologie, Lübeck und Borstel, Deutschland, 2 Universitätsklinikum Köln, Institut für Pathologie, Köln, Deutschland, 3 Universitätsklinikum Köln, Klinik I für Innere Medizin, Köln, Deutschland, 4 Universitätsklinikum Köln, Klinik und Poliklinik für Allgemein-, Viszeral- und Tumorchirurgie, Köln, Deutschland

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Background.  Since new sensitive mutational analysis techniques, such as next-generation sequencing, were developed circulating cell-free tumour DNA carrying somatic mutations can be identified in plasma samples of cancer patients. These liquid biopsies supplement the histological diagno-

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sis of cancer, especially in the context of disease monitoring, resistance to targeted therapy and tumour heterogeneity. As gastro-oesophageal carcinomas are known to exhibit intratumoural heterogeneity molecular diagnostics of these tumours might benefit from the implementation of liquid biopsy mutational analysis. Therefore, we addressed the question whether circulating cell-free tumour DNA can be detected in plasma samples from oesophageal carcinoma patients in primary staging. Methods.  Blood samples and simultaneous tumour biopsies were taken from 25 patients with oesophageal carcinomas at the time of primary staging. Biopsies were histologically characterised by a senior pathologist and cryo-preserved. Plasma was gained from whole blood samples immediately after sampling and stored at -80°C. Genomic DNA from frozen tumour tissue as well as cell-free DNA from plasma samples were extracted. Mutational analysis of 12 genes was performed from all DNA samples using a GeneRead DNAseq Custom Panel V2. Results.  With the gene panel used in more than half of the tumour biopsies at least one somatic mutation was detected with next-generation sequencing. Using the matched cell-free DNA from the time of primary staging these mutations could only occasionally be detected in very low allele frequencies. In contrast, using the same assay a somatic mutation known from tissue based analysis could be easily detected in the circulating cell-free DNA of a patient carrying an oesophageal carcinoma in advanced metastatic stage. Conclusions.  Our study showed that circulating cell-free DNA from patients harbouring oesophageal carcinomas can hardly serve as liquid biopsy in order to detect somatic mutations at the early time of primary staging. However, plasma samples at later time points of advanced tumour disease may provide a valuable source of circulating cell-free tumour DNA, for example to monitor disease progression.

AG13.10 A new method to prevent carry-over contaminations in two-step PCR NGS library preparations V. Seitz*1, S. Schaper2, A. Dröge2, D. Lenze1, M. Hummel1, S. Hennig2 Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland, HS Diagnomics GmbH, Berlin, Deutschland

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Background.  Two-step polymerase chain reaction (PCR) strategies are a convenient approach to generate amplicon libraries suitable for next generation sequencing (NGS). However, there is a high risk of cross-contamination by carry-over of amplicons from first to second amplification rounds, potentially leading to severe misinterpretation of results. Methods.  We developed a new method able to prevent and/or to identify carry-over contaminations by introducing the K-box, a series of three synergistically acting short sequence elements. Our K-boxes are composed of (i) K1 sequences for suppression of contaminations, (ii) K2 sequences for detection of possible residual contaminations and (iii) S sequences acting as separators to avoid amplification bias. In order to demonstrate the effectiveness of our method, we analyzed two-step PCR NGS libraries derived from a multiplex PCR system for detection of T-cell receptor beta gene rearrangements. We used this system since it is of high clinical relevance and may be affected by very low amounts of contaminations. The contaminations were simulated employing spike-in contaminations. Results.  Spike-in contaminations were effectively blocked by the K-box even at high rates as demonstrated by ultra-deep sequencing of the amplicons. Conclusions.  We recommend implementation of the K-box as an additional feature in two-step PCR-based NGS procedures for research and diagnostic applications, where high sensitivity and reliability is mandatory.

AG13.11 Functional assessment of genetic tumor classifications D. Treue1, M. Bockmayr1, D. Heim1, G. Montavon2, J. Budczies1, A. Stenzinger3, C. Denkert1, M. Dietel1, K.-R. Müller2, F. Klauschen*1 Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland, Technische Universität Berlin, Berlin, Deutschland, 3Institut für Pathologie, Universitätsklinikum, Heidelberg, Deutschland 1

2

Background.  An increasing number of genetic alterations found in tumors is being used to complement the conventional organ- and tissue-based tumor classification system and some groups even propose novel genetic tumor classifications based on results from next-generation-sequencing studies. Methods.  Here, we present own experimental proteogenomic profiling data in combination with bioinformatic and machine learning-based modeling of molecular profiling data available through The Cancer Genome Atlas to study and evaluate the functional relevance of mutational profiling. Results.  Our results show the limitations of molecular tumor profiling that solely relies on gene sequencing without integrating histological and functional data and demonstrate how proteomics and computational modeling may be used to assess mutational profiles and identify driver mutations. Our analysis also provides an explanation for the negative outcomes of recent basket trials that used single genetic markers across cancers for therapy selection. Conclusions.  Integration of morphological, genomic and proteomic tumor profiling may help overcome the current limitations of precision oncology and improve therapy design.

AG13.12 Identification of the molecular drivers of endometrial cancer progression E. Bellini*1, N. Valtcheva1, C. Stirnimann2, M. Busquets Lopez2, B. Bodenmiller3, I. J. Frew4, P. J. Wild1 Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz, 2NEXUS Personalized Health Technologies, ETH Zürich, Zürich, Schweiz, 3Institut für Molekulare Biologie, Universität Zürich, Zürich, Schweiz, 4Physiologisches Institut, Universität Zürich, Zürich, Schweiz 1

Background.  Endometrial carcinoma is the most common malignancy of the female genital tract. The characterization of tissue microarrays (TMAs) containing a very large cohort of human endometrial cancer tissues showed that, although PI3K-mTOR pathway activation and TP53 inactivation play different roles in the initiation of different endometrial cancer subtypes, co-occurring alterations in both signaling pathways represent a frequent unifying pathogenic feature of late stage tumors of all subtypes [1]. Methods.  Our aim is to identify and functionally assess the role of the proteins crucial for the survival of endometrial cancer cells with the aforementioned molecular signature. For this purpose, we designed a PI3K pathway-dependent synthetic lethality screen using RNAi technology and used TALEN (TAL-effector nuclease) genome editing tool to knock-out PTEN (an important inhibitor of the PI3K pathway) in TP53-/- endometrial carcinoma cell lines. Results.  The signaling pathways of the identified targets will be further investigated in cell culture and in vivo experiments targeting the corresponding proteins. To confirm the aberrations critical for disease progression and assign them to each step of endometrial cancer development, we will further investigate the obtained results using the samples of our large human TMA cohort. Conclusions. Ultimately, this approach aims at using the knowledge gained from the cell culture model to decipher mechanisms commonly involved in the progression of the endometrial cancer of patients. The final goal is to characterize new prognostic and potentially predictive markers for improving personalised molecular diagnosis and treatment. References Wild PJ et al (2012) p53 suppresses type II endometrial carcinomas in mice and governs endometrial tumour aggressiveness in humans., EMBO Mol Med., doi: 10.1002/emmm.201101063

AG13.13 Imaging mass spectrometry (IMS) to discriminate colon from lung adenocarcinoma using formalin-fixed paraffin-embedded (FFPE) tissue R. Casadonte*1, M. Kriegsmann2, R. Longuespée3, P. Wandernoth3, C. Mohanu3, T. Katzenberger4, D. Aust5, G. Baretton6, J. Kriegsmann1, 7 Proteopath GmbH, Trier, Deutschland, 2Institut für Pathologie, Universitätsklinikum, Heidelberg, Deutschland, 3Molekularpathologie Trier, Trier, Deutschland, 4Klinikum Aschaffenburg, Institut für Pathologie, Aschaffenburg, Deutschland, 5Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Dresden, Deutschland, 6Institut für Pathologie, Universitätsklinikum, Dresden, Deutschland, 7MVZ für Histologie, Zytologie und Molekulare Diagnostik, Trier, Deutschland 1

Background.  There is an increasing need of novel diagnostic and prognostic biomarkers that discriminate between primary tumors. In this study, we specifically addressed the question whether it is possible to discern colon from lung carcinomas at the site of origin using high-throughput imaging mass spectrometry (IMS) technology on formalin-fixed paraffin-embedded (FFPE) tissue microarrays (TMAs). The ability to define proteomic profiles capable of such tumor typing makes IMS a valuable tool in cancer diagnostics and might complement currently used approaches. Methods.  TMAs were collected from patients with primary colon and lung adenocarcinoma. For each TMA specimen, one section was used for hematoxylin/eosin staining and another section was mounted onto an ITO-coated glass slide for IMS analysis. Sections were on-tissue digested with trypsin (0.1 µg/µL), and coated with CHCA matrix solution (7 mg/ ml). IMS data acquisition was performed with an Autoflex Speed TOF/ TOF mass spectrometer (Bruker Daltonik GmbH) at spatial resolution of 100 µm. A classification model was generated and validated with a linear discriminant analysis (LDA) algorithm model using SCiLS Lab software. Results.  The LDA classification model discerned colon from lung primary carcinoma with a sensitivity of 99.17 % and a specificity of 80.31 %. Furthermore, the classification model was applied to colon adenocarcinoma lung metastases. High similarity of protein expression profiles between the primary colon tumors and the colon metastases profiles were consistent with histopathological diagnosis. Conclusions.  Differentiation between colon and lung adenocarcinoma is possible on FFPE tissues by IMS. This expression map analysis may further provide a basis for subtyping metastasis from colon and lung carcinoma.

AG13.14 Mass-Spectrometry based proteomics of formalin-fixed human tissue – Distinction between primary lung tumors and lung-metastases with squamous cell carcinoma histology H. Bohnenberger*1, D. Yepes2, S. Merkelbach-Bruse3, A. Emmert4, K.-T. Pan5, A.-M. Lois1, L. Fischer4, H. Henric-Petri1, J. Strecker1, F. Bremmer1, K. Schmitz1, S. Küffer1, M. Hinterthaner4, M. Sebastian6, J. Kitz1, H.-U. Schildhaus1, H. Wolff7, M. Canis8, B. Danner4, R. Büttner3, P. Ströbel1, H. Serve2, H. Urlaub5, T. Oellerich9 1 Institut für Pathologie, Univeristätsmedizin, Göttingen, Deutschland, 2Klinik für Innere Medizin, Hämatologie/Onkologie, Goethe-Universität, UND Deutsches Krebsforschungszentrum (DKFZ) UND Deutsches Konsortium für translationale Krebsforschung (DKTK), Frankfurt, Deutschland, 3 Institut für Pathologie, Universitätsklinikum, Köln, Deutschland, 4Klinik für Thorax-, Herz- und Gefäßchirurgie, Universitätsmedizin, Göttingen, Deutschland, 5Bioanalytische Massenspektrometrie Gruppe, Max Planck Institut für Biophysikalische Chemie, Universitätsmedizin, Göttingen, Deutschland, 6Klinik für Innere Medizin, Hämatologie/Onkologie, GoetheUniversität, Frankfurt, Deutschland, 7Universitätsmedizin, Göttingen, Deutschland, 8Klinik für Hals-Nasen-Ohrenheilkunde, Universitätsmedizin, Göttingen, Deutschland, 9Abteilung für Hämatologie, Cambridge Institut für medizinische Forschung und Wellcome Trust/MRC Stem Cell Institut, University of Cambridge, UK, Cambridge, Vereinigtes Königreich

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Abstracts Background.  Histological differentiation of tumors in the lung with squamous cell histology is challenging as appropriate diagnostic biomarkers are lacking. Especially patients with head and neck cancer and a smoking history can develop both primary lung cancer and lung metastases. However, the discrimation is clinically important for risk and therapy stratification. Recent genetic studies identified multiple genetic subgroups of squamous cell carcinoma of the lung. However, as the correlation between genetic alterations and protein-expression is strongly influenced by co- and post-transcriptional as well as post-translational regulation, we characterized a broad panel of primary patient-derived formalin-fixed squamous cell carcinomas from lung and head and neck cancer by quantitative mass spectrometry to identify proteomic diagnostic biomarkers and potential novel drug targets. Methods.  Proteins were isolated from formalin-fixed paraffin-embedded (FFPE) patient-derived cancer tissues and subsequently mixed with an isotope labeled quantification standard that allows for quantification of thousands of proteins by high end mass spectrometry even from minimal amounts of microdissected tissue. Using bioinformatics we determined the protein expression patterns. The biomarkers discovered were validated by immunohistochemistry in additional independent tumor tissues. Results.  In this study we characterized the proteomes of squamous cell carcinomas derived from 30 patients with head and neck cancer, who developed lung tumors with similar histology in the course of their disease, of both the primary locus and the potential lung metastasis. Furthermore we characterized 60 pulmonary squamous cell carcinomas. Using the Super-SILAC-based mass spectrometric approach we were able to identify and quantify around 2500 proteins per sample. Unsupervised clustering analyses revealed that the detected protein expression patterns show a strong correlation with the origin of the analyzed carcinomas. Furthermore, secondary lesions in the lung in patients with squamous cell carcinoma of the head-neck-region could be classified as primary or metastatic cancer according to their protein expression profiles. Conclusions.  Collectively, by analyzing formalin-fixed tissue samples by high-end mass spectrometry, this study provides a large set of proteomic biomarkers that can be used to improve the differential diagnosis of squamous cell carcinoma/metastases in the lung.

AG13.15 A laser microdissection-based workflow for FFPE tissue microproteomics: from biomarker discovery to a supportive method for identification from MALDI Imaging analyses R. Longuespée*1, D. Alberts2, C. Pottier3, N. Smargiasso2, G. Mazzucchelli2, D. Baiwir4, M. Kriegsmann5, R. Casadonte1, J. Kriegsmann1, 6, E. De Pauw2 1 Molekular Pathologie Trier, Trier, Deutschland, 2University of Liège, Mass Spectrometry Laboratory, Systems Biology and Chemical Biology, Liège, Belgien, 3University of Liège, Department of Pathology, Liège, Belgien, 4 University of Liège, GIGA Proteomic Facility, Liège, Belgien, 5University of Heidelberg, Institute of Pathology, Heidelberg, Deutschland, 6MVZ for Histology, Cytology and Molecular Diagnostics Trier, Trier, Deutschland

Background.  Formalin-fixed and paraffin-embedded (FFPE) tissues handling has long been a major issue in tissue proteomics. This requires dedicated procedures to access the identity of the largest panel of proteins. Some proteomics experiments need working with restricted tissues sizes, especially when proteomes of cancers of early stages are explored. We designed different procedures in the context of sample amount downscaling. Methods.  Several extraction and digestion approaches, adapted from previous tissue proteomics procedures, were tested on breast cancer FFPE tissue samples obtained by laser micro-dissection (LMD), bearing approximately 2500 cells. The preparations were followed by nanoLC MS/MS analyses using Waters nanoACQUITY UPLC and Thermo QExactive instrumentations. Data processing was performed using MaxQuant and Perseus. Results.  First, experiment sets suggested that raw LMD sample has to be kept during sample preparation. Proteolytic digestion occurs directly on tissues

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and proteins are not fully extracted. The number of steps has to be limited to the minimum to retrieve an adequate number of proteins. Trypsin solution for on-tissue proteins digestion also has to be set to high concentration to compensate the fact that digestion is rather performed on raw samples than on protein extracts. Finally, an adapted procedure of the Citric Acid Antigen Retrieval approach, combined with adequate 2D-UPLC separation, allowed us to retrieve more than 1400 proteins on these samples of limited size. A proof of concept was proposed for the validation of the method, consisting in comparing 5 triple negative breast cancer (TNBC) of no special type (NST), and 5 of invasive lobular carcinoma (ILC). Even though the high similitude of the two cancer types, 10 proteins were found statistically significant more or less abundant between the tissues. These developments were applied to the analysis of proteomes of early stages of cervix cancer, restricted to few thousand of cells. This gave us the first insight of early events occurring for the development of this affliction, supported by the identification of several involved biomarkers. Conclusions.  Our study highlights essential issues for FFPE tissue preparation, regarding sample loss. This will open new opportunities for biomarker discovery from tissues with restricted size. In the next future, this approach would serve as a supportive method for the identification of proteins detected by MALDI imaging.

AG13.16 Molecular, morphological and survival analysis of 177 resected pancreatic ductal adenocarcinomas: Identification of prognostic subtypes A. Segler*1, A. M. Schlitter1, C. Jäger2, N. Pfarr1, K. Steiger1, V. Endris3, I. Regel4, B. Konukiewitz1, B. Kong2, M. Bettstetter1, C. Michalski5, J. Kleeff6, G. Klöppel1, I. Esposito4 Institut für Allgemeine Pathologie und Pathologische Anatomie der Technischen Universität München, München, Deutschland, 2Chirurgische Klinik und Poliklinik, Klinikum Rechts der Isar der Technischen Universität München, München, Deutschland, 3Institut für Humangenetik, Universitätsklinikum, Heidelberg, Deutschland, 4Institut für Pathologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Deutschland, 5 Chirurgische Universitätsklinik Heidelberg, Universität Heidelberg, Heidelberg, Deutschland, 6Department of Surgery, Royal Liverpool and Broadgreen University Hospital, University of Liverpool, Liverpool, Vereinigtes Königreich

1

Background.  Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumor with poor prognosis. Recent data suggest that there are molecular subtypes differing in clinical outcome and drug response. This study examines the association between genetic profile, histopathology and survival. Methods.  Surgical specimens and follow-up data from 177 resected PDAC patients were morphologically reviewed and genetically analyzed. All tumors were classified according to their predominant growth pattern. KRAS (exon 2–4), TP53 (exon 5–9), CDKN2A/p16 and SMAD4 were analyzed by a combination of immunohistochemistry, real-time PCR with high-resolution melting analysis (HRMA), direct Sanger sequencing and loss of heterozygosity (LOH) analyses. Additionally, next generation sequencing (NGS) of a large cancer gene panel was performed in selected cases. Results.  The tumors were classified as typical PDAC (51 %), as PDAC with a predominant (>50 % of the tumor) pattern (cribriform, papillary, clearcell, complex and others) (41 %) and variants 7.3 %. Three of the variants (1.7 %) showed a pure tubular pattern, suggesting a special PDAC subtype. Genetic alterations in at least two out of four genes were found in 95 % of the patients, with highest frequencies in KRAS (93 %), TP53 (79 %) and CDKN2A/p16 (75 %) and lowest in SMAD4 (37 %). Patients with either wild type KRAS or wild type CDKN2A/p16 lived significantly longer than those with altered genes (p = 0.018, median survival >45 vs. 19.7 months; p = 0.006; 36.9 vs. 18.8 months, respectively). Multivariate Cox proportional hazard model identified wild type KRAS (in addition nodal status and grading as independent predictive variable for survival (p = 0.019,

HR 1.8). The 3 patients with tubular PDAC survived for >68.8, >55.3 and 19.3 months. Conclusions.  Our findings suggest that the mutational status of KRAS, but also of CDKN2A/p16, SMAD4 and TP53, correlates with the outcome of patients with PDAC. Survival is also associated with certain morphological subtypes such as the tubular variant. Future pathology reporting of pancreatic ductal adenocarcinoma may include the KRAS status.

AG13.17 Induction of chromosomal instability (CIN) by cooperative activation of yes-associated protein (YAP) and FOXM1 S. Weiler*1, F. Pinna1, T. Lutz1, T. Wolf1, S. Rössler1, S. Wan1, S. Singer1, M. Knaub1, J. Marquardt2, H. Lang3, V. Ustiyan4, J.-S. Lee5, P. Schirmacher1, V. Kalinichenko4, K. Breuhahn1 Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, Innere Medizin I, Universitätsklinikum, Mainz, Deutschland, 3Klinik für Allgemein-, Viszeral- und Transplantationschirurgie, Universitätsklinikum, Mainz, Deutschland, 4Cincinnati Children’s Hospital Medical Center, Cincinnati, Vereinigte Staaten von Amerika, 5MD Anderson Cancer Center, Houston, Vereinigte Staaten von Amerika 1

2

Background.  Chromosomal instability (CIN) is a main characteristic of aggressive tumors and is associated with poor patient prognosis. For different tumor entities Carter et al. [1] defined gene expression signatures (CIN25 and CIN70) which correlate with CIN and poor overall survival of cancer patients. To date, no regulatory mechanisms have been described that link the presence of the CIN signature to altered cellular signaling pathways. In this study we examined the role of the Hippo pathway effector Yes-associated protein (YAP) in the regulation of CIN in hepatocarcinogenesis. Methods.  SiRNA inhibitions, treatment with different inhibitors, Luziferase- and Chromatin-Immunoprecipitation (ChIP) as well as Co-Immunoprecipitation experiments were performed to study the effect of YAP on FoxM1 and CIN genes in different HCC cell lines. Immunofluorescence and immunohistochemistry were used to visualize protein expression in a transgenic mouse model for liver-specific and inducible expression of active YAPS127A and in human tissue samples. Microarray data for human HCC were analyzed for the presence of the CIN signature. Results.  YAP regulated CIN signature genes in different HCC cell lines. Luciferase and ChIP assays showed that YAP induced CIN gene expression by directly regulating the transcription factor FoxM1, which is also part of the CIN signature. Moreover, an interaction between YAP and FoxM1 was detected. In vivo, liver-specific overexpression of YAP led to liver overgrowth and eventually cancer formation [2]. These mice also showed increased expression levels of FoxM1 and CIN signature genes and exhibited histo-morphological signs of chromosomal instability. YAP expression also correlated significantly with FoxM1 and CIN genes in human tissues as assessed by tissue-microarray staining. Analysis of gene expression data from HCC patients illustrated that patients with high expression levels of the CIN signature genes showed the worst overall survival and an early cancer recurrence [3]. Conclusions.  These data demonstrate that YAP and its downstream effector FoxM1 represent master regulators of a CIN signature, which characterizes HCC patients with poor clinical outcome. These patients might benefit from treatment with YAP and/or FoxM1 inhibitors such as Verteporfin (Visudyne®). References 1. Carter SL et al (2006) Nat Genet 38(9):1043–1048 2. Tschaharganeh D et al (2013) Gastroenterol 144(7):1530–1542 3. Roessler S et al (2010) Cancer Res 70(24):10202–12

AG13.18 What’s new – Update parallel sequencing and liquid biopsies S. Merkelbach-Bruse* Institut für Pathologie, Universitätsklinikum, Köln, Deutschland Background.  Update parallel sequencing and liquid biopsies Parallel sequencing on formalin-fixed, paraffin-embedded tissue is already used in several diagnostic laboratories for mutational screening of cancer-related oncogenes across several major cancers. Besides mutational testing, several other applications of parallel sequencing are conceivable. One approach was the concomitant analysis of mutational and microsatellite status in colorectal cancer. This could be achieved by combining amplicon-based targeted parallel sequencing with sequencing of non-coding mononucleotide repeat regions. The nucleotide loss in these regions was quantified and compared to results obtained by using the standard Bethesda primer panel. Amplicon-based parallel sequencing assays do not routinely detect chromosomal rearrangements or copy number changes. Thus, these aberrations are still widely analysed by Fluorescence in situ Hybridization (FISH) and immunohistochemistry (IHC). The development of technologies for the detection of all therapy relevant genomic alteration in one assay is an ongoing process. We tested different technologies enabling the detection of these aberrations using RNA or DNA as source. As one approach for RNA based parallel sequencing, we tested the OncomineTM Solid Fusion Transcript Kit (Thermo Fisher Scientific) on samples previously analysed by FISH and IHC. In addition to the reliable and simultaneous detection of specific translocations, the implemented 3’-5’imbalance assay facilitates the detection of so far unknown translocations. On DNA-level hybrid capture-based parallel sequencing is used for the simultaneous detection of different types of aberration. In this approach, custom designed, biotinylated DNA probes are hybridised to target sequences to allow for sequence enrichment. Libraries containing the target sequences are sequenced on platforms like Illumina’s NextSeq 500. The sequenced data can be analysed for somatic gene mutations, copy number alterations and genomic rearrangements Since new sensitive mutational analysis techniques were developed, circulating tumour DNA carrying somatic mutations can be identified in plasma samples of cancer patients. These liquid biopsies supplement the histological diagnosis of cancer, especially in the context of disease monitoring, resistance to targeted therapy and tumour heterogeneity. Crucial steps in analysis are the preparation of plasma samples and the sensitive detection of mutations with low allelic frequencies.

AG13.20 Molecular pathology update of adrenocortical neoplasms in 2016 H. Sasano* Tohoku University School of Medicine, Department of Pathology, Sendai, Japan Background.  Recently numerous somatic and genomic alterations of adrenocortical neoplasms have been reported but it is important for pathologists to recognize significance and limitations of these reported findings especially related to diagnostic or clinical relevance. In adrenocortical adenomas, somatic mutations of KCNJ5 encoding GIRK4 potassium channels have been reported in 40–60 % of the cases of aldosterone producing adenoma (APA) and those of calcium channels related genes such as ATP1A1, ATP2B3 and CACNA1D also identified in APA. Gain-offunction somatic point mutations in PRKACA, one of the catalytic subunits of protein kinase A have been reported in almost half of Cushing’ s adneoma. However, no distinctive clinicopathological differences have been reported those mutated and non-mutated adrenocortical adenomas. The extensive molecular analysis of adrenocortical carcinoma (ACC) revealed various genetic abnormalities in addition to further identifyicaDer Pathologe Suppl 1 · 2016

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Abstracts tion of the susceptibility of developing ACC in some hereditary disorders. Gene expression profiling studies also defined transcriptional signatures of ACC with IGF2 over-expression as most representative one. MicroRNAs (miRs) as well as DNA methylation profilings, which could be performed in archival materials also classified ACC into low and high risk groups. Integrated genomic characterization revealed p53 mutation dominance in the pediatric ACC cases as well as the frequent and diverse Wnt pathway defects such as CTNNB1 point mutations and ZNRF3 deletions in adult ACC. However, molecular classification of ACC was not that different from that obtained by conventional histopathological study and the relative lack of targetable hotspot mutations made it difficult to evaluate those findings at clinical setting unless employing pan-genomic analysis .

AG13.21 New developments in Molecular Diagnostics of Thyroid Neoplasms K. W. Schmid* Universitätsklinik Essen, Institut für Pathologie, Essen, Deutschland Molecular pathology is playing an increasingly important role in the diagnosis, prognosis and decision making in thyroid pathology. Mutations of the BRAF gene (V600E) as well as rearrangements of RET/PTC and NTRK1 are reliable markers for the diagnosis of papillary thyroid carcinoma (PTC). RAS mutations and PPAR-gamma rearrangements are helpful to distinguish hyperplastic nodule from follicular neoplasia; this can be successfully used to improve fine needle biopsy efficiency. However, so far no molecular markers are available to distinguish follicular adenoma from follicular carcinoma. Mutations of TERT or PIK3CA can be used to identify tumours (follicular carcinoma [FTC], poorly differentiated carcinoma [PDTC]) with a less favourable outcome. Mutations of the p53 gene are also indicating dedifferentiation; almost 40 % of PDTC and 90 % of anaplastic carcinoma (ATC) show p53 muations. Mutations of the RET-protoongene are found in almost all hereditary medullary thyroid carcinoma (MTC); in dependence of the location of the mutation on the RET gene the hereditary MTC show a broad clinical range allowing to subdivide these tumours in four risk classes (ATA A – D) with a strong influence on the clinical management of hereditary MTC. 40–60 % of sporadic MTC also show (somatic) RET mutations; although RET mutations in sporadic MTC is associated with a poorer clinical outcome, recently obtained data have demonstrated, that sporadic MTC with RET a mutation are susceptible to modern targeted therapies.

The aim of this study was to assess the resistance rates to clarithromycin (CLA) and fluorochinolones (FLQ) in H.pylori infections in Germany and hence, to underline the validity of resistance testing prior to first line therapy based on the recommendations of the Maastricht IV/Florence Consensus Report [1]. Methods.  In an unselected cohort (patients with and without prior eradication treatment) of 838 H. pylori positive gastric biopsies submitted to the Institutes of Pathology of the Bundeswehr Hospitals in Koblenz and Ulm between 2008–2015 the potential resistance to CLA and FLQ was evaluated. Therefore, a subset of resistance mediating mutations in the bacterial 23S RNA and gyrA genes were determined using the commercially available GenoTypeHelicoDR®test kit. Results.  In 36.5 % of the unselected sample cohort resistance mediating mutations were found. Specifically, 21 % of the patients harbor H. pylori strains with mutations leading to CLA resistance whereas 20 % contain FLQ resistant strains. Concomitant mutations in the gyrA as well as 23S RNA gene causing double resistance were detected in 4.5 % and predominantly in older patients. In this context, patients containing FLQ resistant H. pylori tended to be significantly older than patients harboring CLA resistant strains. Interestingly, in a subgroup of the cohort (n = 239) a potential primary resistance to CLA could be detected in 13 % and to FLQ in 21 % of the patients, respectively. Conclusions.  The primary resistance rate of 13 % to CLA detected in this study is close to the cutoff value of 15 % recommended by the Maastricht IV/Florence Consensus Report and therefore, suggests a susceptibility testing prior to first line therapy. Considering a primary resistance rate of 21 % to FLQ, a FLQ-based therapy is also not advisable without prior resistance testing. Moreover, the double resistance rate of 4.5 % to CLA and FLQ challenges alternative treatment regimens. Taken together, to avoid increasing resistance and therapy failure rates of CLA and FLQ based therapies a prior susceptibility testing is recommended in Germany. References 1. Malfertheiner P et al (2012) The European Helicobacter Study Group (EHSG), Management of Helicobacter pylori infection–the Maastricht IV/Florence Consensus Report., Gut, 646–664, 61

P01.02 Gastric cancer as first sign of juvenile polyposis H. Geddert*1, F. Flohr2, U. Müller-Reinhartz3, G. Faller1, A. Dimmler1 Institut für Pathologie, St. Vincentius-Kliniken, Karlsruhe, Deutschland, Medizinische Klinik, Endokrinologie und Gastroenterologie, St. VincentiusKliniken, Karlsruhe, Deutschland, 3Klinik für Allgemein-, Viszeral- und Gefäßchirurgie, St. Vincentius-Kliniken, Karlsruhe, Deutschland 1

Postersitzungen der Arbeitsgemeinschaften der DGP Postersitzungen AG Gastroenteropathologie I und II P01.01 Resistance to Clarithromycin and Fluorochinolones in Helicobacter pylori infections in Germany V. G. Umathum*1, A. Ammon1, I. Engels1, K. Kraft2, K. Steinestel2, A. Arndt2 1 Bundeswehrzentralkrankenhaus Koblenz, Institut für Pathologie und Molekularpathologie, Koblenz, Deutschland, 2Bundeswehrkrankenhaus Ulm, Institut für Pathologie und Molekularpathologie, Ulm, Deutschland

Background.  Persistent infection with Helicobacter pylori is a major causative agent for the development of gastric cancer and thus, a successful eradication of this bacterium is mandatory. However, initial eradication failure occurs in a considerable amount of patients due to increasing antibiotic resistance rates.

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Background.  Due to severe anemia in a 49 year old female patient gastroscopy and colonoscopy is performed. Methods.  Two small polyps are removed from the colon. Histologically these polyps are assesed as juvenile polyps. During gastroscopy countless polyps are detected in the gastric mucosa. The largest one nearly occludes the antral lumen. The corpus shows two large suspicious polyps. Histologcal examination of the gastric specimen favours juvenile polyps with dysplasia. Results.  During radical gastrektomie an invasive carcinoma in the corpus is diagnosed by frozen section. The mucinous carcinoma infitrates the muscular layer. Three regional lymph node metastases are present (pT2 pN2 (3/32) L1 V0 G3). The entire gastric mucosa consists of countless juvenile polyps. In the the lung, an arteriovenous malformation is detected within the right lower lobe. Conclusions.  In this patient juvenile polyposis presents mainly with gastric polyps. The neoplastic potential of these polyps is highlighted by the concurrent gastric cancer.

P01.03 Juvenile polyposis with germline SMAD4 mutation and gastric involvement S. Opitz*, C. Wittekind

most conclusive that this tumor derived from some kind of heterotopic tissue. Although some cases of ductal pancreatic carcinoma and insulinoma have previously been described, there has not yet been a description of mucinous carcinoma in this location. It can be speculated that the originator was another kind of heterotopic tissue such as Brunner glands or paraesophageal glands.

Universitätsklinikum Leipzig, Institut für Pathologie, Leipzig, Deutschland Background.  Familial juvenile polyposis (FJP) represent a rare autosomal dominant hereditary disorder that is characterized by the development of multiple juvenile polyps in the upper and lower gastrointestinal tract and an increased risk for cancer. Currently, germline mutations, including mutations within BMPR1A, SMAD4 and PTEN genes, have been described in patients with FJP. Exclusively gastric form of juvenile polyposis, associated with germline SMAD4 mutation, is a rare clinical entity. Methods.  Case Report. Results.  Here we represent a case of a 33 years old woman with FJP with a germline mutation in the SMAD4 gene associated with multiple colorectal polyps among massive gastric polyposis, resulting in a giant stomach with a greater curvature of the stomach of 76 cm. Progressive complications, primarily hematemesis with severe anemia, ultimately necessitated a total gastrectomy. Conclusions.  The case represent the rare association of FJP with SAMAD4-germline mutation and gastric involvement. Especially in cases with SMAD4-alterations, close clinical follow with repeated endoscopic examination is mandatory.

P01.04 Mucinous carcinoma in the gastric wall, probably arising from heterotopic tissue L. Morawietz*1, F. Marusch2 Diagnostik Ernst von Bergmann GmbH, Institut für Pathologie, Potsdam, Deutschland, 2Klinikum Ernst von Bergmann gGmbH, Klinik für Allgemeinund Viszeralchirurgie, Potsdam, Deutschland

1

Background.  Heterotopic pancreatic tissue is a common incidental finding in the gastric wall. Some cases of pancreatic type neoplasms (ductal carcinoma as well as insulinoma) have been described. However, there are as yet no reports of mucinous carcinoma arising in heterotopic tissue. Methods.  This is a case report of an unusual gastric tumor in an 82 year old female. The patient presented with anemia. Endoscopy relvealed a navel shaped retraction in the anterior wall between corpus and antrum, suspicious of an underlying tumor such as GIST. High resolution computed tomography showed a well demarcated round tumor measuring 25 × 21 mm in the gastric wall and no signs of other tumors in any of the abdominal, retroperitoneal or pelvic organs. The patient had no history of cancer. Laparoscopic wedge resection was performed and the specimen was sent in for routine histopathologic evaluation. Results.  The tumor showed clear borders and a gelatinous cut surface. Histologic examination revealed an epithelial neoplasm with papillary growth pattern, small stroma stalks and branches, cylindric tumor cells with abundant mucin and oval shaped, mid-size nuclei as well as numerous atypical mitotic figures. The morphology was mostly reminiscent of mucinous ovarian carcinoma. Most of the tumor was located below the muscularis mucosae and above the muscularis propria. However, a small portion of the tumor showed a more ductular morphology and had a focal connection to the basis of some gastric foveolae. Immunohistochemistry revealed heterogeneous positivity for CK7, CK20 and CDX2, while CA125, CA19.9 and HER2 were negative. Mismatch repair proteins were present. Neither heterotopic pancreatic tissue nor lymph node structures could be detected. Conclusions.  Regarding the undermining growth pattern and the microstructure, the tumor could not simply be regarded as gastric carcinoma. A metastasis of another tumor such as ovarian carcinoma seemed similarly unlikely. Since heterotopic pancreatic tissue is a common finding in this location, it is

P01.05 Hyperplastic gastric polyposis in a patient with a concomitant neuroendocrine tumor of the pancreas and a mucinous neoplasm of the vermiform appendix R. Rottscholl*1, H.-B. Reith2, H. Sostmann3, I. Berger1 Institut für Pathologie, Klinikum Kassel, Kassel, Deutschland, 2Agaplesion Diakonie Kliniken Kassel, Klinik für Allgemein- und Viszeralchirurgie, Kassel, Deutschland, 3Agaplesion Diakonie Kliniken Kassel, Klinik für Innere Medizin, Abteilung für Gastroenterologie, Kassel, Deutschland 1

Background.  Hyperplastic gastric polyposis is a rare entity defined by the presence of 50 or more hyperplastic gastric polyps. Only a few cases have been described and although familiar clustering with a slightly increased incidence of adenocarcinomas does occur, most cases seem to be sporadic. Methods.  We report a case of a 68 year old male with a history of more than two years of repeated iron deficiency anemias with multiple esophagogastroduodenoscopies and polypectomies. The patient also suffered from a seronegative rheumatoid arthritis treated with methotrexate for years. Finally, the patient underwent total gastrectomy with Oesophagojejunoplication Siewert-Pieper and a small conspicuous node inferior to the pancreatic head was also resected during the procedure. On postoperative day four the patient developed a paralytic ileus and relaparotomy was necessary, where the vermiform appendix was removed. Results.  An unopened gastrectomy specimen was submitted for histologic evaluation. The whole mucosal surface was covered by abundant viscous mucus and far more than 50 sessile and pedunculated polyps effaced the gastric mucosa. Microscopic evaluation of the polyps showed tortuous and partly branching glands lined by foveolar epithelium, focal surface erosion was present and at some spots the nuclei were enlarged and the nuclear-cytoplasmic ratio was increased. The diagnosis of hyperplastic gastric polyposis with focal moderate low-grade dysplasia was made. The pancreatic node removed in the same session measured 1.3 cm in diameter. It was cystic and the inner wall was lined by a trabecular arranged epithelium consisting of cuboidal cells with pink cytoplasm and round uniform nuclei with granular “salt and pepper” chromatin. The neoplastic cells were immunohistochemically positive for CD56, Synaptophysin and Chromogranin, the diagnosis of a cystic neuroendocrine tumor was made. The vermiform appendix was removed on postoperative day four. The appendiceal mucosa showed a partly branched epithelial neoplasm lined by a mucinous epithelium with focal pseudostratified nuclei and increased mitotic activity, leading to the diagnosis of an appendiceal low-grade mucinous neoplasm. Conclusions.  Hyperplastic gastric polyposis and its coincidence with a cystic neuroendocrine tumor of the pancreas and a mucinous low-grade neoplasm of the vermiform appendix suggests a syndromal disease in this 68 year old patient.

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Abstracts P01.06 Histological and molecular characteristics of a large pancreatic cancer patient-derived xenograft collection and their association with clinical parameters P. Bronsert*1, 2, 3, T. Kees4, B. Zeitouni4, A.-L. Peille4, U. Wellner5, S. Timme3, M. Werner1, 2, 3, O. Schilling6, J. Schüler4, H. Fiebig4, S. Küsters7, V. Vuaroqueaux4 Tumorzentrum Freiburg, Universitätsklinikum, Freiburg, Deutschland, Deutsches Konsortium für translationale Krebsforschung (DKTK), DKFZ, Heidelberg, Deutschland, 3Institut für Klinische Pathologie, Department für Pathologie, Universitätsklinikum, Freiburg, Deutschland, 4Oncotest GmbH, Freiburg, Deutschland, 5Klinik für Chirurgie (UKSH), Campus Lübeck, Lübeck, Deutschland, 6Institut für Molekulare Medizin und Zellforschung, Universitätsklinikum, Freiburg, Deutschland, 7Chirurgische Abteilung, Universitätsklinikum, Freiburg, Deutschland 1

2

Background.  Pancreatic ductal adenocarcinoma represents one of the leading cancer-related death causes. Stromal content and activation status are strong prognosticators impeding the development of new therapy options. Recent progress in the molecular classification of pancreatic cancer indicates that pancreatic tumors may be grouped into different subtypes, with prognostic and therapeutic implications. To evaluate new therapeutic approaches, good pre-clinical models are needed. Patient-derived xenograft (PDX) models reflect patient tumors very well. Interestingly, only little is known about human stroma replenished by murine stromal components in PDX models and the translation back onto patients’ outcome. Here, we report molecular and stromal characteristics of a pancreatic cancer PDX cohort comprising 42 successfully and 24 non successfully engrafted patient tumor samples. Methods.  All models were characterized for histology features. Whole exome mutations (Hiseq2000), chromosome rearrangements and gene copy number variations (Affymetrix SNP6) and gene expression (Affymetrix U133 Plus2.0) were performed for engrafted models only. Results.  Engraftment success rate correlated with poor patient survival (p = 0.024). Absolute stromal contents in the PDX models were lower than in the primary patient tumors (P0), but relative amounts significantly correlated (p = 0.009). Histological PDX (p = 0.0017) and P0 (p < 0.001) stromal activation status respectively correlated with patients overall survival. Syndetic correlations between PDX and P0 were not detectable (p = 0. 444). Molecular analyses revealed three subtypes transcriptomical subtypes (quasi mesenchymal, classic and exocrine), correlating with stroma activation (p = 0.006), but not with stroma content or patients overall survival. All three subtypes demonstrated different genomic alteration profiles including for mutated genes and amplification/deletions. Contrary to published data, molecular subtype did not correlated with tumor growth inhibition via Erlotinib or Gemcitabine in PDX. Conclusions.  PDX models contain indicators of pancreatic cancer patient outcome and treatment. Pancreatic PDX models preserved capabilities to develop proportional stroma contents compared with P0, correlating with patients survival. Stroma content and activation were associated with gene expression subtypes. Altogether, PDX models reflect primary tumor characteristics and represent an ideally model for translational pre-clinical testing.

P01.07 MYC, EGFR and FGFR2 expression and response and survival in neoadjuvant treated gastric cancer L. Bauer*1, A. Munzig1, J. Slotta-Huspenina1, A. Novotny2, K. Becker1, A. Hapfelmeier3, G. Keller1 Institut für Allgemeine Pathologie und Pathologische Anatomie der Technischen Universität München, München, Deutschland, 2Chirurgische Klinik und Poliklinik, Klinikum Rechts der Isar der Technischen Universität München, München, Deutschland, 3Institut für medizinische Statistik und Epidemiologie, TU München, München, Deutschland 1

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Background.  A three-gene predictor encompassing MYC, EGFR and FGFR2 has been shown to predict survival in cisplatin and fluorouracil – treated metastatic gastric cancer (GC) patients by others [1]. The aim of our study was to determine the value of the expression of these three genes to predict response and/or survival for GC patients treated by a platin/5FU based chemotherapy in the neoadjuvant setting. Methods.  Expression was analysed in pretherapeutic tumour biopsies of 69 patients treated by platinum/5FU based neoadjuvant chemotherapy. Histopathological response was divided into three grades, tumour regression grade 1 (TRG1) corresponding to complete/major response, TRG2 corresponding to partial and TRG3 corresponding to minimal or no response. mRNA was isolated from manually microdissected formalin fixed and paraffin embedded tumor biopsies. Gene expression was quantified by qPCR. Results.  Performing dichotomization of the patients based on median gene expression levels revealed a significant association of high MYC expression with minimal or no response (TRG3) (p = 0.013). No other associations with response were observed for EGFR or FGFR2 expression in the study as a whole or in a separate analysis of patients with intestinal and nonintestinal type tumours. Testing various combinations of the expression of the three genes did not reveal a stronger association with response when compared to MYC expression alone. In the study as a whole, no statistically significant associations with overall survival were found. In the subgroup of nonintestinal-type tumours, univariate Cox regression analysis revealed a significantly increased risk of death for patients with a high expression of MYC (HR 59.56, 95 %CI 1.28–2776.3; p = 0.037). Conclusions.  Our results indicate a prominent role of MYC expression to predict lack of tumour shrinkage after platin/5FU-based neoadjuvant chemotherapy and they suggest a prognostic relevance of this gene in particular for patients with nonintestinal type gastric carcinomas. References 1. Kim HK et al (2012) Three-gene predictor of clinical outcome for gastric cancer patients treated with chemotherapy, The Pharmacogenomics Journal, 12:119–127

P01.08 PIK3CA-mutations are significantly enriched in EBV-positive gastric carcinomas – evaluation of a large Central European patient cohort C. Böger*, H.-M. Behrens, S. Krüger, J. Haag, C. Röcken Institut für Pathologie, Christian-Albrechts-Universität, Kiel, Deutschland Background.  PIK3CA alterations are suggested to be predictive for a therapy with the mTOR-inhibitor Everolimus, which makes PIK3CA an interesting target in the search for potential predictive biomarkers of gastric cancer (GC). Moreover, recent whole-genome sequencing and comprehensive molecular profiling of GC associated PIK3CA-mutations and Epstein-Barr virus (EBV) infection with the CpG island methylator phenotype. Here we aimed to validate the occurrence, phenotype and clinico-pathological characteristics of PIK3CA-mutated and EBV-positive GCs in an independent Central European gastric cancer cohort consisting of 484 patients. Methods.  Mutational analyses of exons 9 and 20 of the PIK3CA gene were performed by pyrosequencing on a PyroMark Q24 instrument. EBV encoded RNA was detected using the in situ hybridization (ISH) EBER-probe. Immunohistochemical stainings regarding the EBV-latency type were carried out with a Bondmax automated slide staining system, using monoclonal antibodies directed against EBNA2 (clone PE2), ZEBRA (clone BZ1) and LMP1 (clone CS1-4). The results were correlated with various clinico-pathological patient characteristics and patient survival. Results.  Twenty-two of 477 (4.6 %) tumors had a PIK3CA mutation in exon 9 [13 (59 %)] or exon 20 [9 (41 %)]. A transition (A>G, G>A) was found in 21 cases, a transversion (A>T) was present in one case in exon

20. No significant correlation was found between the PIK3CA mutational status and any clinico-pathological patient characteristic, except the mucin phenotype. EBV-RNA was found in 22 (4.5 % of 484 valid results) GCs, of which 4 GCs (20 %) showed a heterogeneous EBER-positivity. All 22 EBV-positive GC were immunonegative for EBNA2, LMP1 and ZEBRA, respectively, and were classified as latency type I neoplasms. EBV-infection was significantly more prevalent in men (6.6 % vs. 1.1 %; p = 0.003) and in intestinal and unclassified GCs according to Laurén (p = 0.005). MSI and EBV were mutually exclusive. EBV-positive GCs carried significantly more commonly PIK3CA-mutations compared with EBV-negative GCs (p < 0.001). No significant difference was found in the overall and tumor specific survival between PIK3CA-mutated GCs, EBV-positive GCs, EBV-positive/PIK3CA-mutated and EBV-negative/PIK3CA-wildtype GCs. Conclusions.  Our study confirms the occurrence and clinico-pathological characteristics of EBV-positive GCs, and further confirms the enrichment of PIK3CA-mutations in EBV-positive GCs for the first time for a large Central European patient cohort.

P01.09 Her2 Amplification in a Kindred with Hereditary Diffuse Gastric Cancer L. Wilsberg*1, S. Dango2, A. König3, K. Schmitz1, A. Zibat4, B. Zoll4, M. B. Ghadimi2, H.-U. Schildhaus1 Abteilung für Pathologie, Universitätsmedizin, Göttingen, Deutschland, Allgemein-, Viszeral- und Kinderchirurgie, Universitätsmedizin, Göttingen, Deutschland, 3Gastroenterologie und gastrointestinale Onkologie, Universitätsmedizin, Göttingen, Deutschland, 4Humangenetik, Universitätsmedizin, Göttingen, Deutschland 1

2

Background.  Hereditary diffuse gastric cancer is a dominantly inherited condition which increases the risk of developing diffuse gastric cancer. It is characterized by signet ring cell (diffuse) gastric cancer, usually spreading in the superficial gastric mucosa. The signet ring cells show expression loss of e-cadherin in immunhistochemical staining which is due to germline mutation in CDH1 gene. These mutations can be detected in up to 30 to 40 % of cases. Methods.  We present a family with five confirmed cases of hereditary diffuse gastric cancer.The oldest family member was diagnosed at the age of 53 years, the youngest at the age of 24 years.All five patients underwent total gastrectomy and some received neoadjuvant chemotherapy. Tissue samples were histopathologically processed and stained during our daily routine. Results.  By sequencing of the CDH1 gene a truncating mutation was found. All cancer samples where positive for Her2 immunhistochemical staining and Her2 FISH. One patient was therefore treated with trastuzumab. Conclusions.  Her2 overexpression and/or gene amplification has been shown to be effective in the treatment of advanced gastric cancer. However, Her2 positivity was thought to be predominantly restricted to intestinal type adenocarcinomas. Here we demonstrate the unexpected finding of Her2 positivity in a kindred with genetically proven hereditary diffuse gastric cancer. This underlines the importance of Her2 testing in gastric cancer as a predictive biomarker and could provide evidence that the clinical spectrum of hereditary gastric cancer is larger than previously regarded.

P01.10 The role of the cell adhesion molecule E-cadherin in cetuximab sensitivity of gastric cancer cell lines S. Keller*, B. Mühlthaler, M. Krummhaar, A. Marchetto, J. Platzek, B. Luber Institut für Allgemeine Pathologie und Pathologische Anatomie der Technischen Universität München, Klinikum Rechts der Isar, München, Deutschland Background.  The transition from benign to invasive and metastatic tumors is characterized by loss of cell-cell adhesion. In gastric cancer, the cell adhesion molecule E-cadherin is frequently mutated and consequently functionally inactivated. E-cadherin interacts with the epidermal growth factor receptor (EGFR) in a multicomponent complex and negatively influences the receptor activation, whereas somatic mutation of E-cadherin is associated with increased activation of EGFR [1]. The crosstalk between EGFR and E-cadherin is potentially relevant for tumor progression and response to EGFR-targeted therapy [2]. Cetuximab is a therapeutic monoclonal antibody that targets EGFR. The aim of the project was to clarify the role of E-cadherin in cetuximab sensitivity of gastric cancer cell lines Methods.  Gastric cancer cell lines KATOIII, MKN28, MKN45; overexpression (mutated or wild-type E-cadherin) and silencing of E-cadherin by siRNA transfection; Western blot analysis; life-cell imaging Results.  We previously analyzed the effect of E-cadherin expression and mutation on the cetuximab response in gastric cancer cell lines. Three of five gastric cancer cell lines showed resistance to cetuximab treatment which was associated with the presence of mutations in the E-cadherin gene CDH1 [3]. To determine the influence of E-cadherin on the treatment outcome of molecular therapy with cetuximab, we investigated the underlying molecular mechanisms using a model of three gastric cancer cell lines (KATOIII, MKN28, MKN45), two of them reveal mutations in CDH1 (KATOIII: p.E336E; MKN45: p.G274_P277del). By overexpression or silencing of E-cadherin, we demonstrate the effect of E-cadherin and somatic mutations in CDH1 on the activation of the EGFR pathway and establish a relationship with cetuximab sensitivity of gastric cancer cell lines on the molecular level. Additionally, we analyzed the effect of wild-type E-cadherin, knock-down of E-cadherin and insertion of mutated CDH1 (del8) in MKN28 cells on molecular level as well as on phenotypic level using life-cell imaging. Conclusions.  We demonstrate the influence of E-cadherin on the EGFR signaling network in gastric cancer cell lines which might be relevant for cetuximab sensitivity or resistance. References 1. Bremm et al (2008) Enhanced activation of epidermal growth factor receptor caused by tumor-derived E-cadherin mutations 2. Luber et al (2011) Biomarker analysis of cetuximab plus oxaliplatin/leucovorin/5-fluorouracil in first-line metastatic gastric and oesophago-gastric junction cancer: results from a phase II trial of the Arbeitsgemeinschaft Internistische Onkologie (AIO) 3. Heindl et al (2012) Relevance of MET activation and genetic alterations of KRAS and E-cadherin for cetuximab sensitivity of gastric cancer cell lines

P01.11 Depletion of the long non-coding RNA HOTAIR induces alterations Zof DNA methylation patterns in gastrointestinal stromal tumours (GIST) I. Bure*, S. Geer, M. Roas, J. Knopf, A. Agaimy, A. Hartmann, F. Haller, E. Moskalev Institut für Pathologie, Friedrich-Alexander Universität, Erlangen, Deutschland Background.  Epigenetic silencing of multiple genes that are relevant for tumourigenesis has been reported in virtually all tumour entities. Aberrations of DNA methylation patterns are one of the best documented epiDer Pathologe Suppl 1 · 2016

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Abstracts genetic abnormalities. Their origin in neoplastic cells is, however, only incompletely understood. Certain long non-coding RNAs (lncRNAs) such as the HOX Antisense Intergenic RNA (HOTAIR) enable target specificity of the histone modification complexes. It remains to be defined, however, if lncRNAs may play a role in patterning DNA methylation, too. To ascertain a possible link between lncRNAs and aberrant DNA methylation, we asked by using gastrointestinal stromal tumours (GISTs) as a model if HOTAIR is capable of affecting tumour related DNA methylation patterns. Methods.  Stable knockdown of HOTAIR in the GISTT1 cell line that exhibits high level of endogenous HOTAIR expression was achieved using lentiviral transduction and constitutive expression of two independent shRNAs. Genome-wide DNA methylation patterns were analysed by the Infinium HumanMethylation450 BeadChip platform. Concomitant expression analysis was performed by using the GeneChip Human Transcriptome Array 2.0. Bisulfite pyrosequencing and qRT-PCR were used for validation of array data. Results.  Depletion of HOTAIR in cell line GISTT1 had an opposite effect in different loci and induced genome-wide hypo- and hypermethylation of multiple CpG sites with hypomethylation prevailing. These included potential tumour suppressors, transcription factors, tumour-specific antigens, genes related to angiogenesis or involved in metabolism. Further validation of selected candidates with a potential function in GIST fully confirmed the microarray data. Methylation percentage of CpG sites in the Dipeptidyl-Peptidase 4 (DPP4) was increased up to 52 % with concomitant downregulation of the gene’s expression. Concordant with earlier reports, HOTAIR knockdown led to reduced migration potential in GISTT1 cells. Conclusions.  Taken together, the results suggest that HOTAIR is one of the factors that enable target specificity of DNA methylation in GIST. Specific molecular mechanisms may include HOTAIR dependent recruitment of PRC2 complexes and need to be further elucidated. The results are of potential interest in the context of epigenetic cancer therapy.

P01.12 Early ultrastructural findings in hepatocytes after hydrodynamic tail vein injections in mice S. Ribback*1, K. Evert1, 2, K. Utpatel1, 2, K. Annweiler1, D. Calvisi1, M. Evert1, 2, F. Dombrowski1 Institut für Pathologie, Universitätsmedizin, Greifswald, Deutschland, Institut für Pathologie, Klinikum der Universität, Regensburg, Deutschland

1 2

Background.  Hydrodynamic transfection (HT) is a murine gene transfer model in liver cancer research. The transfected DNA constructs are composed of two plasmids, one containing the gene of interest and the other the Sleeping Beauty-transposase allowing a stable integration of this gene into the hepatocyte genome. Mechanism of HT is based on rapid tail vein injection of a large volume of DNA-solution that induces an acute cardiac congestion, resulting in a reflux into the liver. It is known that the fenestred sinusoidal endothelium is permeabilized by the hydrodynamic force induced by HT. However, the mechanisms of plasmid incorporation into the hepatocyte itself remain unclear. Methods.  2 ml volume of empty vector or saline solution (control) were hydrodynamically injected into the tail vein of C57BL/6J/129Sv mice. Liver tissue was resected at different time points (1, 2, 5, 10, 20, 30 or 60 minutes after HT) and fixed with buffered 1 % osmium tetroxide. For electron microscopic evaluation, ultrathin sections were cut from glycidether embedded blocks. Results.  At earliest time points, hepatocytes in acinar zone 3 near the central venule reveal some small membrane bound vesicles in the cytoplasm. After few more minutes, vesicles increase in number and size, forming larger vacuoles. Vacuoles can often be found in proximity to the nucleus. Vesicles and vacuoles are optically empty and contain no electron dense material. Other hepatocytes neighboring the aforementioned ones contained no vesicle formations but revealed signs of cell damage, i. e. swollen mitochondria, dilated endoplasmatic reticulum and golgi apparatus and

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disrupted plasma membranes. Hepatocytes in azinar zone 1 appeared normal. These findings were similar after the injection of the empty vector and the control saline solution as well. Conclusions.  These ultrastructural findings indicate that hydrodynamic plasmid transfer to the hepatocytes is achieved via active or passive endocytosis with an entrapment of the plasmid in multiple membrane bound vesicles and vacuoles. Hepatocytes containing vacuoles did not show disrupted plasma membranes or other signs of cell damage, therefore mechanisms like i. e. membrane poration are unlikely, but cannot be entirely excluded. The vesicle formation seems to be a non-specific process, independent from the plasmid itself or its enclosed components. It remains to be clarified which active or passive mechanisms are involved in vesicle generation and finally how the DNA enters the hepatocellular nucleus.

P01.13 Endothelial cell type-specific reaction patterns in an oncogene-induced model for hepatocellular carcinoma S. Thomann*1, M. Dittmer2, I. Hausser1, C. Mogler3, C. Ball4, H. Glimm4, K. Heeg5, E. Ryschich6, P. Schirmacher1, K. Breuhahn1 Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, Institut für Parasitologie, Heidelberg, Deutschland, 3Institut für Pathologie und pathologische Anatomie, Technische Universität München, München, Deutschland, 4Nationales Centrum für Tumorerkrankungen (NCT), Heidelberg, Deutschland, 5Institut für Hygiene, Heidelberg, Deutschland, 6 Klinik für Chirurgie, Ruprecht-Karls-Universität, Heidelberg, Deutschland 1

2

Background.  The liver comprises a set of different endothelial cell types, which vary in their degree of differentiation. Next to capillary endothelial cells (CECs), liver sinusoidal endothelial cells (LSECs) with distinct morphological characteristics line the porto-central axis, which spans the distance between portal and central veins. In liver carcinogenesis, a process called capillarization (arterialization) is frequently observed in premalignant and malignant tumors; however, a comparative morphological and biochemical characterization of endothelial cell types in the different stages of hepatocarcinogenesis is missing. In this study we aim to identify the dynamic and cell type-specific reaction patterns of tumor vasculature in a genetic tumor model for hepatocellular carcinoma (HCC). Methods.  Transgenic mice with inducible and liver-specific expression of constitutively active yes-associated protein (YAPS127A) were used as tumor model. YAPS127A induction led to hepatomegaly (around 8–10 weeks) and tumor formation (around 12–15 weeks). Quantitative immunofluorescence was used for spatial expression analysis of endothelial surface markers. MACS-based cell isolation allowed the purification of liver endothelial cells for scanning electron microscopy and FACS analysis. Results.  In liver tissue sections LSECs were characterized by a gradual loss of lymphatic vessel endothelial hyaluronan receptor 1 (Lyve-1) expression along the porto-central axis, while the expression intensity of general endothelial markers did not change (e. g. CD146, CD31). Vice versa absent expression of sinusoidal markers (e. g. Lyve-1 or CD32b) in liver vessels allowed the identification of CEC. Scanning electron microscopy of primary endothelial cells confirmed the heterogeneity of the isolated cell population by visualization of sieve plates (fenestrae). FACS analysis revealed that 4 ± 2 % of the total endothelial cells represented CECs in healthy livers (LSECs: 95 ± 2 %). The number of CEC cells drastically increased in hyperplastic livers (37 ± 16 %) and solid tumors (61 ± 30 %). Importantly, this drastic expansion of CEC numbers is based on increased cell proliferation. Conclusions.  Our endothelial cell population-based results illustrate that the YAP-dependent tumor model is suitable for the analysis of capillarization in hepatocarcinogenesis. In addition, our data emphasize the role of CEC expansion in the process of capillarization and HCC angiogenesis.

P01.14 The role of Endosialin in hepatocarcinogenesis C. Mogler*1, C. König2, M. Wieland2, A. Runge2, P. Schirmacher3, H. Augustin2 Pathologisches Institut, München, Deutschland, 2Deutsches Krebsforschungszentrum, Heidelberg, Deutschland, 3Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland 1

Background.  Endosialin is expressed by tumor-associated pericytes and stromal myofibroblasts, identifying it as a marker of the activated mesenchymal lineage whereas healthy tissues show only weak or no expression of Endosialin. Endosialin is known to play a predominant role in the development and progression of organ fibrosis, especially liver fibrosis, hereby balancing the amount of fibrosis with the hepatocellular proliferative response. Methods.  We consequently hypothesized that Endosialin may be therefore functionally involved in the development and progress of hepatocellular carcinoma (HCC). Results.  Human samples of different HCC show a variable amount of Endosialin especially pronounced in the tumor periphery. Endosialin deficient mice were crossed into a virus inducible mouse model of hepatocellular carcinoma (HCC) to investigate the onset and the progression of hepatocarcinogenesis in vivo. Indeed, Endosialin deficiency resulted in more MRI-detectable tumor nodules, increased liver size and higher overall tumor burden. Candidate-based screens of murine tumor tissue and additional cell culture based experiments identified insulin like growth factor 2 (IGF2) as a main mitogen involved in this process. Conclusions.  Our data therefore indicate that hepatic stellate cell derived Endosialin not only affects liver fibrosis but also seems to display tumor-inhibiting functions in the setting of hepatocarcinogenesis.

P01.15 Mislocalization of polarity protein Scribble promotes oncogenic events in hepatocellular carcinoma S. Wan*1, A.-S. Meyer1, S. Weiler1, T. Lutz1, S. Rössler1, P. Schirmacher1, F. Pinna2, K. Breuhahn1 Pathologisches Institut, Heidelberg, Deutschland, 2Pathologisches Institut, Aachen, Deutschland 1

Background.  Hepatocytes are characterized by distinct luminal, lateral and basal domains that are defined by specific protein complex. Loss of cell polarity is frequently observed in hepatocellular carcinoma. Scribble is one important polarity protein which locates to cell-cell junction of basolateral membrane. It is recognized as a tumor supressor because loss of Scribble induces disoriented cell proliferation. However, overexpression of Scribble has been found to be associated with the progression of tumors from different origins. Methods.  Scribble localization in HCC cell lines and human HCC tissues was analyzed by western immunoblotting and immunofluorescence. Expression vectors containing wildtype Scribble and P305L mutant, which lacks membranous binding capacity, were stably transfected in HCC cells. The impact of these isoforms on PI3K/AKT signaling pathway as well as cell viability were compared. In addition, the interaction between Scribble-P305L and phosphatases inhibiting AKT signaling was identified by Co-IP analysis. Results.  Scribble located to plasma membrane in polarized cells HepG2 and HuH-1, but in the cytoplasm in non-polarized cells HLE and HLF. Cytoplasmic accumulation of Scribble is associated with poor survival of HCC patients. In vitro, the overexpression of Scribble P305L mutant induced AKT phosphorylation. Moreover, the cytoplasmic Scribble promotes HCC cell viability in an AKT-dependent manner. Co-localization of Scribble with the AKT phosphatases PHLPP1 was revealed by Immunofluorescence. Physical interactions between PHLPP1 and both Scribble isoforms were detected.

Conclusions.  Our data demonstrate that the cytoplasmic overexpression of Scribble promotes cell proliferation via the interaction with PHLPP1 and upregulation of AKT signaling. It is indicated that the disturbance of Scribble complex can accelerate the progress of hepatocellular oncogenesis.

P01.16 Role of IQGAP1/IQGAP2 balance in defining β-catenin and YAP activation in hepatocellular carcinoma F. Pinna*, R. Pellegrino, O. Neumann, A. Eberhardt, T. Longerich Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland Background.  Shifting the balance of IQGAP1/IQGAP2 protein levels was recently discovered to be an important feature in human hepatocellular carcinoma (HCC)1. In IQGAP2 knockout mice, autonomous development of liver cancer was characterized by increased IQGAP1 levels, showing a possible inhibitory function of IQGAP2 on IQGAP1 and a potent oncogenic nature driven by IQGAP1 in absence of IQGAP22. The precise mechanism how IQGAP2 exerts its inhibitory effect on IQGAP1 oncogenic properties is still unknown. Considering IQGAP1 and IQGAP2 as specific scaffold proteins at the edge of cell adhesion, actin/cytoskeleton and signaling activation, our study connects the IQGAP1/IQGAP2 balance to the activity of YAP and β-catenin signaling, thus focusing on specific mechanisms connecting cell junctions and activation of signaling pathways. 1. BMC Gastroenterol. 2010 Oct 2. Mol Cell Biol. 2008 Mar Methods.  Different liver cancer cell lines (Huh6, Hep3B, Huh7, HepG2, HLE and SK-Hep1) were used. Analysis of IQGAP1, IQGAP2, β-catenin and YAP expression was examined on protein and gene expression level. Activity of β-catenin and YAP was investigated by subcellular localization upon IQGAP1/2 gene knockout and/or overexpression. Analysis of system components and protein interactions connecting IQGAP1/2 with YAP and/or β-catenin was performed by immunoprecipitation experiments. Results.  We found high IQGAP2 protein levels in cells showing a epithelial-like phenotype (Huh6, Hep3B, Huh7 and HepG2), while cells with a mesenchymal-like phenotype (HLE, SK-Hep1) are low expressors. Total amount of YAP, its activity and main transcription targets were negatively correlating with IQGAP2, whereas IQGAP2 positive cells showed activated β-catenin singalling and intact cell junction formation. Direct interaction between YAP and IQGAP1 was shown as well as an interaction between IQGAP1 and Angiomotin. Conclusions.  Activation of ß-catenin signaling is associated with increased levels of IQGAP2 in epithelial-like HCC cell lines (Huh6, Hep3B, Huh7,HepG2), while mesenchymal-like HCC cells have active YAP signaling. Although ß-catenin mutation/activation occurs in a subset of human HCCs and has been associated with better prognosis, activation of the pathway lacks clinical relevance as β-catenin mutations are currently undruggable. In this context, targeting IQGAP1/2 might be an option to influence the activity of β-catenin and YAP signaling in human HCCs, thereby providing an opportunity for indirect targeting of both pathways.

P01.17 Nuclear ErbB2 Expression of Hepatocytes as Response to Cellular Stress Events in Alcoholic Steatohepatitis P. Döring*, G. M. Pilo, D. F. Calvisi, F. Dombrowski Institut für Pathologie, Universitätsmedizin, Greifswald, Deutschland Background.  ErbB2 is a prominent member of the epidermal growth factor receptor superfamily, a group of transmembrane receptors that mainly attract attention as oncogenic drivers and therapeutic targets in cancer. Der Pathologe Suppl 1 · 2016

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Abstracts Besides transmembrane signaling, ErbB2 may also translocate into the nucleus and mediate distinct nuclear signaling effects, including DNA repair and cell cycle arrest. Methods.  The immunohistochemical pattern of ErbB2 staining and several metabolic markers and signaling molecules were analyzed in 476 liver biopsy samples from patients with hepatic dysfunction. Additionally, ErbB2 expression upon ethanol exposure was tested in cell culture experiments. Results.  We found a cytoplasmic and nuclear ErbB2 expression in hepatocytes from different disease conditions, with the strongest expression being detected in alcoholic steatohepatitis. These ErbB2 overexpressing hepatocytes revealed peculiar metabolic and hormonal changes: enhanced lipid turnover (increase of fatty acid synthase and acyl-CoA-dehydrogenase), downregulation of estrogen receptor, cell cycle alterations (increase of p21 and heat shock protein 27), and an increase of phospho-Stat3, a downstream effector of nuclear ErbB2 signaling. Notably, ballooned hepatocytes, often interpreted as degenerative cells, exhibited a particularly strong ErbB2 expression and appeared to be highly metabolically active as described above. Of note, an increased ErbB2 expression as well as metabolic alterations and signs of cell cycle deregulation similar to that described in human steatohepatitis specimens were detected when HepG2 cells or cultivated Cynomolgus hepatocytes were subjected to 48-hour ethanol treatment. Furthermore, alternative stress events like hyperosmolarity or increase of pH seem to enhance ErbB2 expression levels of HepG2 cells, too. Conclusions.  These novel observations on hepatocellular ErbB2 expression with evidence of nuclear receptor signaling and metabolic alterations imply a so far unknown mechanism in hepatocytes upon cellular stress events, particularly ethanol exposure, presumably leading to resistance to cell death and metabolic reprogramming.

P01.18 Fatty acid synthase is indispensable for hepatocarcinogenesis driven by the AKT protooncogene D. Calvisi*1, 2, L. Li3, G. M. Pilo2, X. Li3, A. Cigliano1, G. Latte2, S. Ribback1, R. M. Pascale2, C. F. Semenkovich4, K. Utpatel5, K. Evert5, F. Dombrowski1, X. Chen3, M. Evert5 Institut für Pathologie, Universitätsmedizin, Greifswald, Deutschland, Abteilung für Klinische und Experimentelle Medizin, Universität Sassari, Sassari, Italien, 3Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, Vereinigte Staaten von Amerika, 4 Abteilung für Zellbiologie und Physiologie, Washington, Vereinigte Staaten von Amerika, 5Universität Regensburg, Institut für Pathologie, Regensburg, Deutschland 1

2

Background.  Cumulating evidence underlines the crucial role of aberrant lipogenesis in human hepatocellular carcinoma (HCC). Here, we investigated the oncogenic potential of fatty acid synthase (FASN), the master regulator of de novo lipogenesis, in the mouse liver. Methods.  FASN was overexpressed in the mouse liver, either alone or in combination with activated N-Ras, c-Met, or SCD1, via hydrodynamic gene delivery. Activated AKT was overexpressed via hydrodynamic injection in livers of conditional FASN or Rictor knockout mice. FASN was suppressed in human hepatoma cell lines via specific small interfering RNA or the FASN chemical inhibitor, C75. Results.  Overexpression of FASN, either alone or in combination with other genes associated with hepatocarcinogenesis, did not induce histological liver alterations. In contrast, genetic ablation of FASN resulted in the complete inhibition of hepatocarcinogenesis in AKT-overexpressing mice. In human HCC cell lines, FASN inactivation led to a decline in cell proliferation and a rise in apoptosis, which were paralleled by a decrease in the levels of phosphorylated/activated AKT, an event controlled by the mammalian target of rapamycin complex 2 (mTORC2). Downregulation of AKT phosphorylation/activation following FASN inactivation was associated with strong inhibition of rapamycin-insensitive companion of

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mTOR (Rictor), the major component of mTORC2, at post-transcriptional level. Finally, genetic ablation of Rictor impaired AKT-driven hepatocarcinogenesis in mice. Conclusions.  FASN is not oncogenic per se in the mouse liver, but is necessary for AKT-driven hepatocarcinogenesis. Pharmacological blockade of FASN might be highly useful in the treatment of human HCC characterized by activation of the AKT pathway.

P01.19 THSD7A overexpression in a mixed adenoneuroendocrine carcinoma of the gall bladder P. R. Stahl*, E. Hoxha, R. A. K. Stahl, T. Wiech Universitätsklinikum Hamburg-Eppendorf, Institut für Pathologie, Hamburg, Deutschland Background.  Thrombospondin Type-1 Domain-Containing 7A (THSD7A) is a novel antigen that has recently been characterized as a potential target in patients with membranous nephropathy (MN). We describe a case of a 40-year-old woman with a THSD7A-positive mixed adenoneuroendocrine carcinoma (MANEC) of the gall bladder who primarily presented with a THSD7A-associated MN. Methods. Immunohistochemistry Slides were deparaffinized, pretreated in citrate buffer (pH 6.1) for 15 min at 120 °C and cooled down in iced water (10 min). After rinsing in 100 % ethanol, slides were incubated 10 min with normal serum (Vector S2000) followed by THSD7A [Sigma, rabbit, 1:500) in antibody diluent (Invitrogen) over night at 4 °C. The slides were then washed in PBS and incubated with polymer 1 (Zytomed Zytochem-Plus AP Polymer-Kit POLAP), rinsed in PBS and incubated with polymer 2 (Zytomed Zytochem-Plus AP Polymer-Kit POLAP). After washing in PBS, slides were stained in new fuchsin naphthol As-Bi phosphate substrate mixture (10 min) followed by 1 min nuclear staining in hemalaun. FISH analysis A 4 µm section was used for FISH analysis. For proteolytic slide pre-treatment, a commercial kit was utilized (paraffin pre-treatment reagent kit; Vysis-Abbott). A Spectrum Green-labeled THSD7A-probe (EmpireGenomics) was used together with a Spectrum Orange-labeled CEP7probe (Vysis-Abbott). Before hybridization, sections were deparaffinized, air-dried, dehydrated and then denatured at 72 °C for five minutes in a ThermoBrite (Vysis-Abbott). After overnight hybridization at 37 °C in a ThermoBrite, slides were washed and counterstained with 10 µl DAPI I (Vysis-Abbott). Results.  Three weeks after renal biopsy and diagnosis of MN, surgery was performed to treat a tumor in the gall bladder. Histopathologic examination revealed a MANEC of the gall bladder with three corresponding lymph node metastases. The infiltrated lymph nodes were non-regional and therefore considered distant metastases. Tumor classification was as follows: G3, pT2, M1 (LYM), L1, V0, Pn0 – tumor-free resection margin. Both, the primary tumor of the gall bladder, as well as the corresponding metastases were positive for THSD7A by immunohistochemistry. FISH analysis revealed a polysomy of chromosome 7 with up to six copies of the THSD7A gene and chromosome 7 centromere (CEP7), respectively. Conclusions.  This is the first report on a THSD7A-positive malignant tumor. The role which this novel antigen might play in tumor development has to be clarified in further investigations.

P01.20 miRNA and HDAC expression in pancreatic neuroendocrine tumors S. Swierczynski1, E. Klieser2, R. Urbas2, S. Stättner1, F. Primavesi1, T. Jäger1, T. Kiesslich3, 4, R. Kemmerling5, P. DiFazio6, D. Neureiter*2 Universitätsklinik für Chirurgie, Salzburger Landeskliniken, Paracelsus Medizinische Privatuniversität, Salzburg, Österreich, 2Universitätsinstitut für Pathologie, Salzburger Landeskliniken, Paracelsus Medizinische Privatuniversität, Salzburg, Österreich, 3Universitätsklinik für Innere Medizin I, Salzburger Landeskliniken, Paracelsus Medizinische Privatuniversität, Salzburg, Österreich, 4Institut für Physiologie und Pathophysiologie, Labor für Tumorbiologie und Experimentelle Therapien (TREAT), Paracelsus Medizinische Privatuniversität, Salzburg, Österreich, 5Medizinisches Versorgungszentrum für Histologie, Zytologie und Molekulare Diagnostik, Trier, Deutschland, 6Institut für Chirurgische Forschung, Philipps-Universität Marburg, Marburg, Deutschland 1

Background.  Epigenetic factors are essentially involved in carcinogenesis, tumor promotion and chemo resistance [1][2]. Major key players in these processes are miRNAs and histone deacetylases (HDACs). As shown by theoretical databank analysis [3], the crosstalk between miRNAs and HDACs are essential in different human chronic diseases and cancerogenic pathways. Here, we investigated a well-defined subset of “pro-proliferative” miRNAs, which are related to expression of HDACs and clinical parameters in pancreatic neuroendocrine tumors (pNETs). Methods.  We analyzed the expression levels of miR132-3p, miR145-5p, miR183-5p, miR34a-5p and miR449a in 57 PNETs resected between 1997 and 2015. These findings were linked to the immunhistochemical expression pattern of members of the four HDAC classes (Class I, IIA, IIB, III & IV) analyzed on human tissue microarrays (TMA) platform covering these 57 pNETs. All pNET cases were clinically and pathologically characterized according to published guidelines. Results.  57 cases (32 (56.1 %) female versus 25 (43.9 %) male) with pNET (47.4 % immunohistochemically endocrine positive) showed following detailed clinic-pathological characteristics: G1-3 56.1–29.9–14.0 %; pT14 42.0–21.0–30.0–7.0 %; pN0-1 64.9–35.1 %; pM0-1 80.7–19.3 %. The expression levels of the miRNAs declined in the following order: miR145-5p (HDAC11) >> miR34a-5p (HDAC1) > miR132-3p (SIRT1) >> miR183-5p (HDAC2) >> and miR449a (HDAC1). Correlation analysis revealed a significant association of miRNAs with most of the HDACS and their known regulative HDAC (shown in parentheses). Additionally, a linkage between the expression of miRNAs and clinic-pathological parameters like grading and nodal infiltration could be statistically established. Conclusions.  Overall, we could demonstrate that specific miRNAs could be linked to HDAC expression in pNETs. These first data could help, to improve our knowledge of the complex interactions of these epigenetic drivers in pNETs for further therapeutic approaches in future. References 1. Taby R, Issa JP (2010), Cancer epigenetics, CA Cancer J Clin, 376–392, 60(6):376, PMID: 20959400 2. Huang J, Plass C, Gerhauser C (2011), Cancer chemoprevention by targeting the epigenome, Curr Drug Targets, 12(13), 1925–1956, PMID: 21158707 3. Swierczynski S, Klieser E, Illig R, Alinger-Scharinger B, Kiesslich T, Neureiter D (2015), Histone deacetylation meets miRNA: epigenetics and post-transcriptional regulation in cancer and chronic diseases, Expert Opin Biol Ther, 15(5), 651–664, PMID: 25766312

P01.21 „Pros“ of pancreas processing with pancreatic cancer by axial slicing techniques E. Klieser*1, R. Urbas1, S. Stättner2, F. Primavesi2, T. Jäger2, S. Swierczynski2, D. Neureiter1 Universitätsinstitut für Pathologie, Salzburger Landeskliniken, Paracelsus Medizinische Privatuniversität, Salzburg, Österreich, 2Universitätsklinik für Chirurgie, Salzburger Landeskliniken, Paracelsus Medizinische Privatuniversität, Salzburg, Österreich 1

Background.  Patients suffering from pancreatic cancer (PC) have poor prognosis due to limited chemo- and radiotherapy options. Up to now, only surgical resection is the suitable curative treatment. But diagnosis and prognosis as well as clinical and experimental studies could be misdirected through inconsistent pathological preparation and consecutive evaluation based on heterogeneous intra- and inter-institutional technical approaches. Methods.  Comparison of 32 pancreatoduodenectomy (PDE) specimens with PC were completely processed by axial slicing (AS, as introduced by Verbeke et al. [1][2]) and were correlated to 39 PDE specimens processed with traditional/conventional grossing (TG) techniques matched by gender, age and localizations as well as tumor entity. Results.  Tumor grade G3 was the most observed histo-morphological entity, and assessed lymph node number in AS was twice as high compared to TG. Consecutively, number of infiltrated lymph nodes in AS was half times higher than in TGs. Additionally, total number of assessed lymph nodes correlated with FFPE-embedded specimen and indirectly with tumor infiltrated lymph nodes. At least, no differences in tumor resection status were observed. Conclusions.  The AS-technique of PC is easy to perform and yields to a more precise assessment concerning tumor grading and total lymph node status. Despite of more technical afford, complete tumor processing helps to characterize the complete pathological potency of these highly malignant tumor entity. References 1. Verbeke CS, Menon KV (2009) Redefining resection margin status in pancreatic cancer, HPB (Oxford), 11(4), 282–289, PMID: 19718354 2. Verbeke CS, (2013), Resection margins in pancreatic cancer, Pathologe Suppl 34 (Supp 2): 241–247, PMID: 24196622

P01.22 Genomic and proteomic landscape of pancreatic primary tumors and their metastases M. Kriegsmann*1, A. Muckenhuber2, R. Casadonte3, R. Longuespée4, N. Pfarr2, J. Kriegsmann3, 5, W. Weichert2 1 Institut für Pathologie, Ruprecht-Karls Universität Heidelberg, Heidelberg, Deutschland, 2Institut für Allgemeine Pathologie und Pathologische Anatomie der Technischen Universität München, München, Deutschland, 3 Proteopath GbR, Trier, Deutschland, 4Molekularpathologie Trier, Trier, Deutschland, 5MVZ für Histologie, Zytologie und Molekulare Diagnostik, Trier, Deutschland

Background.  Pancreatic cancer is among the most aggressive cancer types with a median survival of about 3–6 month for inoperable disease (80– 85 %). The genetic landscape for primary tumors has been described in the last decades. However, little is known on the genome and proteome of metastases which is surprising against the background that the majority of patients are not suitable for surgical therapy. Thus, we investigated 25 primary tumors and their corresponding metastases by massive parallel sequencing (MPS) and imaging mass spectrometry (IMS) in order to reveal their genetic and proteomic properties. Methods.  A total of 25 formalin fixed and paraffin embedded primary tumors and their corresponding metastases were analyzed. MPS studies

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Abstracts have been conducted as previously described with a custom build pancreatic cancer panel that covers all pancreatic mutations detected with a frequency of more than 1 % according to the COSMIC database. IMS studies were performed as previously described on an AutoflexSpeed mass spectrometer. Approximately 500 different peptides were detected per sample. Results.  Both, MPS and IMS studies revealed highly concordant results in primary tumors and their corresponding metastases. In few primary tumors and also in several metastases additional mutations could be demonstrated. As with the mutational status, only few aberrant peptide profiles were detected in the two subgroups. Conclusions.  Overall the genetic and the proteomic landscape of pancreatic primaries is similar to that of their corresponding metastases.

P01.23 Molecular characterisation of invasive intraductal papillary mucinous neoplasms of intestinal type in the pancreas B. Neumayer* , M. Nieser , G. Klöppel , F. Bergmann , N. Giese , D. Aust , R. Grützmann6, B. Sipos1 1

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Institut für Pathologie, Universitätsklinikum, Tübingen, Deutschland, Institut für Pathologie und pathologische Anatomie, Technische Universität München, München, Deutschland, 3Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, 4Klinik für Viszeral- und Transplantationschirurgie, Ruprecht-Karls-Universität, Heidelberg, Deutschland, 5Institut für Pathologie, Universitätsklinikum Carl Gustav Carus, Dresden, Deutschland, 6Chirurgische Klinik, Universitätsklinikum Carl Gustav Carus, Dresden, Deutschland 1

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Background.  Intestinal type intraductal papillary mucinous neoplasia (intIPMN) of the pancreas follows an adenoma-carcinoma sequence that is phenotypically identical to the adenoma-carcinoma sequence in colorectal carcinomas. However, molecular alterations of invasive intIPMN are poorly understood. We addressed the question whether molecular alterations in intIPMN show more similarities to colorectal carcinomas rather than to classical ductal pancreatic adenocarcinomas. The molecular profiling may aid in selecting targeted treatment options for this rare tumor type. Methods.  Formalin-fixed paraffin embedded tissues of 20 invasive intIPMN have been analysed for the expression of lineage markers and tumor suppressor proteins by immunohistochemistry. Whole exome sequencing and targeted sequencing have been performed in ten intIPMN. Results. The intestinal differentiation in invasive intIPMN has been shown by immunohistochemical detection of cytokeratin 20 (17/20), CDX2 (17/20) and Muc2 (14/17). Loss of tumor suppressor proteins has been detected in 12 % of the cases (SMAD4 – 2/17: p16 -2/17). IHC revealed aberrant expression of TP53 in 35 % (6/17) of the cases. Whole exome and targeted sequencing identified GNAS mutations in 81 % (13/16) in the invasive component of intIPMN. Single cases exhibited missense point mutations and/or inDels in 81 genes, e. g. in K-ras (1/12, B-raf (1/7), TP53 (1/11), c-MET (1/11), VHL (1/11) und Myc (1/11). No other alterations in 649 genes related to human carcinogenesis could be detected. Conclusions.  Invasive intIPMN exhibits a unique molecular profile, which is dominated by GNAS mutations. The dissimilarity of this profile to molecular alterations in pancreatic ductal adenocarcinomas indicates that invasive intIPMN of the pancreas should be treated by different protocols than conventional pancreatic adenocarcinomas.

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P01.24 Selective inhibition of the p38 alternative activation pathway in infiltrating T cells inhibits pancreatic cancer progression M. M. Gaida*1, 2, M. S. Alam2, F. Bergmann1, F. Lasitschka1, T. Giese3, N. Giese4, T. Hackert4, U. Hinz4, S. P. Hussain5, S. V. Kozlov6, J. D. Ashwell2 Pathologisches Institut, Ruprecht-Karls-Universität, Heidelberg, Deutschland, 2National Cancer Institute, National Institutes of Health, Labor für Immunzellbiologie, Bethesda, Vereinigte Staaten von Amerika, 3 Institut für Immunologie, Ruprecht-Karls-Universität, Heidelberg, Deutschland, 4Klinik für Allgemein-, Viszeral- und Transplantationschirurgie, Universitätsklinikum, Heidelberg, Deutschland, 5National Cancer Institute, National Institutes of Health, Labor für Humane Karzinogenese, Bethesda, Vereinigte Staaten von Amerika, 6Nationales Krebsforschungsinstitut, Labor für Tumor- und Entwicklungsbiologie, Frederick, MD, Vereinigte Staaten von Amerika 1

Background.  Pancreatic ductal adenocarcinoma (PDAC) displays a tumor-promoting inflammatory microenvironment that contains a large number of tumor infiltrating T cells (TIL), which are a major source of cytokines, many of which are regulated by the MAPK p38. In antigen-activated T cells, p38 is exclusively activated via a MAPK cascade independent mechanism (alternative pathway; p38Y323) that is specifically blocked by GADD45α. Methods.  Human PDAC biopsies (n = 192) were tested for the presence of alternatively activated TIL, p38-downstream cytokines, and factors promoting tumor angiogenesis and epithelial-to-mesenchymal transition, using immunohistochemistry, immunofluorescence, and qRT-PCR. KPC mice (a genetically engineered K-ras-mediated PDAC mouse model) were sublethally irradiated, followed by transplantation with bone marrow from mice that can or cannot activate p38 via the alternative pathway. A peptide derived from GADD45α comprising its residues 71–85 was synthesized and made plasma membrane–permeable by 11R-fusion. KPC mice were treated in a prevention model and in a therapeutic model with this alternative p38 inhibitor by i. v. injection, followed by the characterization of TIL using flow cytometry. The pancreata of these mice were evaluated by computer-based tissue analysis. In vivo tumor growth was determined by ultrasound. Toxic effects of the inhibitor were monitored by serum analysis of liver enzymes. Results.  In all human samples, p38 pY323+ TIL were found. PDAC with dense infiltrates revealed high amounts of Th cells expressing TNFα, IL17A, and IL-21. These tumors showed high levels of VEGF and a dense vascularization. These patients had a shorter survival. In KPC mice, the ablation of this pathway in bone marrow chimeric mice significantly prolonged their survival. Injection of KPC mice with (11R)71–85 reduced the amounts of T cell-derived tumor promoting cytokines in TIL(TNFα, IL10, and IL-17A), decreased the intratumor inflammation, and in line delayed significantly the progression of early PanIN to late PanIN and invasive cancer. In a therapeutic model in KPC mice with established PDAC, the injections with (11R)71–85 slowed the tumor growth. Moreover, no toxic effects were found. Conclusions.  High amounts of alternatively activated TIL were associated with neoangiogenesis and a poor prognosis for PDAC patients. Alternative p38 activation in TIL enhanced tumor progression in murine models. Its inhibition showed a preventive and a therapeutic benefit in mice without toxic effects.

P01.25 Characterization of murine models with colonic inflammatory bowel disease for translational studies N. Gaßler*1, 2, E. Basmah3, K. Else3, M. Glymenaki3, M. Travis3, J. Worthington3, S. Levison3, E. Kaemmerer1, 2, T. Longerich1, J. Pennock3, M. Pils4 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, Institut für Pathologie, Klinikum Braunschweig gGmbH, Braunschweig, Deutschland, 3Institute of Inflammation and Repair, Manchester, Vereinigtes Königreich, 4Helmholtz Zentrum Braunschweig, Braunschweig, Deutschland 1

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Background.  Inflammatory bowel disease (IBD) encompasses a spectrum of inflammatory gastrointestinal disorders. The large intestine is affected by the most important entities, Crohn’s disease (CD) and ulcerative colitis (UC). Etiology and pathogenesis of both are not fully elucidated as yet. For better understanding of inflammatory bowel disease and translational work several murine models have been already established. In the present study murine models were analyzed for specific pathological hallmarks of colonic CD and UC. Methods.  In mice colitis were chemically (dextran sodium sulphate) or infectiously (Trichuris muris) induced. Models of spontaneous colitis were IL10-ko, T-cell transfer and MDR1a-ko animals. Murine colonic tissues were analysed following routine procedures and compared to human CD and UC colonic tissues. Results.  Histomorphological hallmarks of human UC are crypt architectural distortion, mucosal inflammation, cryptitis, crypt empyema, and Paneth cell metaplasia. In human colitis Crohn discrete foci of inflammation, aphthous erosions, lymphoid aggregates and architectural changes adjacent to histologically normal crypts are found. In the murine models investigated erosive-ulcerous lesions were found accompanied with transmural inflammation. However, transmural lymphofollicular hyperplasia, goblet cell depletion, eosinophilia, granulomas or restriction of the inflammatory infiltrate to the mucosal layer were rare. Conclusions.  Murine models of chemical induced, spontaneous or infection-related colitis demonstrate quite different histomorphological features of inflammatory bowel disease. The inflammatory phenotype of all models investigated is closer to human UC than CD. In conclusion the use of the murine models of colonic inflammatory bowel disease is probably limited in translational studies.

P01.26 Lymph node hypoplasia is associated with adverse outcomes in node-negative colon cancer using advanced lymph node dissection methods P. Mayr*1, G. Aumann2, T. Schaller1, G. Schenhirsch3, M. Anthuber2, B. Märkl1 Institut für Pathologie, Klinikum Augsburg, Augsburg, Deutschland, Insitut für Viszerale Chirurgie, Klinikum Augsburg, Augsburg, Deutschland, 3 Tumorzentrum Augsburg, Klinikum Augsburg, Augsburg, Deutschland

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Background.  Until this day lymph node count is one of the most important aspects in the clinic-pathologic description of colon carcinomas, e. g. UICC staging requires at least 12 lymph nodes for N-stage description for a sufficient staging. In opposite to the lymph node number itself lymph node size as a prognostic parameter in nodal negative colon carcinomas has not been investigated well in the past. Recent data, however, have indicated that this parameter could be even more important in node negative disease than lymph node count itself. Methods.  Based on the results and data of earlier studies, we analyzed lymph node size and number of node-negative colon cancer patients with regard to survival. Data from 115 node-negative cases of colon cancer were analyzed. All cases were categorized according to the number of lymph nodes with a diameter of at least 5 mm (LN5). Lymph nodes with diameters ≤ 5 mm were defined as small, whereas we defined all other cases of lymph nodes (diameter > 5 mm) as intermediate/large in size. The LN-

5vl (very low) group included 34 cases with no or just one LN5. All other cases with two and more LN5 (n = 81) were assigned to the LN5 l/h (low/ high) group. Results.  The overall survival analysis revealed significantly worse outcomes for the LN5vl group, with a mean survival of 34 months, compared to the LN5 l/h group, with a survival of 40 months (P = 0.022). After adjusting these results toward their T-stages (pT1/2 vs. pT3/4), we still found a significant outcome difference (P = 0.012). Multivariate analysis identified LN5vl and T-stage as independently correlated with outcome. The vast majority of LN5vl cases (91 %) were located in the left colon. Location itself, however, was not prognostic (P = 0.478). Conclusions.  The count of lymph nodes with a diameter of 5 mm or larger could be shown as being prognostic in node-negative colon cancer. Lymph node size is described as a possible marker of immunologic response by some publications, the larger the more active. Patients with a small number of LN5 showed poor outcomes and may be regarded as a high risk group. In analogy to other high risk situations they could perhaps profit from adjuvant chemotherapy. Outcome in chemotherapy of this node negative high risk group (LN5vl) and differences in survival compared to the more favorable LN5 l/h group should be regarded as a subject of future studies. This may show a superiority of lymph node size over lymph node number in colon carcinomas.

P01.27 New macroscopically and microscopically aspects improving evaluation of tumor regression grading of neoadjuvant treated rectal cancer R. Urbas*1, E. Klieser1, T. Jäger2, A. Dinnewitzer2, D. Neureiter1 Universitätsinstitut für Pathologie, Salzburger Landeskliniken, Paracelsus Medizinische Privatuniversität, Salzburg, Österreich, 2Universitätsklinik für Chirurgie, Salzburger Landeskliniken, Paracelsus Medizinische Privatuniversität, Salzburg, Österreich 1

Background.  Up to now, only semi-quantitative methods of histo-pathlogical regression grading for neo-adjuvant treated rectal cancer (NTRC) relating vital tumor content to fibrosis according four to five tier scheme (see AJCC [1], Rödel [2] and Mandard [3] were used to predict patients survival. Here, we investigate possible further macroscopically and microscopically criteria to improve these grading systems. Methods.  147 cases of clinico-pathological characterized NTRC of the single center Salzburg were retrospectively analyzed according to the current TNM-classification as well as to the current regression grading systems. Additionally, the number of macroscopically embedded tumor blocks was linked to the finally resulting number of microscopically evaluated tumor blocks. Furthermore, the budding of cancer cells was quantified according to Hase et al. [4]. All embedded lymph nodes were specified for tumor infiltration, signs of regression as well as perinodal infiltration. These findings were correlated with defined time-end-points of disease (e. g. recurrence, metastasis and survival). Results.  Overall, the three investigated grading systems correlated significantly among themselves (abs(r)>0.932, p < 0.001). The ratio of embedded tissue versus embedded tumor as well as the quantitative budding of the remaining tumor could be significantly related to the grading schemes (in decreasing significant levels: Mandard, Rödel, AJCC). Additionally, analysis of ANOVA revealed a significant stratification scheme. Finally, these macro- and microscopically aspects were linked to patients’ outcome parameters like recurrence, disease-free and overall survival. Conclusions.  Here, we present new features for easy assessing macro- and microscopically aspects supporting and improving the tumor regression grading systems of neoadjuvant treated rectal cancer essentially. References 1. Mace AG, Pai RK, Stocchi L, Kalady MF (2015) American Joint Committee on Cancer and College of American Pathologists regression grade: a new prognostic factor in rectal cancer, Dis Colon Rectum, 58(1): 32–44, PMID: 25489692

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Abstracts 2. Rödel C, Martus P, Papadoupolos T, Füzesi L, Klimpfinger M, Fietkau R, Liersch T, Hohenberger W, Raab R, Sauer R, Wittekind C (2005) Prognostic significance of tumor regression after preoperative chemoradiotherapy for rectal cancer, J Clin Oncol 23(34):8688–8696, PMID: 16246976 3. Mandard AM, Dalibard F, Mandard JC, Marnay J, Henry-Amar M, Petiot JF, Roussel A, Jacob JH, Segol P, Samama G, et al (1994) Pathologic assessment of tumor regression after preoperative chemoradiotherapy of esophageal carcinoma. Clinicopathologic correlations, Cancer 73(11):2680–2686, PMID: 8194005 4. Hase K, Shatney C, Johnson D, Trollope M, Vierra M (1993) Prognostic value of tumor „budding“ in patients with colorectal cancer, Dis Colon Rectum 36(7): 627–635, PMID: 8348847

P01.28 Prognostic factors in colorectal caricnoma: correlation of lymph node numbers with extent of local immune response M.-T. Wand1, N. Koniezny1, G. Schröder1, M. Barros2, G. Niedobitek*1 Sana Klinikum Lichtenberg, Institut für Pathologie, Berlin, Deutschland, Unfallkrankenhaus Berlin, Institut für Pathologie, Berlin, Deutschland

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Background.  The number of detected regional lymph nodes is an independent prognostic parameter in colorectal carcinomas independent of nodal status. Furthermore, it has been shown that large numbers of tumour infiltrating T-cells are associated with improved outcome. We hypothesise that the local immune reaction may induce lymph node hyperplasia thus facilitating the detection of larger numbers of regional lymph nodes. To test this hypothesis, we have investigated if the degrees of local immune reactions in colorectal cancer correlate with sizes and numbers of regional lymph nodes. Methods.  175 colorectal carcinomas were studied, excluding cases with regional lymph node metastases. Numbers and sizes of dissected regional lymph nodes were recorded. The degrees of local inflammation at the invasive margin and in the tumour centre, as well as of Crohn’s like reaction were scored semi-quantitatively. The numbers of intraepithelial CD3+ T-cells were determined in relation to 100 tumour cells, and the numbers of stromal CD3+-T-cells were quantitated at the invasive margin per mm2. Results.  There were significant associations between pT stage and the degree of local immune reactions; low pT stages showed a correlation with larger overall numbers of inflammatory cells at the invasive margin and with larger numbers of CD3+ T-cells in the stroma at the invasive margins as well as with larger numbers of intraepithelial CD3+ T-cells. The numbers of detected lymph nodes correlated significantly with lymph node sizes. Furthermore, significantly more lymph nodes were found in cases with more advanced pT stage. Of all immune cell parameters investigated, only the presence and degree of local Crohn’s like reaction correlated significantly with increased sizes and larger numbers of detectable regional lymph nodes. Conclusions.  Our results do not support the hypothesis that there is a direct relationship between the intensity of local immune reactions and the numbers of regional lymph nodes detected in colorectal cancer resection specimens.

P01.29 p21 as a predictive biomarker for neoadjuvant therapy of rectal cancer S. Elezkurtaj*1, A. Müller2, M. Kruschewski3, 4

P01.30 Human colon fibroblasts inhibit CRC cells‘ EMT by down-regulating the canonical Wnt signaling pathway H. Deng*, Z. Zhu, Q. Nie Pathologisches Institut, Hangzhou, China, Volksrepublik Background.  The aim of the research is to study the effect of human colon fibroblasts on colorectal cancer (CRC) cells’ epithelial–mesenchymal transition (EMT) and to investigate the mechanism. Methods.  We used Western blot and Immunofluorescence assay to identify the normal fibroblasts (NFs) and and cancer associated fibroblasts (CAFs). β-Galactosidase staining was used to detect the degree of senescence of the cells. Q-PCR assay was to detect the activation level of them. Colorectal cancer (CRC) cells (SW620) were co-cultured with NFs or cultured in NFs supernatant to detect the expression of epithelial and mesenchymal markers and relative proteins of Wnt/β-catenin signaling by Western blot, and the migration, invasion and the proliferation of CRC cells by transwell motility assay and CCK8 assay respectively. Results.  We isolated and identified successfully primary NFs and CAFs from patients with colorectal cancer. β-Galactosidase staining showed that NFs had a lower degree of senescence than CAFs. The expression of α-SMA and FAP in NFs, the activated markers, was decreased. The supernatant of NFs up-regulated the expression of E-Cadherin, while down-regulated N-Cadherin detected by Western Blot. The supernatant of NFs inhibits the migration and invasion of CRC cells, but promote the proliferation. The canonical Wnt/β-catenin signaling pathway was down-regulated in CRC cells co-cultured with NFs or their supernatant. Conclusions.  The normal colon fibroblasts from patients with colorectal cancer inhibit CRC cells’ epithelial–mesenchymal transition through suppressing the canonical Wnt/β-catenin signaling pathway.

P01.31 Crypitits confined to basal crypts – a distinct histological feature associated with Familial Mediterranean Fever E. Ellert*1, N. Blank2, M. Andrulis1, 3

Charité, Institut für Pathologie, Berlin, Deutschland, 2Charité, Zahnärztliche Prothetik, Alterszahnmedizin und Funktionslehre, Berlin, Deutschland, 3 Charité, Chirurgische Klinik und Hochschulambulanz I, Campus Benjamin Franklin, Berlin, Deutschland, 4Städtisches Klinikum Solingen, Klinik für Allgemein-, Viszeral- und Thoraxchirurgie, Solingen, Deutschland

Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, Universitätsklinikum Heidelberg, Medizinische Universitätsklinik V, Heidelberg, Deutschland, 3Institut für Pathologie, Universität Ulm, Ulm, Deutschland

Background.  Neoadjuvant chemoradiation is a central modality of care for locally advanced rectal cancer but a considerable share of patients dis-

Background.  Monogenic autoinflammatory syndroms are rare diseases caused by abnormal activation of innate immune system. Familial

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play poor response to treatment. It would therefore be desirable to have straightforward measures of prediction to treatment which have so far not been established in routine despite extensive research on a variety of biomarkers. We have chosen to validate the cell cycle protein p21 for correlation with histopathological regression grading in a retrospective cohort. Methods.  Immunohistochemical expression of p21 was assessed quantitavely in cases of rectal cancer before and after treatment with 5-FU based chemoradiation. Histopathological regression grading was performed by Dworaks method and correlated with expression data in a stepwise fashion by nonparametric testing followed by parametric testing and receiver operating characteristics. Results.  Nuclear expression of p21 correlates positively with regression grading of chemoradiated rectal cancer for a broad range of cutoffs. Interestingly cytoplasmic expression of p21 is upregulated after neoadjuvant therapy. Conclusions.  We have demonstrated by retrospective analysis, that p21 deserves further appreciation as a biomarker for multimodal management of rectal cancer. Furthermore p21 appears to be involved in the cellular response to chemoradiation therapy.

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Mediterranean Fever (FMF) is one of the most frequent and best studied monogenic autoinflammatory syndrome caused by mutations MEFV gene mutations and transmitted by autosomal recessive inheritance. Because FMF is rare in central European countries and FMF symptoms can be non-specific or imitate other diseases, the definitive diagnosis can be delayed for many years. Recurrent episodes of abdominal pain are frequently followed by diarrhea. In this particular clinical setting the differential diagnoses include inflammatory bowel disease (IBD) and infectious enteritis. Therefore, many FMF patients undergo biopsies of gastrointestinal tract (GI-tract) before the final diagnosis is made. The commonest histological findings include mucosal defects, edema, and mixed inflammation, which can be found in IBD or infectious enteritis. Except for AA Amyloid deposition no other histological features typical for FMF have been described yet. Methods.  14 patients with FMF and available intestinal histology (biopsies n = 12, resection specimens n = 2; small bowel n = 2, large bowel n = 12) as well as clinical and genetic data were identified among a total of 129 FMF patients from our center and included in the study. Histological features like chronic inflammation, distortion of crypt architecture, ulcera and cryptitis. A Kongo Red stain was performed in all cases to confirm/ exclude amyloidosis. Results.  We systematically reviewed histological large bowel samples taken from fourteen FMF patients at the episode of FMF attack. Histology revealed multiple foci of cryptitis. Erosions of the surface epithelial cells were absent, except the two resection specimens performed due to deep chronic mucosal ulcera. Remarkably, cryptits was exclusively confined to basal crypts in three FMF cases and none of the assessed biopsies showed significant crypt distortions or signs of chronic inflammation i. e. infiltration by lymphocytes or plasma cells. Basal cryptits was associated with higher disease activity score, AA Amyloidosis, and high penetrance MEFV mutations. Conclusions.  In the present work we performed an systematic analysis of colon histology from a series of FMF patients and correlated the data with clinical and genetic characteristics. We found that cryptits confined to basal crypts of colon is associated with cases of severe FMF and can be a possible indicator for the diagnosis of FMF.

P01.32 Neuroendocrine tumors of the appendix are probably harmless neoplasms B. Sipos*1, A.-S. Weysser1, M. Nieser1, G. Klöppel2 Institut für Pathologie, Universitätsklinikum, Tübingen, Deutschland, Institut für Pathologie und pathologische Anatomie, Technische Universität München, München, Deutschland

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Background.  Appendiceal neuroendocrine tumors (aNETs) have a five years survival rates between 74 % and 95 % according to recent data from Cancer Registries of Norway and the United States, respectively. However, in the daily clinical practice one almost never encounters patients suffering from aNETs with distant metastasis or dying of the disease. In order to resolve this contradiction, we performed a systematic search in PubMed to collect well-documented aNETs. Methods.  Systematic search was carried out using key words “appendix or appendiceal”, “carcinoid OR neuroendocrine tumors”, “survival” and “registry or register”. Studies were considered only if they clearly distinguished between goblet cell carcinoids and “well differentiated carcinoids/ neuroendocrine tumors. We accessed to the row data from Surveillance, Epidemiology and End Results Program (SEER) and received recent data from Norway Cancer Registry. Results.  26 papers from institutional series were identified that reported 976 patients presented with appendiceal NETs. 3 disease related cases of death have been recorded which accounted for a calculated mortality rate of 0.31 %. No additional aNET with lethal outcome could be identified among single case reports in PubMed. Analysis of the data from SEER

revealed 95 % and 89 % survival rate of aNETs for 5 years and 40 years, respectively. The Cancer Registry of Norway registered a 5 years survival rate of 80.1 % for aNETs. Conclusions.  Published institutional series of aNETs revealed a very low, almost negligible mortality rate of 0.31 %. Data from large cancer registries demonstrate strikingly higher mortality rates, which indicate that several thousands patients died of aNET. However, these cases have not been documented in the medical literature. The reason for this discrepancy is not known, but could be partly explained by the data acquisition of cancer registries that provide comprehensive data sets but may be compromised by low accuracy of disease coding and imprecise data on causes of death. In summary, aNETs are probably less aggressive neoplasms than previously thought.

P01.33 Volvulus management of the intestine – rare cause of an acute abdomen in adult with unusual „twister“-like appearance on computed tomography F. Meyer*, C. Wex Klinik für Allgemein-, Viszeral- und Gefäßchirurgie, Universitätsklinikum, Magdeburg, Deutschland Background.  Small bowel volvulus is a rare cause of mechanical obstruction of the small intestine in adults. Nevertheless, any suspicion requires immediate clinical diagnostics since an acute torsion of the mesenteric vessels may result in the consequence of (partial) irreservible mesenteric ischemia, followed by the (in-)complete gangrene of the bowel or bowel segments. Methods.  Exemplary case presentation on an unusual case of a 67-year old patient with CT-proven, novelly completed through sectional image sequence and intraoperatively confirmed small bowel volvulus. Results.  The patient reported a killing pain and since many days consisting constipation at the time of emergency presentation. Clinically, he showed a local tenderness and palpation pain in the lower abdomen (therapy-refractory pain) and changing peristalsis in all four abdominal quadrants in the sense of an acute abdomen (but normal laboratory findings [inflammatory paramteres] and transabdomina sonogram). The CT scan revealed, impressively by using video sequence of thin-layer transversal-scans, a proving twister-like figure of the small bowel volvulus with spirally arranged consolidations. The thus derived indication for laparotomy and immediately followed execution approved the finding, which promptly induced to an essential derotation. Secondary but very likely promoting findings were an elongated sigmoid, moderately distinctive mobile cecum and over long (sagging) transversal colon. As there was no visible cause, a primary bowel voluvulus was most likely assumed whereby a predisposition by an over-long root of the mesentery is supposed. Using untangling of the small intestine, the reperfused detorqued bowel segment (extension, approximately 80 cm) was replaced in the abdominal cavity. The postoperative course was uneventful and the patient could be discharged on the 11th postoperative day. Conclusions.  The shown, even in the clinical daily routine under emergency conditions nearly real time achievable image processing made it possible to find quickly the correct diagnosis and, subsequently, to initiate the adequate therapy via prompt laparatomy for untwisting of the intestinal torsion. The CT scan herewith underlines the early place value in the differential diagnostic clarifying course of diagnostics by the acute abdomen, amongst other things with findings-associated accentuation through further timely image-processing according to the given technical potential as in the presented case.

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Abstracts P01.34 Detection of KRAS, NRAS, BRAF and PIK3CA by Mass Spectrometry: A flexible Technology for Routine Diagnostics M. Kriegsmann*1, P. Wandernoth2, N. Arens2, L. Houben2, R. Casadonte3, R. Longuespée2, J. Kriegsmann2 Institut für Pathologie, Ruprecht-Karls Universität Heidelberg, Heidelberg, Deutschland, 2Molekularpathologie Trier, Trier, Deutschland, 3Proteopath GbR, Trier, Deutschland 1

Background.  It has long been recognized that the epidermal growth factor receptor (EGFR) pathway is frequently activated in colorectal cancer. Therefore targeted therapies against EGFR such as cetuximab and panitumumab have been developed and are currently approved for the treatment of metastatic disease. It has been shown that activating mutations of KRAS or NRAS lead to a consecutive activation of the RAS-RAF pathway downstream of EGFR and consequently result in resistance to anti-EGFR therapy. Therefore mutational analysis for KRAS and NRAS is recommended prior to EGFR-directed therapy. Moreover BRAF status in conjunction with microsatellite testing adds prognostic information and is increasingly demanded by clinical collegues. As patients with PIK3CA mutations may benefit from nonsteroidal anti-inflammatory drugs, testing may further be extented for PIK3CA. Thus, reliable, fast, sensitive and cost effective methods for routine tissue based molecular diagnostics are required that allow the assessment of the CRC mutational status in a high throughput fashion. Methods.  We developed a custom designed assay for routine mass-spectrometric (MS) (MassARRAY, Agena Bioscience) analysis to test for the presence/absence of KRAS, NRAS BRAF and PIK3CA mutations. We applied this assay to 371 samples from patients with CRC, compared the results to Sanger sequencing and a chip hybridization assay (KRAS LCD-array Kit, Chipron). Results.  MS detected KRAS mutations in 160 (43 %), NRAS mutations in 7 (2 %), BRAF mutations in 34 (9 %) and PIK3CA mutations in 57 (15 %) out of 371 cases. Interestingly, Co-mutations of PIK3CA occured in 68 % of cases. Conclusions.  Mutational analysis by MS is a reliable method for routine diagnostic use, which can be easily extended for testing of additional mutations.

P01.35 Gender-specific differences of the perioperative management, early postoperative and oncosurgical long-term outcome in rectal cancer – data obtained in a prospective multicenter observational study J. Katzenstein*1, F. Meyer2, H. Ptok2, S. Wolff2, R. Albrecht3, R. Otto4, I. Gastinger4, H. Lippert4 AMEOS-Klinikum Aschersleben, Klinik für Allgemein- und Viszeralchirurgie, Aschersleben, Deutschland, 2Klinik für Allgemein-, Viszeral- und Gefäßchirurgie, Universitätsklinikum, Magdeburg, Deutschland, 3HELIOS Kliniken Aue, Klinik für Allgemein- und Viszeralchirurgie, Aue, Deutschland, 4 AN-Institut für Qualitätssicherung in der operativen Medizin, Otto-vonGuericke-Universität, Magdeburg, Deutschland 1

Background.  To investigate impact of gender-specific differences (primary end point) & alterations over various time periods (secundary end point) onto the outcome of surgical treatment of rectal cancer. Methods.  Using observational study design, patient-, tumor- & treatment-associated data of periop. management of consecutive patients with histological diagnosis of rectal cancer were documented in 2005/06 & 2010/11 incl. follow-up. Results.  Overall, 10,657 patients were evaluated, in the majority males (60.9 %). Risk factors: Males have (same in both study periods) a significantly (P < 0.001) higher risk by alcohol/nicotine abuse; No. of obese patients increased (other exogenic risk factors were stable over the years). The No. of MRI/EUS investigations increased rapidly through the years (P < 0.001) with greater proportion in men than women (trend). Based on

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the greater No. of EUS in 2010/11 (males: 65.8 %/females: 60.3 % versus 0.5 % within 2005/2006), a connection with the male gender was found (P < 0.001). There was no significant association between tumor site (cm from anus) & gender as between histopathological criteria & gender. Neoadjuvant radiochemoTx was significantly more often initiated in males (P < 0.001; no difference between study periods). Males underwent significantly more often rectum exstirpation (2005/2006: P < 0.001; 2010/2011: P = 0.05) & experienced significantly more often specific complications (2005/2006: P < 0.001; 2010/2011: P < 0.001). In contrast, in females Hartmann’s procedure & palliative creation of colostoma were significantly more often performed in both study periods. Early postop. outcome showed a significant difference between males & females in both study periods with regard to 30-day morbidity (P < 0.001) (hospital lethality: no difference). Overall survival was not significantly different between males & females in both study periods. Local recurrence rate increased through the postoperative course; after 5 years, it is 5 % in both genders with no significant difference as it was the same for tumor-free survival in both study periods meaning that local tumor recurrence not metachronous metastases determines tumor-free survival. The 5-year survival rate was appr. 60 % in both study periods with no significant association to genders. Conclusions.  The gender contributes to the partially different characteristics of rectal cancer but also providing a substantial impact onto the type of diagnostics & therapeutic procedure as well as early postoperative results but less onto the prognosis.

P01.36 Heterogeneous nuclear ribonucleoprotein K (hnRNP K) as a potential therapeutic target to increase radiosensitivity of RAS-mutant colorectal cancer K. Steinestel*1, W. Daskalaki1, M. Port2, S. Huss1, E. Wardelmann1, S. Eder2 Gerhard-Domagk-Institut für Pathologie, Universitätsklinikum, Münster, Deutschland, 2Institut für Radiobiologie der Bundeswehr, München, Deutschland

1

Background.  Mutations in the RAS oncogene are associated with enhanced radioresistance in a variety of malignancies and occur in about 50 % of colorectal carcinomas (CRC). We have previously shown that heterogeneous nuclear ribonucloprotein K (hnRNP K) confers radioresistance to malignant melanoma cells, while expression of the protein can be targeted by MAPK pathway inhibition. Methods.  We assessed hnRNP K expression in human CRC surgical specimens and in CRC cell lines with known KRAS mutation status. Furthermore, we employed cell culture methods to analyze the effect of ionizing radiation on cellular hnRNP K levels, subcellular distribution, cell survival and clonogenic viability and apoptotic index as assessed by FACS analysis. Finally, we employed a chicken chorioallantoic membrane model to analyze the effect of ionizing radiation and/or hnRNP K knockdown on CRC tumor growth and tumor cell proliferation in vivo. Results.  We show that hnRNP K is significantly overexpressed in human CRC surgical specimens compared to healthy mucosa and in KRAS-mutant compared to KRAS wild-type CRC cell lines. Ionizing radiation quickly leads to enhanced ERK phosphorylation together with upregulation and cytoplasmic accumulation of hnRNP K, while KRAS-mutant CRC cells with high endogenous hnRNP K levels display a higher baseline radioresistance in vitro and in vivo. SiRNA-mediated knockdown of hnRNP K abrogates the radioprotective effect of KRAS mutation in clonogenic survival assay and in the chorioallantoic membrane model, but this effect is not associated with enhanced IR-induced apoptosis; moreover, hnRNP K expression and clonogenic survival is significantly impaired upon MEK inhibition with Binimetinib (MEK162)in vitro. Conclusions.  Taken together, our results indicate MAPK-mediated overexpression of hnRNP K as a potential therapeutic target to overcome intrinsic radioresistance of KRAS-mutant CRC.

P01.37 An endoscopy-based orthotopic model of colorectal cancer resulting in distant metastasis in mice M. M. Mücke*1, P. Lenz2, 3, D. Bettenworth3, K. Schwegmann4, A. Faust4, M. Schäfers4, 5, D. Domagk6, C. Poremba7 1 Universitätsklinikum Münster, Münster, Deutschland, 2Universitätsklinikum Münster, Stabsstelle Palliativmedizin, Münster, Deutschland, 3 Universitätsklinikum Münster, Medizinische Klinik B, Münster, Deutschland, 4 University of Münster, European Institute for Molecular Imaging, Münster, Deutschland, 5Universitätsklinikum Münster, Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), Münster, Deutschland, 6Josephs-Hospital Warendorf, Münster, Deutschland, 7Pathologie München-Nord, München, Deutschland

Background.  Despite recent advances in colorectal cancer (CRC) management the prognosis is still limited, especially in advanced stages of the disease. Only a few mouse models described orthotopic tumor growth and distant metastases. Most of them involve surgical procedures that may influence tumor immunogenicity. Aim of this study was to establish an endoscopy-based murine orthotopic model that simulates advanced stages CRC. Methods.  The implantation of CRC tumor cells (Caco-2 and HT-29) performed either subcutaneously or orthotopic by submucosal injection during murine colonoscopy (coloview miniendoscopic system) in CD1 nude mice (n = 24) and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice (n = 10). For monitoring of tumor development, matrixmetalloproteinases (MMP) expression of tumors was assessed 24 h after i. v.-injection of a Cy5.5-labeled MMP-selective tracer (Cy5.5-AF443) by Fluorescence Reflectance Imaging (FRI) and Fluorescence Endoscopy. Tumors and metastases were histologically evaluated. Results.  Subcutaneaously implanted HT-29 cells resulted in a marked tumor growth 14 days after implantation. Orthotopic implantation in the colon of CD1 mice lead to decelerated tumor development after 17 weeks. In the NOD/SCID mice, distinct tumor growth could already be detected beginning at day 14 after submucosal cell injection. Subsequently, rapid tumors growth with occupation of the entire colonic circumference could be observed. Post mortem analysis revealed two liver and one lymph node metastasis which were confirmed by histological evaluation and immunohistochemistry (tumor cells both in the primary tumor as well as in the metastases positiv for CK20 and CDX2). As opposed to HT-29 cells, successful implantation of Caco-2-cells could not be achieved. FRI revealed only a discreet tracer uptake in s. c. implanted tumors with a target-to-background ratio of 1.55 ± 0.49), confirmatively MMP-2/-9 expression was not significantly increased. In contrast, MMP-tracer uptake was significantly enhanced in orthotopic implanted tumors. Conclusions.  We successfully employed a metastatic, endoscopy based CRC model in immunodeficient mice. To date, this is the first minimal invasive murine model with minimal influence on the tumor microenvironment reporting high rates of distant metastasis. Therefore, this model appears to be promising for examination of CRC biology even and preclinical evaluation of novel diagnostic and therapeutic approaches in advanced stages of the disease in the future.

P01.39 The new approach to find and identify MSI-associated genes in colorectal cancer patients F. Han*, Y. Zhang, Q. Huang, H. Zhang, E. Xu, Y. Ke, J. Chen, M. Lai The First Affiliated Hospital, Zhejiang University Medical College, Department of Pathology, Hangzhou, China, Volksrepublik Background.  Microsatellite instability (MSI), caused by DNA mismatch repair (MMR) deficiency in many cancers, could be observed with a high rate of insertion/deletions in cancer-related genes. The Cancer Genome Atlas (TCGA) aims to generate comprehensive, multi-dimensional maps of the genomic changes in 20 different cancer types. Our study was aimed

to identify some MSI-associated genes in Chinese colorectal cancer (CRC) patients by combined the MSI detection in clinical samples and data analysis from TCGA database. Methods.  Based on MSI status, TCGA colon cancers samples had been divided into MSI-H and MSI-L/MSS group. The frequency of frameshift mutations from the two groups has been accumulated, and then we searched simple repeat microsatellite sequences in the target frameshift mutations sites, especially focus on exon region of gene in the TCGA database. Genomic DNA was extracted from macroscopically dissected slides in 660 paraffin-embedded, paired with primary tumors and normal colorectal tissues. Microsatellite instability status was determined by analyzing a comparable panel of five microsatellite markers (BAT25, BAT26, D2S123, D17S250 and D5S346). Sanger sequencing and specific antibodies for the truncated proteins had been used to confirm the mutation status in CRC samples. Results. The frequency of frameshift mutation panel in MSI-H and MSI-L/MSS group was different. Among MSI-H colon cancer the top eight frequently frameshift mutated genes were RNF43, TTN, BCL9L, SETD1B, DOCK3, HNRNPL, MBD6 and BMPR2. While, APC, ZFPM1, SOX9, B3GNT6, TGIF1, COL18A1, MAL2 and GRIN3B are the top eight frameshift mutated genes in MSI-L/MSS colon cancer group. The frameshift mutation frequency was 218.9/sample in the MSI-H group, which was much higher than it in the MSI-L/MSS group (5.96/sample). Furthermore, the mutation sites of SETD1B, MBD6, BMPR2, TEAD2 and AXIN2 were all comprised mononucleotide repeats. Conclusions.  MSI-associated genes have some unique features. They have some special mononucleotide or dinucleotide repeats in their exon regions. These nucleotide sites have a higher frequency for mutation, especially for frameshift mutation. Moreover, some frameshift mutations are specially detected in MSI-H group in our samples. Some online databases, like TCGA, provide us a new approach of data mining from the clinical samples. We can take advantage of these databases and combine with our own clinical data to identify new MSI-associated genes.

P01.40 Number of Tumour-Infiltrating Lymphocytes is Associated with Lymph Node Size, Lymph Node Harvest and Outcome in Node-Negative Colon Cancer B. Märkl*1, J. Wieberneit1, H. Kretsinger1, P. Mayr1, M. Anthuber2, H. Arnholdt1, G. Schenkirsch3 Institut für Pathologie, Klinikum, Augsburg, Deutschland, 2Insitut für Viszerale Chirurgie, Klinikum Augsburg, Augsburg, Deutschland, 3 Tumorzentrum Augsburg, Augsburg, Deutschland 1

Background.  Colon cancer is one of the most common malignant tumors in the western world. The outcome strongly depends on the UICC stage. Stage I and II cancers show a favorable clinical course. Nevertheless, it is well known that about 20 % of stage II cases show an aggressive behavior. Up to know no prognostic system could be established to identify such cases in a sufficient way with general acceptance. The microenvironment and immune response gain attention in many different tumor entities. Recently, we postulated lymph node (LN) size as a prognostic marker in node negative colon cancer. We assumed that LN size is associated we the immunogenicity of a tumor. Therefore, it could perhaps serve as a surrogate marker for the immune response. In order to identify an association between LN size and the inflammatory reaction against colon cancer we performed a retrospective study measuring the densitity of intratumoral T-lymphocytes and its association with LN size. Methods.  We included 170 node-negative colon cancer cases to evaluate the density of tumor infiltrating lymphocytes (TILs). Immunohistochemically CD3- and CD8-positive T-cells were counted within the tumor center (CT) and the invasion front (IF) using an open source digital system (Image J) according to the description by Vayrynen et al. A score was built including the densities of the T-cell populations at the two tumor regions (IM score). The MMR-status was determined in all cases.

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Abstracts Results.  The TIL density was significantly increased in cases with sufficient LN harvest and higher numbers of LNs larger than 5 mm (LN5). The mean TIL densities were higher in the sub-group of MMR-deficient cases. There was a non-significant trend towards an increasing rate of MMR-deficient cases in higher IM categories. High TIL numbers were associated with improved cancer-specific survival. The analysis of the immune (IM)score revealed a significantly different cancer-specific outcome (P = 0.024) with no cancer-related death in the group with the highest score. IM score and pT stage were independently prognostic. Conclusions.  We were able to show an association between immune response, LN size and LN harvest. The density of TIL is a robust independent prognostic marker in colon cancer. Our data provide further indications that the supposed prognostic effect of LN harvest is confounded by immune response. LN size could serve as a surrogate marker for immune response that is very easy to determine without need for special equipment.

P01.41 CYB5R1 links EMT and poor prognosis in colorectal cancer C. Woischke*1, C. Blaj1, E. M. Schmidt1, S. Lamprecht1, J. Engel2, H. Hermeking1, T. Kirchner1, D. Horst1 Pathologisches Institut, Ludwig-Maximilians-Universität, München, Deutschland, 2Tumorregister München (TRM) und Institut für medizinische Informationsverarbeitung, Biometrie und Epidemiologie, LudwigMaximilians-Universität, München, Deutschland 1

Background.  Colon cancers show significant tumor cell heterogeneity within the same core genetic background. Epithelial-mesenchymal transition (EMT) is an important functional aspect of this heterogeneity and a hallmark of colon cancer progression. Here, we identify NADH-cytochrome b5 reductase 1 (CYB5R1), which is part of an enzyme family involved in oxidative stress reactions and drug metabolism, as an indicator of EMT in colon cancer. Methods.  Immunostainings and immunofluorescence were used for in situ localization of CYB5R1 in colon cancer tissues. Gene Set Enrichment Analyses (GSEA) were applied to gauge for associations of CYB5R1 expression and EMT. Prospectively collected survival data were used to determine clinical impact of CYB5R1 and data were validated in a large data set from The Cancer Genome Atlas (TCGA). Results.  CYB5R1 strongly labelled tumor cells at the infiltrative tumor edge of colon cancers undergoing EMT. GSEA analyses revealed an extraordinarily strong association of CYB5R1 expression and two core EMT gene expression signatures. High CYB5R1 expression significantly indicated poor prognosis in colorectal cancer patients on the protein and mRNA level. Conclusions.  CYB5R1 strongly links EMT and colon cancer progression, a finding that might be exploited for diagnostic and eventually therapeutic purposes for colorectal cancer patients.

Postersitzung AG Uropathologie P02.01 Telangiectatic Oncocytoma – Two Cases of a Rare Variant of Classic Oncocytomas M. M. Gerlach*1, D. Krause1, D. Wilbert2, F. Bauknecht3, B. Rost4, M. Rössle1, B. Padberg-Sgier1 1 Institut für Pathologie, Kantonsspital Graubünden, Chur, Schweiz, 2Klinik für Urologie, Spital Linth, Uznach, Schweiz, 3Chirurgische Klinik, Kantonsspital Glarus, Glarus, Schweiz, 4Klinik für Radiologie, Kantonsspital Glarus, Glarus, Schweiz

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Background.  Telangiectatic oncocytomas are rare epithelial tumors of the kidney. Unlike classic oncocytomas they present with a spongy appearance without typical central scarring. Therefore, they are often highly suspicious for malignancy in preoperative medical imaging and differentiation against renal cell carcinomas with cystic growth can be difficult. Methods.  We present two cases of this entity infrequently found in daily diagnostic work-up and autopsy. First case: A 54 years old male patient who underwent total nephrectomy because of a large tumor of the left kidney. Macroscopically, a 50 mm measuring tumor with spongy cut surface was seen, surrounded by clearly delimitable renal tissue. Second case: During autopsy of a 75 years old patient who died of cardial decompensation and suffered from renal insufficiency, a multicystic, bloodfilled and 20 mm measuring tumor was found next to focal oncocytosis, several papillary adenomata and multiple cysts of the renal parenchyma. Results.  In both cases, microscopic examination of the tumors revealed cystic, blood-filled spaces surrounded by eosinophilic oncocytes. Oncocytic tumor cells lacked cytogical atypia and were negative for cytokeratin (CK) 7, vimentin, CD10 and CD31. In the first case, a sporadically developed telangiectatic oncocytoma was diagnosed. Whereas, the second case might represent a hereditary variant as various oncocytic cell nests were found. Conclusions.  In summary, this rare variant of renal oncocytomas should be kept in mind when being faced with renal tumors with a hemorrhagic spongy or multicystic appearence. Particularly, the exclusion of a malignant renal neoplasia by means of morphological analysis and immunohistochemical stainings is indispensable to ensure appropriate subsequent clinical management.

P02.02 CD73 Predicts Favorable Prognosis in Patients with Nonmuscle-Invasive Urothelial Bladder Cancer M. S. Wettstein1, L. Buser*2, T. Hermanns1, F. Roudnicky3, D. Eberli1, P. Baumeister1, T. Sulser1, C. Poyet1, P. Wild2 1 Klinik für Urologie, UniversitätsSpital, Zürich, Schweiz, 2Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz, 3Institute of Pharmaceutical Sciences, ETH Zürich, Zürich, Schweiz

Background.  CD73 is a membrane associated 5’-ectonucleotidase that has been proposed as prognostic biomarker in various solid tumors. The aim of this study is to evaluate CD73 expression in a cohort of patients with primary bladder cancer in regard to its association with clinicopathological features and disease course. Methods.  Tissue samples from 174 patients with a primary urothelial carcinoma were immunohistochemically assessed on a tissue microarray. Associations between CD73 expression and retrospectively obtained clinicopathological data were evaluated by contingency analysis. Survival analysis was performed to investigate the predictive value of CD73 within the subgroup of pTa and pT1 tumors in regard to progression-free survival (PFS). Results.  High CD73 expression was found in 46 (26.4 %) patients and was significantly associated with lower stage, lower grade, less adjacent carcinoma in situ and with lower Ki-67 proliferation index. High CD73 immunoreactivity in the subgroup of pTa and pT1 tumors (n = 158) was significantly associated with longer PFS (HR: 0.228; p = 0.047) in univariable Cox regression analysis. Conclusions.  High CD73 immunoreactivity was associated with favorable clinicopathological features. Furthermore, it predicts better outcome in the subgroup of pTa and pT1 tumors and may thus serve as additional tool for the selection of patients with favorable prognosis.

P02.03 Association of the G/​G-SNP309 variant in the MDM2 gene with earlier tumor onset in female renal cell carcinoma patients C. Stöhr*1, R. Stöhr1, A. Wenners2, A. Hartmann1, S. Bertz1, V. Spath3, B. Walter4, 5, K. Junker6, H. Moch7, R. Hinze8, S. Denzinger9, E. E. Bond10, G. L. Bond10, K. Blümke11, K. Weigelt4, V. Lieb4, E. Nolte4, P. Fornara12, B. Wullich4, S. Wach4, H. Taubert4 1 Institut für Pathologie, Friedrich-Alexander-Universität ErlangenNürnberg, Erlangen, Deutschland, 2Klinik für Gynäkologie und Geburtshilfe, Universitätsklinikum Schleswig-Holstein, Campus Kiel, Kiel, Deutschland, 3 Institut für Pathologie, Caritas-Krankenhaus Bad Mergentheim, Bad Mergentheim, Deutschland, 4Klinik für Urologie, Universitätsklinikum Erlangen, Erlangen, Deutschland, 5Klinik für Urologie und pädiatrische Urologie, Kreiskliniken Altötting Burghausen, Altötting, Deutschland, 6 Klinik für Urologie und Kinderurologie, Universitätsklinikum des Saarlands, Homburg/Saar, Deutschland, 7Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz, 8Institut für Pathologie, Helios Kliniken Schwerin, Schwerin, Deutschland, 9Klinik für Urologie, University of Regensburg, Regensburg, Deutschland, 10The Ludwig Institute of Cancer Research, University of Oxford, Oxford, Vereinigtes Königreich, 11Institut für Rechtsmedizin, Martin Luther Universität Halle-Wittenberg, Halle, Deutschland, 12Klinik für Urologie, Martin Luther Universität HalleWittenberg, Halle, Deutschland

Background.  Mdm2 (Human mouse double minute 2) is an important opponent of the tumor suppressor p53. The G/G variant of SNP309 in the MDM2 promoter can increase Mdm2 mRNA/protein expression and is associated with an increased risk and earlier onset of different cancers especially in Asian populations. The frequency and impact of the G/G variant has not been studied yet in Caucasian renal cell carcinoma (RCC) patients. Therefore, we analyzed 197 consecutive Caucasian RCC patients and 205 age-selected causcasian RCC patients for MDM2 SNP 309 genotype distribution. Methods.  DNA was isolated from formalin-fixed and paraffin-embedded normal renal tissues of RCC patients or from blood lymphocytes. Depending on the participating institution, genotyping was performed by restriction fragment length polymorphism or Sanger sequencing, Age at tumor onset and tumor characteristics were tested for associations to MDM2 SNP 309 genotype by two-sided exact χ²-test using IBM SPSS statistics 20. P-values <0.05 were regarded as significant. Results.  In the cohort of 197 consecutive RCC patients, we detected the G/G variant in 18 (9.1 %) patients, the G/T variant in 116 (58.9 %) patients and the T/T variant in 63 (32.0 %) patients. There was no correlation detected between age at tumor onset and SNP309 genotypes among the entire cohort or among male RCC patients. However, female G/G patients (median age 59.5 years) were diagnosed 13.5 years earlier than T/T females (median age 73 years). After separating all females into two groups at their median age (68 years), 7 patients and 1 patient with the G/G variant and 9 patients and 13 patients with the T/T variant were noted in these age groups (P = 0.024) Conclusions.  Female caucasian RCC patients carrying the MDM2 SNP 309 G/G variant showed significantly earlier tumor onset than patients with the wildtype T/T genotype.

P02.05 Histological-cytological prerequisites for the therapeutic option of active surveillance in prostate cancer according to the new ISUP grading system D. Ringli*1, H. Gevensleben2, G. Kristiansen2, B. Helpap1 1 Institut für Pathologie, HBH-Kliniken, Singen, Deutschland, 2Institut für Pathologie, Universitätsklinikum, Bonn, Deutschland

Background.  Aims: It is well known that not all cases of prostate cancer (PCa) have to be actively treated but so called insignificant tumours can alternatively be monitored under active surveillance (AS). Criteria that

need to be fulfilled for the therapeutic option of AS include prostate specific antigen (PSA) levels < 10 ng/ml, 1 to 3 tumour-positive core needle biopsies in one lobe, a tumour volume < 50 %, and a Gleason score (GS) of 6 or 7a (3 + 4). In the case of radical prostatectomy, these cancers present with small GS 6 malignancies and an organ-confined tumour stage pT2a without metastases or PSA-relapse even after years of monitoring. Unfortunately, not all GS 6 cancers behave this way. In some cases, the tumour advances, presents with an increased tumour volume (stage pT2c or pT3a), and elevated PSA levels under AS. Courses of disease that may also occur in PCa with GS 7a. Methods and Results.  According to the Gleason-Helpap grading, we subdivided GS 6 PCa into GS 6/2a and 2b tumours. Ninety-five percent of GS 6/2a cancers exhibited an involvement of < 10 % in one positive core biopsy, whereas GS 6/2b and GS 7a/2b PCa presented with an infiltration of > 10 % of least two biopsies, partly involving both prostate lobes. We subsequently applied the recently published prognostic grade grouping according to Epstein and colleagues: grade group 1 PCa being designated as insignificant very low grade type(GS 6/2a), grade group 2 tumours as low and early intermediate type (GS 6/2b and 7a), grade group 3 including the late intermediate or early high grade type (GS 7b), grade group 4 the high grade type (GS 8),and grade group 5 the very high grade type (GS 9–10). Conclusions.  The classification according to the prognostic grade grouping corresponds with the new grading of ISUP grade 1 to 5 which has been declared obligatory for the grading of PCa by the ’Deutsche Gesellschaft für Pathologie (DGP)’ as of 1. 1. 2016. The histological classification complies with the previous GS 6–10.

P02.06 Tumor-associated macrophages in prostate cancer and their potential role in tumor progression H. Hessel*1, M. Athelogou2, A. Buchner3, N. Harder2, N. Brieu2, M. Yigitsoy2, R. Schönmeyer2, G. Schmidt2, G. Binnig2, C. Stief3, T. Kirchner1 1 Pathologisches Institut, Ludwig-Maximilians-Universität, München, Deutschland, 2Definiens AG, München, Deutschland, 3Urologische Klinik und Poliklinik, Klinikum der Universität, München, Deutschland

Background.  Tumor-associated macrophages (TAM) are relevant components of the tumor microenvironment (TME). Based on the macrophages polarization model TAM can be differentiated in vitro into M1 macrophages with tumoricidal and M2 macrophages with tumorigenic capacity. TAM have been associated with tumor progression in different tumor entities. In this study we investigated in vivo the prognostic relevance of TAM within low and intermediate risk prostate cancer (PCa) specimens by comparative analyses of TAM-related features as well as histopathological and clinical diagnosis parameters. Methods.  Analysis and quantification were performed on formalin-fixed paraffin-embedded tissue sections of 50 low to intermediate risk PCa specimens from radical prostatectomies with Gleason-Score ≤ 7a. Thirty of all carcinoma patients showed a Prostate Specific Antigen (PSA) recurrence. M1- and M2-like macrophages were characterized by immunohistochemical duplex staining for CD68 and CD163, while CK18 and p63 duplex staining was used to enable automatic annotation of tumor and non-tumor regions. CD68/CD163 virtual slides were automatically co-registered with the corresponding CK18/p63 stained serial slides and macrophages were quantified separately in tumor regions, non-tumor regions, and TME. Both, overall macrophages and the fraction of M1 and M2 macrophages were analyzed using the Tissue Phenomics technology, which combines co-registration, image analysis and data mining methods for finding correlations between histopathological and clinical data. Statistical analyses were carried out to analyze the correlation between the macrophages signatures within different regions of interest (tumor, non-tumor, TME), the tumor grading and size, and clinical parameters (degree of PSA recurrence, overall- and disease-free survival).

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Abstracts Results.  Prostate cancer patients without PSA recurrence showed a significant higher proportion of M1 vs. M2 macrophages, in particular, in the TME in contrast to patients with PSA recurrence (two-sided t-test p < 0.01). Additionally, the fraction of M1 and M2 macrophages turned out to be related to the disease-free survival time (log-rank test, p-value < 0.05). Conclusions.  We presented promising results which indicate a considerable prognostic potential of macrophages in PCa. Furthermore, we demonstrated that the Tissue Phenomics technology enables the investigation and the evaluation of new prognostic tissue-based biomarkers for accurate and reliable patient stratification.

P02.07 HER2 and TOP2A gene amplification and protein expression in Upper Tract Urothelial Carcinomas K. Aumayr*1, T. Klatte2, B. Neudert1, P. Birner1, S. Shariat2, M. Schmidinger3, M. Susani1, A. Haitel1 Medizinische Universität Wien, Klinisches Institut für Pathologie, Wien, Österreich, 2Medizinische Universität Wien, Universitätsklinik für Urologie, Wien, Österreich, 3Innere Medizin, Medizinische Universität Wien, Wien, Österreich 1

Background.  HER2, a potential target for therapy, has been described to be amplified in urothelial carcinomas. As the topoisomerase II alpha (TOP2A) gene is located close to the HER2 gene on chromosome 17q12-q21, it is frequently either co-amplified or deleted with HER2 amplification. The purpose of this study was to assess the impact HER2 and TOP2A gene amplification as well as protein expression on outcomes of upper tract urothelial carcinoma (UTUC). Methods.  HER2 and TOP2A gene amplification and protein expression were assessed in 81 patients with radical nehroureterectomy for UTUC. Immunohistochemistry and chromogenic in-situ hybridization was performed on formalin-fixed, paraffin-embedded samples Results.  HER2 protein expression was observed in 27/81 (33 %) cases, of which 8 cases exhibited amplification of HER2. One of them had an additional polysomy 17, whereas 6/67 HER2 non-amplified cases revealed a polysomy 17. Coamplification of HER2 and TOP2A was found in 4 cases, whereas 3 cases showed only HER2 amplification and 20 cases only TOP2A amplification. HER2 IHC overexpression was associated with higher-grade tumors (p = 0.001), non-organ confined carcinomas (p = 0.017), HER2 amplification (p < 0.00001), TOP2A amplification (p = 0.016). HER2 amplification was association with higher tumor grade (p = 0.001) and lymphnode metastasis (p = 0.003). TOP2A IHC positivity was significantly associated with higher tumor grade (p = 0.0004), TOP2A amplification (p = 0.0003), polysomy 17 (p = 0.035) and HER2 IHC overexpression (p = 0.28), whereas all categories of tumor stage and HER2 amplification remained not related. TOP2A amplification was significantly more frequent in tumors with higher grade, higher tumor stage, polysomy 17 and distant metastasis (p = 0.015; p = 0.042; p = 0.032; p = 0.011). In univariable analyses HER2 IHC positivity, TOP2A amplification, and polysomy 17 were associated with poor clinical outcome after surgery. Conclusions.  HER2 IHC overexpression and TOP2A amplification are associated with features biologically aggressive UTUC. Overexpression and/ or amplification of HER2 and TOP2A could help identify patients who may benefit from targeted therapy.

P02.08 Adenocarcinoma of the bladder, colorectal and urothelial cancer: similar or diverse? A. Maurer*1, H. Reis2, S. Stoerkel3, R. Golz3, D. Lazica4, K. Schwamborn5, F. Kurtz6, T. Grimm7, S. Siegert8, C. Lüders9, J. Ellinger10, F. Bremmer11, A. Zimpfer12, S. Bertz13, N. Gassler14, R. Knuechel1, N. T. Gaisa1 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, Institut für Pathologie, Universitätsklinikum Essen, Universität DuisburgEssen, Essen, Deutschland, 3Institut für Pathologie, HELIOS Klinikum Wuppertal, Universität Witten/Herdecke, Wuppertal, Deutschland, 4Klinik für Urologie und Kinderurologie, HELIOS Klinikum Wuppertal, Universität Witten/Herdecke, Wuppertal, Deutschland, 5Institut für Pathologie und pathologische Anatomie, Klinikum rechts der Isar, Technische Universität München, München, Deutschland, 6Klinik für Urologie, Technische Universität München, München, Deutschland, 7Klinik für Urologie, Klinikum der Universität München, München, Deutschland, 8Pathologie MünchenNord, München, Deutschland, 9Institut für Pathologie, Universitätsklinikum, Bonn, Deutschland, 10Klinik und Poliklinik für Urologie und Kinderurologie, Universitätsklinikum, Bonn, Deutschland, 11Abteilung für Pathologie, Universitätsmedizin, Göttingen, Deutschland, 12Institut für Pathologie, Universitätsmedizin Rostock, Rostock, Deutschland, 13Institut für Pathologie, Universitätsklinikum Erlangen, Universität Erlangen-Nürnberg, Erlangen, Deutschland, 14Institut für Pathologie, Klinikum Braunschweig, Braunschweig, Deutschland 1

2

Background.  Primary adenocarcinoma of the bladder is a rare malignancy accounting for less than 2 % of bladder cancers. Since some primary bladder and colorectal adenocarcinoma show similar morphologies, we compared the mutation frequencies of 20 genes in order to reveal similarities and differences in tumorigenesis. Results of our comparison of primary bladder adenocarcinoma (BAC), urothelial carcinoma (UC) and colorectal adenocarcinoma (CAC) are presented. Methods.  Clinically validated formalin-fixed, paraffin embedded primary bladder adenocarcinoma samples were obtained from different German centers. N = 27 suitable samples were sequenced using a self-designed amplicon panel covering all exons of 20 genes on a benchtop sequencer. The BAC mutation frequencies were compared to mutational data for UC and CAC generated by the TCGA Research Network. Additionally N = 7 samples of glandular lesions (cystitis cystica et glandularis and intestinal metaplasia) were analyzed. Results.  All BACs showed a higher frequency of KRAS mutations than expected for invasive UC and an elevated frequency of TP53 mutations compared to CAC and UC. No activating HRAS mutation could be found in any sample. More than half of the BACs showed either a PIK3CA or RAS mutation. In benign glandular lesions of the bladder no PIK3CA, TP53 or RAS mutations could be detected. Conclusions.  Our results show elevated TP53 and RAS mutations in BACs compared to UCs arguing for a different tumorigenesis pathway of UCs and BACs. The investigated benign glandular lesions display no somatic TP53, RAS or PIK3CA mutations, encouraging their classification as benign and non-precancerous.

P02.09 Precision Medicine in Patients with Genitourinary Tumors V. Sailer*1, J. Cyrta1, 2, C. Pauli1, 2, J. M. Mosquera1, H. Beltran1, 2, A. Sboner1, 2, M. A. Rubin3 Weill Cornell Universität für Medizin, Englander Institut für Präzisionsmedizin, New York, Vereinigte Staaten von Amerika, 2Weill Medical College of Cornell University, New York, Vereinigte Staaten von Amerika, 3 Weill Medical College of Cornell University, Meyer Cancer Center, New York, Vereinigte Staaten von Amerika 1

Background.  Precision Medicine (PM) denominates the tailored treatment of patients not only with malignant tumors but also with benign

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conditions. Next generation sequencing of tumor tissue can detect actionable somatic molecular alterations that might have a direct clinical impact on patient care. This data can contribute to the better understanding of molecular mechanisms that underlie a disease and fuel translational research. Here we report our workflow in a cohort of patients with genitourinary tumors. Methods.  More than 200 patients with genitourinary tumors have been enrolled in the Englander Institute for Precision Medicine (IPM) at Weill Cornell Medicine so far, enabling pathologists to collect fresh (or archived) tumor tissue for whole exome sequencing (WES) and for the development of patient-derived organoids. A computational team summarizes the genomic data in a clinical report (. Fig. 1). All IPM data can be accessed through cBioPortal. Additionally, a Rapid Autopsy Program is implemented in the IPM workflow. In a biweekly tumor board selected cases are presented and discussed in a multidisciplinary team. Results.  Clinical reports from 115 patients with a total of 184 tumor samples and germline control (71/104 prostatic carcinomas, 30/63 urothelial carcinomas, 10/10 renal cell carcinomas, 4/7 other) were issued revealing a multitude of somatic copy number alterations and mutations.

Conclusions.  In patients with genitourinary tumors WES can reveal actionable mutations. Examples are Trastuzumab for a patient whose urothelial carcinoma overexpressed HER2 or targeting the PI3K pathway in tumors with PTEN loss. WES data can also enable the clinician to include the patient in a basket trial where molecular alterations rather than the disease type define inclusion criteria. Furthermore, the development of patient-derived organoids provides an opportunity for individual in vitro drug testing. All data can be accessed and visualized through cBioPortal and thus reviewed in context of published data, e. g. from The Cancer Genome Atlas (TCGA). The pathologists expertise in obtaining, processing and reviewing fresh as well as archival tissue for WES is indispensable and this task can not be delegated.

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Abstracts P02.10 Mammalian (mechanistic) target of Rapamycin (mTOR) signaling pathway in papillary renal cell carcinoma I. Polifka*1, C. Stöhr1, V. Spath2, L. Trojan3, P. Ströbel3, F. Becker4, V. Jung5, C. Wülfing6, A. J. Schrader7, 8, P. Barth9, M. Stöckle5, M. Staehler10, C. Stief10, H. Höfler11, A. Haferkamp12, M. Hohenfellner13, S. Macher-Göppinger14, V. Lieb15, B. Wullich15, C. Bolenz16, T. Klein17, 18, J. Noldus18, S. Bierer8, L. Hertle8, W. Brenner19, F. Roos19, B. Walter20, W. Otto21, M. Burger21, E. Herrmann8, A. Hartmann1 Institut für Pathologie, Friedrich-Alexander Universität, Erlangen, Deutschland, 2Institut für Pathology, Bad Mergentheim, Deutschland, 3 Abteilung für Pathologie, Universitätsmedizin, Göttingen, Deutschland, 4 Urologisches Zentrum am Boxberg, Neunkirchen, Deutschland, 5Klinik für Urologie und Kinderurologie, Universitätsklinikum des Saarlandes, Homburg, Deutschland, 6Klinik für Urologie, Asklepios Klinik Altona, Hamburg, Deutschland, 7Klinik für Urologie, Pediatrische Urologie und Andrologie, Universitätsklinikum Gießen und Marburg, Marburg, Deutschland, 8Klinik für Urologie, Universitätsklinikum Münster, Münster, Deutschland, 9Gerhard-Domagk-Institut für Pathologie, Universitätsklinikum, Münster, Deutschland, 10Klinik für Urologie, Universitätsklinikum München, München, Deutschland, 11Pathologisches Institut, TU München, Klinikum Rechts der Isar, München, Deutschland, 12 Klinik für Urologie und Kinderurologie, Universitätsklinikum, Frankfurt am Main, Deutschland, 13Urologische Klinik, Universitätsklinikum, Heidelberg, Deutschland, 14Institut für Pathologie, Ruprecht-Karls-Universität, Heidelberg, Deutschland, 15FAU Erlangen-Nürnberg, Institut für Urologie, Erlangen, Deutschland, 16Institut für Urologie, Pediatrische Urologie, Universitätsklinikum, Ulm, Deutschland, 17Urologische Gemeinschaftspraxis, Bielefeld, Deutschland, 18Klinik für Urologie, Marien Hospital Herne, Ruhr Universität Bochum, Herne, Deutschland, 19Urologische Klinik und Poliklinik, Universitätsmedizin Mainz, Mainz, Deutschland, 20Klinik für Urologie, Kreiskliniken Altötting-Burghausen, Burghausen, Deutschland, 21Klinik für Urologie, Klinikum der Universität, Regensburg, Deutschland 1

Background.  The mammalian (mechanistic) target of rapamycin (mTOR) pathway plays an important role in protein translation, metabolism and cell growth. In clear cell renal carcinoma mTOR inhibitors such as temsirolimus and everolimus are an efficient therapy. There are some clinical studies that hint to a potential of mTOR targeted therapy in papillary renal cell carcinoma (pRCC). The pRCC is currently divided in two subtypes (type 1 and type 2), which display a distinct histology and outcome. In this study we screened a large cohort of pRCC for the expression of mTOR related proteins to estimate the potential of mTOR targeted therapies and to uncover differences between the two subtypes. Methods.  A cohort of pRCC tumors and clinical data were collected from the participating pathologies and staged according to the 2002 TNM classification. Tissue micro arrays were constructed. All tumors were reclassified to identify type 1 and type 2 pRCCs according to the WHO, the International Society of Urological Pathology (ISUP) and Vancouver Classification of Renal Neoplasia. Then mTOR-related immunohistochemical stainings (p-mTOR, AKT1, p-MEK1/2, PTEN, p-S6 Ribosomal Protein, phospho-p70 S6 Kinase, p-4E-BP1, FKHR, HIF1-α, Cyclin D1) were performed on the TMA blocks in order to detect the expression of these proteins and to show differences between the two subtypes. The staining intensity and the percentage of positive tumor cells were merged into an immunoreactive score. The influence on the survival of the patients was be investigated in univariate and multivariate cox analysis. Results.  From the cohort of pRCC tumors 161 were classified as type 1 and 93 were categorised as type 2. The expression of all mTOR pathway related proteins showed always some activation of the mTOR signalling pathway, with especially phosphorylated mTOR in 56.3 % of all tumors. In most cases the both subtypes displayed no difference, but in the case of FKHR and Cyclin D1 there was a significant higher frequency of the expression in type 2 tumors (p < 0.001, p = 0.002, respectively). Conclusions.  pRCC frequently showed a mTOR signalling activity (especially in pmTOR) with a potential of mTOR inhibitor therapy at least in

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a subpopulation. Both pRCC subtypes showed slightly different mTOR pathway profiles. This hints to different molecular alterations which could also influence the suitability for mTOR targeted therapies according to the subtype. The influence of mTOR expression on the survival of patients will be reported.

P02.11 Are environmental and genetic risk factors of bladder cancer different among early-and late-onset patients? V. Weyerer*1, 2, M. Rava2, E. López De Maturana2, M. Márquez2, A. Hartmann1, R. Stöhr1, A. Carrato3, A. Tardón4, S. Chanock5, M. Garcia-Closas6, N. Rothman5, D. T. Silverman5, M. Kogevinas7, F. X. Real8, N. Malats2 Institut für Pathologie, Friedrich-Alexander Universität, Erlangen, Deutschland, 2Genetic and Molecular Epidemiology Group, Spanish National Cancer Research Centre, Madrid, Spanien, 3Servicio De Oncología Médica, Hospital Ramón Y Cajal, Madrid, Spanien, 4Faculty of Medicine, University of Oviedo, Oviedo, Spanien, 5Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, Vereinigte Staaten von Amerika, 6 Division of Genetics and Epidemiology, Institute of Cancer Research, Sutton, Vereinigtes Königreich, 7Centre For Research In Environmental Epidemiology, Barcelona, Spanien, 8Epithelial Carcinogenesis Group, Spanish National Cancer Research Centre, Madrid, Spanien 1

Background.  The majority of patients with urothelial bladder cancer (UBC) are diagnosed after age 50. Therefore, most studies of risk factors reflect associations with late onset of the disease. Here we aim to assess whether early-onset UBC shares the same etiological factors as late-onset UBC. Methods.  We investigated 51 early- (aged ≤45 years) and 1163 late-onset UBC patients newly-diagnosed during 1998–2001 and 1270 matched controls from the Spanish Bladder Cancer/EPICURO Study. Epidemiological information was collected using computer-assisted personal interviews. 21 genetic variants previously identified by GWAS were considered. Using a case-case-control design we assessed associations in early- and late-onset patients with respect to controls. We also compared early-onset cases with young controls to rule out the cohort effect. We used unconditional logistic and multinomial regression to estimate odds ratios (OR), relative risk ratios (RRR), and 95 % confidence intervals (95 %CI). Results.  “High-risk occupational exposures” was associated with risk in both groups of patients with a slightly higher magnitude of effect in early-onset (RRR = 3.51, 95 %CI 1.45–8.48) compared to late-onset (RRR = 2.00, 95 %CI 1.58–2.51). We observed a similar pattern for family history of cancer, the RRR being 3.46 (95 %CI 1.62–7.39) and 1.26 (95 %CI 1.06–1.50), respectively. In addition, high vitamin D3 levels decreased the risk of early- and late-onset UBC. However, high daily intake of coffee and no use of nonaspirin non-steroidal anti-inflammatory drugs increased the risk only in individuals >45 years. After comparing early-onset cases with young controls, smoking was only a risk factor for late-onset UBC. At a nominal p-value ≤ 0.05 the variant CBX6-APOBEC3A-rs1014971 was associated with risk in both groups with a stronger effect in early-onset (RRR = 0.43, 95 %CI 0.22–0.85) than in late-onset (RRR = 0.84, 95 %CI 0.73–0.96). Genetic variants in TACC3-FGFR3-rs798766 and SLC14A1-rs1058396 increased significantly (p-value ≤ 0.05) the risk of UBC only in early-onset, whereas variants mapped to NAT2, UGT1A, TERC, CLPTM1L, LSP1, GSTM1, and JAG1 were associated only with the risk of late-onset UBC. Conclusions.  Our results suggest that early-onset UBC has a different etiological scenario than late-onset UBC. While statistical power limits the interpretation of some of the associations, these results indicate that both genetic and xenobiotic effects may account for the observed differences.

P02.12 High frequency of TERT promoter mutations in HPV-negative penile squamous cell carcinoma R. Stöhr*1, R. Weisser1, O. Wendler2, J. Giedl1, K. Daifalla1, N. Gaisa3, G. Richter4, M. Burger5, B. Wullich6, A. Hartmann1 Institut für Pathologie, Universitätsklinikum Erlangen, Erlangen, Deutschland, 2Hals-Nasen-Ohren-Klinik, Universitätsklinikum Erlangen, Erlangen, Deutschland, 3Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, 4Institut für Pathologie Dr. Richter, Hameln, Deutschland, 5Lehrstuhl für Urologie, Universität Regensburg, Regensburg, Deutschland, 6Lehrstuhl für Urologie, Universitätsklinikum, Erlangen, Deutschland

1

Background.  Penile squamous cell carcinoma (SCC) is a rare disease with an often aggressive biological behavior. Apart from a causative HPV infection in a part of the cases and involvement of p53 only limited data on molecular alterations in penile SCC are available. Recently, recurrent point mutations within the core promoter of the human telomerase reverse transcriptase (TERT) gene were described in various cancer entities (eg. malignant melanoma, bladder cancer). These mutations have a functional consequence and lead to a 2–4 fold increased transcriptional activity in vitro. To date, no data on TERT promoter mutations in penile SCC are available. Methods.  So far, sections from 98 archival formalin-fixed and paraffin-embedded penile SCCs were used for DNA isolation. After precise microdissection the mutation hotspot region within the TERT core promoter (–260 to +60) was analysed by direct Sanger sequencing or SNaPshot analysis. HPV detection was done by usage of GP5+/6+ primers followed by subtype-specific PCR. Results.  Overall, 83/98 case were analysed successfully for TERT promoter mutations. In 30 % of the cases (25/83) a mutation was found. Forty per cent (36/90) of the analysed cases showed positivity for HPV. There was a significant association between occurrence of TERT promoter mutation and negative HPV status (mutation frequency of 45 % in HPV-negative cases vs. mutation frequency of 9 % in HPV-positive cases, p < 0,001). There were no additional associations between TERT mutation status and histopathological characteristics of the tumors. Conclusions.  The TERT core promoter mutations reported from several other malignancies could be detected with a high frequency in HPV-negative penile SCCs. Enhanced TERT promoter activation caused by these alterations might be an important mechanism in HPV-independent penile carcinogenesis.

P02.13 Clinical impact of Gleason score underestimation at prostate biopsy grading in an academic and community setting E. X. Keller*1, A. Mortezavi1, C. Poyet1, 2, T. Hermanns2, K. Saba2, M. Randazzo2, C. D. Fankhauser2, P. Wild2, H. Moch2, T. Sulser1, D. Eberli1 Klinik für Urologie, UniversitätsSpital Zürich und Universität Zürich, Zürich, Schweiz, 2Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz

1

Background.  Gleason score undergrading of prostate biopsy (PB) specimens may lead to inappropriate management of patients with prostate cancer (PC). We evaluated whether the rate of Gleason score (GS) upgrade on final pathology, the rate of positive surgical margins (PSM) and the rate of biochemical recurrence (BCR) after radical prostatectomy (RP) were different if PBs were graded by community pathologists (CP) as compared to specialized uro-pathologists (UP) in a tertiary-care academic center. Methods.  Data of all patients undergoing RP in our institution between 2005 and 2013 were reviewed retrospectively. Any GS higher or lower in RP specimen as compared to PB GS was defined as GS upgrade or downgrade, respectively. A stepwise logistic regression model was used to determine independent predictors of a GS upgrade and of PSMs. Multivariate Cox regression analyses were performed to assess prognostic parameters for BCR.

Results.  A total of 786 patients were available for analysis and the median follow-up was 36 months (1–101 months). Of those, 487 (62 %) had their biopsy graded by a CP and 299 (38 %) by a UP. A GS upgrade was found in 345 patients (44 %) and a GS downgrade in 91 patients (12 %). Discordance between PB GS and RP GS was significantly more frequent when grading had been performed by a CP (51 % upgrade, 9 % downgrade) than by a UP (33 % upgrade, 16 % downgrade, P < 0.001). In a logistic regression analysis adjusting for preoperative parameters, CP evaluation was an independent prognostic factor for GS upgrade (OR 1.91, 95 % confidence interval [CI] = 1.40–2.61, P < 0.001) and for PSMs (OR 1.69, 95 % CI = 1.20–2.38, P = 0.003). The origin of the PB pathology report remained an independent predictor of BCR in multivariate Cox regression analyses adjusting for pre- and postoperative parameters (OR 1.65, 95 % CI 1.06–2.60, P = 0.028). Conclusions.  Pathologic evaluation of PBs by a dedicated UP should be recommended to reduce the rate of GS undergrading, PSM and BCR after RP.

P02.14 Prognostic relevance of PITX3 DNA methylation in Prostate Cancer Patients E. E. Holmes*, D. Goltz, G. Kristiansen, D. Dietrich Institut für Pathologie, Universitätsklinikum, Bonn, Deutschland Background.  PITX3 is a homeobox transcription factor that is known to play a key role in the development, function, and maintenance of midbrain dopamineric neurons. In cancer, especially prostate cancer, there is very little known about PITX3. The methylation of PITX3 was previously analysed in breast cancer and showed a higher methylation in breast cancer compared to normal ducts and adenosis [1]. The aim of this study was to analyse PITX3 methylation as a potential biomarker in prostate cancer. Methods.  The DNA methylation of PITX3 was analysed in a test-series of 66 paired specimens of normal, hyperplastic and carcinomatous prostate tissue and a validating cohort, including radical prostatectomy patients (n = 268) operated for primary prostate cancer at the University Hospital Bonn. The PITX3 methylation was detected by means of a methylation specific quantitative real-time PCR. Methylation results were correlated to clinicopathological parameters. The prognostic value for biochemical recurrence-free (BCR) survival was analysed by Kaplan-Meier analyses and Cox proportional hazards regression. Results.  Carcinomatous prostate tissue revealed a significantly higher PITX3 methylation compared to normal and hyperplastic tissues. In the radical prostatectomy cohort, mean PITX3 methylation was 56.6 %. PITX3 methylation showed a significant positive correlation with the initial PSA (p = 0.013), Gleason Score (GS, p < 0.001) and tumor size (pT, p < 0.001). The univariate Cox Analyses for BCR-free survival showed a significant prognostic value for PITX3 methylation as a continuous variable (Hazard Ratio: HR = 1.01 [95 %CI: 1.00–1.03], p = 0.030) and also as median dichotomized variable (Hazard Ratio: HR = 1.82 [95 %CI: 1.03–3.25], p = 0.041). This could be confirmed in a Kaplan-Meier survival analysis (p = 0.037). Conclusions.  PITX3 DNA methylation is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients.

P02.15 New molecular aspects, biomarker insights and PD1/PDL1 immune-checkpoint-analysis in urachal carcinoma H. Reis*1, C. Niedworok2, O. Módos3, A. Szendröi3, A. Szasz4, P. Hollosi5, K. Baghy5, I. Kovalszky5, K. Okon6, T. Golabek6, P. Chlosta6, B. Peyronnet7, R. Mathieu7, F. vom Dorp2, 8, P. Nyirady3, K. W. Schmid1, S. F. Shariat9, H. Rübben2, T. Szarvas2, 3 Universitätsklinikum Essen, Universität Duisburg-Essen, Institut für Pathologie, Essen, Deutschland, 2Universitätsklinikum Essen, Universität Duisburg-Essen, Klinik für Urologie, Essen, Deutschland, 3Semmelweis 1

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Abstracts Universität, Klinik für Urologie, Budapest, Ungarn, 4Semmelweis Universität, 2. Abteilung für Pathologie, Budapest, Ungarn, 5Semmelweis Universität, 1. Abteilung für Pathologie, Budapest, Ungarn, 6Jagiellonen-Universität, Klinik für Urologie, Krakau, Polen, 7Universitätsklinikum Rennes, Klinik für Urologie, Rennes, Frankreich, 8Helios Klinikum Duisburg, Klinik für Urologie, Duisburg, Deutschland, 9Medizinische Universität Wien, Universitätsklinik für Urologie, Wien, Österreich Background.  Urachal Carcinoma (UrC) is a rare cancer entity accounting for <1 % of all bladder cancer cases with predominantly adenocarcinoma (ADC) histology. Due to its rarity, biomarkers of UrC are poorly studied and targeted therapy regimes are not established. We therefore analyzed potential biomarkers formerly shown to be prognostic in both urothelial bladder and colorectal cancer, the most commonly occurring mutations in the targetable EGFR-pathway and the expression of immune-checkpoint targets PD1/PDL1 in UrC-ADC. Methods.  Expression of hyaluronan mediated motility (RHAMM), biglycan (BGN), matrix metalloproteinase-7 (MMP-7), insulin-like growth factor II mRNAbinding protein 3 (IMP3), Ki67 and p53 was evaluated in 15 cases and PD1/PDL1-expression in 22 cases of UrC-ADC by immunohistochemistry (IHC). Mutational hot spots of EGFR, KRAS, NRAS, PIK3CA and BRAF were analyzed by pyrosequencing. Uni-/multivariable analyses regarding associations with clinico-pathological and follow-up data were conducted. Results.  Immunoexpressions of RHAMM, IMP3, Ki67 and p53 were significantly enhanced in UrC-ADC compared to controls (p = 0.043, p = 0.005, p = 0.001, p = 0.002). No specific tumorous membranous IHC-reactivity was noted in PD1-/PDL1-analyses. IMP3 immunoexpression was significantly higher in Sheldon stage IIIA vs. ≥IIIB UrC (p = 0.005). Overall, 11 mutations were detected in 10 of 22 cases (45 %; KRAS: n = 6, NRAS: n = 1, BRAF: n = 4) with one concurrent NRAS/BRAF mutation. No further correlations of mutational or IHC results with clinico-pathological and/or survival data (OS, PFS) were detected. Conclusions.  As UrC is a rare cancer entity, data on biomarkers is sparse. We added more evidence to the field and detected enhanced expressions levels but no prognostic value for Ki67, p53, RHAMM and IMP3. In lack of clinical trials, no evidence-based recommendations can be made regarding adjuvant (tailored) therapy regimes of progressed UrC, which is therefore based on individual decisions. Our results demonstrated high mutation rates of the EGFR-signaling pathway, underlining the importance of its analysis when considering an anti-EGFR targeted therapy for UrC-ADC. Additionally, as immune-checkpoint inhibitors (ICI) like Nivolumab are emerging in several cancer entities, we tested the PD1/PDL1 expression in UrC-ADC and did not detect any specific membranous reactivity. This result, however, does not preclude potential ICI efficacy in UrC-ADC as for example seen in advanced renal cell carcinoma.

P02.16 PD-L1 expression in aggressive primary prostate cancer H. Gevensleben1, D. Dietrich*1, J. Ellinger2, C. Stephan3, K. Jung3, P. Brossart4, G. Kristiansen1 Institut für Pathologie, Universitätsklinikum, Bonn, Deutschland, 2Klinik und Poliklinik für Urologie und Kinderurologie, Universitätsklinikum, Bonn, Deutschland, 3Charité Universitätsklinikum Berlin, Department für Urologie, Berlin, Deutschland, 4Universitätsklinikum Bonn, Medizinische Klinik III (Onkologie), Bonn, Deutschland 1

Background.  Anti-PD-L1 immunotherapies promote anti-tumor immunity and have shown promising results in several tumors. Immunohistochemically detected PD-L1 may be predictive for anti-PD-1 therapy. Methods.  Immunohistochemical analysis of PD-L1 expression was performed in two independent cohorts of primary prostate cancer patients who underwent radical prostatectomy. PD-L1 expression was correlated to clinicopathological parameters and outcome. Results.  In the training cohort (209 patients), 52 % of cases showed moderate to high PD-L1 expression levels. PD-L1 correlated with proliferation

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(Ki67, p < 0.001), Gleason score (p = 0.004), and androgen receptor (AR) expression (p < 0.001). In addition, PD-L1 expression was prognostic for biochemical recurrence (BCR; p = 0.004; HR = 2.37 [95 %CI = 1.32–4.25]). In the test cohort (611 patients) elevated PD-L1 expression was found in 62 % of patients. High PD-L1 expression remained prognostic for BCR (p = 0.011; HR = 1.49 [95 %CI = 1.10–2.02]. Furthermore, the correlation of Ki-67 and AR with PD-L1 expression was confirmed in the test cohort (p < 0.001). In multivariate Cox analysis of all patients from both cohorts, PD-L1 was established as independent prognostic factor for BCR (p = 0.007; HR = 1.46 [95 %CI = 1.11–1.92]). Conclusions.  This study showed that expression of the therapy target PDL1 is highly prevalent in primary prostate cancer cells and an independent indicator of BCR, suggesting a biological relevance in primary tumors. A PD-1/PD-L1 targeted immunotherapy might be a treatment option for hormone-naive prostate cancers.

P02.17 Uncovering the proteome of primary prostate cancer and corresponding lymph node metastases – a clinical proteomics approach A.-K. Müller1, C. A. Jilg2, M. Föll1, M. L. Biniossek1, U. Wetterauer2, W. Schultze-Seemann2, M. Werner3, 4, 5, O. Schilling1, 3, V. Drendel*5 Institut für Molekulare Medizin und Zellforschung, Albert-LudwigsUniversität, Freiburg, Deutschland, 2Klinik für Urologie, Universitätsklinikum, Freiburg, Deutschland, 3Deutsches Konsortium für translationale Krebsforschung (DKTK), DKFZ, Heidelberg, Deutschland, 4Tumorregister CCCF Freiburg, Universitätsklinikum, Freiburg, Deutschland, 5Institut für Klinische Pathologie, Department für Pathologie, Universitätsklinikum, Freiburg, Deutschland 1

Background.  Considering Grade Group (GG)/Gleason Score (GS) and TNM stage as risk factors, metastases are associated with poorer outcome in prostate cancer (PCa) patients. New potential biomarkers for disease progression and local or metastatic recurrence are currently intensively investigated. This study characterizes the proteome signature of primary tumors compared to corresponding lymph node metastases (LNM) to identify proteins that are involved in metastasis formation. Methods.  We performed a quantitative proteome comparison on formalin-fixed paraffin-embedded (FFPE) specimens of 5 patients with PCa. In radical prostatectomy (RP), T-categories were pT2c (1/5), pT3a (2/5) and pT3b (2/5) with GG 3/GS 7b (1/5) and grading was GG 4/GG 8 (1/5), each with a tertiary pattern 5, and GG 5/GS 9 (3/5). During follow up (range 1–6 years) all individuals developed nodal relapse followed by salvage lymphadenectomy (SLA) and histological validation. Proteins were extracted from FFPE tumor tissue of RP and SLA, digested into peptides and analyzed by mass-spectrometry (MS) with subsequent label-free protein quantification and bioinformatics analysis comparing primary tumors (n = 5) versus corresponding LNM (n = 5). Results.  In total, 1833 proteins were quantified, whereof 75 were significantly altered (p-value < 0.01) and met additional inclusion criteria (1.5 fold change in 4/5 samples). MS-analysis showed increased abundance of proteins associated with proliferation and protein turnover in all LNM compared to primary tumors, whereas proteins associated with cytoskeleton organization, cell adhesion and extracellular matrix organization were decreased. Pathway analysis revealed an underlying upregulation of MYC/ MYCN gene expression and RhoGDI-signaling together with downregulated of TGFB1 and RICTOR gene expression as well as reduced integrin-and actin-signaling. On individual protein level, proteins such as NDRG3, ALCAM (CD166) or Fascin-1 were increased in LNM while biglycan, AZGP1 or ITGb1 showed lower abundances in the LNM. Conclusions.  We were able to apply quantitative proteomic analysis in FFPE samples of PCa tissue with excellent proteome coverage. Our results demonstrate that primary PCa and patient-matched LNM exhibit dramatically different proteome motifs. Proteins involved in the regulation

of the extracellular-milieu and cell-matrix-interaction have been identified as potential key players in metastasic tissue.

P02.18 SDK1:AMACR fusion transcript and NKAIN2 functions in Chinese prostate cancer G. Ren*, Y. Zhang, Y.-J. Lu The First Affiliated Hospital, Zhejiang University Medical College, Department of Pathology, Hangzhou, China, Volksrepublik Background.  Four high-frequency CaP specific fusion genes, SDK1:AMACR, RAD50:PDLIM4, CTAGE5:KHDRBS3 and USP9Y:TTTY15 have been reported in Chinese CaP samples through a transcriptome sequencing study. We previous reported that USP9Y:TTTY15 is a transcription-mediated chimeric RNA, which is expressed in both tumor and non tumor samples, we also reported that NKAIN2 may be a putative tumor suppressor gene (TSG) in Chinese prostate cancer patients. Methods.  Here, We detected SDK1:AMACR fusion transcript in Chinese CaP samples. However, fluorescence in situ hybridization analysis did not detect genomic rearrangement of SDK1 gene, indicating that SDK1:AMACR is also a transcription-mediated chimeric RNA. Quantitative analysis demonstrated that high level AMACR expression was associated with SDK1:AMACR fusion. These data suggest that SDK1:AMACR fusion transcript is Chinese CaP specific, which was neither detected in adjacent non-malignant prostate tissues from Chinese patient nor in CaP samples from UK patients, and SDK1:AMACR fusion transcript may promote prostate carcinogenesis through increasing AMACR expression. We further investigated the genomic, and expression changes of NKAIN2 and determined the functional role of NKAIN2. We found that the genomic truncation of NKAIN2 is specific for prostate cancer in Chinese men. Results.  The identification of the SDK1:AMACR fusion transcript in CaP cases from China but not UK further supports our previous observation that different genetic alterations contribute to CaP in China and Western countries. And that NKAIN2 is a novel TSG commonly down-regulated in prostate cancer. Conclusions.  Further studies are required to establish if CaPs with SDK1:AMACR represent a distinct subtype and NKAIN2 is a novel therapy target for prostate cancer

P02.19 Serum ferritin as a risk factor and prognostic biomarker for prostate cancer: a case-control study X. Wang*, G. Ren The First Affiliated Hospital, Zhejiang University Medical College, Department of Pathology, Hangzhou, China, Volksrepublik Background.  Prostate cancer is one of the top causes of cancer death in men worldwide and there has been evidence indicating that serum ferritin to be a biomarker for cancer from studies in several malignancies, which supports ferritin as having diagnostic and prognostic significance for cancer. Methods.  A case-control study consisting of 2030 prostate cancer patients and 960 benign prostatic hyperplasia patients were performed. Immunohistochemical staining of ferritin was performed in prostate tissues from 130 prostate cancer patients and 31 benign prostatic hyperplasia patients. The relationship among serum ferritin, PSA and prostate cancer risk, as well as the possibility of ferritin being used as diagnostic or prognostic biomarker was analyzed. Results.  Individuals with abnormally high serum ferritin was significant higher in prostate cancer patients (P < 0.0001). Serum ferritin was positively associated with serum PSA and prostate cancer risk (OR 1.20, 1.13– 1.36; per 100 ng/mL). Using serum ferritin alone or combined with serum

PSA, lower sensitivity was found than total PSA or free/total PSA ratios for prostate cancer diagnosis. But serum ferritin significantly increased by prognostic Gleason grade (P for trend < 0.0001) and improved prognosis accuracy was found using serum PSA for patients with hyperferritinemia (serum ferritin > 300 ng/mL). In prostate cancer patients with serum ferritin > 400 ng/mL, correlation coefficientbetween Gleason score and total PSA was 0.38, free PSA 0.49. Consistent with serum findings, prostate tissue ferritin concentration well correlated with serum ferritin levels (R = 0.56). And higher frequency of elevated ferritin was observed in prostate tumor tissues according to cancer staging (P< 0.0001). Conclusions.  In summary, our study emphasized the substantial but indistinct characteristics of the relationship between serum ferritin and prostate cancer risk and prognosis. We provide novel and strong evidence for the significant roles of serum ferritin in terms of prostate cancer prognosis rather than diagnosis, in addition to compelling clinical implications for utilizing serum ferritin as a prostate cancer biomarker supplementary to PSA in order to guide further treatment and improve clinical prognosis.

P02.20 Detection and Classification of Prostate Cancer by Geometric Measurements T. Greim*1, U.-D. Braumann1, M. Muders2, M. Löffler3, G. Baretton2 Universität Leipzig, Medizinische Fakultät, Interdisziplinäres Zentrum für Bioinformatik, Leipzig, Deutschland, 2Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden, Institut für Pathologie, Dresden, Deutschland, 3Medizinische Fakultät, Institut für Medizinische Informatik, Statistik und Epidemiologie, Universität Leipzig, Leipzig, Deutschland

1

Background.  Grading of Prostate Cancer mainly applies the Gleason scheme depending on morphological and architectural aspects. Intra- and inter-observer reproducibility remains the main problem – even after revisions [1]. To further improve and standardize malignancy analyses, computer-assisted approaches should be integrated. Unlike in our previous work [2] using specimens of radical prostatectomy with ROIs we are now working with prostate needle biopsies containing only few glands. Prior to classifying malignant glands they need to be localized, whereas a set of characteristics addressing general prostate tumor growth is used. To classify located tumor glands two measures quantifying their morphology were applied: inverse compactness and number of holes. Methods.  H&E stained specimens are color-separated since for further processing only hematoxylin stained basophil structures (e. g. nuclei, endoplasmic reticuli) are important. Next, an edge-preserving image smoothing was applied. Nuclei of epithelial cells are then combined as connected chains. Resulting images are binarized (hysteresis thresholding). Malignant glands lose their basal epithelial cell layer. Using thinning and distance transform the epithelial thickness is measured, so tumor glands can be distinguished from physiological and hypertrophic glands. Further, other tumor features useful for detection are both circular and monomorphic glandular lumen, as well as spatial tumor gland aggregations. Results.  102 prostate needle biopsies with 29 tumor pattern of Gleason grade (GG) 3 and 73 with pattern GG 4 were considered. Biopsies were graded (blinded to the image analysis persons) by a trained uropathologist. As the inverse compactness is defined as ratio of the area of an isoperimetric circle and the area of the epithelium, it is high for GG 4 with lobulated structures. High grade tumor is characterized by fusion of several glands and hence they exhibit multiple holes. Counting them gives large values for GG 4. Combining both measures has reached 92 % for biopsies with single tumor pattern. Conclusions.  Our computer-assisted approach to standardize and support the pathologist’s grading combining detection and classification reaches a remarkable accuracy above 90 %. Soon about 6000 specimens will be available for testing and improving. Additionally we will provide malignancy “heat maps”, and we will distinguish different patterns within a specimen.

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Abstracts References 1. I. Epstein J (2010) An Update of the Gleason Grading System, The Journal of Urology 183(2):433–440, dx.doi.org/10.1016/j.juro.2009.10.046 2. Loeffler M, Greulich L, Scheibe P, Kahl P, Shaikhibrahim Z, Braumann UD, Kuska JP, Wernert N (2012) Classifying Prostate Cancer Malignancy by Quantitative Histomorphometry, The Journal of Urology 187(5):1867–1875, dx.doi.org/10.1016/j. juro.2011.12.054

P02.21 Hepatocyte Differentiation Markers in Adenocarcinoma of the Prostate: HepPar-1 but not Arginase-1 is specifically expressed in a Subset of Prostatic Adenocarcinoma J. Giedl*1, M. Büttner-Herold2, S. Wach3, B. Wullich4, A. Hartmann1, A. Agaimy1 1 Pathologisches Institut, Erlangen, Deutschland, 2Pathologisches Institut, Abteilung für Nephropathologie, Erlangen, Deutschland, 3 Universitätsklinikum Erlangen, Molekulare Urologie, Erlangen, Deutschland, 4Universitätsklinikum Erlangen, Klinik für Urologie, Erlangen, Deutschland

Background.  Prostate adenocarcinoma and hepatocellular carcinoma (HCC) are frequent malignancies. Both may present with bone metastases and are known to lack expression of CK7/CK20. Thus for diagnosis of poorly differentiated tumors at metastatic sites immunohistochemical confirmation is mandatory. However, no sufficient data exists on the expression of the main two markers of hepatocytic differentiation, HepPar-1 and arginase-1 (Arg-1), in prostatic adenocarcinoma. Methods.  We investigated a total of 557 prostate carcinoma specimens for expression of these two markers using tissue microarrays. Results.  A total of 64/557 (11.5 %) cases showed highly variable expression of HepPar-1 in 1 to 75 % of tumor cells with characteristic strong granular “mitochondrial” pattern. Of those only 13 cases (2.3 %) expressed HepPar-1 in >10 % of the tumor cells. No correlation was seen with Gleason grade. With regard to Arg-1 a variable expression was seen in 19/557 cases (3.4 %) which was almost exclusively (18/19) with strict cytoplasmic pattern, in contrast to the combined nucleo-cytoplasmic staining of normal liver and HCC. This cytoplasmic Arg-1 pattern was seen only using one antibody lot and not another, suggesting cross reactivity. Only one case (1/557, 0.2 %) showed specific combined nucleo-cytoplasmic positivity for Arg-1 in 20 % of tumor cells; this case was HepPar-1 negative. Conclusions.  Specific granular cytoplasmic staining for HepPar-1 is occasionally observed in prostatic adenocarcinomas (11.5 %), but usually very focal limited to <5 % of tumor cells. This should not be misinterpreted as evidence of HCC, particularly in solid-pattern neoplasms. In contrast, specific Arg-1 expression is hardly ever seen, highlighting the value of Arg-1 in distinguishing HepPar-1 positive prostatic carcinoma from HCC at metastatic sites or in cases of liver metastasis of prostate carcinoma.

P02.22 CDO1 DNA Methylation is a Prognostic Biomarker for Biochemical Recurrence-free Survival in Prostate Cancer Patients S. Meller*1, L. Zipfel1, J. Ellinger2, M. Jung1, K. Jung3, 4, G. Kristiansen1, D. Dietrich1 1 Institut für Pathologie, Universitätsklinikum, Bonn, Deutschland, 2Klinik und Poliklinik für Urologie und Kinderurologie, Universitätsklinikum, Bonn, Deutschland, 3Universitätsmedizin Berlin, Charité, Abteilung für Urologie, Berlin, Deutschland, 4Berliner ForschungsInstitut für Urologie, Berlin, Deutschland

Background.  Prostate cancer is the most common cancer in the Western world among men. The high incidence coupled with a low mortality requires a careful clinical management in order to save lives on the one hand and avoid overtreatment on the other hand. Molecular biomarkers could

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help to distinguish between aggressive tumors and clinically insignificant carcinomas which pose little or no threat to the patient’s life. Aberrant DNA methylation is a common molecular alteration in malignant tumors and a chemically stable mark. Hence, DNA methylation is a powerful analyte for molecular diagnostics. Methods.  A cohort comprised of 303 patients with localized prostate cancers who underwent radical prostatectomy at the University Hospital Bonn between 1998 and 2008 was retrospectively enrolled. A quantitative methylation-specific real-time PCR (qRT-PCR) assay was used to measure the promoter methylation of the putative tumor suppressor gene CDO1 (cysteine dioxygenase 1) in bisulfite-converted DNA from formalin-fixed and paraffin-embedded tumor specimens. In a subset of the cohort as well as in prostate cancer cell lines the correlation between CDO1 methylation and CDO1 expression was investigated by qRT-PCR. Results.  The CDO1 promoter region was significantly higher methylated in the cancerous tissues compared to normal adjacent tissues or benign prostate hyperplasia (p < 0.001). In univariate Cox proportional hazards analysis, CDO1 hypermethylation significantly correlated with biochemical recurrence-free survival (HR = 2.321, p = 0.003, CI 95 % [1.34–4.02]). This finding was confirmed by a Kaplan-Meier survival analysis (p = 0.002). Furthermore, high CDO1 DNA methylation was associated with a reduced expression of the gene. Conclusions.  CDO1 is epigenetically silenced by DNA methylation of the CDO1 promoter region which is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients and therefore could help to identify patients who might benefit from a radical treatment.

Postersitzung AG Hämatopathologie P03.01 Bone Marrow Morphology Predicts Response to Hypomethylating Agents L. M. Hillen*1, 2, L. Zukerberg2, V. Deshpande2, A. H. Cleven3, A. T. Fathi4, A. zur Hausen1, R. P. Hasserjian2 Maastricht University Medical Center, Department of Pathology, Maastricht, Niederlande, 2Massachusetts General Hospital, Harvard Medical School, Department of Pathology, Boston, Vereinigte Staaten von Amerika, 3Leiden University Medical Center, Department of Pathology, Leiden, Niederlande, 4 Massachusetts General Hospital, Harvard Medical School, Department of Medicine, Hematology Oncology, Boston, Vereinigte Staaten von Amerika 1

Background.  Hypomethylating agents (HMAs) are cytidine nucleoside analogues and are an important therapy for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). An area of active research is the prediction of response to HMA in order to identify patients who may benefit from this therapy. In this context, bone marrow (BM) morphology has been rarely studied. Methods.  We examined sequential BM biopsies and aspirates from 39 patients with high-risk MDS or MDS/MPN (n = 24) and AML (n = 15) receiving HMA monotherapy (. Fig. 1). Post-treatment biopsies were performed after a mean of 3 HMA cycles (range 1–8). Results.  The mean patient age was 70 years (range 35–88). According to IWG-2006 criteria, 21 % showed complete or partial remission (R), 49 % showed stable disease (SD) and 30 % showed failure (F). There was no significant difference in the IPSS-R scores between the three response groups. Pre-treatment blast counts were significantly higher (p = 0.04) and degree of erythroid (p = 0.01) and megakaryocyte (p = 0.04) dysplasia were lower in the R/SD group compared to the F group. Compared to the pre-treatment samples, the post-treatment samples in the R group showed an increase in maturing myeloids (from 24 to 39 %, p = 0.06), whereas in the F group maturing myeloids decreased (from 42 to 34 %, p = 0.16). BM cellularity post-treatment decreased in the R/SD group (mean 59 to 47 %), which was not statistically significant. There was no significant change in

Fig. 1 I P03.01 7 BM morphologic changes observed during HMA therapy for high risk MDS. a: Pre-treatment BM from a patient with REAB bordering on AML, show­ ing 90% cellularity. Circles show dysplastic micromegakaryocytes (H&E, original magnification x40). b: Post-treatment BM from the same patient after 4 cycles of HMA, with complete remission according to the IWG-2006 response criteria. Cellularity decreased to 40 % and there is maturing hematopoiesis. Circles show persistent dysplastic megakaryocytes, despite the clinical remission (H&E, original magnification x40) the degree of dysplasia between pre- and post-treatment samples in any of the groups. Conclusions.  We found that high-risk MDS and AML patients with higher blast counts were more likely to respond to HMAs than those with lower blast count; this may reflect the ability of HMAs to inhibit methylation of rapidly dividing cells. Conversely, patients with more severe dysplasia in maturing elements had less likelihood of responding. Persistent dysplasia in post-treatment samples did not appear to correlate with response to HMA.

P03.02 Nodal Marginal Zone Lymphomas: Sequence analysis of CD79A, CD79B and MYD88 L265P M. Gurth*1, V. Bernard2, H.-W. Bernd2, J. Schemme2, C. Thorns1

Analyses concerning MYD88 L265P showed wild type sequences with the exception of three samples: Two cases are positive for T>C (COSM85940, c.794T>C, p.L265P) and one sample could not be evaluated. Conclusions.  In this study, we found no evidence for specific mutations that could indicate the association of NMZL with a chronically active NFκB signaling pathway on the basis of activating mutations on CD79A, CD79B or MYD88. Therefore this signaling pathway seems to play a minor role in the lymphomagenesis of this entity compared to its extranodal counterpart.

P03.03 Analysis of microenvironmental dynamics in classical Hodgkin Lymphoma using gene expression profiling A. Schnitter*1, M. Szczepanowski1, C. Kohler2, R. Spang2, W. Klapper1

Institut für Pathologie, Universitätklinikum Schleswig-Holstein, Lübeck, Deutschland, 2Hämatopathologie Lübeck, Konsultations- und Referenzzentrum für Lymphom- und Knochenmarkdiagnostik, Lübeck, Deutschland

1 Institut für Pathologie, Sektion Hämatopathologie, Universitätsklinikum Schleswig-Holstein, Kiel, Deutschland, 2Institute of Functional Genomics, Regensburg, Deutschland

Background.  Nodal marginal zone lymphoma (NMZL) is a rare B-cell neoplasm, whose pathogenesis and diagnostic features still remain widely elusive. A typical characteristic of extranodal MZL is the constantly activated NF-κB pathway on the basis of specific mutations or translocations. An overexpression of certain NF-κB-related and -binding genes has also been described in NMZL, however, it lacks evidence for typical mutations or translocations. CD79A and CD79B are transmembrane proteins that form a complex with the B-cell-receptor, which initiates the NFκB pathway upon antigen interaction. Activating mutations of CD79 can be found in activated B-cell like diffuse large B-cell lymphoma and are associated with a chronically activated B-cell-receptor signaling pathway. MYD88 plays an essential role in a variety of inflammatory pathways. Being an adaptor molecule between TLRs, Interleukin1 receptor and Il-1R associated kinase (IRAK), it enables the transduction of numerous signaling pathways including NFκB. The gain-of-function variant L265P on in MYD88 is frequently found in Waldenström’s macroglobulinemia. Methods.  In our study, we analyzed hotspot regions of CD79A, CD79B and MYD88 in order to find out, whether specific genomic lesions can be detected in these areas, that can constantly activate the NFκB pathway. Using Next Generation Sequencing and Sanger Sequencing, we analyzed two exons of CD79A and CD79B in 58 samples of lymph nodes diagnosed as NMZL. Through Pyrosequencing, we quantitatively evaluated the variant MYD88 L265P in the same 58 samples. Results.  DNA sequencing of CD79A exon 4 and 5 and CD79B exon 5 and 6 revealed mostly wild type sequences and one tumor sample with a known somatic mutation on CD79B exon 5.

Background.  The classical Hodgkin Lymphoma (cHL) is a so called paucicellular tumor that consists of tumor cells, the Hodgkin and Reed-Sternberg cells and an abundant tumor microenvironment. The non-neoplastic microenvironment represents the predominant part of the tumor tissue. The composition of the microenvironment has been associated with the course of the disease if assessed by gene expression profiling. The aim of our study was to compare the composition of the microenvironment in cHL between first diagnosis and a later relapse by gene expression analysis in order to understand the mechanisms associating microenvironment and outcome in cHL. Methods.  We identified 18 patients in the files of the Lymph Node Registry, of which formalin fixed and paraffin-embedded specimens at first diagnosis and one or more specimens at relapse were available (total number of specimens n = 39). The lymphoma samples were analyzed histologically for morphological subtype and immunophenotype by immunohistochemistry (CD20, CD30, CD15, CD3, Pax5, LMP1). nanoString digital gene expression profiling for 594 immune system-associated genes was performed. We used the ExpressArt FFPE Clear RNA ready kit (AmpTec, Hamburg, Germany) for RNA extraction. Results.  The cohort shows a distribution of Hodgkin lymphoma subtypes typical for Central Europe (60.7 % nodular sclerosis, 32.1 % mixed cellularitiy, 3.6 % lymphocyte depletion and 3.6 % others). In 7/11 (63.6 %) the subtype between first diagnosis and relapse remained unchanged. In 3/11 (27.3 %) the morphology changed from mixed cellularity (at diagnosis) to nodular sclerosis (at relapse). Only 4 genes showed a statistically significant fold change between the first samples from diagnosis and from

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Abstracts relapse. Among the genes with differential expression T-cell markers CD3, CD247, and CD7 were identified. Furthermore, expression of S100 protein decreases from first diagnosis to relapse. Validation of the findings by immunohistochemistry is currently ongoing. Conclusions.  Our data suggests that the extent of microenvironmental T-cells might increase at relapse. Future studies will have to characterize the subtype of T-cells that is more abundant at relapse. We believe that characterizing the dynamics of microenvironment in cHL will help to understand the aggressive clinical course at relapse. Furthermore, our findings might help to define microenvironmental characteristics that allow for identification of cHL with risk for relapse already at first diagnosis.

P03.04 The composition of FL microenvironment at primary diagnosis and relapse N. Böhling*1, K. Koch1, R. Spang2, M. Altenbuchinger2, A. Staiger3, G. Ott3, W. Klapper1 1 Institut für Pathologie, Sektion Hämatopathologie und Lymphknotenregister, Universitätsklinikum Schleswig-Holstein, Kiel, Deutschland, 2Institut für funktionelle Genomik, Universität Regensburg, Regensburg, Deutschland, 3Institut für klinische Pathologie, RobertKoch-Krankenhaus, und Dr. Margarete Fischer-Boch Institut für klinische Pharmakologie, Universität Tübingen, Stuttgart, Deutschland

Background.  Histologically Follicular Lymphoma (FL) shares the specific microenvironment of reactive germinal center. The composition of the microenvironment (non-neoplastic bystander cells) at diagnosis was associated with prognosis in previously published studies using gene expression profiling. In these studies a high content of macrophages was associated with a favorable and an expression signature of dendritic cells was associated with an unfavorable prognosis. However, these results are still controversial. We have previously shown that the composition of the microenvironment correlates with the stage of disease at diagnosis suggesting that in this slowly progressing disease the microenvironment changes over time. We have observed, that the microenvironment was more similar to physiological germinal centers in early stage (follicular T-helper cells high, regulatory T-cell low) compared to later stages of the disease. [1] Methods.  Sequential biopsies obtained at the primary diagnosis and the relapse from the same patients (n = 20) were selected from the files of the Lymph Node Registry. Immunohistochemical staining and analysis were performed on formalin fixed and paraffin-embedded for components of the microenvironment such as FoxP3 and PD-1 to detect regulatory T-cells or follicular T-helper cells, respectively. Furthermore gene expression profiling the FL microenvironment using NanoString technology was performed. All patients had been treated in the time between the biopsies. Results.  The content of macrophages, follicular T-helper cells and regulatory T-cells did not change between primary diagnosis and relapses. To gain a more complete picture of the microenvironment, the cohort will be expanded. NanoString analysis for gene expression is currently ongoing. Conclusions.  We have previously shown that the microenvironment at the time of diagnosis differs according to stage of the disease. Using immunohistochemistry to quantify non-neoplastic bystander cells we did not detect a change in the microenvironment of FL between primary diagnosis and relapse. A possible explanation might be that the therapy between the two biopsies interferes with the natural course of microenvironment evolution. To understand the interaction of FL with its microenvironment will ultimately help to understand the mechanisms of disease progression. To gain a more complete picture of the microenvironment, the cohort will be expanded and completed with gene expression data. References 1. Koch et al (2012) The composition of the microenvironment in follicular lymphoma is associated with the stage of diesease, Hum Pathol, 43(12):2274–2281

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P03.05 Morphological and cellular transformation of chronic myelomonocytic leukemia (CMML) is accompanied by altered mutation profiles S. Bartels*, B. Hasemeier, E. Schipper, G. Büsche, J. Schlue, H. H. Kreipe, U. Lehmann-Mühlenhoff Institut für Pathologie, Medizinische Hochschule Hannover, Hannover, Deutschland Background.  In order to elucidate the molecular changes underlying the morphological and cellular transformation of chronic myelomonocytic leukemia (CMML) into MDS/MPN-like conditions the mutation profile of serial biopsies was assessed employing next generation sequencing. Methods.  Using a customized next generation sequencing gene panel comprising 23 genes most frequently mutation in MDS and MPN serial biopsies from four patients (4–6 biopsies from each patient) were analyzed on a Ion Torrent/PGM. For library preparation 30 ng of genomic DNA were extracted from routinely processed bone marrow trephines. 8 samples were analysed in parallel on a PGM 318 chip. For data evaluation the Ion Torrent Variant caller, Cartagenia Bench Lab NGS software version 4.0.2, and the IGV browser were used. Results.  Each biopsy under study showed two to four mutations in the following genes: TET2, SRSF2, JAK-2, MPL, CBL, and CSF3R. The TET2 gene harbored two different mutations in three out of four patients, but did not display any changes in allele frequency or occurrence of mutation. In contrast to this the JAK-2, SRSF2, MPL and CBL genes displayed marked differences in allele frequency and occurrence of mutations in all patients of this small series. In three cases the transformation into an MPN-like condition is underlined by occurrence and/or dramatic increase in allele frequency of the well-defined hotspot mutation JAK-2 p.V617F. The case which does not show gain of or increase in JAK-2 p.V617F shows instead gain of MPL p.W515L and displays a near complete loss a CBL mutation (from 44 % down to 1 %) indicating similar function of JAK-2 and MPL in this setting and opposite functions of CBL in the transformation of CMML cases. Conclusions.  The morphological and cellular transformation of CMML is accompanied by specific qualitative and quantitative changes in the mutational profile. The details of the underlying molecular mechanisms have to be addressed in future studies.

P03.06 Risk Stratification in Early-Stage and Advanced Follicular Lymphoma Patients enrolled in the German Low-Grade Lymphoma Study Group A. M. Staiger*1, 2, H. Horn1, E. Hoster3, E. Leich4, T. Barth5, H.-W. Bernd6, A. Feller6, W. Klapper7, M. Hummel8, H. Stein9, D. Lenze7, M.-L. Hansmann10, S. Hartmann10, K. Koch7, P. Möller5, M. Engelhard11, M. Dreyling3, W. Hiddemann3, A. Rosenwald4, G. Ott2 Dr. Margarete Fischer Bosch Institut für Klinische Pharmakologie, Stuttgart, Deutschland, 2Robert-Bosch-Krankenhaus, Abt. für Klinische Pathologie, Stuttgart, Deutschland, 3LMU München, Abteilung für Innere Medizin III, München, Deutschland, 4Institut für Pathologie, Universität Würzburg, Würzburg, Deutschland, 5Institut für Pathologie, Universität Ulm, Ulm, Deutschland, 6Institut für Pathologie (UKSH), Campus Lübeck, Lübeck, Deutschland, 7Institut für Pathologie, Christian-Albrechts-Universität, Kiel, Deutschland, 8Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland, 9Pathodiagnostik, Berlin, Deutschland, 10Institut für Pathologie, Goethe Universität, Frankfurt, Deutschland, 11Abteilung für Radiatherapie, Universitätsklinik Essen, Essen, Deutschland 1

Background.  Follicular lymphoma (FL) represents an indolent B-cell non-Hodgkin lymphoma and is characterized by heterogeneous morphological, genetic and clinical features. An accurate risk stratification at the time of diagnosis is of high importance. Using gene expression analyses, the Immune Response (IR) signature was developed allowing for an ac-

curate biological risk stratification of FL patients [1]. Until now, however, this signature, which is based on the usage of fresh tumor material, was not validated. Since the availability of fresh-frozen tissue samples is often limited in routine diagnostics, it is of pivotal interest to establish an appropriate gene expression based risk stratification tool also for FL samples obtained from FFPE material. Methods.  A large FL-cohort of two prospectively randomized trials of the German Low Grade Lymphoma Study Group (GLSG) (early stages n = 160; advanced stages n = 707) was assessed using targeted gene expression (GE) profiling (n = 184 candidate genes, associated with prognosis and/or progression in FL) using the nCounter® platform. Results.  With the target genes selected it was not possible to generate a GE-based prognostic model within the samples of advanced stages. Furthermore, the previously published IR signature could not be confirmed. Intriguingly however, the GE profile between early and advanced FL tumor stages differs significantly. Moreover, applying this signature it was possible to identify advanced FL-samples showing a GE profile of early stages. Interestingly, in these cases, a smaller number of lymph nodes was affected as judging by image analysis, and the clinical course was more favorable. Conclusions.  It is possible to reliable distinguish FL-samples from early and advanced clinical stages based on gene expression profiling. This might be of interest in the optimization of therapy decisions. References 1. Dave SS, Wright G, Tan B, Rosenwald A, Gascoyne RD, Chan WC et al (2004) Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells. N Engl J Med; 351:2159–2169.

P03.07 Is there a feedback loop between LITAF and BCL6 in B cell Non-Hodgkin’s- lymphoma? Y. Kuai*, Y. Shi, Y. Weng, L. Lei, R. Zhou Zhejiang University Medical College, Institute of Pathology and Forensic Medicine, Hangzhou, China, Volksrepublik Background.  Lipopolysaccharide-induced tumor necrosis factor-alpha transcription factor (LITAF) exerts transcription factor activity and is initially identified as a p53-inducible gene, which is implicated as a tumor suppressor. B cell lymphoma (BCL) 6 plays an important role in lymphomagenesis. LITAF is recently demonstrated as a novel target of BCL6. Here we evaluated the effects of LITAF in B cell Non-Hodgkin’s- lymphoma, and the downstream effects in diffuse large B-cell lymphoma (DLBCL). Methods.  We used quantitative real time PCR (qRT-PCR) to calculate mRNA relative expression levels; lentivirus-based and electro-transfection-based LITAF gain and loss of function experiments; to examine the feedback loop between LITAF and BCL6 we performed Chromatin immunoprecipitation (ChIP) assays and Luciferase reporter assay; to check the relationship between LITAF and BCL6 we performed Co-Immunoprecipitation (Co-IP) and Immunofluorescence staining; to examine the level of LITAF and BCL6 in patient tissues, we performed immunohistochemistry; We used the flow cytometry to check the function of LITAF in cell lines. All the experiments were performed in triplicate. Results.  Overexpression or knockdown of LITAF significantly increased or decreased apoptosis of B cell Non-hodgkin’s lymphoma cell lines. LITAF, therefore as stimuli of cell apoptosis, was supported by a rise in BaX and fall in Bcl-XL (although not significant), releasing Cytochrome C, cleaving PARP and caspase 3, which eventually induce apoptosis. ChIP assays and Luciferase reporter assay showed that LITAF could repress expression of BCL6 by binding to Region A (−87 to +65) containing putative LITAF-binding motif (CTCCC) of BCL6 promoter. Immunohistochemistry in patient tissues showed the level of LITAF correlates inversely with BCL6 in B-NHL (p < 0.05). Co-IP and Immunofluorescence staining showed that LITAF interacts with BCL6 in vivo. Conclusions.  Our study for the first time showed that there is a feedback loop between LITAF and BCL6. LITAF positively regulates apoptosis through intrinsic mitochondrial pathway in B-NHL cell lines. LITAF in-

teracts with BCL6 in vivo. LITAF may function as an anti-oncogene in B cell Non-hodgkin’s lymphoma. Our study is expected to provide a possible biomarker for clinical therapies by targeting cell apoptosis.

P03.08 The molecular mechanism of UPR and effects on prognosis in DLBCL X. Jiang*, Y. Weng, L. Lei, Y. Kuai, R. Zhou Zhejiang University Medical College, Institute of Pathology and Forensic Medicine, Hangzhou, China, Volksrepublik Background.  The UPR is fundamentally a cytoprotective response, which mitigates normal and unusual levels of ER stress to promote cell survival and growth by enhancing the capacity of the secretory apparatus and by altering transcriptional and translational programs of the secretory pathway. Recently reports described that UPR activation plays a critical role in normal B cell development and lymphomagenesis. Here, we want to assess the influence of UPR pathway and target genes expression on development and prognosis of DLBCL. Methods.  We first focused on 2 major UPR sensors (IRE1α and PERK) phosphorylation status by IHC analysis in 154 de novo DLBCL biopsies and 30 normal lymphoid tissues. Next, we generated DLBCL lines (OCI-LY10 and SU-DHL4) that stably express lenti-shRNA targeting IRE1α, ATF6 or PERK, and observed that TG-induced apoptotic cell death occured in DLBCL lines with IRE1α or ATF6 depletion, but not in PERK deletion lines. However, PERK knockdown could partially rescued TG-induced apoptosis in DLBCL lines with IRE1α or ATF6 shRNA. Immunoblot also showed that the expression levels of pro-aopototic ATF4 and CHOP protein were higher in DLBCL lines with IRE1α or ATF6 shRNA than in DLBCL lines with IRE1α/PERK or ATF6/PERK shRNA co-transfection after induction of ER stress by TG induced apoptosis. Results.  The results fully support our hypothesis that a chronic yet non-lethal UPR activation presented in DLBCL cells without apparent signs of ER stress. Conclusions.  Together, our results conclude a substantial but variable basal activation of UPR-related components in clinical DLBCL cases and cell lines without apparent signs of ER stress. We also demonstrate that UPR activation is critical for survival of DLBCL cells undergoing TG-induced ER stress, indicating that persistent activation of the pathways is an important adaptive mechanism to ER stress in DLBCL cells, and suggest that interruption of IRE1α and ATF6 signaling may be useful in combination with drugs that induce ER stress to kill DLBCL cells. Furthermore, the elevated levels of UPR activation correlate with poor DLBCL patient prognosis, which has implications for therapeutic interventions.

Postersitzung AG Thoraxpathologie P04.01 Cystatin A is silenced by DNA hypermethylation in human lung cancer Y. Ma*, Y. Chen, I. Petersen Institut für Pathologie, Friedrich-Schiller-Universität, Jena, Deutschland Background.  Cystatin A (encode by CSTA), a cysteine proteinase inhibitor, is a cornified cell envelope constituent expressed in the upper epidermis, and form tight complexes with papain and cathepsin B, H, and L. Loss or decreased expression of CSTA was found in breast neoplasms, squamous cell carcinomas of the head and neck and brain tumors. However, its role in lung cancer has not yet been elucidated. Methods.  The expression of CSTA in lung cancer cell lines was analyzed by northern blotting, RT-PCR and western blotting. Methylation status of

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Abstracts CSTA was examined by demethylation tests and bisulfite sequencing (BS). In primary lung tumors, the protein expression of CSTA was evaluated by immunohistochemistry on tissue microarray. For the functional assay, a CSTA expression vector was constructed and transfection was performed. Results.  CSTA was lost in more than 8 lung cancer cell lines at both mRNA and protein levels compared to normal bronchial epithelial cells (HBEC). The expression of CSTA was restored in 5 lung cancer cell lines after treatment with demethylation agent 5’-Aza-2’-DC. The results from BS revealed a detailed methylation pattern of CSTA in the promoter and exon 1 region. In primary lung tumors, lung squamous cell carcinoma (SCC) significantly expressed more CSTA compared to adenocarcinoma (ADC), and the protein level of CSTA was significantly associated with lung metastasis (P < 0,01). The transfection and functional assays are still in progress; however, overexpression of p53 resulted in an increased level of CSTA in lung cancer cells. Conclusions.  CSTA Gene silencing was correlated with DNA hypermethylation in lung cancer cell lines. CSTA has a potential diagnostic value in subclassification of lung cancer. CSTA could be upregulated by p53.

P04.02 Differential diagnostic value of CD5 and CD117 expression in thoracic tumors: a large scale study of 1465 non-small cell lung cancer cases M. Kriegsmann*1, T. Muley2, A. Harms1, T. Goldmann3, L. Tavernar1, H. Dienemann4, E. Herpel1, A. Warth1, 5 Institut für Pathologie, Ruprecht-Karls Universität Heidelberg, Heidelberg, Deutschland, 2Thoraxklinik Heidelberg, Heidelberg, Deutschland, 3 Clinical and Experimental Pathology, Research Center Borstel, Borstel, Deutschland, 4Thoraxklinik am Universitätsklinikum Heidelberg, Abteilung Thoraxchirurgie, Heidelberg, Deutschland, 5Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Borstel, Deutschland 1

Background.  Thoracic pathologists are frequently faced with tissue specimens from mediastinal tumors and the differentiation between thymic and pulmonary squamous cell carcinomas (SqCC) may be challenging. However, differentiation of both is of high clinical importance since therapies differ substantially and thymic carcinomas are also associated with a better prognosis. In order to clarify the differential diagnostic value of CD5 and CD117 in this setting, we performed a large scale expression study of both markers in 1465 non-small cell lung cancer (NSCLC) cases. Methods.  Tissue microarrays of formalin-fixed paraffin-embedded resection specimens of 1465 NSCLC were stained with CD117 and CD5. Expression of both markers was correlated with clinicopathological variables. Results.  CD117 was positive in 145 of 1457 evaluable cases (9.9 %) and CD5 was positive in 133 of 1427 evaluable cases (9.3 %). 28 cases (1.9 %) showed coexpression of CD117 and CD5. Among the 145 cases that were positive for CD117, 97 cases (66.8 %) were adenocarcinomas (ADC), 34 cases (23.4 %) were SqCC, 5 cases (3.4 %) were adenosquamous carcinomas (ADSqCC), 8 cases (5.5 %) were large cell carcinomas (LC), and one case (0.6 %) was a pleomorphic carcinoma (PC). In the CD5 positive group consisting of 133 cases, 123 cases (92.4 %) were ADC, 0 cases (0 %) were SqCC, 4 cases (3.0 %) were ADSqCC, 3 cases (2.2 %) LC and 3 cases (2.2 %) were PC. None of the 586 SqCC showed expression of CD5. No association of CD117- or CD5 positivity to patients’ age, any pathological stage or to T-, N-, or M- categories was observed. Conclusions.  A substantial subset of NSCLC exhibit expression of CD117 and CD5. Since CD5 positivity was not observed in pulmonary SqCC but in the majority of thymic squamous cell carcinomas the application of this immunomarker is a valuable tool in the differential diagnosis of thoracic neoplasms.

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P04.03 NUT carcinoma of the thorax A. Harms*1, E. Herpel1, N. Pfarr2, R. Penzel1, C. P. Heussel3, F. Herth4, H. Dienemann5, W. Weichert6, A. Warth1 Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, Technische Universität München, München, Deutschland, 3Thoraxklinik Heidelberg, Abteilung für diagnostische und interventionelle Radiologie mit Nuklearmedizin, Heidelberg, Deutschland, 4Thoraxklinik Heidelberg, Abteilung für Pneumologie und pneumologische Intensivmedizin, Heidelberg, Deutschland, 5Thoraxklinik Heidelberg, Abteilung für Thoraxchirurgie, Heidelberg, Deutschland, 6Technische Universität München, Institut für Pathologie, München, Deutschland 1

2

Background.  NUT (nuclear protein in testis) carcinomas (NC) are exceedingly rare neoplasms with specific molecular alterations and often follow a devastating course. Thus, a precise early diagnosis is of utmost importance. Known from the sinonasal region for years, the new 2015 WHO classification now also recognizes the existence of this entity in the thorax, specifically the lungs and the mediastinum. However, yet available data on this entity are sparse. Here, we report on a 31 years old female patient with an aggressively growing tumor localized in the median line that was initially sampled by endobronchial ultrasound-guided transbronchial biopsies. Pathological assessment of the biopsy specimens revealed a NUT carcinoma with typical morphological characteristics and an uncommon NUT translocation variant with a NSD3-NUT fusion. Methods.  Due to its morphological and immunohistochemical features NC diagnosis in biopsy material can be challenging. In the published literature initial diagnosis from biopsy material in 14 cases was a poorly or undifferentiated carcinoma, which points out that the well differentiated tumor cell population must not be necessarily evident in small biopsies or can be completely lacking. Focal keratinization at the biopsy was only described in 6 cases. In other cases the tumors were initially classified as basaloid carcinoma, carcinoma not otherwise specified, dedifferentiated tumor with mixed phenotype, round cell sarcoma, Ewing sarcoma, diffuse large cell lymphoma, desmoplastic round cell tumor or germ cell tumor. Results.  In 25 cases NUT immunohistochemistry was performed and was positive in all cases. In the other 12 cases FISH analyses confirmed the diagnosis. Overall, in 31 cases FISH was performed to support the diagnosis. Conclusions.  NC is a rare, aggressively growing cancer occurring predominantly in young adults and children. Due to its histomorphological resemblance to squamous cell carcinoma, NC may be underdiagnosed; diagnosis can be strongly suspected on the basis of morphological appearance and confirmed by immunohistochemistry against NUT or detection of the rearrangement of specific NUT variants. Outcome is poor and there is no standard treatment regimen available so far. However, there are promising targeted therapies which are currently tested in clinical trials. Both clinicians and pathologists should be aware of this rare entity in order to make the appropriate diagnosis and to establish effective treatment algorithms in the future.

P04.04 Effect of the autophagy inhibitor chloroquine on gefitinib-resistant lung cancer cells and clinical relevance of autophagy-associated proteins K. L. Schnitzler*, Y. Chen, I. Petersen Institut für Pathologie, Friedrich-Schiller-Universität Jena, Jena, Deutschland Background.  ERGR targeted therapy with EGFR-tyrosine kinase inhibitors (EGFR-TKIs) has improved clinical outcome in patients with advanced non-small cell lung cancer (NSCLC). However, the development of secondary resistance inevitably leads to treatment failure. Autophagy is a degradative process associated with resistance of anti-cancer drugs. Therefore, the aim of this study was to investigate the effects of autophagy inhibitor chloroquine (CQ) on EGFR-TKI gefitinib (Gef)-resistant NS-

CLC cells and to investigate whether autophagy-associated proteins may have clinical relevance. Methods.  The cell viability was determined by MTT assay. The expression of autophagy-associated and apoptosis-associated proteins was analyzed by western blotting. Flow cytometry was used to detect apoptosis. Immunohistochemistry (IHC) on lung tissue microarrays (TMAs) was applied to analyze the expression of autophagy-associated proteins. Results.  MTT assay showed decreased viability after treatment with Gef and Gef-CQ-combination in EGFR-TKI sensitive cell line H322. In EGFR-TKI resistant cell line H1650 and H1975 the viability was reduced after treatment with CQ or combination. Western blotting could not show alterations of LC3II levels after treatment with Gef. In H322 cells, p62 levels were decreased after treatment with Gef and drug combination, a sign for autophagy. Flow cytometry revealed that H1650 cells underwent apoptosis after treatment with Gef and combined treatment. In primary lung tumors, IHC analysis revealed a significant correlation between Beclin-1 expression and lymph node metastasis (low expression of Beclin-1 in pN0, p = 0.041) and between LC3A expression and the tumor type (high expression in adenocarcinoma, p = 0.002) and tumor grade (higher expression in G1-2, p = 0.032). No significant correlation with clinical parameters was found for other autophagy-associated proteins such as LC3B, ATG5 and p62. None of the investigated proteins had impact on clinical outcome. Conclusions.  The combination treatment with EGFR-TKI and autophagy inhibitor may effectively suppress tumor cell growth. Autophagy-associated proteins (LC3A and Beclin-1) may have clinical relevance in patients with NSCLC. Investigation of underlying mechanisms (pathways) responsible for tumor-inhibitory effects will provide further evidence for the utility of autophagy inhibition in overcoming EGFR-TKI resistance.

P04.05 Expression patterns of DNA methytransferases (DNMT) correlate with histological subtype and smoking habits in non-small cell lung cancer (NSCLC) patients U. Fehrenbach*1, 2, A. Csanadi3, S. Wiesemann4, J. Rawluk5, U. Nestle6, C. Kayser3, M. Werner3, 7, G. Kayser2 Charité – Universitatsmedizin, Department für diagnostische und interventionelle Radiologie, Berlin, Deutschland, 2Department für Pathologie – Ludwig-Aschoff-Haus – Institut für Klinische Pathologie, Universitätsklinikum Freiburg, Freiburg, Deutschland, 3Department für Pathologie – Ludwig-Aschoff-Haus – Institut für Klinische Pathologie, Universitätsklinikum, Freiburg, Deutschland, 4Abteilung für Thoraxchirurgie, Universitätsklinikum, Freiburg, Deutschland, 5Abteilung für Onkologie und Hämatologie, Universitätsklinikum, Freiburg, Deutschland, 6Abteilung für Strahlentherapie, Universitätsklinikum, Freiburg, Deutschland, 7Deutsches Konsortium für Translationale Krebsforschung und DKFZ, Heidelberg, Deutschland 1

Background.  Among malignant diseases, lung cancer holds the highest incidence and mortality rates worldwide. Recent molecular diagnostic techniques and treatment options are nearly all based on classical genetic mutations. However, epigenetic changes of tumor tissue, as hypermethylation in promotor regions of tumor suppressor genes, play a crucial part in tumor development and maintaining. A major role in epigenetic alterations is methylation of DNA bases which is performed by DNA-methyltransferases (DNMT). For this reason we studied the expression levels of DNMT1, DNMT3a and DNMT3b in non-small cell lung cancer patients (NSCLC). Methods.  We performed immunohistochmical analyses in tumor tissue and corresponding normal lung tissue of 454 NSCLC patients which were hospitalized at the University Medical Center Freiburg for surgical treatment of their lung cancer between 1989 and 2008. The expression levels were correlated to clinical and pathological patient characteristics. Results.  We could reveal statistically significant correlations between the expression level of DNMT3a and histological subtype. Squamous cell carcinoma showed higher levels of DNMT3a expression than lung adeno car-

cinoma. Large cell neuroendocrine carcinoma had the highest expression of DNMT3a. Expression patterns of the investigated DNMTs were also different in specific histological subtypes. Expression levels of DNMT in tumor tissue of smokers was higher than in tissue of non-smokers. For DNMT1 and DNMT3a these findings were statistically significant. Expression of DNMT3a also correlated with the consumed tobacco dose measured in packyears. Conclusions.  Our findings lead to the hypothesis that there are not only cancer specific methylation patterns but also specific expressions patterns of DNMTs in the different histological NSCLC subtypes. The connection between tobacco smoking and DNMT expression suggests that epigenetic alterations occur early in carcinogenesis, so that early detection of lung cancer through epigenetic tissue tests may be possible.

P04.06 Identification of potential genes regulated by tumor suppressor HOPX in human lung cancer cells Y. Chen*, Y. Li, I. Petersen Institut für Pathologie, Friedrich Schiller Universität, Jena, Deutschland Background.  HOPX is a tumor suppressor inactivated through epigenetic mechanism in various types of cancer. In lung cancer, it inhibits tumor cell growth through ras-induced cellular senescence. However, its downstream genes (pathways) have not yet been analyzed. Therefore the aim of the study was to identify genes potential regulated by HOPX in lung cancer cells. Methods.  Affymetrix cDNA microarray analysis was performed to compare gene expression between HOPX-transfectants and mock control. The cDNA microarray data were validated by real-time RT-PCR analysis, western blotting, and immunohistochemistry on tissue microarrays (TMAs) containing more than 100 primary lung tumors. Transfection with genes of our interest will be carried out and cell-based functional assays will be performed. Results.  cDNA microarray analysis showed that at least 502 genes were differentially expressed in HOPX-transfected cells or in control cells, and among them, 40 genes showed more than 4-fold changes (increased or decreased). The downstream genes (pathways) are involved in cell adhesion, cell cycle, apoptosis, differentiation, and metabolism. Real-time RT-PCR analysis confirmed the utility of microarray data with 8 out of 10 selected genes (CORO1A, SEPT6, EMP2, LEPREL2, EDG8, SLC34A2, Fibulin2, HOXD10, ARHGEF10, and Moesin) being differentially expressed. Downregulation of one of the candidate genes EDG8 (endothelial differentiation gene 8) was detected by IHC in primary lung tumors. Transfection and functional analysis are still in progress. Conclusions.  HOPX might take part in multiple cellular activities including cell adhesion, cell cycle, apoptosis, differentiation, and metabolism. The characterization of its downstream genes (pathways) may shed light on the mechanisms through which it regulates the multiple cellular events.

P04.07 Screening of pleural mesotheliomas for DNA-damage repair players by digital gene expression analysis can support clinical management of patients receiving platinbased chemotherapy R. F. H. Walter1, C. Vollbrecht2, R. Werner3, J. Wohlschlaeger1, D. C. Christoph4, K. W. Schmid1, F. D. Mairinger*2 1 Institut für Pathologie, Universitätsklinikum, Universität Duisburg-Essen, Essen, Deutschland, 2Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland, 3Abteilung für Pathologie, Heliosklinikum Emil von Behring, Berlin, Deutschland, 4Universitätsklinikum Essen, Abteilung für Onkologie, Westdeutsches Tumorzentrum, Essen, Deutschland

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Abstracts Background.  Malignant pleural mesothelioma (MPM) is a rare, predominantly asbestos-related and biologically highly aggressive tumour leading to a dismal prognosis. Multimodality therapy consisting of platinum-based chemotherapy is the treatment of choice. The reasons for the rather poor efficacy of platinum compounds remain largely unknown but may origin from differences in DNA damage repair and recognition (. Fig. 1). Methods.  24 FFPE tumour specimens were screened by digital gene expression analysis. Based on data from preliminary experiments and recent literature, 366 mRNAs were investigated using a Custom CodeSet from NanoString. Results.  CDC25A and PARP1 gene expression were correlated with lymph node spread, BRCA1 and TP73 expression levels with higher IMIG stage. NTHL1 and XRCC3 expression was associated with TNM stage. CHECK1 as well as XRCC2 expression levels were correlated with tumour progression in the overall cohort of patients. CDKN2A and MLH1 gene expression influenced overall survival in this collective. In the adjuvant treated cohort only, CDKN2A, CHEK1 as well as ERCC1 were significantly associated with overall survival. Furthermore, TP73 expression was associated with progression in this subgroup. Conclusions.  DNA-damage response plays a crucial role in response to platin-based chemotherapeutic regimes. In particular, CHEK1, XRCC2

and TP73 are strongly associated with tumour progression. ERCC1, MLH1, CDKN2A and most promising CHEK1 are prognostic markers for OS in MPM. TP73, CDKN2A, CHEK1 and ERCC1 seem to be also predictive markers in adjuvant treated MPMs (. Fig. 2). After a prospective validation, these markers may improve clinical and pathological practice, finally leading to a patients’ benefit by an enhanced clinical management.

P04.08 Folic Acid Phenotype (FAP) as Superior Marker to Predict Pemetrexed Response in Pleural Mesothelioma R. F. H. Walter1, C. Vollbrecht2, D. C. Christoph3, K. W. Schmid1, J. Kollmeier4, H. H. Popper5, T. Mairinger6, F. D. Mairinger*2 Institut für Pathologie, Universitätsklinikum, Universität Duisburg-Essen, Essen, Deutschland, 2Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland, 3Universitätsklinikum Essen, Abteilung für Onkologie, Westdeutsches Tumorzentrum, Essen, Deutschland, 4Abteilung für Pneumologie, Heliosklinikum Emil von Behring, Berlin, Deutschland, 1

Fig. 1 I P04.07 8 Overview of the DNA-damage response pathway-network. Green Arrows indicate activating signals, red cross-marks indicate inhibitory signals. Blue bold lines indicate complexes. Turquoise octagons indicate the different pathway end-points

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Fig. 2 I P04.07 9 Prognostic and predictive results are illustrated. The upper line (a–d) show results from the overall collective, the lower line (e–h) results from the adjuvant cohort only

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Abstracts

Fig. 1 I P04.08 8 Kaplan-Meier curves for the different ratios are shown. In a), the ratio between SLC19A1 and in b) the ratio between FPGS and TYMS is shown. Last shown in c), the low-risk group defined by the gene expression of folic-acid pathway members Medizinische Universität Graz, Institut für Pathologie, Graz, Österreich, Abteilung für Pathologie, Heliosklinikum Emil von Behring, Berlin, Deutschland

5 6

Background.  Malignant pleural mesothelioma (MPM) is a rare and biologically highly aggressive tumour leading to a dismal prognosis. Multimodality therapy consisting of antifolates is the treatment of choice. The reasons for the rather poor efficacy of these remain largely unknown. Methods.  63 MPM specimens treated with pemetrexed were used for qPCR analysis. Based on this data, phenotypes and risk groups were defined and correlated to survival and objective response. Results.  Phenotypes were significantly associated with progression (p = 0.0279) and remission (p = 0.0262). Cox-regression revealed significant association between SLC19A1/TYMS-ratio (p = 0.0076) as well as FPGS/TYMS-ratio and OS (p = 0.0026) (. Fig. 1). For the differentiation by risk-groups, the COXPH calculates a strong correlation (p = 0.0008). Conclusions.  Our results indicate that a determination of the balance between folic acid uptake, activation and metabolism plays a crucial role in response to pemetrexed-based chemotherapy. Adapting this marker profile will have, in our opinion, a clear benefit for the affected patients through individualization of MPM-therapy.

P04.09 Proteasome – a potential drug target in pleural mesothelioma? S. R. Sydow* , R. F. H. Walter , C. Vollbrecht , E. Berg , A. Sommerfeld , E. Oker , J. Kollmeier4, D. C. Christoph5, T. Mairinger6, K. W. Schmid2, M. Hummel3, F. D. Mairinger3 1

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Beuth Hochschule für Technik, Biotechnologie, Berlin, Deutschland, 2 Universitätsklinikum Essen, Universität Duisburg-Essen, Institut für Pathologie, Essen, Deutschland, 3Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland, 4Abteilung für Pneumologie, Heliosklinikum Emil von Behring, Berlin, Deutschland, 5Universitätsklinikum Essen, Abteilung für Onkologie, Westdeutsches Tumorzentrum, Essen, Deutschland, 6Abteilung für Pathologie, Heliosklinikum Emil von Behring, Berlin, Deutschland 1

Background.  Malignant pleural mesothelioma (MPM) is a rare, predominantly asbestos-related and biologically highly aggressive tumour leading to a dismal prognosis. Multimodality therapy consisting of platinum-based chemotherapy is the treatment of choice. The reasons for the rather poor efficacy of platinum compounds remain largely unknown. An increased

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proteasome activity can possibly serve as a marker for sensibility against proteasome inhibitors. Methods.  106 patient samples of MPM were analyzed for their gene expression level of the proteasome-subunits (β1, β2, β4, β5, D1, α1, α5) via Taqman qPCR. Additionally as a reference group, 20 benign samples of spontaneous pneumothoraxes were analyzed in the same way. To prove a potential effect of proteasome inhibition in vitro, three mesothelioma celllines were treated with different concentrations of bortezomib in a time dependent manner. Results.  Malignant pleural mesotheliomas exhibit a high expression of the proteasome. All investigated proteasome-subunits show a clear stronger gene expression in comparison to the benign control tissue (. Fig. 1). Furthermore the α5-subunit seems to represent a limiting factor. A dependence between the clinical-pathological data and the proteasome-subunits could not determine. Of note, proteasome-subunit expression may potentionally influence patients survival (. Fig. 2) Preliminary results of proteasome inhibitionin vitro render a higher induction of apoptosis, even compared to cisplatin and pemetrexed as single agent or in combination. Conclusions.  The ubiquitin-proteasome pathway plays a crucial role in the reduction of misfolded proteins. A strong proteasome overexpression is essential for the survival of the tumour cells. Consequently the proteasome is an attractive target for the development of targeted therapies in malignant pleural mesothelioma.

P04.10 ALK-Translocations in pulmonary adenocarcinomas – comparison of immunohistochemistry and fluorescence in situ hybridization S. Stallmann*1, M. Demes2, N. Schröder1, A. Fisseler-Eckhoff1 Gemeinschaftspraxis für Pathologie Wiesbaden, Wiesbaden, Deutschland, Gemeinschaftspraxis für Pathologie Wiesbaden, Molekularpathologie, Wiesbaden, Deutschland

1

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Background.  Patients with advanced adenocarcinoma of the lung (­pAdCa) and ALK alterations may benefit from an ALK inhibitor, Crizotinib. Currently, the time-consuming and expensive fluorescence in situ hybridization (FISH) is the gold standard in routine diagnostics. Several antibodies for the detection of ALK expression are controversially discussed. Methods.  The ALK expression (anti-ALK1 Primary Antibody, Roche) pattern of 97 pAdCa were retrospectively assessed and compared to ALK translocations (ZytoLight SPEC ALK/EML4 TriCheckProbe, Zytomed) detected by FISH. Discrepant individual cases were taken into account regarding the histomorphology, too. A strong granular cytoplasmic expres-

Fig. 1 I P04.09 9 Comparison of proteasomal-subunit gene expression levels between tumour samples and benign controls

2 FISH-positive cases were ALK IHC negative: One pAdCa mixed type (micropapillary/solid) with 12 % del and 6 % inv and a micropapillary one with 22 % inv/del as well as a polysomal character (53 %). One pAdCa mixed type (solid/micropapillary) was ALK positive detected by IHC and FISH, respectively. The ALK IHC positive cases showed an inv/del (detected by FISH) ranging from 21 to 59 % (mean 33 %). 44 % of ALK positive cases showed an inv/del of 20 to 30 %. 56 % of ALK positive tumors showed 30 % altered cells with an inv/del. Conclusions.  The concordance of the final results obtained by FISH (Zytomed) and IHC (Roche) were only 75 %. According to our results, a cut-off value of 15 % inv/del appear too low which could be explained by discrepant immunohistochemical stainings. Growth pattern-specific features can also affect the final FISH result. For instance overlaying nuclei found within a micropapillary subtype can mimic ALK positive cells. In routine diagnostics ALK-EML4 testing could be improved by applying an immunohistochemical screening method. Under the aspect of discrepant IHC and FISH cases, the response to a Crizotinib therapy should be assessed in future. Fig. 2 I P04.09 8 High expression of PSMA5 leads to a clear shortened overall survival of MPM patients sion in at least one tumor cell as well as 15 % inversions and/or deletions (inv/del) were assessed as ALK IHC and ALK FISH positive, respectively. Results.  12 of 97 pAdCa (12 %) were FISH and/or IHC-positive. The cases showed a concordance of 75 % (9/12) and a disconcordance of 25 % (3/12). 7 of 12 ALK-positive pAdCa (58 %) were classified as mixed type, 4 (33 %) as solid and 1 (8 %) as micropapillary. 9 of 12 ALK-positive pAdCA (75 %) were low (G3) and 3 (25 %) moderate (G2) differentiated. High differentiated pAdCa (n = 3) showed no ALK alteration.

Postersitzung AG Gynäko- und Mammapathologie P05.01 PSCA protein expression in breast cancer is associated with Her2/neu status and proliferation F. Kuithan*1, T. Link2, A. Ehninger3, M. Kramer4, J. Kuhlmann2, B. Richter5, A. Gatzweiler6, A. Werner7, P. Wimberger2, G. Baretton1, K. Friedrich1 Universitätsklinikum Dresden, Institut für Pathologie, Dresden, Deutschland, 2Universitätsklinikum Dresden, Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe, Dresden, Deutschland, 3GEMoaB 1

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Abstracts Monoclonals GmbH, Dresden, Deutschland, 4Universitätsklinikum Dresden, Medizinische Klinik 1, Dresden, Deutschland, 5Elblandklinikum Radebeul, Klinik für Gynäkologie und Geburtshilfe, Radebeul, Deutschland, 6 Krankenhaus St. Josephstift, Klinik für Gynäkologie und Geburtshilfe, Dresden, Deutschland, 7Evangelisches Diakonissenkrankenhaus, Klinik für Gynäkologie und Geburtshilfe, Dresden, Deutschland Background.  Prostate stem cell antigen (PSCA) is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein, which can be up-regulated in various human malignancies such as prostate, bladder or pancreatic cancer. Its over-expression was primarily described in prostate cancer and it has been suggested to be a prognostic biomarker and a putative therapeutic target. Up to now, however, only little is known about PSCA expression and its clinical relevance in breast cancer. Therefore, we analyzed PSCA protein expression in a large cohort of primary breast carcinomas and correlated the results with clinico-pathological parameters and outcome of the patients. Methods.  PSCA protein expression was studied by immunohistochemistry on tissue microarrays/TMAs with FFPE tissues from 405 patients with breast carcinomas. The main histological was subtype “invasive carcinoma of no special type (NST)” (84.4 %), the second most common variant was invasive lobular carcinoma (8.4 %). The PSCA protein expression was evaluated statistically by Chi-square-test for clinic-pathological features and correlated with patient’s survival, using Kaplan-Meier analysis. Results.  PSCA expression in at least 10 % of the tumor cells was observed in a subgroup of 94/405 patients (23 %), thereof a weak PSCA expression was detected in 66/407 patients (16 %) and a moderate or strong expression in 28/405 patients (7 %). Interestingly, the PSCA expression was significantly associated with worse histopathological grade (p = 0.038) and with Ki67 proliferation index (p = 0.015). Furthermore we could verify a very strong correlation between PSCA protein expression in primary breast cancer tissue and Her2/neu status (p = 0.00003). The first analysis for outcome indicated a shorter overall survival (p = 0.038) for patients with PSCA positive breast cancers. A more detailed survival analysis is in progress. Conclusions.  To the best of our knowledge, the present study is the first comprehensive analysis of PSCA expression in breast carcinomas of different histological types. We were able to show a correlation of PSCA expression with unfavorable prognostic markers and -in a first approachalso with unfavorable outcome. Therefore, PSCA might be an attractive target for novel targeted treatment strategies alone or in combination with HER2 directed therapies.

P05.02 Histo-pathological changes in a high-risk breast cancer screening cohort and their impact on patient care K. Tendl*1, P. Baltzer2, M. Bernathova2, M. A. Marino2, E.-M. Langthaler1, T. Helbich2, M. Rudas1, Z. Bago-Horvath1 Medizinische Universität Wien, Klinisches Institut für Pathologie, Wien, Österreich, 2Medizinische Universität Wien, Universitätsklinik für Radiologie und Nuklearmedizin, Wien, Österreich

1

Background.  Women with high familial risk for breast cancer, including BRCA mutation carriers are known to benefit from intensive multimodal screening. Due to increased sensitivity of combined imaging techniques, however, the rate of core needle biopsies (CNB) increases. In the present study, the association between radiological and histological findings and their significance in further patient care was investigated. Methods.  This retrospective study included 93 core needle biopsies from 65 high risk patients after multimodal imaging procedures. Classification of radiological changes was performed according to current BI-RADS guidelines. CNB was carried out either stereotactically, ultrasound- or MRI-guided. Histological workup was conducted according to the current European Guidelines for Breast Cancer Screening, including B-classification. Comparison of histological diagnoses in CNB and surgical specimens (SR) was conducted with regard to epithelial atypia and neoplastic

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lesions. Interrelation of radiological and histological findings was evaluated using chi-square test. Results.  Fibroadenomatous hyperplasia (12.9 %), UDH (11.8 %), fibroadenomas (8.6 %) and pseudoangiomatous stromal hyperplasia (6.5 %) represented the most frequent benign histological changes. ADH was found in 6.5 % and DCIS was diagnosed in 8.6 % of biopsies. Invasive carcinoma was present in 11.8 % of CNB specimens. We found a significant association of BI-RADS and B-classification of CNB with the reproducibility of histological diagnoses in subsequent SR specimens. While excellent reproducibility was found in B5 cases, this did not apply for B2 and B3 cases. In these cases a discordance between CNB and SR (33.3 %) was noted. Furthermore, significant association with specific imaging modalities and particular histological changes was demonstrated (p = 0.029). 10.7 % of included patients carried proven BRCA 1/2 mutations. While mutation status was of marginal significance regarding association with younger age (p = 0.069) and subsequent breast surgery (p = 0.187), independent of CNB findings, no significant relation to the specific histological changes listed above, was found. Conclusions.  We identified characteristic histological changes frequently associated with particular imaging findings in a high-risk screening cohort. Improved radiological and pathological identification of these lesions might reduce avoidable CNBs. Our results should be further validated in a larger cohort.

P05.03 Impact of histopathological tumor parameters on diagnostic accuracy of intraoperative margin assessment by frozen section in breast cancer C. Kornauth*1, K. Tendl1, F. Rössler2, E.-M. Langthaler1, J. Pammer1, H. Wiener1, M. Bernathova3, R. Bartsch4, P. Dubsky5, R. Horvat1, M. Rudas1, Z. Bago-Horvath1 Medizinische Universität Wien, Klinisches Institut für Pathologie, Wien, Österreich, 2Universitätsspital Zürich, Klinik für Viszeral- und Transplantationschirurgie, Zürich, Schweiz, 3Medizinische Universität Wien, Universitätsklinik für Radiologie und Nuklearmedizin, Wien, Österreich, 4 Medizinische Universität Wien, Universitätsklinik für Innere Medizin I, Wien, Österreich, 5Medizinische Universität Wien, Universitätsklinik für Chirurgie, Wien, Österreich

1

Background.  Reliable intraoperative assessment of surgical resection margins (RM) in breast cancer is rapidly gaining importance due to increased use of oncoplastic surgical techniques. Our study analysed the influence of tumor-related factors on diagnostic accuracy of intraoperative RM assessment by frozen section (IFS). Methods.  Our study comprised 563 patients with invasive breast cancer treated by surgical resection at the Breast Cancer Centre of the Medical University of Vienna. In all patients, core needle biopsy (CNB) was performed to confirm invasive breast cancer. Factors analysed included CNB method, BI-RADS class of radiological anomaly, tumor size, multifocality, histological tumor grade (including separate grading components), lymph node status, presence of in situ tumor component and/or lymphovascular invasion and immunohistochemically defined intrinsic subtype. Association of these characteristics with RM status and presence of residual tumor in subsequent extended resection specimens was analysed by Chi square test. Results.  437 of 563 cases (77.6 %) were submitted to IFS analysis, with RM being positive for tumor cells in 115 cases (20.4 %). In 253 cases (44.9 %), additional tissue was resected, of which 98 (17.4 %) contained residual invasive or in situ carcinoma. In 29 cases (5.2 %), subsequent resection specimen contained residual tumor in spite of negative RM in frozen section. IFS was more often requested in cases with BIRADS 4 classification compared to BIRADS 5 (85.9 % vs. 72.5 % p = 0.024). Resection margins were significantly more frequently positive in larger (p = 0.001) and multifocal (p = 0.001) primary tumors. In subsequent resection specimens, histological grade (p = 0.013), tumor size (p = 0.022), multifocality (p < 0.001)

and HER2 amplification status (p = 0.024) were significant predictors of residual invasive or in situ carcinoma. In HER2 amplified breast cancer, 50 % of subsequent resection specimens contained residual tumor tissue, whereas in HER2 negative disease, residual carcinoma was detected in 37.2 % of cases. CNB method, presence of an in situ component, intermediate grade of differentiation and lymphovascular invasion were associated with false negativity of IFS (p < 0.05, respectively). Conclusions.  We conclude that intraoperative frozen section is a safe and effective method for RM assessment. Pathological tumor size, tumor grade, multifocality, HER2 status as well as CNB method and the presence of an in situ component impact diagnostic accuracy of frozen section analysis.

P05.04 Abundant NDRG2 expression is associated with aggressiveness and unfavorable patient outcome in basal-like breast cancer V. Kloten*, M. Schlensog, J. Eschenbruch, J. Tiedemann, J. Mijnes, T. Braunschweig, R. Knüchel, E. Dahl Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland Background.  NDRG2, a member of the N-myc downstream-regulated gene family, is thought to be a putative tumor suppressor gene with promising clinical impact in breast cancer. Since breast cancer comprises heterogeneous intrinsic subtypes with distinct clinical outcome we investigated the pivotal role of NDRG2 in basal-type breast cancers. Methods.  Based on subtype classified tumor (n = 45) and adjacent normal tissues (n = 17) we examined NDRG2 mRNA expression and CpG-hypermethylation, whose significance was further validated by independent data sets from The Cancer Genome Atlas (TCGA). In addition, NDRG2 protein expression was evaluated immunohistochemical using a tissue micro array (TMA, n = 215). In vitro, we investigated phenotypic effects caused by NDRG2 silencing or over-expression in the basal-like breast cancer cell lines HCC1806 and BT20, respectively. Results.  Our tissue collections demonstrated an overall low NDRG2 mRNA expression in breast cancer subtypes compared to normal breast tissue in line with an increased CpG-hypermethylation in breast cancer tissue. Independent TCGA data sets verified a significant (P < 0.001) expression loss of NDRG2 in breast tumors. Of interest, basal-like tumors more frequently retained abundant NDRG2 expression concordant with a lower CpG-hypermethylation. Unexpectedly, basal-like breast cancer revealed an association of NDRG2 expression with unfavorable patients’ outcome. In line with this observation, in vitro experiments demonstrated reduced proliferation and migration rates (~20 %) in the highly aggressive basal A cell line HCC1806 following NDRG2 silencing. Conclusions.  Until now, this is the first study investigating in depth the putative role of NDRG2 in basal-type breast cancer. Our data indicate that the described putative tumor suppressive function of NDRG2 may be confined to luminal-type breast cancers.

P05.05 Absence of CD34+ stromal fibrocytes in invasive lobular breast carcinomas correlates with lymph node metastasis C. C. Westhoff*1, S. Ebrahimsade2, H. Daniel3, A. Ramaswamy1, P. J. Barth4, R. Moll1 Institut für Pathologie, Philipps-Universität Marburg, UKGM GmbH, Marburg, Deutschland, 2Facharzt-Praxis für Pathologie, Berlin, Deutschland, 3 Institut für Medizinische Biometrie und Epidemiologie, Philipps-Universität Marburg, Marburg, Deutschland, 4Gerhard-Domagk-Institut für Pathologie, Westfälische Wilhelms-Universität Münster, Universitätsklinikum Münster, Münster, Deutschland 1

Background.  CD34-positive fibrocytes (CD34+Fc) are constitutive elements of the normal stroma of most organs including the breast, playing a role in matrix synthesis, antigen presentation and tumor-associated stro-

mal remodeling. We have previously described the partial maintenance of CD34+Fc in invasive lobular breast carcinomas (ILCs) [1], contrary to e. g. invasive breast carcinomas of no special type. In this study, we analyzed the prognostic relevance for the patients. Methods.  The clinical data of 32 ILC patients (including 10 tubulo-lobular carcinomas) from the prior study were reassessed until February 2014 and the local residents’ registration offices were contacted for information on vital status and date of death, resulting in follow-up periods of up to 14 years. An association between the immunohistochemical results from the previous study concerning the presence of CD34+Fc and α-smooth-muscle actin-positive myofibroblasts (SMA+Mfb) and overall survival (OS) and disease-free survival (DFS) were assessed by Kaplan-Meier curves and log-rank tests. Differences in tumor size, pT stage (pT), grading (G), lymph node status (LN, pN0 vs. pN+), status of distant metastasis until February 2014 (dmet) and tumor type (ILC versus tubulo-lobular) between groups with and without CD34+Fc or SMA+Mfb were evaluated with Mann-Whitney-U-, Χ2- and Fisher’s exact tests where appropriate. Results.  Regarding CD34+Fc, no statistically relevant correlation was found with tumor size, pT, G, LN, dmet and tumor type. However, a statistically relevant correlation was proven for positive LN and absence of CD34+Fc (p < 0.001). No statistically relevant correlation with the above-mentioned parameters was shown for SMA+Mfb. OS was not significantly correlated to the presence of CD34+Fc or SMA+Mfb, to LN, G, tumor size or pT but was significantly worse for patients with dmet (p < 0.001). Kaplan Meier curves showed a trend for worse OS and DFS rates with negative tumor stroma for SMA+Mfb (p = 0.393 and p = 0.214). DFS correlated significantly with positive LN (p = 0.029) and dmet (p < 0.001). Conclusions.  The maintenance of CD34+Fc in ILCs is contrary to the situation in most other carcinomas and suggests distinctive stroma characteristics. Absence of stromal CD34+Fc may serve as a prognostic surrogate marker for the development of lymph node metastasis in ILCs. Further, larger studies may help to clarify the prognostic relevance of presence and number of CD34+Fc with regard to survival. References 1. Ebrahimsade S, Westhoff CC, Barth PJ (2007) CD34+ fibrocytes are preserved in most invasive lobular carcinomas of the breast, Pathology Research & Practice, Pathol Res Pract. 2007; 203(9):695–698

P05.06 TMEM26 is a promising predictive biomarker for chemotherapeutically treated patients with ER-negative mamma carcinoma N. Nass*1, A. Dittmer2, V. Hellwig2, T. Lange2, J. M. Beyer2, B. Leyh2, C. Weißenborn3, A. Ignatov3, A. Roessner1, T. Kalinski1, J. Dittmer2 Institut für Pathologie, Universitätsklinikum, Otto-von-Guericke-Universität, Magdeburg, Deutschland, 2Martin Luther Universität Halle-Wittenberg, Klinik und Poliklinik für Gynäkologie, Halle/Saale, Deutschland, 3Otto von Guericke Universität Magdeburg, Universitätsfrauenklinik, Magdeburg, Deutschland 1

Background.  TMEM26 is a transmembrane protein that has been described as a marker for beige adipose cells in mice. Little is known about the function of this protein. Recently, is has been discussed as a factor involved in resistance to endocrine therapy of breast cancer. We here analyzed the expression of TMEM26 in breast cancer cell lines and breast cancer specimens and correlated TMEM26 abundance with the presence of the estrogen receptor α and the outcome of treatment with antiestrogens and chemotherapy. Methods.  Gene expression in cell cultures was analyzed by qRT-PCR. Protein detection was performed by Western blotting and immunohistochemistry. Kaplan Meyer analysis was used to determine the probability of relapse free – and overall survival. Results.  Estrogen receptor positive cell lines exhibited a high abundance of TMEM26 mRNA but protein was also present in ER-negative cell lines,

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Abstracts however the distribution of isoforms varied between different cellular compartments. The expression of TMEM26 in the ER positive cell line MCF-7 was downregulated in response to fulvestrant and tamoxifen, indicating TMEM26 regulation by estrogen. In breast cancer samples, the expression of TMEM26 was associated with the expression of ERα and PR, and high abundance of TMEM26 protein was correlated with a lower probability of relapse free – and overall survival especially after chemotherapy. Conclusions.  TMEM26 expression is associated with ER and PR expression and might become a valuable predictive marker for the success of chemotherapy in breast cancer.

P05.07 Differentially expression of SOX2 is strongly related to HPV status of vulvar carcinomas A. Gut*, H. Moch, M. Choschzick Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz Background.  SRY box 2 protein (SOX2) is a transcription factor mainly implicated in stem cell differentiation. The gene was found to be amplified in squamous cell carcinomas of various origins. The aim of our study was to analyze SOX2 expression in a large set of vulvar cancers with clinical follow up data. Methods.  The vulvar cancer tissue microarray used for this study contains a total of 55 formalin fixed, paraffin-embedded tumors. For immunohistochemical detection of SOX2, we used a rabbit monoclonal anti-SOX2 antibody (clone EPR3131, Epitomics Inc.). The SOX2 staining intensity and the percentage of stained tumor cells were recorded for each tissue spot and compared with p16 expression. A final 2-step scoring result was made, distinguishing low and high SOX2 expression levels. Analysis of the tumor HPV status was done with RNA in situ hybridization, utilizing specific probes against pan HPV and HPV 16 (ViewRNA, Affymetrix Inc.). A Cox proportional-hazards model was used in survival analysis. Results.  SOX2 was detectable in 30/55 (54.5 %) of all interpretable specimens. 15 (27 %) samples exhibited high SOX2 expression levels. Increased SOX2 expression was associated with histological tumor type. Basaloid vulvar carcinomas exhibited significantly more frequent high SOX2 expression levels (83.3 %) than keratinizing or non keratinizing tumors (19 % and 22 %, respectively). High levels of SOX2 were significantly associated with strong p16 overexpression (p < 0.01). Furthermore SOX2 overexpression was strongly related to positive HPV status of vulvar carcinomas. Tumors which were positive with pan HPV probes in RNA in situ hybridization showed significantly more frequent high SOX2 expression levels than tumors not associated to HPV in this test (57 % vs. 18 %, p < 0.01). SOX2 expression was unrelated to tumor stage, grading and nodal status of vulvar carcinomas. However, nodal stage but not SOX2 expression was related to patient survival in univariate Cox regression analysis (p < 0.05 and p = 0.24, respectively). Conclusions.  SOX2 is overexpressed in a substantial proportion of vulvar cancers and strongly related to HPV associated tumors. Our results may give new insights in the molecular pathogenesis of HPV related vulvar cancers. Further studies could elucidate possible mechanisms of SOX2 overexpression in these tumors.

P05.08 Never enough lymph nodes in gynecological specimen? What are the relevant influence factors for lymph node counts? J. Andruszkow*1, I. Meinhold-Heerlein2, B. Winkler2, B. Bruno3, R. Knüchel1, J. Jäkel1 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, Klinik für Gynäkologie und Geburtsmedizin, Uniklinik der RWTH Aachen, Aachen, Deutschland, 3Klinik für Frauenheilkunde, Luisenhospital Aachen, Aachen, Deutschland

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Background.  Quality requirements in gynecological surgery demand increased lymph node (LN) numbers in lymphadenectomy specimen. Apart from surgical procedures, pathology has to render efforts in time consuming LN preparation (LNP) to fulfil clinically expected LN counts. Recently, we have shown that complete tissue embedding (CTE) of submitted adipose tissue could increase LN numbers significantly without changing the examined total LN area or the pN stage (pN0 vs. pN1) considerably. Therefore this study aims to reveal relevant factors influencing LN numbers in lymphadenectomy specimen. Methods.  Our study was performed in lymphadenectomy specimens of patients with invasive ovarian and uterine carcinomas. Between 2011 and 2013, LNP was performed; LN count was determined and confirmed by histological analysis. Then, CTE was added followed by histological evaluation. Differences in LN counts and the covered area of LN according to LNP and CTE were detected. Then, clinical data of patients (age, size, and weight) and surgical procedures were analysed in order to identify factors for low or high LN numbers in LNP and CTE. Results.  We received 106 lymphadenectomy specimens of invasive ovarian (40.6 %), endometrial (30.2 %) and cervical (29.2 %) carcinomas. The median patients’ age was 59 years. In 75.5 % of lymphadenectomy specimens, discrepancies between CTE and LNP occurred with an increase of 3.5 % of LN area in CTE specimen. In laparoscopic surgery, more LN were missed during LNP than in laparotomies (p = 0.23). Patients’ size or weight did neither affect the rate of missed LN nor the amount of total LN (p > 0.05). Conclusions.  CTE revealed a significant increase in LN numbers in contrast to LNP although the covered area of additional LN remained low, still raising the question if whole LN were missed or if merely small fragments of larger nodes were registered in CTE. Interestingly, patients’ body mass did neither influence the total amount of LN nor the rate of missed LN in LNP. However, the surgical technique seems to have an important impact of missed LN in our cohort of gynaecological tumours.

P05.09 MALDI imaging approach for the diagnosis of squamous carcinoma of the cervix R. Casadonte*1, R. Longuespée2, M. Kriegsmann3, M. Becker4, S.-O. Deininger4, J. Kriegsmann1, 5 1 Proteopath GmbH, Trier, Deutschland, 2Molekularpathologie Trier, Trier, Deutschland, 3Institut für Pathologie, Universitätsklinikum, Heidelberg, Deutschland, 4Bruker Daltonik GmbH, Bremen, Deutschland, 5MVZ für Histologie, Zytologie und Molekulare Diagnostik, Trier, Deutschland

Background.  Cervical cancer it is one of the leading causes of cancer death in women occurring worldwide. The main difficulties encountered in the diagnosis of these neoplasms are in differentiating them from high-grade squamous intraepithelial neoplasia (HSIL). Thus, there is a need to provide information on the malignant potential of dysplastic changes to estimate the biological risk. In this study, we applied imaging mass spectrometry (IMS) in an attempt to reveal distinctions between non-neoplastic, preneoplastic and neoplastic changes. Methods.  Formalin-fixed paraffin-embedded (FFPE) tissue sections were collected from a patient with cervical cancer. Sections were analyzed by histochemical- and immunotechniques. One section was sprayed with trypsin solution (0.5 µg/µL) and subjected to matrix deposition (7 mg/ ml in 50/50 acetonitrile/0.5 %TFA) using an automatic spray device. Tissue section was subsequently analyzed with a high speed mass spectrometer. Spectra were acquired from a raster of laser spots with 50 µm spacing between each spot. From the image data set, mass spectra were exported from regions of interest and statistically compared through hierarchical cluster analysis. Results.  We use IMS technology to reveal in one single section and in one singular analysis test distinct histological features through differentially detected tryptic peptide ions. Exploring the IMS spectral data integrated

with histology, we were able to correlate peptide signatures with malignant, preneoplastic and non-neoplastic squamous cells. Conclusions.  Our findings provide a new molecular strategy to supplement the current histopathological analysis of cervical cancer, and may be helpful for better understanding aggressiveness and progression of the disease. Further studies will be carried out to validate our results.

P05.10 Immunoprofile of Gestational Trophoblastic Disease (GTD) A. Jungbluth*1, R. Soslow1, D. Frosina1, M. Fayad1, D. Zamarin2, C. Aghajanian2, S. Chiang1 Memorial Sloan-Kettering Cancer Center, Abteilung für Pathologie, New York, Vereinigte Staaten von Amerika, 2Memorial Sloan Kettering Cancer Center, Department of Medicine, New York, Vereinigte Staaten von Amerika

1

Background.  GTD consists of several entities. The underlying cellular component is the trophoblast whose variable malignant potential determines the biology of the disease. Normal placental trophoblast is immunologically distinct from most other cells and tissues due to the repertoire of immunomodulatory molecules at the maternal/fetal interface. The role of these molecules in GTD is poorly understood; moreover novel therapies based on immunomodulatory molecules or T-cell targets such as Cancer Testis (CT) antigens MAGE-A or NY-ESO-1 may offer options for treatment of GTD. For example, MAGE-A is a tumor associated antigen eliciting autologous T and B cell responses in affected patients and has been used as a vaccine target for cancer therapy Methods.  Archival paraffin blocks were available for 13 cases: 2 hydatiform moles (HM), 1 placental site trophoblastic tumor (PSTT), 6 invasive hydatiform moles (IHM), 4 choriocarcinomas (CCa). Immunohistochemistry was performed for the following molecules: HLA-I, HLA-II, HLA-G, beta-2- microglobulin (B2M), PD-L1, MAGE-A, NY-ESO-1. Extent of staining was graded based on extent of tumor cell staining. Results.  MHC-I was strongly expressed in 4/6 IHMs, 1/1 PSTT and negative or only focally expressed in HM and CCa. MHC-II was negative in most lesions except focal presence in 1/6 IHM and 1/4 CCa. Both HLA-G and B2M were strongly expressed in all cases except 2/4 CCa cases. PD-L1 was present in all tested cases and most homogenously expressed in CCa. CT antigen NY-ESO-1 was negative in all cases while MAGE-A was positive in all analyzed cases except 1/6 CCa. Conclusions.  Here we describe the heterogeneity of molecules in the immune-environment in GTD. Not surprisingly, HLA-G, present in normal trophoblast, is abundantly present in GTD. MHC-II, absent in normal placenta, is also absent in GTD. Interestingly, B2M and MHC-I, not expressed in normal placenta, are present in GTD, except CCa. Not surprisingly, PD-L1, expressed in normal trophoblast, is also abundantly present in GTDs and in particular in CCa, where its expression is homogeneous. The same applies to MAGE-A, which is expressed in all analyzed lesions. The abundant presence of PD-L1 and MAGE-A suggests possible therapeutic usefulness in patients with GTD. However, since both therapies work via T cell mediated immune responses, lacking expression of MHC-I casts doubt on the effectiveness of PD-L1 blockade and/or MAGE-A vaccine based therapies in CCa.

Background.  In the present case report of a 33 year old female patient we used targeted amplicon resequencing for clarifying the origin of synchronous endometrioid adenocarcinoma of the corpus uteri and of the ovary. A lung metastasis was found four years after hyster- and adnexectomy, vaginal brachytherapy and treatment with the synthetic steroid tibolon. Removal of the lung metastasis and continuous megestrol treatment led to a complete remission of the patient for seven years. Methods.  409 genes were resequenced with the Ion Ampliseq Comprehensive Cancer Panel (Thermo Fisher) in all three lesions. In parallel, a comprehensive immunohistochemical analysis of the lesions was performed. Results.  Mutations in ten different genes were identified and confirmed by Sanger sequencing, three of which were found in all lesions (CTNNB1, PIK3CA, SYNE1), suggesting a common origin. Moreover, the identified mutations were shown or predicted to affect the function of the corresponding proteins but not necessarily their expression levels and thus, were not detected in the comprehensive immunohistochemical analysis. Conclusions.  In summary, synchronous endometrial and ovarian cancer displayed a similar mutational landscape. The mutations leading to activation of AKT-mTORC1 signaling, previously reported to be a target of megestrol, might explain the long-term complete remission of the patient.

P05.12 Placental Chorangioma. Case report with Review of the Literature M. Gajda*1, T. Groten2, U. Schneider2, E. Schleußner2, I. Petersen1 1 Institut für Pathologie, Jena, Deutschland, 2Klinik für Frauenheilkunde und Geburtshilfe – Abteilung Geburtshilfe, Jena, Deutschland

Background.  Chorangioma, originally described by Clarke in 1798, is the most common tumor of the placenta, with a reported prevalence of approximately 0.5–1.0 %. As the name indicates, itis an hamartoma of the primitive chorionic mesenchymal thataris es from angioblastic tissue and is defined as an expansible nodular lesion composed entirely of capillary vascular channels, intervening stromal cells, and surrounding trophoblasts. Methods.  Detailed clinical and histopathologic review of a clinical case and review of the literature using PUBMED. Results.  The tumor was observed in the placenta of a 32-year-old gravida I. The pregnancy was terminated at 28 + 1 weeks by caesarean section. Gross examination of the placenta showed a well-demarcated mass, bulging paracentrally from the fetal surface. On gross examination, as well as microscopically, umbilical cord and amniotic membranes were unremark-

P05.11 Tracking the origin of synchronous endometrial and ovarian cancer by next-generation sequencing. A case report N. Valtcheva*1, F. Lang2, A. Noske3, E. Samartzis2, A.-M. Schmidt2, D. Fink2, H. Moch1, K. Dedes2, P. Wild1 Institut für Klinische Pathologie, UniversitätsSpital, Zürich, Schweiz, UniversitätsSpital Zürich, Klinik für Gynäkologie, Zürich, Schweiz, 3 UniversitätsSpital Zürich, Institut für Klinische Pathologie, Zürich, Schweiz 1

2

Fig. 1 I P05.12 8 Histological appearance of the chorangioma (H&E×100) Der Pathologe Suppl 1 · 2016

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Fig. 2 I P05.12 8 Chorangioma VEGF-R able. Microscopy showed capillary proliferation resembling fetal vessels in enlarged villi covered by trophoblastic epithelium (. Fig. 1). Immunohistochemical staining for CD31 showed strong intensity; CD 34 places also VEGF-R2 demonstrated (. Fig. 2). The proliferation index, measured by Ki67, was 10–15 %. Conclusions.  Chorangiomas are hamartoma malformations of the placenta which in some cases may be large enough to influence the course of pregnancy and associated complications are hydramnion, gestosis or hemorrhage. Chorangioma is the most common benign tumor of the placenta. Its recognition and diagnosis is often made without difficulty. Chorangiomas are rather frequent neoplasms encountered on placental examination but in rare cases they present some worrisome histological features that could alarm the pathologist and be misinterpreted as a malignant neoplasm, even if their biological behavior is favorable. Rarely, a chorangioma may show many mitoses and exhibit histologic features of a sarcoma. References 1. A Selmin et al (2014) An epidemiological study investigating the relationship between chorangioma and infantile hemangioma, Pathol Res Pract. 210(9):548-553 2. T Faes et al (2012) Chorangiocarcinoma of the placenta: A case report and clinical review, Placenta 33(8):658–661 3. US Andola et al (2014) Chorangioma of placenta with high risk pregnancy: A case series, J Basic Clin Reprod Sci 3:71–73 4. M Guschmann (2002) Wachstumsfaktoren und Apoptoserate in einem ungewöhnlichen Chorangiom, Pathologe 23(5):389–391 5. VG Vellone et al (2015) Atypical cellular chorangioma: A potential diagnostic pitfall with worrisome aspects but a favorable prognosis, Int J Surg Pathol. 23(5):364–368

P05.13 Anogenital papillary hidradenoma shows recurrent and frequent mutations in PIK3CA and AKT1 N. Pfarr*1, H.-P. Sinn2, F. Klauschen3, C. Flechtenmacher2, M. Bockmayr3, K. Ridinger2, M. von Winterfeld3, A. Warth2, K. Lorenz2, J. Budczies3, R. Penzel2, V. Endris2, W. Weichert1, 2, A. Stenzinger2 Institut für Pathologie, Technische Universität München, München, Deutschland, 2Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, 3Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland

1

Background.  Papillary hidradenoma is a benign neoplasm of the anogenital region that almost exclusively arises in middle-aged Caucasian women. These tumors may recur and rare cases of malignant development have been re-

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ported. While from a phenotypic point of view these tumors are well defined, the genetic underpinnings of papillary hidradenoma are currently unknown. Methods.  We employed targeted high-coverage next generation sequencing interrogating 50 cancer-related genes to investigate the mutational landscape in a cohort of 15 cases. Confirmatory conventional Sanger sequencing was performed for validation of detected mutations. Additionally, we analyzed the HPV status of these tumors. Results.  Thirteen cases (87 %) harbored mutations in cancer-related genes. Recurrent mutations in PIK3CA (p.H1047R, p.H1047L and p.E545K) were most prevalent (8 of 13 mutated cases (62 %), 8 of 15 cases of the whole cohort (53 %)). One PIK3CA mutated case had a concomitant STK11 mutation. The other 5 mutated papillary hidradenomas displayed mutually exclusive missense mutations in AKT1 (2 cases, p.E17K), APC (p.A1358S), ERBB4 (p.P270L) and BRAF (p.V600E). All mutations were independently validated by Sanger sequencing (concordance: 100 %). The remaining 2 cases showed no mutations. None of the cases harbored DNA of human papilloma virus. Conclusions.  Our data suggest that the majority of PH (67 %) are driven by mutated PIK3CA or AKT1 while the remaining mutated cases mainly showed mutations in driver genes encoding MAPK signaling. Moreover, our results provide evidence that a mutated driver gene does not imply malignant behavior per se but may set the basis for malignant transformation. The latter point may explain why rare cases of papillary hidradenoma have been reported to take a malignant course. Lastly, the genetic data may suggest treatment avenues beyond conventional surgery for some of these tumors.

Postersitzung AG Knochen-, Gelenk- und Weichgewebspathologie P07.01 Calcitonin-related polypeptide beta (CALCB) is an EWSR1-FLI1 target gene contributing to proliferation of Ewing sarcoma cells M. Dallmayer*1, M. C. Baldauf1, P. Gilardi-Hebenstreit2, R. Alba Rubío1, J. Musa1, A. Marchetto1, M. M. Kiran1, M. F. Orth1, G. Sannino1, C. Vokuhl3, I. Leuschner3, J. Alonso4, U. Dirksen5, T. Kirchner6, T. G. P. Grünewald1 1 Labor für Pädiatrische Sarkombiologie, Pathologisches Institut der LMU München, München, Deutschland, 2Institut de Biologie de l’ENS (IBENS), U1024 INSERM, UMR8197 CNRS, École Normale Supérieure (ENS), Paris, Frankreich, 3Institut für Paidopathologie, Kinder-Tumorregister, Christian-Albrechts-Universität, Kiel, Deutschland, 4Instituto de Investigación de Enfermedades Raras, Instituto de Salud Carlos III, Madrid, Spanien, 5Abteilung für pädiatrische Hämatologie und Onkologie, Universitätsklinikum Münster, Münster, Deutschland, 6Pathologisches Institut der LMU München, München, Deutschland

Background.  Ewing Sarcoma (EwS) is a highly aggressive pediatric cancer caused by chromosomal translocations generating fusions of the EWSR1 gene and a member of the ETS family of transcription factors (mostly FLI1). EWSR1-FLI1 alters the expression of many genes via binding to enhancer-like GGAA-microsatellites, whose activity increases with the number of GGAA-repeats. This study investigates the role of calcitonin-related polypeptide beta (CALCB) – a neuropeptide with likely vasodilatory function – in EwS. Methods.  Microarrays, ChIP-Seq, luciferase reporter assays, qRT-PCR, immunohistochemistry, RNA interference and functional assays. Results.  Analysis of 2792 gene expression microarrays comprising 50 tumor entities and 71 normal tissue types revealed that CALCB is specifically and highly overexpressed in EwS relative to other cancers and normal tissues. Time-course knockdown experiments showed that CALCB expression is tightly linked to that of EWSR1-FLI1. ChIP-seq data and epigenetic data generated in EwS cells demonstrated that EWSR1-FLI1 occupies a GGAA-microsatellite close to the CALCB gene, which exhibits

epigenetic characteristics of an active enhancer. Luciferase reporter assays confirmed the strong and EWSR1-FLI1-dependent enhancer activity of this GGAA-microsatellite. In accordance with the inter-individual variability of GGAA-microsatellites concerning repeat numbers, we observed generally high but variable CALCB expression across 27 EwS cell lines by qRT-PCR and in primary EwS by immunohistochemistry. Since CALCB expression levels correlate significantly with clinical outcome of EwS patients, we tested the effect of siRNA-mediated CALCB knockdown on the phenotype of EwS cells. While short-term suppression of CALCB had no effect on proliferation of EwS cells, its long-term knockdown decreased clonogenic growth. We observed that the gene CALCRL, which encodes a central part of the physiological CALCB receptor-complex, is only lowly expressed in EwS. In contrast, we detected high CALCRL expression in endothelial cells, suggesting that EwS cells might influence their microenvironment through CALCB-secretion. Therefore, we currently analyze the effect of CALCB in co-culture assays of EwS cells and endothelial cells. Conclusions.  CALCB is an EWSR1-FLI1 target gene, which is highly overexpressed in primary EwS tumors and which contributes to proliferation of EwS cells. Future experiments will determine whether CALCB additionally affects the tumor microenvironment.

P07.02 Bioinformatic identification of novel prognostic biomarker signatures for Ewing sarcoma J. S. Gerke*1, C. C. Friedel2, M. F. Orth1, T. Kirchner3, T. G. P. Grünewald1 Pathologisches Institut der LMU München, Labor für Pädiatrische Sarkombiologie, München, Deutschland, 2Institut für Informatik, LMU München, München, Deutschland, 3Pathologiches Institut der LMU München, München, Deutschland 1

Background.  Identification of prognostic biomarkers is key for personalized anti-cancer therapy. High-throughput ‘omics’-technologies generated comprehensive sets of matched clinical and gene expression data providing the opportunity to scan for novel biomarker signatures. Methods.  To enable hypothesis-free and data-driven identification of biomarker signatures we developed an application dubbed ‘GenEx’. Our software implements annotation tools, statistical and predictive methods, as well as machine-learning algorithms such as prediction analysis of microarrays, which identifies minimal lists of genes that best characterize a given classifier, and ’WEKA’, which enables integration of patient-specific features. Results.  We applied GenEx to an Affymetrix dataset comprising 239 samples from primary Ewing sarcoma. Microarray data were simultaneously normalized with RMA using customized brainarray chip description files yielding one optimized probe-set per gene. All genes were analyzed individually with the Kaplan-Meier method on patients grouped by quartile calculation with respect to their intratumoral gene expression values, and by an optimized cut-off for each gene. The statistical significance regarding survival probability was calculated with a log-rank test corrected for multiple testing (Bonferroni). In addition, we applied machine-learning algorithms to predict a set of genes, whose expression levels are highly correlated with outcome. Combining the results of unsupervised and supervised analyses and implementing additional random-forest and information-gain algorithms, we identified a prognostic gene signature comprising 9 genes from the totality of 19,674 genes, which can predict dismal outcome with a specificity of >90 % (accuracy 87 %; diagnostic odds ratio 39.7). These genes are mainly involved in non-redundant functions such as telemore stabilization, adaptation to hypoxia and RNA-splicing. Of note, analyzing the prognostic effect in patients without metastasis at time of diagnosis yielded similar results, indicating that our signature can predict outcome in patients with localized disease. Conclusions.  GenEx is a user-friendly application that simplifies and accelerates the identification of potential biomarkers and biomarker signatures. Applying this tool to matched gene expression and clinical data for

a large Ewing sarcoma cohort identified a promising prognostic gene signature prompting further validation in additional datasets and by independent methods.

P07.03 MYBL2: A pro-proliferative EWSR1-FLI1 target gene associated with poor outcome in Ewing sarcoma J. Musa*1, M.-M. Aynaud2, O. Mirabeau2, P. Gilardi-Hebenstreit3, M. F. Orth1, M. M. L. Knott1, M. Dallmayer1, M. Baldauf1, R. Alba Rubío1, A. Marchetto1, G. Sannino1, C. Vokuhl4, I. Leuschner4, U. Dirksen5, J. Alonso6, T. Kirchner7, O. Delattre2, T. G. P. Grünewald1 Pathologisches Institut der LMU München, Labor für Pädiatrische Sarkombiologie, München, Deutschland, 2Institut Curie Research Center, INSERM U830 Genetics and Biology of Cancers, Paris, Frankreich, 3Institut de Biologie de l’ENS (IBENS), École Normale Supérieure (ENS), Paris, Frankreich, 4 Institut für Pathologie des UKSH Kiel, Sektion Kinderpathologie, KinderTumorregister, Kiel, Deutschland, 5Klinik für Kinder- und Jugendmedizin der Universität Münster, Pädiatrische Hämatologie und Onkologie, Münster, Deutschland, 6Instituto de Investigación de Enfermedades Raras, Instituto de Salud Carlos III, Madrid, Spanien, 7Pathologisches Institut der LMU München, München, Deutschland 1

Background.  Ewing sarcoma is a pediatric bone-associated cancer, characterized by specific EWSR1-ETS fusion-oncogenes, most commonly EWSR1-FLI1. EWSR1-FLI1 is an aberrant transcription factor driving the expression of pro-proliferative genes by diverting nearby GGAA-microsatellites as enhancers. At such GGAA-microsatellites, which underlie natural germline variability, the number of GGAA-repeats positively correlates with their enhancer activity. MYBL2 belongs to the Myb-familiy of transcription factors and is an important regulator of cell cycle, stemness, differentiation and apoptosis. Methods.  DNA microarrays, gene-set enrichment analysis, ChIP-Seq, qRT-PCR, RNA interference in conjunction with in vitro assays, xenografts, immunohistochemistry, luciferase reporter assays. Results.  Analysis of published gene expression data from primary Ewing sarcomas revealed that MYBL2 is overexpressed in about one third of tumors. Interestingly, patients with high intratumoral MYBL2 expression have a far worse prognosis than those with low expression, suggesting an oncogenic function of MYBL2 in Ewing sarcoma. In support of this notion, RNA interference-mediated knockdown of MYBL2 strongly reduced proliferation and clonogenic growth of Ewing sarcoma cell lines, while prolonged MYBL2 suppression induced cell death. Moreover, geneset enrichment analysis of MYBL2 co-expressed genes in primary Ewing sarcoma tumors demonstrated that MYBL2 expression is associated with transcriptomic signatures related to stemness and drug-resistance. The generally high, but variable expression of MYBL2 was validated on protein level by immunohistochemistry in Ewing sarcoma cell line xenografts and primary tumors. Using time-course knockdown experiments of EWSR1-FLI1 in an Ewing sarcoma cell line, we found that MYBL2 expression is tightly linked to that of EWSR1-FLI1. In accordance, ChIP-Seq and epigenetic data revealed two GGAA-microsatellites close to the MYBL2 gene, which showed characteristics of active enhancers. Using luciferase reporter assays, we found that one of them had strong and strictly EWSR1-FLI1-dependent enhancer activity. We now investigate whether the interindividual differences in repeat-length of this GGAA-microsatellite translate into differential EWSR1-FLI1 binding and thus MYBL2 expression levels and patient outcome. Conclusions.  MYBL2 is an EWSR1-FLI1 target gene, whose overexpression confers a pro-proliferative phenotype and poor outcome in Ewing sarcoma.

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Abstracts P07.04 High AMPD2 expression is correlated with worse clinical outcome in undifferentiated pleomorphic sarcoma (UPS) M. F. Orth*1, J. S. Gerke1, M. Baldauf1, T. Kirchner2, T. G. P. Grünewald1 Pathologisches Institut der LMU München, Labor für Pädiatrische Sarkombiologie, München, Deutschland, 2Pathologiches Institut der LMU München, München, Deutschland

1

Background.  Undifferentiated pleomorphic sarcoma (UPS) is a common and aggressive soft tissue sarcoma, which most frequently occurs in the extremities and retroperitoneal space during later adulthood. Besides surgical resection, treatment comprises adjuvant irradiation with good tumor response, while chemotherapy is usually less effective. UPS is characterized by high local relapse rates and early metastasis. Metastasis is a catastrophic event in the clinical course of UPS patients. However, currently no bona fide prognostic biomarkers are available that can predict outcome and which may thus help tailoring personalized therapy. Methods.  We analyzed publicly available multi-dimensional ’omics’ datasets with matched clinical annotation to scan transcriptome-wide for novel candidate biomarkers for UPS. Results.  As a discovery cohort we used 51 UPS samples from The Cancer Genome Atlas (TCGA) in which we assessed the correlation of the expression levels of each gene with overall survival in patients stratified by the corresponding median gene expression levels. Significant candidates were validated in an independent UPS cohort (n = 136). Among 11 validated genes, whose high expression significantly correlated with worse outcome, AMPD2 (adenosine monophosphate deaminase 2) – a regulator of purine biosynthesis – was most promising: High AMPD2 expression is significantly associated with clinical outcome (long-term overall survival 38 % in AMPD2-high vs. 92 % AMPD2-low tumors, P = 0.0083; long-term metastasis-free-survival 52 % in AMPD2-high vs. 68 % AMPD2-low tumors, P = 0.0034). Indeed, most events accumulated in those individuals who exhibited high intratumoral AMPD2 expression. AMPD2 expression was not correlated with known relevant clinical parameters such as age, gender, tumor site, metastasis at diagnosis, margin status after resection and adjuvant treatment (P = 0.206 to 0.944), suggesting that AMPD2 may serve as an independent biomarker in UPS. Gene-set enrichment analysis of AMPD2 co-expressed genes revealed that high AMPD2 levels significantly (P < 0.001) correlated with transcriptomic signatures involved in proliferation and invasion. However, high AMDP2 expression appeared not to be caused by copy-number changes as determined by TCGA. Conclusions.  Our data indicate that AMPD2 is as an attractive novel biomarker for dismal outcome in UPS, prompting further validation in additional cohorts and analyses whether AMPD2 in conjunction with other candidates can even improve outcome prediction.

P07.05 Evaluation of PSMA expression in soft tissue tumors B. Heitkötter*1, M. Trautmann1, M. Bögemann2, K. Rahbar3, B. Heitplatz1, K. Steinestel1, I. Grünewald1, W. Hartmann1, E. Wardelmann1, S. Huss1 Gerhard-Domagk-Institut für Pathologie, Universitätsklinikum, Münster, Deutschland, 2Klinik und Poliklinik für Urologie und Kinderurologie, Universitätsklinikum Münster, Münster, Deutschland, 3Klinik für Nuklearmedizin, Universitätsklinikum, Münster, Deutschland 1

Background. PSMA (prostata specific membrane antigen) is highly expressed in prostate cancer cells and is suggested as a target for antibody-based therapy. Furthermore PSMA expression has been described in the neovasculature of a variety of solid cancers and potentially inhibiting neoangiogenesis by targeting tumor-associated blood vessels is a promising therapeutic strategy. As PSMA expression has not been systematically analyzed in soft tissue tumors the current study aims at investigating a large cohort of different entities.

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Methods.  Immunohistochemistry was used to detect PSMA expression in a total of 451 samples of soft tissue tumors. CD34 coexpression was used to study the expression in the neovasculature. Results.  PSMA expression in the tumor-associated neovasculature was found positive in a subset of 451 tumors of the soft tissue in twenty different entities: 2/77 well differentiated liposarcomas; 17/79 dedifferentiated liposarcomas, 5/10 pleomorphic liposarcomas, 1/32 myxoid liposarcomas, 3/14 schwannomas, 0/2 neurofibromas, 0/2 ganglioneuromas, 7/23 malignant peripheral nervous tumors, 9/17 synovial sarcomas, 2/6 hemangiomas, 3/30 angiosarcomas, 1/6 myxoid fibrosarcomas, 17/36 undifferentiated sarcomas, 21/67 leiomyosarcomas, 12/39 solid fibrous tumors, 1/4 endometrial stromal sarcomas, 1/3 gastrointestinal stromal tumors, 0/2 inflammatory fibroid polyps, 0/2 desmoids. Normal tissue cells were all negative. Conclusions.  PSMA is expressed in the neovasculature of different soft tissue tumors potentially offering the possibility for anti-angiogenic therapy.

P07.06 CDK4, MDM2 and TP53 overexpression correlates with recurrence free survival in high risk gastrointestinal stromal tumours M. A. Ihle*1, S. Huss2, H. Pasternack3, M. Trautmann2, B. Heitkötter2, S. Merkelbach-Bruse1, H.-U. Schildhaus4, W. Hartmann2, K. Steinestel2, R. Büttner1, H. Sihto5, K. Sundby Hall6, M. Eriksson7, P. Reichardt8, H. Joensuu5, E. Wardelmann2 Institut für Pathologie, Universitätsklinikum Köln, Köln, Deutschland, Gerhard-Domagk-Institut für Pathologie, Universitätsklinikum, Münster, Deutschland, 3Institut für Pathologie, Universitätsklinikum Schleswig Holstein, Lübeck, Deutschland, 4Abteilung Pathologie, Universitätsmedizin (UMG), Göttingen, Deutschland, 5Universitätsklinikum Helsinki, Helsinki, Finnland, 6Oslo Universitätskrankenhaus, Oslo, Norwegen, 7Lund University, Lund, Schweden, 8Heliosklinikum Emil von Behring, Berlin, Deutschland 1

2

Background.  Little is known about altered expression of apoptosis and cell cycle key regulators as prognostic biomarkers for the recurrence of high risk GIST. We therefore aimed at evaluating the prognostic relevance of different cell cycle regulators and apoptosis modulators. Methods.  In the Scandinavian Sarcoma Group XVIII trial, 400 patients with a high estimated risk for GIST recurrence were randomly assigned for adjuvant imatinib either for 1 or 3 years. Of these, 320 primary tumour samples were available and were immunohistochemically analysed for the expression of CCDN1, CDKN1A, CDKN2A, CDK4, E2F1, MDM2, TP53 and p-RB1 on tissue microarrays. In addition, TP53 mutational analyss was carried out (exons 4–8). Results.  High expression of CDKN2A was detected in 44.4 % of the samples, CCDN1 in 44.4 %, TP53 in 35.3 %, MDM2 in 12.2 %, CDK4 in 32.8 %, p-RB1 in 24.7 %, CDKN1A in 42.2 % and E2F1 in 47.8 % of the cases. The expression of individual markers could be correlated to the mutational status of KIT or PDGFRA, tumour size, age at study entry and recurrence free survival. Especially, the expression of CDK4 was associated with favourable recurrence free survival (RFS), whereas high expression of MDM2 and TP53 or TP53 mutations were associated with unfavourable RFS. 7.1 % of the GIST with high TP53 expression showed a mutation in the TP53 gene as compared with 3.5 % in the whole cohort. Conclusions.  Our study suggests that the expression of CDK4, MDM2 and TP53 and the mutational status of TP53 may be supportive to predict the RFS in treatment naïve, high risk GISTs.

P07.07 H3F3A and IDH1/2 mutations in different giant cell lesions I. Sändig*1, F. Haller2 Universitätsklinikum Leipzig, Institut für Pathologie, Leipzig, Deutschland, FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland

1 2

Background.  Giant cell tumors of bone (GCT) represent about 5 % of all primary bone tumors, usually effecting young adults between the ages of 20 and 45. Although GCT generally take a benign course, local recurrences and distant metastasis occur in rare cases. Since their tumorigenesis is poorly understood and the number of effective treatment options is limited particular for malignant GCT, they are an interesting target for sequencing analysis. Lately specific mutations of the H3F3A gene, a gene encoding a so called replacement histone subclass, were identified in the stromal compartment [1]. In addition IDH2-mutations, which are present in quite a few neoplasms, such as glioblastoma, astrocytoma or acute myeloid leukemia, were reported in a small number of GCT cases from japan [2]. Since there are numerous other giant cell lesions known, whose biology is still unenlightened, we compared different types of giant cell lesions regarding the presence of H3F3A and IDH1/2 mutations. Methods.  Formalin-fixed paraffin-embedded tissue specimens from 69 different patients with giant cell lesions were analyzed, including GCT, giant cell tumor of soft tissue, central giant cell granulomas, peripheral giant cell granulomas, giant cell tumors of tendon sheath and pigmented villonodular synovitis. Following microdissection, isolation and amplification of DNA bidirectional Sanger sequencing of hotspot regions of H3F3A and IDH1/2 was performed. Results.  17 of 22 GCTs held mutations in H3F3A (77.3 %, exclusively p.G34 W). No IDH1 or IDH2 mutations were observed. Also no other entity showed H3F3A or IDH1/2 mutations. Conclusions.  The high prevalence of H3F3A mutations in GCT supports the often discussed assumption of their neoplastic nature. Since there was no IDH1/2 mutation found, the ethnicity possibly plays a role in the tumorigenesis of GCT. The fact, that no other entity held mutations either in H3F3A or in IDH1/2 could be of diagnostic use and may be giving an answer to the question, whether there is a relation between GCT and other giant cell lesions, especially central giant cell granuloma. References 1. Behjati S, Tarpey PS, Presneau N et al (2013) Distinct H3F3A driver mutations define chondroblastoma and giant cell tumor of bone. Nat Genet 45:1479-1482 2. Kato Kaneko M, Liu X, Oki H et al (2014) Isocitrate dehydrogenase mutation is frequently observed in giant cell tumor of bone. Cancer Sci 105:744-748

P07.08 Establishing and validating the fluorescent amyloid ligand h-FTAA (heptamer formyl thiophene acetic acid) to identify amyloid deposits in the carpal tunnel ligament K. Hahn*1, P. Nilsson2, P. Hammarstrom2, P. Urban3, R. Meliß4, H.-M. Behrens1, C. Röcken1 Institut für Pathologie, Christian-Albrechts-Universität, Kiel, Deutschland, IFM-Department of Chemistry, University of Linkoping, Linkoping, Schweden, 3Institut für Pathologie, Gehrden, Deutschland, 4Institut für Dermatopathologie, Hannover, Deutschland 1

2

Background.  Congo red-stain is not very sensitive for the detection of tiny amyloid spots with the pitfall of staining collagen fibers. The stain h-FTAA is supposed to detect tiny amyloid spots without staining collagen. Here, we aimed to prove the hypothesis that h-FTAA is more sensitive than Congo red-stain for the detection of amyloid Methods. From the Amyloid Registry of the Institute of Pathology, Christian-Albrechts-University, we retrieved 53 formalin-fixed and paraffin-embedded samples from 50 patients with ATTR amyloid of the carpal tunnel ligament. Serial sections were stained with Congo red, h-FTAA

(dilution 1:500; 30 min.), and an antibody directed against transthyretin (TTR). Congo red- and h-FTAA-stained sections were mounted using DAPI medium in order to enable scanning using a Hamamatsu scanner (fluorescent light, 40x resolution, 4x exposure time, DAPI as focus filter). Corresponding areas of the three different stains were identified and the amount of amyloid was detected by the color threshold function of Image J. Thresholds were determined by identifying the amount of amyloid in 20 randomly chosen samples. The mean value for Congo red and h-FTAA was calculated and then used for all areas of the collective. The percentage of pixels containing amyloid in relation to the whole chosen area was calculated and compared in between the three different stains. Results.  Amyloid was found in all samples with a characteristic yellow-green-birefringence and typical fluorescence color after Congo red-staining. Amyloid stained intensely with h-FTAA and an anti-TTR-antibody. Tiny amyloid deposits were barely visible or not identified by Congo red-staining as compared with h-FTAA and TTR-immunostaining. This observation was confirmed by computational quantification of the scanned tissue sections: the highest percentage area was found in TTR-immunostained sections (44 %), followed by h-FTAA (31 %) and Congo red (26 %). The mean amyloid content was 6.7 % for TTR, 6.5 % for h-FTAA and 3.9 % for Congo red. Using linear regression analysis, the results were statistically significant (p < 0.001). The Pearson correlation coefficient was 0.8 (Congo red vs. h-FTAA) and 0.9 (TTR vs. h-FTAA). Conclusions.  This study shows that h-FTAA is a highly sensitive method to detect even small amounts of TTR-amyloid in the carpal tunnel ligament. The staining protocol is easy and h-FTAA may be a much more sensitive procedure to detect amyloid at an earlier stage.

P07.09 Tumour heterogeneity within a sacral chordoma persists in the derived chordoma cell lines D. Jäger*1, A. von Baer2, M. Schultheiß2, P. Möller1, K. Mellert1, T. F. Barth1 Universität Ulm, Institut für Pathologie, Ulm, Deutschland, 2Universität Ulm, Klinik für Unfallchirurgie, Ulm, Deutschland 1

Background.  Chordomas are rare malignancies with a broad spectrum of histological differentiation. We present histological heterogeneity within a sacral chordoma and its related metastases and the cell lines derived from the various lesions. Methods.  We analysed a sacral chordoma and two metastases derived from a patient using routine histopathology and immunohistochemistry. Furthermore, we established 4 new chordoma cell lines originating from the peripheral and the central region of the primary tumour and from a skin and a soft tissue metastasis. Results.  The primary tumour showed partly chondroid and spindle cell differentiation, but also areas with classical physalipherous cells. Occasionally, there were sections with dedifferentiated cell morphology and especially in peripheral regions of the tumour pleomorphic cells were found. The soft tissue metastasis showed chondroid and spindle-shaped areas, in the skin metastasis a classical and spindle-shaped morphology with necrosis were detected. Immunohistochemical analyses showed positive Brachyury staining in all variously differentiated parts of the tumour. Ki-67 index was significantly higher at peripheral regions (up to 10–15 %) and at the invasion front than in central regions (<5 %). Especially dedifferentiated areas of the tumour showed a higher Ki-67 index (up to 20 %), whereas the pleomorphic cells in the peripheral parts showed virtually no proliferation, possibly representing senescent cells. Classical differentiated parts generally showed a lower Ki-67 index than dedifferentiated areas, while a high Ki-67 index was detected in the metastases. We observed loss of tumour suppressor p16 protein in all parts of the tumour, while CDK4, CDK6, Rb and pRb expression varied in intensity, being higher in areas with a higher Ki-67 index.

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Abstracts Cytological analyses of the 4 cell lines reproduced morphological differences between the primary tumour and the metastases, including higher proliferation in cell lines derived from the metastases. Conclusions.  Chordomas can have a broad spectrum of intratumorous differentiation patterns. This tumour heterogeneity may persist in the derived tumour cell lines and may help to address studies on tumour heterogeneity in in vitro experiments.

P07.10 Extraskeletal myxoid chondrosarcoma: A clinicopathologic and molecular analysis reveals novel genetic aberrations M. Trautmann*, M. Cyra, I. Isfort, S. Huss, I. Grünewald, E. Wardelmann, W. Hartmann Universitätsklinikum Münster, Gerhard-Domagk-Institut für Pathologie, Münster, Deutschland Background.  Extraskeletal myxoid chondrosarcomas (EMCs) are rare mesenchymal neoplasms comprising <3 % of all soft tissue tumors. EMCs arise mainly from the deep soft tissues of the extremities, accompanied with high rates of recurrence and metastases. The molecular hallmarks of EMCs are various cytogenetic NR4A3 rearrangements, generating chimeric -NR4A3 proteins. The most common reciprocal translocation t(9;22)(q22;q12), results in a fusion of the EWS RNA-binding protein 1 gene (EWSR1) to the nuclear receptor subfamily 4, group A, member 3 gene (NR4A3 or TEC; approximately 75 % of cases). Further cytogenetic t(9;17)(q22;q11) rearrangements involve TAF15 RNA polymerase II, TATA box binding protein (TBP) associated factor (TAF15; approximately 15 % of cases). The less frequent reciprocal translocations t(9;15)(q22;q21) and t(9;3)(q22;q12), result in transcription factor 12 (TCF12)-NR4A3 and TRK fused gene (TFG)-NR4A3 fusion proteins. Although the oncogenic -NR4A3 fusion transcripts seem to have a crucial role in EMC tumorigenesis and progression, the specific biological function and the mechanism of action remain to be defined. Methods.  We characterized the cytogenetic rearrangements of 25 comprehensive EMC tumors by RT-PCR and/or fluorescence in situ hybridization (FISH). Next generation sequencing (NGS) was performed (Illumina MiSeq platform) to reveal additional genetic alterations besides the known chromosomal translocation. Therefore, a comprehensive cancer panel was designed, comprising 31 cancer-related genes known to be frequently mutated across various malignancies. Results.  Overall, fusion transcripts were detected in 22 of 25 samples (88 %). Sixteen were positive for the EWSR1-NR4A3 and six for the TAF15-NR4A3 fusion gene. The t(9;15) and t(9;3) translocations, resulting in TCF12-NR4A3 and TFG-NR4A3 fusion proteins were not identified in any EMC case. In Addition, several known oncogenic mutations were detected which have not been reported previously in EMC. Conclusions.  The combination of RT-PCR and FISH on paraffin-embedded tissue is a sensitive and specific method for the molecular detection of the pathogenic translocations to be applied in the differential diagnosis of extraskeletal myxoid chondrosarcomas. Our results emphasize that cytogenetic NR4A3 rearrangements are the initiating events in the pathogenesis of EMC. Furthermore, our results indicate the occurrence of additional genetic aberrations.

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P07.11 Functional characterization of IGF-IR and PI3K/AKT/GSK3-beta signaling in myxoid/round cell liposarcoma M. Trautmann*, J. Menzel, S. Huss, I. Grünewald, E. Wardelmann, W. Hartmann Universitätsklinikum Münster, Gerhard-Domagk-Institut für Pathologie, Münster, Deutschland Background.  Myxoid/round cell liposarcoma (MRCLS) is the second most common type of liposarcoma (accounts for 30–35 % of all LS cases). One third of MRCLS cases will become metastatic with tumors spreading to unusual bone and soft tissue locations. Ninety percent of MRCLS lesions are characterized by a specific reciprocal translocation t(12;16) (q13;p11), leading to the pathogenic gene fusion of FUS and DDIT3. The resulting chimeric FUS-DDIT3 fusion protein is suggested to play a crucial role in MRCLS pathogenesis, although the specific biological function and the mechanism of action remain to be defined. Aiming at the preclinical identification of novel therapeutic options, we here investigate the functional and therapeutic relevance of IGF-IR and PI3K/AKT/GSK3-beta signaling in primary human MRCLS and tumor-derived cell lines. Methods.  Immunohistochemical analyses of IGF-IR and PI3K/AKT/ GSK3-beta signaling components were performed in a comprehensive cohort of primary MRCLS specimens. FUS-DDIT3 -dependent activation of the PI3K/AKT/GSK3-beta signaling cascade were analyzed in vitro by siRNA and immunoblotting. Cell proliferation assays were performed in two MRCLS cell lines. Results.  In a significant subset of MRCLS specimens, immunohistochemical staining revealed elevated phosphorylation levels of various signaling components, representing a strong indication of activated IGF-IR and PI3K/AKT/GSK3-beta signaling to be a frequent feature in MRCLS. IGF-IR inhibition significantly blocked the PI3K/AKT/GSK3-beta downstream cascade, associated with reduced phosphorylation levels of several signaling components and reduced MRCLS cell viability. Furthermore, siRNA-mediated knockdown of FUS-DDIT3 lead to dephosphorylation of PI3K/AKT/GSK3-beta signaling components, implying that the FUSDDIT3 fusion protein is involved in the IGF-IR regulated signaling cascade. Conclusions.  In summary, FUS-DDIT3 – regulated IGF-IR and PI3K/ AKT/GSK3-beta signaling appears to be of crucial biological importance in MRCLS tumorigenesis and progression, representing a potential molecular target for the development of novel therapeutic strategies.

P07.12 CD31 heterogeneity in angiosarcoma determines intracellular redox status and malignant behavior – Linking Hippopathway to oxidative-mediated DNA damage response V. Venkataramani*1, S. Küffer2, L. Trümper1, G. G. Wulf1, P. Ströbel2 Abteilung Hämatologie und Onkologie, Universitätsmedizin, Göttingen, Deutschland, 2Abteilung für Pathologie, Universitätsmedizin, Göttingen, Deutschland

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Background.  Angiosarcomas (AS) are a rare sarcoma subgroup that originate from vascular endothelial cells (EC) and represent less than 2 % of all sarcomas. AS are associated with a poor clinical outcome due to a moderate chemo-response rate and a high propensity for local and distal recurrences. Several studies have demonstrated that CD31 is highly expressed in AS, implicating that CD31 is still preserved in malignant cells marking their evolutionary origin. We demonstrate that AS contain a small but reproducible amount of CD31low cells that lost the capability to form vasuclar tubes and present a significant more aggressive and stress-resistant phenotype. Methods.  A collective of human AS patients were used for CD31 expression. Following cell lines were analyzed for CD31 heterogeneity: ASM,

ISO-HASc.1, HAMON (all AS), HUVEC, HDMEC cells (all EC) and EA.hy926 (a transformed HUVEC cells). To dissect the CD31high and CD31low cells we used a single-cell-cloning and parallel also a FACS-based approach. To validate the biological function of CD31 we isolated EC from CD31-KO and WT mice. Functional assays such as cell growth, colony formation, tube formation, western blot, angiogenesis array and xenograft analysis were performed. Results.  Immunhistochemical analysis revealed that well differentiated AS predominantly express CD31, whereas more undifferentiated cells present a significant lower amount. CD31high ASM cells typically formed vascular tubes in contrast to CD31low cells in which the capacity to form tubular structures was lost. Surprisingly, CD31low cells presented a significantly increased cell growth, higher methylcellulose-survival, chemoresistance and tumor growth in vivo. We discovered that CD31low cells contain lower levels of reactive oxygenous species (ROS) and endogenous DNA double strand breaks, consistent with a significant increased antioxidative stress response. On the molecular level, we show that the ablation of CD31 results in a decreased VE-Cadherin stability which leads to an increased nuclear accumulation of the Hippo inhibitor YAP, a co-transcription factor critically involved in the anti-oxidative stress response. Conclusions.  Our study highlights the novel connection between CD31, YAP and oxidative-stress mediated DNA damage response. These findings are likely to encourage further research into targeting the Hippo inhibitor YAP as a new treatment opportunity against the small but chemoresistant CD31low subfraction of AS.

Conclusions.  In most cases the pathologist confirmed correctness of the extracted information. Not covered entries were immoderately erroneous or ambiguous and hard for the pathologist to derive encoded information. This fact states a promising performance of the applied approach. We also prepared TNMs to import to the analytical system to provide a search on samples using attributes of TNMs as search criterion in ontology based data warehouse I2B2 [3].

Postersitzung AG Informatik, innovative Bildgebung und Biobanking

Background.  The centralized Biomaterial Bank of RWTH (RWTH cBMB) is built on top of a solid IT infrastructure, consisting of a variety of software components for a wide range of applications. Registration of the received medical samples, managing biobank workflow, performing patients data pseudonymization and depseudonymization are just a few examples of activities that require development of specific software tools. While offering interfaces for the different tasks and being physically separated due to privacy policies, these applications are expected to be integrated to share data in special cases. Methods.  One of the examples of such an integration is the sample search tool. From the point of view of a RWTH cBMB client, this tool should offer an interface for sample search by criteria like sample material and/or patient diagnosis. This tool has to provide both pseudonymized data like IDs for the later sample request, as well as access to sample metadata like diagnosis and material type. In this particular example three software systems are involved: the pseudonymization service for ID retrieval, the patient diagnosis database and the biobank organizational database for sample metadata. All these data sources have to be queried in an organized and secure way. Results.  I2b2R (Informatics for Integrating Biology and the Bedside at RWTH cBMB) is our search tool for biobank clients. I2b2R provides a software infrastructure that allows researchers to access medical data associated with samples. It constitutes a flexible database schema that can store and query almost any kind of data in an effective way by allowing user to build custom queries when search concepts are defined. It also provides knowledge organization in a clear way using an ontology tree, so search criteria selection can be made in an intuitive way. A tool prototype has been developed to populate the i2b2 database with the data collected from our systems: diagnoses are associated with the metadata from samples archivation software using sample identifiers provided by the identification tool. Conclusions.  Dynamically imported high-level concepts like diagnoses and material types all together form the knowledge base concepts for the i2b2 ontology and can be used as sample search query criteria. To satisfy the special needs of a biomaterial bank, a dedicated data view plugin is in development to support sample-oriented search results inspection.

P08.01 Processing of authentic TNM classifications for automated evaluation I. Shumeiko*1, C. Spreckelsen2, E. Dahl1, P. Leusmann1 Uniklinik RWTH Aachen, RWTH cBMB, Institut für Pathologie, Aachen, Deutschland, 2RWTH Aachen, Institut für Medizinische Informatik, Aachen, Deutschland 1

Background.  In Pathology TNM codes are regularly used to encode cancer stages of cancer tumors [1]. It is defined during microscopic examinations of tumor samples by pathologists. Pathological records are imported to clinical information systems to enable data search and analysis. Unfortunately, this information is not standardized due to different styles and structures of pathological reports, e. g. some entries contain more than one TNM code without clear delimiters, making automatic separation difficult. Thus data must be cleaned and normalized before it can be imported to outside analytical systems. Methods.  D’Avolio et. al. extracted T, N and M stages of TNM codes [2] using regular expressions (REs) from medical reports. This good idea was improved in our work to parse not only for T, N and M stages but also the seven supplementary components described by the UICC. Apart from stage identifiers, each component provides additional properties of the tumor containing material, that were mostly ignored in D’Avolio et al. We developed an algorithm that can separate sequential TNM records for two or more samples stored in one entry. It discovers succeeding TNMs on basis of detecting TNM components, that are met in the inspected entry more than once, e. g. one entry contains two T components, indicating a presence of several TNMs. A Java program was designed to perform parsing. Results.  We tested our program on a set of 1600 pathological records. Each of the records could contain from 1 to 4 TNMs. The TNMs were automatically separated and their properties were parsed to be represented in an object oriented manner. A table with results in a printed form was produced and directed to a pathologist for assessment.

References 1. D’Avolio LW, Litwin MS, Rogers SO, Bui AA (2008) Facilitating Clinical Outcomes Assessment through the Automated Identification of Quality Measures for Prostate Cancer Surgery, Journal of the American Medical Informatics Association, 341–348 3. Murphy SN, Weber G, Mendis M, Gainer V, Chueh HC, Churchill S, Kohane I (2010) Serving the enterprise and beyond with informatics for integrating biology and the bedside (i2b2),). Journal of the American Medical Informatics Association 17(2):124–130 2. Sobin LH, Gospodarowicz MK, Wittekind C (2011) TNM classification of malignant tumours, John Wiley & Sons

P08.02 Using a clinical data warehouse for biomaterial query A. Devaykin*1, C. Spreckelsen2, E. Dahl1, P. Leusmann1 Uniklinik RWTH Aachen, RWTH cBMB, Institut für Pathologie, Aachen, Deutschland, 2RWTH Aachen, Institut für Medizinische Informatik, Aachen, Deutschland

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Abstracts P08.03 Integrating Starlims with a BPMN-based sample request workflow S. Pavlitskaya*1, C. Spreckelsen2, E. Dahl1, P. Leusmann1 1 Uniklinik RWTH Aachen, RWTH cBMB, Institut für Pathologie, Aachen, Deutschland, 2RWTH Aachen, Institut für Medizinische Informatik, Aachen, Deutschland

Background. The purpose of the RWTH centralized Biomaterial Bank (RWTH cBMB) is the cryopreservation of biomaterial samples and association of medically relevant data to them. Providing this information for researchers is a crucial task of a biobank. Consequently, the demand for a flexible tool facilitating the interaction of biomaterial requesters with biobank is high. Methods.  In order to automate sample requests processing at RWTH cBMB, a web-based portal is being developed. It provides a flexible interface enabling researchers to submit requests for specific biomaterials. The portal then helps to process requests at different organizational levels at RWTH cBMB, thus passing all stages of assessment and approval. For this, workflows for sample requests and approval at all biobank levels have been formalized and modeled using BPMN (Business Process Model and Notation) making the portal the central sample request data sink. Results.  One of the central tasks in sample request processing includes search for suitable samples and check of their availability and status in biobank. Laboratory staff traditionally performs this task in the laboratory information management system Starlims. Additional sample query is performed using different data sources such as the data warehouse returning Excel tables of sample candidates, which can be imported into the RWTH cBMB request portal. Both ways of data retrieval need to be synchronized. Therefore a SOAP (Simple Object Access Protocol)-based interface between the request portal and Starlims has been developed. It provides authorized users with access to information on biomaterials such as sample availability, material and status in the request portal. Information on running requests has also been made available in Starlims by a dedicated interface. It presents an overview of the requests and provides transactions to manage samples within request before its completion. The laboratory users are thus informed about the demanded biomaterials. Conclusions.  The described solution offers sample query interface in the currently developed RWTH cBMB portal. It helps to retrieve organizational data on biomaterials from the LIMS, thus facilitating search for the requested samples in the portal. Process automation using Business Process Modeling helps to increase process transparency, reduce costs and improve quality through elimination of manual tasks and possible errors.

P08.04 Bioinformatic Identification of novel targets for Sarcoma Immunotherapy M. C. Baldauf*1, M. Dallmayer1, M. F. Orth1, A. Kirschner2, J. Gerke3, J. Musa1, R. Alba Rubío1, M. M. Kiran1, A. Marchetto1, G. Sannino1, T. Knösel4, C. C. Friedel3, U. Thiel2, T. Kirchner4, T. G. P. Grünewald1 Pathologisches Institut der LMU, Laboratory for Pediatric Sarcoma Biology, München, Deutschland, 2Technische Universität München, Kinderklinik, Forschungszentrum für Krebskranke Kinder, München, Deutschland, 3 Institut für Informatik, LMU, München, Deutschland, 4Pathologisches Institut der LMU, München, Deutschland 1

Background.  Conventional sarcoma therapies often have limited effect despite high toxicity. Thus, more specific and in particular less toxic therapies are required to improve patient outcome and to reduce the toxic burden of cure. Due to its specificity, adoptive T cell therapy emerged as a promising therapeutic alternative. Methods.  We combined bioinformatic analysis of DNA microarrays with in vitro and in situ experiments to identify novel and highly specific immunogenic targets for multiple cancer entities with a focus on human sarcoma and pediatric cancers.

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Results.  Publicly available gene expression data generated on the same microarray platform for >2700 samples comprising 50 tumor entities and 71 normal tissue types were rigorously quality-checked, clinically annotated and normalized simultaneously. An in-house developed algorithm was used to scan transcriptome-wide in each cancer entity for highly and specifically expressed genes that are virtually not expressed in normal tissues. As a proof-of-concept we identified established tumor-associated antigens, but also many novel candidates for each tumor entity of which some appear suitable for targeting several tumor types. In addition, we tested the association of these specific transcripts with patient outcome across tumor entities and we are currently validating their specific expression on the protein level in a comprehensive tissue microarray. Subsequently, we identified immunogenic peptides encoded by the most specific transcripts for each tumor entity by predicting the strength of their peptide-MHC-binding to HLA-A02:01. Next, the peptides with the highest affinity were crosschecked with protein databases to exclude sequence similarity with otherwise abundantly expressed proteins. Specific T2-cell peptide-binding assays were employed to validate the high affinity of the identified peptides and to determine their relative efficacy in stabilizing MHC molecules on the cell surface. In addition, we developed a user-friendly web-browser interface to make our transcriptomic data and immunogenic peptide libraries easily accessible to other researchers. Conclusions.  Collectively, we provide a comprehensive, exquisitely curated and validated catalogue of cancer-specific, immunogenic and high-affinity peptides across 50 cancer entities. We anticipate that our data will constitute a rich resource for further development of specific cancer immunotherapies that shall ultimately help improving patient outcome.

P08.05 Systems biology approach identifies a critical role of the Wnt-pathway deregulation in Ewing sarcoma – implications for targeted therapy M. M. L. Knott*1, M. F. Orth1, J. Gerke1, R. A. Rubio1, J. Musa1, T. Kirchner2, T. G. P. Grünewald1 1 Institut für Pathologie der LMU München, Laboratory for Pediatric Sarcoma Biology, München, Deutschland, 2Institut für Pathologie der LMU München, München, Deutschland

Background. Ewing sarcoma (EwS) is a highly aggressive pediatric bone-associated cancer. It is characterized by EWSR1-ETS fusion-oncogenes – most commonly EWSR1-FLI1. EWSR1-FLI1 is an aberrant transcription factor, which massively deregulates the transcriptome of the putative cell of origin of EwS (mesenchymal stem cells) and arrests these cells in a largely undifferentiated, but highly proliferative state. Several reports show that EWSR1-FLI1 interferes with differentiation pathways. However, this has not been investigated in a systematic and coherent way yet. Methods.  We performed a systematic network analysis of differentiation pathways modulated by EWSR1-FLI1. First, we catalogued EWSR1-FLI1-modulated genes being annotated with the gene ontology term ‘regulation of cell differentiation’ across six different EwS cell lines. Then, we carried out an in silico network prediction analysis on these genes supplemented with expression data from primary tumors. Identified pathways and hubs were assessed for their effect on patient outcome and validated in vitro using RNA-interference, qRT-PCR, functional as well as pharmacologic dose-response assays. Results.  We identify the Wnt-pathway as a key regulator of cell differentiation and stemness in EwS, whose activity is suppressed on multiple levels by EWSR1-FLI1. Network analysis pointed to a central role of the Wnt-associated transcription factors LEF1, TCF7L1 and TCF7L2, which are downregulated by EWSR1-FLI1. Indeed, downregulation of TCF7L1 was correlated with very poor outcome of EwS patients. In accordance, the GSK3 inhibitor Chir99021, which activates Wnt-signaling, strongly reduced proliferation and clonogenic growth of different EwS cell lines. Notably, knockdown of EWSR1-FLI1 significantly decreased the sensitivity of EwS cells

toward this inhibitor, suggesting a potential synthetic lethality of Chir99021 and EWSR1-FLI1. We currently explore the effect of TCF7L1 re-expression on transcriptomic network rewiring in EwS cells and whether Chir99012 shows synergistic effects in combination with conventional chemotherapy. Conclusions.  Collectively, our data suggest that EWSR1-FLI1-mediated deregulation of the Wnt-pathway plays a pivotal role in maintaining a pro-proliferative and stem cell-like state in EwS, and we identified TCF7L1 as a key player with strong prognostic relevance in this network. Moreover, our data indicate that pharmacological restoration of Wnt-signaling may provide a new therapeutic option in EwS.

P08.06 Implementing the PAXgene Tissue stabilization system for a dedicated clinical routine workflow K.-F. Becker*1, J. Slotta-Huspenina1, K.-P. Janssen2, A. Brandtner3, C. Beese1, A. Lehmann4, K. Kuhn4, R. M. Schmid3, W. Weichert1, M. Quante3 1 Technische Universität München, Institut für Pathologie, München, Deutschland, 2Klinikum rechts der Isar, Chirurgische Klinik, München, Deutschland, 3Klinikum rechts der Isar, II. Medizinische Klinik, München, Deutschland, 4Klinikum rechts der Isar, Institut für Medizinische Statistik und Epidemiologie, München, Deutschland

Background.  We aimed to implement the PAXgene tissue fixation system for a prospective pilot study for the morphological and molecular characterization of Barrett’s esophagus (BE) and cancers of the gastroesophageal junction. The ultimate aim is to identify patients who will progress to esophageal adenocarcinoma (EAC) via low grade dysplasia (LGD) and high grade dysplasia (HGD). Methods.  In this ongoing study patients with BE, LDG, HGD and EAC are being included and are objected to surveillance with follow up endoscopies and biopsies according to the national guidelines. Per visit one blood sample, the standard biopsies for formalin fixation (FFPE) and up to six biopsies for PAXgene fixation and paraffin embedding are collected. Inclusion and documentation of demographic data are recorded via a new mobile iPad-based questionnaire. The data protection concept contains a multi-level pseudonymisation procedure. Data is automatically uploaded to the BarrettNet database. Results.  We established parallel tissue processing pathways for either routine formalin or PAXgene fixation. The physician treating the patient receives the routine FFPE report but also an additional report for the PAXgene-fixed samples. So far we evaluated 1085 PAXgene-fixed samples from 241 patients, including 363 samples from 76 patients from external hospitals or doctor’s offices. We classified 187 cases as normal, 19 patients with LGD, 18 patients with HGD, and 17 patients with adenocarcinoma. From 10 of the cases with adenocarcinoma we isolated DNA and RNA from the PAXgene-fixed tissue samples for whole exome next generation sequencing. The analysis of the data has not been finished yet but typical mutation spectra, such a mutations in p53, wnt-pathway, EGFR-pathway, were detected. We established and operated an IT system for the BarrettNet that combines iPad-based questionnaires with a biobank system. Conclusions.  The PAXgene tissue stabilization workflow ensures standardization of the entire process from sample collection to analyte extraction, i. e. the pre-analytical phase. The successful implementation of the PAXgene tissue stabilization process in a specific clinical workflow as demonstrated with our pilot study on BE can easily be transferred to other scenarios where excellent morphology and comprehensive molecular analyses are required, e. g. biopsy collection before and after therapy for treatment monitoring or molecular characterization of colon polyps for prediction of surveillance intervals after endoscopic resection.

P08.07 Robust and accurate quantification of immune cells in the prostate cancer microenvironment using deep convolutional neural networks in AUGURA platform L. Aprupe*1, 2, 3, 4, 5, G. Litjens4, 6, M. Zauser2, 3, 4, 5, K. Safferling4, 7, T. Sütterlin4, 6, 7, B. Lahrmann4, 6, F. Lasitschka5, N. Grabe4, 5, 6, 7 1 DKFZ/DKTK, TIGA Center/AG Grabe, Heidelberg, Deutschland, 2Deutsches Konsortium für Translationale Krebsforschung (DKTK), Heidelberg, Deutschland, 3Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Deutschland, 4Hamamatsu TIGA Center, BioQuant, Ruprecht-KarlsUniversität Heidelberg, Heidelberg, Deutschland, 5Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, 6Steinbeis Center for Medical Systems Biology (STC-MSB), Heidelberg, Deutschland, 7Nationales Centrum für Tumorerkrankungen Heidelberg (NCT), Medizinische Onkologie, Heidelberg, Deutschland

Background.  Prostate cancer is the most common cancer in men and the third cancer-related male death cause in Germany. Immune cell infiltration in tumor regions plays an important role in prostate cancer disease progression and survival. However, the precise role of T-cell infiltration in prostate cancer remains unclear. As such, to better understand the role of infiltrating T-cells, immune cell quantification has attracted significant research interest. The goal of this work was to improve accuracy and reproducibility of image-analysis-based immune cell counting in immunohistochemtically-stained (IHC) digital slides through our new automated deep learning platform AUGURA. Obtained T-cell counts could potentially help to stratify patients with respect to their responsiveness to the immune therapy. Methods.  Digital tissue classification was achieved by applying deep convolutional neural network (DCNN) algorithms to prostate cancer digital slides, which were stained with an anti-CD3/pan-cytokeratin stain. We used 11 digital slides from 7 patients, which were annotated and tiled in small images (patches). The DCNN was trained on the labeled patches with a network of three convolutional and two pooling layers. The network’s performance was compared with manual counting by human observers and with counts by Definiens Tissue Studio. Results.  We found that the DCNN in AUGURA was able to successfully count CD3+, CD4+ and CD8+ stained cells in the prostate tissue. DCCN was insensitive to staining variations and whether immune cells are intraor extra-epithelial. Resulting counts were equivalent to the average of human. Definiens Tissue Studio provided highly inaccurate results in cases of within-slide staining variation. Conclusions.  Our results show that the application of deep learning on the histopathological images is able to automatically and robustly count IHC stained immune cells. Due to staining variations the use of current off-theshelf-software like Tissue Studio cannot be recommended for automatic counting. Deep learning based analysis of the tumor microenvironment provides the necessary technical basis for overcoming problems raised by IHC staining variations. As such, a high-throughput, fully automatic analysis of the tumor microenvironment, also in tumors exposing a difficult morphology, appears possible.

P08.08 Measuring the spatial intratumoral organization of proliferation in immunostains of pancreatic neuroendocrine neoplasms N. Valous1, B. Lahrmann1, N. Halama1, F. Bergmann2, D. Jäger1, N. Grabe*1 Nationales Centrum für Tumorerkrankungen (NCT) Heidelberg, Medizinische Onkologie, Heidelberg, Deutschland, 2Pathologisches Institut, Heidelberg, Deutschland 1

Background. Intratumoral proliferative heterogeneity is common in many neoplasms and contributing factors may reflect on expression of biomarkers and their utility in prognosis/stratification. Hence, a rigorous approach for assessing the spatial intratumoral heterogeneity of histological Der Pathologe Suppl 1 · 2016

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Abstracts biomarker expression with accuracy and reproducibility is required. The critical bottleneck has become the development of computational methods to analyze, integrate, and connect data to prognostic and actionable clinical information. Methods.  The aim is to investigate – in whole sections of primary pancreatic neuroendocrine neoplasms (pNENs) – the spatial intratumoral heterogeneity of Ki-67 immunostains. Tissue samples are obtained and images are acquired using a whole-slide imaging system. Descriptive statistics on single or variable foci cannot capture the complete picture since single measurements are often insufficient. Lacunarity as a multiscale second-order statistical measure assesses spatial heterogeneity in a robust manner. Results.  Inhomogeneity of distribution depends not only on percentage content of proliferation phase, but also on how the phase fills the space. Some pNENs have an increased degree of spatial heterogeneity comparing to others with similar grade. The approach provides multiscale information about Ki-67 distribution that corresponds to degree of spatial organization of proliferation. Whether this is a sign of different tumor biology, and association with a benign or malignant clinical course needs to be assessed further. The extent and range of proliferative heterogeneity has the capacity to be evaluated as a prognostic marker. The association with tumor grade, as well as the rationale that the methodology reflects true tumor architecture (pertinent to proliferation) supports the technical soundness of the approach. Conclusions.  Drawing upon the richness of histopathological information and merits of computational biomedicine, the approach removes qualitative ambiguities and uncovers salient features for use in future studies of clinical relevance.

P08.09 DEEPATH, a software tool for managing machine learning training data in digital pathology M. Zauser*1, 2, 3, G. Litjens2, 4, L. Aprupe1, 2, 3, T. Sütterlin2, 4, B. Lahrmann2, 4, F. Lasitschka3, N. Grabe2, 3, 4, 5 DKFZ/DKTK, Deutsches Krebsforschungszentrum, Heidelberg, Deutschland, Hamamatsu TIGA Center, Universität Heidelberg, Heidelberg, Deutschland, 3 Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, 4 Steinbeis-Transferzentrum Medizinische Systembiologie (STC-MSB), Heidelberg, Deutschland, 5Medizinische Onkologie, Nationales Centrum für Tumorerkrankungen, Universitätsklinikum Heidelberg, Heidelberg, Deutschland 1

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Background.  Various computational algorithms and software tools are available to support the pathologist in the quantitative histological analysis of digital slides. A powerful new technology in image analysis is Deep Learning, an advanced form of machine learning allowing to robustly analyze morphological images. Deep Learning algorithms process image data on increasing levels of complexity to recognize complex morphological structures in tissue. Methods.  We are showing the current state in the development of the webbased software DEEPATH for managing training data for Deep Learning in digital pathology. Images are imported from a fileserver or uploaded by the user. Additional information like tissue origin and staining protocol can be managed. Tissue slides are processed in the background fully automatically. Based on a built-in library of deep convolutional neural networks (DCNN) the software thereby detects e. g. the tumor area and the immune cells in the tissue. After finishing the analysis the results are provided in a compact and clearly arranged form to the user. For obtaining new morphological tissue classifiers (DCNNs) DEEPATH offers a developer mode. Exemplary networks were trained with histological samples from the BioBank of the Heidelberg University Hospital. Results.  DEEPATH allows the systematic management of Deep Learning training data. Examples from immunotherapy are shown in which automatically tumor regions (tumor central, invasive margin, tumor far, stroma, background) and different immune cell populations (CD3, CD4, CD8) in solid tumors like prostate cancer are detected (. Fig. 1). Conclusions.  DEEPATH provides the first technical framework for Deep Learning in digital pathology easing the management of training data and revision of automatic classification results for Deep Learning. Classification results can be digitally validated through multiple users and compared to the automatic classification.

P08.10 A new way of long-term preservation of gross specimens for student teaching and academic study A. Obliers*, A. Erbersdobler Institut für Pathologie, Universitätsmedizin, Rostock, Deutschland

Fig.1 I P08.09 8 Automated detection of tumor region (orange) and different sizes of invasive margin (yellow)

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Background.  Long-term preservation of gross specimens for student teaching and academic study as an alternative to fresh specimens and formalin-fixed macroscopic specimens in glass containers. Establishment of a systematic collection of pathologically altered organs and tissues. Methods.  Organs and tissues with characteristic pathologic changes from the surgical pathology and autopsy service of our institute are preserved using a fixation technique that has already been utilized in veterinary medicine for several years. After fixation the tissue is put into a clear plastic bag, sealed with vacuum and labelled. The material is then stored at 4C in the refrigerator Results.  Our fixation technique results in tissues with maintained original colour, consistency and shape. As opposed to formalin, the fixative we used

is non-toxic. The vacuum-sealed specimens keep their original properties over a long time and can be used for “hand-on” demonstration for medical students. I addition, the specimen preservation does not destroy the tissue and it can be used for subsequent histologic examination. Conclusions.  The method is a cheap way of long-term preservation of gross specimens to provide additional didactic material for student teaching. Through visualization and direct handling of original non-biohazardous material students’ practice-based learning in pathology can be improved.

P08.11 A digital IHC companion diagnostic assay for the quantification of the immunotherapy target B7-H3 in breast cancer K. Safferling1, B. Lahrmann1, F. Lasitschka2, C. Ernst1, T. Sütterlin1, P. Schirmacher2, D. Jäger3, N. Grabe*1 1 Tissue Imaging and Analysis Center, Universitätsklinikum, Heidelberg, Deutschland, 2Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, 3Nationales Centrum für Tumorerkrankungen (NCT), Heidelberg, Deutschland

Background.  Clinical trials of therapeutics targeting the type I transmembrane glycoprotein B7-H3 will benefit through provision of standardized, objective quantification of target availability as a major determinant of a patient’s stratification. As membranous B7-H3 expression intensity was found to be significantly correlated with tumor infiltrating lymphocytes (TILs) and lymph node metastasis, we developed a digital diagnostic IHC assay capable of objectively, reproducibly and automatically scoring membraneous B7-H3 in breast cancer. Methods.  168 patient tissue samples were analyzed by using a B7-H3 and collagen 6 double staining protocol discriminating between tumor and stroma regions. Two computational B7-H3 scoring methods have been developed: i) the membrane intensity method measures solely the membrane expression of the target based on IHC intensity values. ii) the membrane connectivity method measures membrane expression and the degree of how the cell is encircled by the target protein. Both methods were compared to the scoring of the study pathologist in terms of robustness to staining variations and intra-observer variability. Results.  Breast tumor cells were characterized by a membranous B7-H3 staining pattern, whereas specimens with benign breast tissue morphologies showed no or only weak cytoplasmic staining. Performance comparison of the different B7-H3 scoring methods showed that the pathologist could moderately compensate staining variations (ĸ-value: 0.32) as compared to the membrane intensity (ĸ-value: 0.00) and the connectivity (ĸ-value: 0.20) methods. Using a color correction algorithm however improved the connectivity method (ĸ-value: 0.35) thereby outperforming the study pathologist. Regarding reproducibility, the membrane intensity (ĸ-values: 0.61; 0.81) and the connectivity method (ĸ-values: 0.78; 0.72) showed higher intra-observer agreements in comparison to the study pathologist (ĸ-values: 0.55; 0.52) pointing to a more reproducible scoring. As the connectivity method, is measuring the cell border surrounded by the target protein our results indicate a loss of polarity for B7-H3. Conclusions.  Our digital diagnostic assay enables fast, objective and robust B7-H3 status assessment in patients with breast cancer. Measuring connectivity in immune-check-point blockade could provide a new robust, quantitative biomarker analysis outperforming human observer.

P08.12 Computer-assisted diagnosis of Monoclonal Gammopathy of Undetermined Significance (MGUS) based on image analysis of co-registered serial virtual slides from bone marrow biopsies M. Athelogou1, H. Hessel*2, H.-P. Horny2, R. Schoenmeyer1, M. Dillmann1, G. Schmidt1, K. Sotlar2, T. Kirchner2, G. Binnig1 Definiens AG, München, Deutschland, 2Pathologisches Institut, LudwigMaximilians-Universität, München, Deutschland

1

Background.  The histopathological differential diagnosis of MGUS includes plasmacytosis as a purely reactive state but also plasma cell myeloma as a hematological malignancy. The focus of this study was to develop a new method and the corresponding software systems, which allows synchronous navigation and quantification of the of κ:λ light chain ratio and the percentage of CD38 stained plasma cells in routinely processed bone marrow biopsies. Methods.  Bone marrow biopsy sections of 16 patients with histopathological diagnosis of MGUS (n = 15) and multiple myeloma (n = 1) were re-analyzed by digital image analyses using the following immunostains: kappa-/ lambda light chain double and single staining and the plasma cells-associated antigen CD38. Co-registration and quantification of the digitalized slides were performed by “Tissue Phenomics” methods (Definiens AG) and visualized by heatmaps for each parameter. A comparison between co-registered stained slides and the visualized expression profiles highlighted by the established heatmap was used for a synchronously and detailed visual inspection of the diagnostic parameters in the slides locally. The calculated κ:λ light chain ratio and the overall percentage of CD38 stained plasma cells for each patient were compared to the corresponding diagnostic findings. Results.  Based on our digital image analyses 15 out of 16 patients did show similar κ:λ light chain ratios and overall percentages of CD38 positive plasma cells as documented in the pathological findings and therefore confirmed diagnosis of MGUS. One sample could not be analyzed due to technical artefacts while preventing digitalization. Conclusions.  Our results clearly show that our system based on Tissue Phenomics methods allows reliable quantification of plasma cells and κ:λ light chain ratio in bone marrow sections. With regard to our results we could reproduce the pathological diagnoses for MGUS and plasma cell myeloma according to the diagnostic findings.

P08.13 Automated evaluation of p16/Ki-67 stained cervical and anal cytology slides using digital pathology B. Lahrmann1, 2, N. Wentzensen3, N. Grabe*1, 2, 4 Hamamatsu Tissue Imaging and Analysis Center, Universität Heidelberg, Bioquant, Heidelberg, Deutschland, 2Steinbeis-Transferzentrum Medizinische Systembiologie (MSB), Heidelberg, Deutschland, 3National Cancer Institute, Division of Cancer Epidemiology and Genetics, Rockville, MD, Vereinigte Staaten von Amerika, 4Nationales Centrum für Tumorerkrankungen, Medizinische Onkologie, Universität Heidelberg, Heidelberg, Deutschland 1

Background.  p16/Ki-67 dual stain (DS) cytology has been evaluated as a biomarker for cervical and anal cancer screening. DS evaluation requires manual detection of DS cells on stained slides. Automated slide evaluation could improve accuracy and throughput of DS cytology. Methods.  DS cytology was performed in cervical samples from a colposcopy clinic and in anal samples from HIV-positive men who have sex with men. Thinprep slides were stained using CINtec Plus (Roche) and evaluated manually by an expert cytotechnologist. All slides were scanned on a Hamamatsu Nanozoomer. 193 slides representing the whole spectrum of cervical disease were used to train classifiers for DS-positive cells detection, with and without morphological features. Classifiers were evaluated

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Abstracts in an independent set of 341 cervical slides (including 45 CIN3) and 349 anal slides (including 28 AIN3). Results.  Sensitivity and specificity for CIN3 were 88.9 % and 38.5 %, respectively, for manual evaluation of cervical slides. Automated detection of DS-positive cells showed equal sensitivity (86.7 %, p = 1.0) and increased specificity (48.0 %, p = 0.002) compared to manual evaluation. Adding morphological features further increased the specificity (52.4 %, p < 0.0001). Sensitivity and specificity for AIN3 were 89.3 % and 31.4 %, respectively, for manual evaluation of anal slides. Automated detection of DS-positive cells showed equal sensitivity (92.9 %, p = 1.0) and specificity (33.4 %, p = 0.6) compared to manual evaluation; morphological features did not increase specificity in anal specimens. Conclusions.  Automated evaluation of DS slides achieves equal or better performance compared to manual evaluation. Classifiers developed for detection of DS-positive cervical cells were robust, showing comparable performance in anal specimens.

P08.14 Form Follows Function – Morphological and Immunohistological Insights into Tumor Architecture P. Bronsert*1, 2, 3, K. Enderle-Ammour3, T. Ahrens3, A. Csanadi3, S. Timme3, U. Wellner4, M. Werner1, 2, 3 Tumorzentrum Freiburg, Universitätsklinikum, Freiburg, Deutschland, 2 Deutsches Konsortium für translationale Krebsforschung (DKTK), DKFZ, Heidelberg, Deutschland, 3Institut für Klinische Pathologie, Department für Pathologie, Universitätsklinikum, Freiburg, Deutschland, 4Klinik für Chirurgie (UKSH), Campus Lübeck, Lübeck, Deutschland 1

Background.  In cancer cell biology, the architectural concept “form follows function” is reflected by cell morphology, migration and EMT protein pattern. In vivo, features of EMT have been associated with tumor budding, which correlates significantly with tumor aggressiveness and overall survival. We described previously that the majority of tumor buds are not truly detached tumor cell groups, but rather protrusions still connected to a major tumor mass. For more detailed insights into the different tumorbud-compartments and the process of tumor budding we quantified tumor cells according to histomorphological and immunohistological EMT characteristics. Methods.  Three-dimensional reconstruction was performed as published. From four different carcinoma entities, comprising three patients, two strictly serial tissue series were prepared (n = 24). Two research approaches were performed. The first was stained for Cytokeratin AE 1/3 for characterization of tumor cell morphology. The second was stained for Cytokeratin AE 1/3, ZEB1 and e-Cadherin for tumor cell EMT and morphology characteristics. Thereof visionfields of 200x magnification reconstructed. Tumor buds were classified into main tumor mass (MTM), connected tumor buds (CTB) and isolated tumor buds (ITB). Cell morphology and EMT marker expression were assessed from each tumor cell. Results.  In 9662 tumor cell clusters, 143930 tumor cells were characterized for ZEB1, 163,939 cells for e-Cadherin and 490969 tumor cells for histomorphological characteristics. EMT characteristics between ITB and CTB demonstrated no significances or trends (p > 0.15). Tumor-cell-count correlated significantly with EMT (e-Cadherin p = 0.03; ZEB1 p < 0.001) and histomorphological characteristics (p < 0.001). Regression curve analysis revealed initially a loss of membranous E-Cadherin (9 cells), followed by expression of cytoplasmic E-Cadherin (8 cells) and subsequent an expression of nuclear ZEB1 (7 cells). Morphologic changes followed later in this sequence. Conclusions.  The idea of “form follows function” is transferable onto the histo-morphological aspects of migration – form – and EMT – function. Tumor buds connected and not-connected with the main tumor mass are similar in form. Translated to function, EMT marker expression and tumor cell configuration does not differ in both types of tumorbuds. Quantita-

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tively, the EMT cascade is initiated with a membranous E-Cadherin loss, followed by cytoplasmatic E-Cadherin shuttling. ZEB1 increase can only be detected at lower cell counts.

P08.15 Determination of a miRNA-mRNA network promoting lung adenocarcinoma spread M. González-Vallinas*1, M. Albrecht2, A. Pitea3, M. Rodríguez-Paredes4, 5, D. Stichel2, S. Sass3, J. Gutekunst5, J. Schmitt1, T. Muley6, M. Meister6, A. Warth1, P. Schirmacher1, F. J. Theis3, N. S. Müller3, F. Matthäus2, K. Breuhahn1 Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, Zentrum für Modellierung und Simulation in den Biowissenschaften (BIOMS), Universität Heidelberg, Heidelberg, Deutschland, 3Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt, Neuherberg, Deutschland, 4Universitätstumorzentrum Düsseldorf, Universität Düsseldorf, Medizinische Fakultät, Düsseldorf, Deutschland, 5Abteilung Epigenetik, DKFZ-ZMBH Allianz, Deutsches Krebsforschungszentrum, Heidelberg, Deutschland, 6Thoraxklinik am Universitätsklinikum Heidelberg, Sektion Translationale Forschung, Heidelberg, Deutschland 1

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Background.  Lung adenocarcinoma (LUAC) is one of the most deadly tumors, and emerging evidences indicate that microRNAs (miRNAs) are important regulators of LUAC spread. Due to each miRNA is able to regulate the expression of genes belonging to different molecular pathways, they are ideal targets to develop drugs that produce efficient therapeutic effect with low resistance. However, the key miRNA-mRNA network promoting LUAC dissemination has not been well established yet. Methods.  Firstly, we compared the miRNA expression of tumor samples from LUAC patients with and without lymph node metastasis (N1, N2 and N3 vs. N0) in a cohort of The Cancer Genomic Atlas (TCGA) database (n = 449). To validate the results, fresh-frozen samples from an independent patient cohort (n = 108) were analyzed by qRT-PCR. The involvement of selected miRNAs in tumor dissemination was tested by migration and invasion assays after transient miRNA modulation in LUAC cells (timelapse microscopy). Potential miRNA targets were identified by “miRlastic”, a novel algorithm that integrates miRNA-mRNA data by a multiple regression model. Additionally, differential methylation of the miRNA genomic locations were studied in a TCGA patient cohort (n = 29) as a possible mechanism of miRNA dysregulation. Results.  We identified 135 miRNAs differentially expressed in LUAC patients with lymph node metastasis (p ≤ 0.05). Among them, the miRNA cluster located at 14q32.31 was highly represented (22/135). Increased expression of miR-323b, miR-487a and miR-539, located in the 14q32.31 cluster, was significantly associated with poor patient survival. Functional studies showed that these miRNAs induced migration (assessed by timelapse and quantitative analysis) without affecting cell proliferation. Moreover, miRlastic analysis indicated several metastasis-associated genes as potential targets of these miRNAs, and some predicted targets were confirmed in LUAC cell lines (e. g. Pumilio RNA-Binding Family Member-2; PUM2). Furthermore, hypomethylation of the 14q32.31 locus in tumors might be responsible for the overexpression of these miRNAs. Conclusions.  Our study shows that several members of the miRNA cluster encoded at 14q32.31 are inducing LUAC spread. Hence, we postulate that they are coordinately up-regulated as part of a genetic network that promotes tumor dissemination, pointing to these miRNAs as promising cancer biomarkers and targets for antitumor therapy.

P08.16 A Novel MALDI Imaging Technology for High-Speed Analysis of FFPE Tissues R. Casadonte*1, R. Longuespée2, M. Kriegsmann3, M. Becker4, D. Trede5, S.-O. Deininger4, M. Toma6, G. Baretton6, J. Kriegsmann1, 7 1 Proteopath GmbH, Trier, Deutschland, 2Molekularpathologie Trier, Trier, Deutschland, 3Institut für Pathologie, Universitätsklinikum, Heidelberg, Deutschland, 4Bruker Daltonik GmbH, Bremen, Deutschland, 5SCiLS GmbH, Bremen, Deutschland, 6Institut für Pathologie, Universitätsklinikum, Dresden, Deutschland, 7MVZ für Histologie, Zytologie und Molekulare Diagnostik, Trier, Deutschland

Background.  MALDI imaging is a label-free analytical technique for direct analysis of samples that revolutionized biological mass spectrometry, and especially proteomics, as ionized biomolecules maintain their spatial integrity. For large tissue sections and/or tissue microarrays (~4 cm2), MALDI imaging analysis at high spatial resolution was previously impaired by the prohibitively slow acquisition speed of existing platforms. In this study, we present the application of a novel MALDI instrument for the analysis of tryptic peptides from FFPE tissue sections. In addition to allowing acquisition speed of >20 pixel/second, the newly developed system allows quasi-square, discrete pixels and continuous stage movement, contributing to consistent data quality even at small pixel size. Methods.  FFPE kidney and lung tumor tissues were subjected to deparaffination and heat-induced epitope retrieval. Trypsin solution was sprayed on the tissue sections using an automatic spray instrument. After 1.5 h digestion, alpha-cyano-4-hydroxycinnamic acid matrix was deposited using the same sprayer device and the tissues were analyzed for peptides with an AutoflexSpeed MALDI TOF/TOF mass spectrometer and a novel rapifleX MALDI TissueTyper (Bruker Daltonik GmbH, Bremen, Germany). Ion images were visualized using flexImaging software at 50 µm spatial resolution. Results.  MALDI data generated from the two platforms (AutoflexSpeed and rapifleX TissueTyper) were evaluated and compared based on the acquisition speed, time, and maintenance of the tissue integrity. Analyses of multiple large tissue sections were analysed with the novel high-speed instrument, enabling extremely fast acquisition speeds (~50 pixel/sec). A single tissue section allowed a measurement of 160,694 spectra (pixels) at 50 µm pixel size with a total acquisition time of ~1.5 h. Similar manageable timeframe was used for the analysis of a lung cancer tissue microarray including 99 tissue cores, that generated 51,932 spectra data. Conclusions.  A novel MALDI imaging platform has been used for the direct analysis of large FFPE tissue sections with high-speed (up to 50 pixel/ sec), high resolution, and high throughput. The statistical analysis of spectral data from several hundred thousand pixels represents another challenge which we have addressed using new software architecture.

P08.17 Investigation of neutrophilic proteins/peptides in periprosthetic tissue by matrix-assisted laser desorption/ionization (MALDI) imaging R. Longuespée*1, R. Casadonte1, M. Kriegsmann2, M. Otto3, S. Gravius4, T. Randau4, S.-O. Deninger5 Molekular Pathologie Trier, Trier, Deutschland, 2University of Heidelberg, Institute of Pathology, Heidelberg, Deutschland, 3MVZ for Histology, Cytology and Molecular Diagnostics Trier, Trier, Deutschland, 4University Clinic of Bonn, Department of Orthopedics and Trauma Surgery, Bonn, Deutschland, 5Bruker Daltonik GmbH, Bremen, Deutschland 1

Background.  The diagnosis of infection in patients with arthritis and/or in joint prostheses requires interdisciplinary cooperation and the application of up-to-date methods. Histological evaluation of the synovial membrane is made by counting neutrophils per area of tissue, giving a highly

imprecise and unsatisfactory diagnosis. The aim of this study is to establish an automated MALDI imaging method for the detection of neutrophilic aggregates. Methods.  Periprosthetic formalin-fixed paraffin-embedded tissue samples containing a very high number of neutrophils from patients with proven infections were on-tissue digested with trypsin and sprayed with a matrix solution for MALDI analysis. MALDI spectra from regions with high number and lower number of neutrophils were statistically compared. In parallel, peptide digests were extracted from the tissue and analyzed by LC-MS/MS for the identification of peaks that were specifically mapped by MALDI imaging in the regions containing a high number of neutrophils. Results.  MALDI imaging allowed us to specifically localize 20 m/z peaks in the region containing a high number of neutrophils. The subsequent LC-MS/MS analyses allowed to retrieve the identification of proteins containing peptides with the same monoisotopic mass analyzed in the MALDI images. Among the identified proteins, some were known to be related to neutrophil biological activities. Myeloperoxidase, endoplamins, 60S ribosomal protein, tenascin, protein disulfide isomerase, 78 kDa glucose-regulated proteins are some of the candidate proteins that we found specifically located in neutrophil areas. Conclusions.  In the present study we compared joint tissue with high- and low neutrophilic counts. The different peptide patterns observed may be markers for a joint infection. In the next future, tissues from infected and non-infected patients will be analyzed by MALDI imaging and compared in order to determine the peaks that are specifically correlated to infection. A final step of the workflow will be the validation of the infection biomarkers by immunohistochemistry. MALDI imaging may then be used as a routine technique for the discrimination of infected and non-infected patients.

P08.18 A web-based Tool for Tissue Microarray (TMA) Evaluation and Data Storage V. Sailer*1, T. MacDonald1, J. Tang2, R. Kim1, 3, M. A. Rubin4 Weill Cornell Universität für Medizin, Englander Institut für Präzisionsmedizin, New York, Vereinigte Staaten von Amerika, 2Brigham and Women’s Hospital, Boston, Vereinigte Staaten von Amerika, 3Weill Medical College of Cornell University, New York, Vereinigte Staaten von Amerika, 4 Weill Medical College of Cornell University, Meyer Cancer Center, New York, Vereinigte Staaten von Amerika 1

Background.  TMA technology is indispensable for the validation of biomarkers in translational research. However, evaluating TMA slides by light microscopy is time-consuming and prone to errors. We have developed and updated an internet based data management platform (Profiler) using improved scanning technology to upload TMA scanned images, evaluate TMA histospots and archive and export annotated data. The robust Profiler system provides a wide array of features integrated into functional modules that can be adapted to serve the researcher’s need. Methods.  TMA slides are scanned and spot data extracted using the Aperio scanner and eslide-manager software (Leica Biosystems). The data is uploaded into the Profiler system and linked to the TMA punchfile, which contains distinct identifiers for each core. Working folders are assigned to authorized persons for managing different projects. Results.  The workflow from TMA glass slide to Profiler database results in a spot-by-spot interface ready for evaluation of H&E, immunohistochemistry or in situ hybridization. Evaluation data is automatically assigned to the appropriate case in the punchfile and can be exported. Data management of biobanking cohorts was validated in a large prostate cancer-screening cohort with prostatectomy specimen from 333 patients. Conclusions.  Profiler allows the pathologist to evaluate TMA slides using an internet compatible device. It eradicates any errors that stem from moving the TMA slide at the microscope and entering data into a spreadsheet manually. It can be adapted for a variety of different organ systems

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Abstracts

Fig. 1 I P08.18 8 and evaluation criteria. The system can also be used to store data including molecular data thus creating evolving and valuable datasets. Importantly, as a web-based application it is accessible from virtually anywhere thus enabling investigators to share selected data with collaborators. User registration provides a high level of security. Apart from the function as a TMA sample and data storage system Profiler can also manage biobank cohorts through a sample tracking database. To the best of our knowledge, Profiler is the most up to date and validated web based centralized sample and data management system available.

P08.19 Tie2 expression during tumor progression in a syngeneic orthotopic murine breast carcinoma model and human breast specimens S. von Stillfried*1, A. Rix2, S. Arns2, M. Bartneck3, C. Jonas2, F. Tacke3, R. Knüchel1, F. Kiessling2, W. Lederle2 RWTH Aachen, Institut für Pathologie, Aachen, Deutschland, 2RWTH Aachen, Experimentelle Molekulare Bildgebung, Aachen, Deutschland, 3 RWTH Aachen, Medizinische Klinik III, Aachen, Deutschland

1

Background.  Tumor angiogenesis is important for tumor progression and metastasis. The expression of the angiopoietin-receptor Tie2 is upregulated on endothelial cells at the invasive front of human breast carcinomas1. Additionally, Tie2 positive monocytes/macrophages have been identified as important players in tumor-associated inflammation, contributing to angiogenesis and metastasis. The aim of our study was to investigate the changes in Tie2 expression in an orthotopic mouse mammary carcinoma model and in human mammary tissue specimens in order to better understand the role of Tie2 for progression from primary tumor growth to metastasis. Methods.  Syngeneic metastatic mammary carcinomas were induced by injecting 4T1 tumor cells into the mammary fat pad of female BALB/c mice. Over a period of 5 weeks, endothelial Tie2 expression was monitored longitudinally in vivo with molecular ultrasound. To determine the link between tumor hypoxia and metastatic progression, tumor oxygenation was analyzed with photoacoustic measurements. Metastases were

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detected with longitudinal µCT imaging. In vivo data were validated and complemented by FACS and immunohistochemistry. To compare our data from the animal model with the situation in humans, Tie2 expression in human mammary carcinomas and precursor lesions was investigated using immunohistochemistry. Results.  In the murine breast carcinoma model, highest endothelial Tie2 levels were measured with molecular ultrasound imaging from day 10 to 18 after inoculation, shortly before lung metastases were detected in vivo starting at day 21, followed by a decline. FACS and immunohistochemistry revealed differential Tie2 expression on monocytes/macrophages during tumor progression. Photoacoustic measurements showed a decrease in tumor oxygenation with ongoing growth and progression. In human specimens of mammary tissue, we found a strong upregulation of Tie2 in invasive carcinoma in comparison to normal mammary tissue. Conclusions.  Our preclinical and clinical findings further substantiate the crucial role of Tie2 in breast tumor progression from localized disease to metastasis. Future studies will dissect the roles of Tie2 expressing monocytes/macrophages versus endothelial cells for tumor progression and metastasis. References 1. Peters KG et al (1998) Expression of Tie2/Tek in breast tumour vasculature provides a new marker for evaluation of tumour angiogenesis. Br. J. Cancer 77, 51–56.

P08.20 Comparative analyses on bronchuswall-thickness by histologic and computed tomographic measurements – an experimental study in porcine lungs V. Schmitt*1, C. Shmitt1, D. Hollemann2, O. Weinheimer3, W. Roth2, C. J. Kirkpatrick1, C. Brochhausen4, 5 REPAIR-Lab, Institut für Pathologie, Universitätsmedizin, Mainz, Deutschland, 2Institut für Pathologie, Universitätsmedizin, Mainz, Deutschland, 3Klinik für diagnostische und interventionelle Radiologie, Universitätsklinik Heidelberg, Heidelberg, Deutschland, 4REPAIR-Lab, Institut für Pathologie, Universität Regensburg, Regensburg, Deutschland, 5Zentrum für Rheumapathologie, Universitätsmedizin Mainz, Mainz, Deutschland 1

Background.  Histologic slides are commonly used as template in the evaluation and development of medical imaging methods. Diseases like Asthma and COPD show characteristic changes in airway morphology and airway measurement by computed tomography is a promising diagnostic approach. However, tissue shrinkage caused by fixation and histological processing is known in lung tissue. In the present study, the thickness of bronchus walls performed in paraffin and frozen sections as well as via CT and MicroCT were compared. Methods.  Airway measurements of swine lungs were performed after freezing in ventilated condition in liquid nitrogen by measuring the wall thickness of seven bronchi via CT and MicroCT as well as in frozen and paraffin sections. Statistical analysis was performed using Wilcoxon test with significance at p < 0.05. Results.  Airway measurement distinguished significantly between the groups. Bronchus wall thickness was smallest in frozen sections (median 0.71 mm) followed by paraffin sections (median 0.75 mm), MicroCT

(median 0.84 mm) and CT (median 1.69 mm). Statistical significance was given (p < 0.05). Conclusions.  Striking differences were found in airway measurements performed in frozen and paraffin sections, MicroCT and CT. The diversity between bronchial wall size measured is paraffin and frozen sections is important regarding the comparison of radiologic data with histologic measurements in the further development of medical imaging. Further studies are necessary to assess the best representative histological method for this purpose. Regarding radiology, CT has become a useful tool for airway imaging but MicroCT represented more adequate results of airway measurements in the present study and was superior compared to CT.

P08.21 First high-resolution synchrotron X-ray microtomograph of a biomaterial-induced multinucleated giant cell W. Wagner*1, 2, F. Barsch3, M. Benckendorff3, M. Evert1, S. J. Mentzer4, C. J. Kirkpatrick3, M. Ackermann2, C. Brochhausen1 1 REPAIR-Lab, Institut für Pathologie, Universität Regensburg, Regensburg, Deutschland, 2Institut für Funktionelle und Klinische Anatomie, Universitätsmedizin, Mainz, Deutschland, 3REPAIR-Lab, Institut für Pathologie, Universitätsmedizin, Mainz, Deutschland, 4Harvard Medical School, Laboratory for Adaptive and Regenerative Biology, Boston, Vereinigte Staaten von Amerika

Background.  Biomaterial-induced multinucleated giant cells are found at the implantation sites of many different biomaterials. However, their

Fig. 1 I P08.21 7 a) Reconstruction of a segmented biomaterial induced multinucleated giant cell (BIMGC) in porcine subcutaneous tissue. The synthetic biomaterial filaments (dark grey), as well as BIMGC (white) can be individually segmented from the original 3D dataset (b1). Crossvalidation with same specimen, semi-thin cut, stained with methylenblue. Scanning (c) and transmission (d) electron microscopy reveal organellecontaining fingerlike extensions of BIMGC into the blind endings of biomaterial (d2 high mag.) Der Pathologe Suppl 1 · 2016

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Abstracts three-dimensional (3D) extend and their orientation alongside the biomaterials, remain mostly unclear. The classical reconstruction of multiple serial sections involves the detriments of stereological approaches to volumetric morphological information. Methods.  Porcine tissue was analyzed 30 days after the subcutaneous implantation of a filamentous biomaterial. Specimens were formalin fixed, dehydrated and critical point dried according to standard protocols. The contrast was enhanced with osmium tetroxide. Synchrotron radiation x-ray tomographic microscopy (SRXTM) was performed at the TOMCAT Beamline at the Swiss Light Source, Villigen, Switzerland. Specimens were scanned at an energy of 18keV, a magnification of 20x and absorption contrast, resulting in a voxel size 0.325³µm³. BIMGCs have been identified based on their morphology, 3D-texture and spatial dependence to biomaterial filaments. Cross-validation was performed using conventional histology of semi-thin cut tissue samples stained with methylenblue. Results.  To our knowledge, in this study, we present the first X-ray tomographic characterization of BIMGC. We were able to show the entire cellular extensions alongside the biomaterial filaments (. Fig. 1). The morphological information was derived from rotational projection images, which can be reconstructed to true tomographic data. Even though the BIMGC surround the cylindrical filaments, they proved to be largely non-convex with a shape factor around 5.5. The volumes of the BIMGC were mostly below 60.000 µm³. These findings are in line with our histology-derived stereological estimates. SRXTM allows the non-destructive, volumetric morphological study of biological tissue on a true cellular level. Conclusions. The 3D morphometric information, combined with a sub-micron resolution, obtainable by SRXCT have a high impact on the characterization of BIMGC, and therefore pave the way for a better understanding of the foreign body response towards synthetic biomaterials, used in regenerative medicine.

dered an important anatomical variation with regard to operative tactic of surgical intervention. 3. Venous aneurysmatic malformation of the thoracic wall as rare cause of pulmonary embolism (66-year old man) as already native manifestation of an aneurysmatic alteration of a venous bundel at the proximal segment of the right brachial/axillary/subclavian vein. 4. Successful 2-side hybrid approach (image-guided/vascularsurgical intervention) because of paraneoplastic thrombosis of the superior vena cava caused by BroCa-induced compression (65-year old patient), in particular, open vascularsurgcial intervention with access via left jugular vein including open surgery comprising thrombectomy & transfemoral placement of a stent within the stenotic segment of the superior vena cava & left brachiocephalic vein). 5. In case of venous retention after previous creation of an av-fistula, Vena communicans can be ligated as a reliable and safe approach with limited efforts of open surgery. 6. Rare aneurysm of the popliteal vein as cause for recurrent episodes of pulmonary embolism (to be included as venous and aneurysmatic source for differential diagnosis) warrant a vascularsurgical approach. Conclusions.  The presented complex cases demonstrate impressively the challenge, which need competent interdisciplinary management in diagnostics, decision-making & therapeutic splitting not rarely planned as hybrid procedure and mostly performed within a center of vascular medicine.

P09.02 Visceral artery aneurysm(VAA) within the spectrum of vascularmedical diseases with need of image-guided and vascularsurgical intervention V. Scholtz, F. Meyer*, A. Udelnow, Z. Halloul

Postersitzung AG Herz-, Gefäß-, Nieren- und Transplantationspathologie P09.01 Various non-varicose venous diseases and their need of an image-guided approach or vascularsurgical intervention – a representative selection of cases with their challenging management F. Meyer*, Z. Halloul Klinik für Allgemein-, Viszeral- und Gefäßchirurgie, Universitätsklinikum, Magdeburg, Deutschland Background.  Venous diseases can vary diversely, in particular, those of non-varicose profile which need an image-guided or vascularsurgical approach. Aim.  Practice-relevant demonstration of case-specific characteristics, in particular, the pathoanatomical, degenerative, inflammatory & malignant profile of non-varicose venous diseases with need of any therapeutic approach. Methods.  Based on single cases, diagnostic and therapeutic management including follow-up aspects of such venous diseases is decribed. Results.  Specific cases: 1. 54-year old woman: Mid-term, relatively tumor-stable outcome (5 years & 6 months) of a leiomyosarcoma of the inferior vena cava with hepatic infiltration and pulmonary metastases as well as successful resection (segmental resection of the venacava inferior, liver resection, tumor thombectomy in hepatic veins). 2. CT-A in a 76-year old woman prior to open vascularsurgical intervention of an infrarenal aortic aneurysm revealed extraordinary mouth of the left renal vein into the left common iliac vein, which can be consi-

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Klinik für Allgemein-, Viszeral- und Gefäßchirurgie, Universitätsklinikum, Magdeburg, Deutschland Background.  VAA is a relatively rare disease & considered a challenge for the practicing vascular surgeon because of its size- & configuration-dependent potential of rupture, which requires an interdisciplinary therapeutic management. Methods.  Through a defined time period & using a consecutive patient cohort from a hospital case load of vascularmedical patients with diagnostic imaging of a VAA as part of a systematic single-center prospective observational study, spectrum (symptomatology, frequency) & diagnostics of the different VAA sites as well as the postinterventional course of the various therapeutic options used according to local finding & patient’s clinical status as well as risk factors were analyzed to contrast the different procedures (conservative, image-guided radiological intervention, open vascular surgery) in consideration of their decision-making criteria & their early postinterventional outcome. Results.  During a time period of 14 years, 22 patients (sex ratio: 12 males/10 females; mean age: 54.3 [range: 22–76] years) were registered. Most frequently, VAA occurred in the splenic artery (50 %). Gastroduodenal artery, hepatic artery & right renal artery were affected with 13.6 % (n = 3/22) each, superior mesenteric artery in 9.1 % (n = 2/22). The majority of patients (54.5 %) were treated with image-guided radiological intervention, whereas in 31.8 %, patient underwent open vascular surgery & 13.6 % of cases were managed with “watchful waiting”. While morbidity was 21.1 % (n = 4/19), overall lethality was 9.1 % (n = 2/22). Conclusions.  Decision-making for a specific therapeutic approach should be made i) after adequate diagnostic measures (ultrasound, MRA, Duplex ultrasonography, CTA/DSA if required) ii) on an individual case-adapted base, iii) in a vascular surgical centre, iv) case-associated to the specific local finding (in particular, according to size/specific probability of rupture [CAVE: gravidity]) & v) according to the individual risk profile using the whole spectrum of therapeutic options (conservative vs. interventional; image-guided radiological intervention [endovascular repair such as embolization, stent or stent graft] vs. open vascular surgery [according to a

step-up approach]; open vascular ligation vs. reconstruction after exclusion of the aneurysm) including sufficient quality assurance of the treatment results as well as control investigations (Duplex ultrasonography; MRA if required) in a specialized vascularsurgical out-patient center within appropriate time intervals.

P09.03 Macrophage infiltration quantified by digital approach shows high density in humoral renal transplant rejection A. Khalifa*, J. Schmitz, H.-H. Kreipe, F. Feuerhake, J. H. Bräsen Institut für Pathologie, Medizinische Hochschule Hannover, Hannover, Deutschland Background.  Macrophages are important mediators of kidney injury, homeostasis, inflammation and fibrosis. Their divergent roles at different locations and timepoints during injury and disease of the kidney are still poorly understood. In this study quantity and spatial localization of kidney macrophages were investigated in kidney biopsies (bx) taking advantage of a pixel based image analysis approach. Methods.  472 kidney biopsies (control and indicated biopsies after renal transplantation and indicated bx from native kidneys) were stained for CD68 (clone PGM1) with an automated routine stainer (Ventana Benchmark Ultra). Subsequently, whole slide images were scanned (20x, AT2, Leica Microsystems) and analyzed for immunopositively stained area (separately for cortex, medulla and extrarenal area) using a commercial image analysis platform (Definiens Tissue Studio). Results.  The mean patient age was 51 years (minimum 2 years, maximum 86 years). 72 % of the investigated bx came from renal transplants. Immunopositively stained area for CD68 was 2.7+/–4.5 % in cortex, 2.6+/–4.3 % in medulla and 2.1+/–3.3 % in extrarenal area including all studied cases. Correlation (Spearman) of values for CD68-positive area (%) was 0.76 (p < 0.001) between cortex and medulla, 0.63 between medulla and extrarenal tissue (p < 0.001) and 0.48 (p = 0.001) between cortex and extrarenal tissue. Obtained values (%) were in general higher in transplants than in native kidneys (cortex 2.8 vs. 0.97, p < 0.001; medulla 2.71 vs. 0.71, p < 0.001; extrarenal tissue 1.92 vs. 0.71, p < 0.001). Highest numbers of macrophages in transplanted kidneys were measured in humoral rejection vs. all other entities, including cellular rejection (cortex 5.91 vs. 2.96, p < 0.001; medulla 7.02 vs. 2.66, p < 0.001). In transplanted kidneys the area of CD68-positively stained tissue increased over time (<8 days vs. <90 days vs. <1 year vs. <1 year) in cortex and medulla (p < 0.05). Conclusions.  The data analysis shows that macrophages are important contributors to inflammatory infiltrates, particularly in transplanted kidneys culminating in humoral rejection. Their numbers increase over time and in atrophic/fibrotic areas. These findings confirm the importance of innate immunity in kidney tissue injury and rejection.

P09.04 Expression of Keratin 17 in renal injury indicates tubular stress and dedifferentiation R. D. Bülow*1, S. Djudjaj1, 2, P. Strnad3, J. Floege2, P. Boor1, 2 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, Abteilung für Nephrologie, RWTH Universität, Aachen, Deutschland, 3 Medizinische Klinik III und IZKF/RWTH Aachen, Aachen, Deutschland 1

2

Background.  Keratins (K), the cytoskeletal intermediate filaments of epithelial cells, are upregulated and display cytoprotective functions under stress situations. We have recently shown that the major renal keratins, i. e. K7, K8, K18 and K19 can serve as markers of renal tubular epithelial cell injury. Currently, no data on other keratins in kidneys exist. In particular, nothing is known on K17, which is described to have only lim-

ited expression under basal conditions, being induced under stress and directly mediating inflammation, e. g. in the skin. Here we comprehensively analyzed the expression of K17 in healthy and diseased murine and human kidneys. Methods.  Five murine models were analyzed for K17-expression by means of immunohistochemistry, immunofluorescence, qRT-PCR and Western blotting. Additionally, both human non-fibrotic and fibrotic renal tissue was investigated using immunohistochemistry and immunofluorescence. A tissue microarray representing major renal tumors was also stained for K17. Results.  K17 was not detected in healthy murine kidneys, but was de novo expressed exclusively in tubular epithelial cells of collecting ducts and distal tubules in all five models of primary or secondary tubular cell injury. Early in disease single tubular cells expressed K17, whereas later on groups of neighbouring cells and whole tubules were K17-positive. In glomerular injury models, K17 was also de novo expressed in a subset of parietal epithelial cells. In healthy human kidneys, K17 was expressed at the basolateral side of distal tubular cells. In injured kidneys, K17 was expressed circumferentially both in single cells and entire tubular cross-sections. No glomerular K17 positivity was found in healthy kidneys, whereas glomeruli with prominent periglomerular fibrosis showed de novo expression of K17 in parietal epithelial cells. In human fetal kidneys, circumferential K17 expression was found in entire tubular segments of ureteric bud. In renal tumors, K17 was only expressed in metanephric adenomas but not in renal cell carcinomas or oncocytomas. Conclusions.  We show that K17 is de novo expressed in injured renal epithelial cells and lends itself as a more sensitive marker of tubular cell stress compared to more commonly expressed renal keratins. Given the resemblance to the embryonic pattern of expression, K17 might indicate dedifferentiation of renal tubular epithelial cells in response to injury.

Postersitzung AG Kinder- und Fetalpathologie P10.01 OEIS complex – associated malformations in 12 cases M. R. Mallmann1, H. Reutter2, U. Gembruch1, A. M. Müller*3 Abteilung für Geburtshilfe und Pränatalmedizin, Universitätsklinik, Bonn, Deutschland, 2Institut für Humangenetik, Universitätsklinik, Bonn, Deutschland, 3Zentrum für Kinderpathologie und Pathologie, MVZ Uniklinik, Bonn, Deutschland

1

Background.  OEIS complex refers to a combination of defects consisting of omphalocele, exstrophy of the cloaca, imperforate anus, and spinal defects. It is a rare variant of the bladder-exstrophy–epispadias complex. Its etiology is still debated. The complete spectrum of associated malformations is unknown due to the rarity of disease and the fact that large series describing the spectrum of disease or additional malformations are rare. Due to the variety of urogenital and spinal malformations diagnosis of the OEIS complex is challenging. Methods.  The morphological findings, found prenatally and by autopsy in a series of 12 OEIS cases were analysed. Results.  All fetuses showed exstrophy of the bladder, 9/12 an omphalocele, 9/12 anal atresia, 10/12 neural tube defects, 4/12 vertebral defects, 5/12 lower extremity defects including clubfeet and 4/12 a single uterine artery. Additional malformations included, hydrocephalus, hypertelorism, aplasia of the gall bladder, heart defects and kidney malformations. All 11 fetuses with chromosomal analysis showed a normal karyotype. Conclusions.  The study adds cases with associated malformations, helping to make the OEIS diagnosis even when there are additional findings which might distract from this diagnosis and explores further the broad spectrum of this disease. Der Pathologe Suppl 1 · 2016

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Abstracts P10.02 Asymmetric omphalopagus; a rare case of conjoined twinning: a case report S. Jabari*1, M. Besendörfer2, R. Carbon2, A. Hartmann3, S. Söder3 FAU Erlangen-Nürnberg, Institut für Anatomie I, Erlangen, Deutschland, Kinder- und Jugendklinik, Friedrich-Alexander-Universität ErlangenNürnberg, Erlangen, Deutschland, 3FAU Erlangen-Nürnberg, Institut für Pathologie, Erlangen, Deutschland 1

2

Background.  Asymmetric omphalopagus is a rare situation of conjoined twinning, in which a grossly defective twin (the parasite) is attached to the thorax and upper abdomen of the main twin (the autosite). We describe a case of an omphalopagus and tried to analyze the inner and outer aspects of the twins, in order to further characterize and describe the asymmetric twinning. Methods.  Pre- as well as postoperative diagnostic imaging was carried out and analyzed followed by an autopsy to evaluate the outer and inner aspects of the parasite. Furthermore conventional histological examination of the organ systems found accompanied by immunhistochemical staining was performed. Results.  The parasite had well developed lower extremities and pelvis as well as upper extremities with a cleft hand syndrome and syndactyly. From the outer aspect, the sex was non-determinable and we couldn’t find any testis or ovaries. There was no sign of any axial skeleton or central nervous system. We found a rudimentary rectum with a non-pervious anus, a kidney, ureter, urinary bladder and a blind ending urethra. The blood supply of the parasite was connected to the vessel system of the autosite, which on the other side had an associated omphalocele and an atretic duodenum. Conclusions.  To our knowledge, only 50 cases of parasitic omphalopagus have been described to date, most of the parasites, as in our case, were successfully separated from the autosite.

P10.03 Evaluation of CD163+ Hofbauer cells in the placenta of patients with HELLP syndrome F. Gründahl*1, K. Hussein2 St. Franziskus Hospital, Department of Gynaecology and Obstetrics, Münster, Deutschland, 2Institut für Pathologie, Medizinische Hochschule Hannover, Hannover, Deutschland 1

Background.  During pregnancy the maternal immune system is in a state of tolerance of the semiallogenetic fetus. Immunmodulatory cells are regulatory T cells and histiocytes in the decidua and the villous Hofbauer cells (HBC). HBCs express CD163, which indicate a M2 stage with regulatory and immunosuppressive functions. Preeclampsia (PE) and HELLP syndrome are a result of decidual and immune cell dysfunctions which lead to a failure of remodeling the spiral arteries, causing higher residence, maternal hypertension and other complications. PE and HELLP ususally lead to unspecific changes in the placenta but there are contrasting reports on HBC, either a decrease or an increase of HBC in PE/HELLP. Therefore, we performed a characterization of the immunophenotype, which indicates the functional stage of HBC (general marker CD68, M2 marker CD163, reticulum cell marker CD103, dendritic cell marker CD11c). Furthermore, we quantified the number of HBC and evaluated the localisation within the villi. The aim was to verify if evaluation of HBC could be an additional tool for placenta diagnostics of PE and HELLP. Methods.  Formalin-fixed and paraffin-embedded tissue samples from placentas with and without HELLP syndrom cases were analysed with immunohistochemistry (n total = 40; 2. and 3. trimenon). Positive cells were counter in 4 representative high powever fields of a representative placenta tissue section.

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Results.  In general, HBC were mainly CD163+ and partially CD68+ but negativ for CD103 and CD11c. We found no significant difference between PE/HELLP and control placentas regarding the number or the immunphenotype of HBC or decidual cells. Conclusions.  In genral, our analyses showed that CD163 immunostaing can be used to visualize HBC more accurate than CD68. PE/HELLP is not associated with a significant quantitative change of placental HBC and HBC remain mainly in a M2 stage of differentiation. Therefore, no PE/ HELLP-specific placental changes could be verified.

P10.04 Gonadal anomalies in the fetus K. Schoner*1, H. Barth2, B. Arabin3, R. Axt-Fliedner4, S. Schweighoefer5, W. Coerdt6, I. Kennerknecht7, H. Rehder1, 8 1 Universitätsklinikum Gießen und Marburg, Institut für Pathologie, Fetalpathologie, Marburg, Deutschland, 2Universitätsklinikum Gießen und Marburg, Standort Marburg, Zentrum für Kinder- und Jugendmedizin, Neonatologie, Kinderkardiologie, Marburg, Deutschland, 3 Universitätsklinikum Gießen und Marburg, Frauenklinik, Pränatale Diagnostik und Therapie, Marburg, Deutschland, 4Universitätsklinikum Gießen und Marburg, Frauenklinik, Pränatale Diagnostik und Therapie, Gießen, Deutschland, 5Praxis für Frauenheilkunde und Geburtshilfe Baier, Plassmann, Praxis für Praenatalmedizin, Dortmund, Deutschland, 6Johannes Gutenberg-Universität Mainz, Institut für Pathologie, Kinderpathologie, Mainz, Deutschland, 7Westfälische Wilhelms-Universität Münster, Institut für Humangenetik, Münster, Deutschland, 8Medizinische Universität Wien, Institut für Medizinische Genetik, Wien, Österreich

Background.  Gonadal anomalies are rare and rather detected if there are associated malformations or syndromal disorders. Thus we know e. g. by quantitative studies in chromosomal syndromes (Coerdt et al. 1983) that reduction of germ cells is present already in fetal testes of trisomy 21, 13 and 18 at midterm of pregnancy. We have seen mostly unilateral string gonads in association with urogenital malformations. Reduction of ovarian follicles may occur in fetuses with XY-gonadal dysgenesis such as in Campomelic dysplasia or Frasier syndrome. An increasing number of observations of agonadism and gonadal anomalies in the same sib ship indicates that a-/hypogonadism syndromes are not always distinct entities but might manifest a phenotypic spectrum of the same disorder. Methods.  Autopsies including radiological, histological and photographic documentation were performed. The cases were selected from a cohort of almost 800 fetal autopsies. Here we report on three fetuses showing distinct and syndromic gonadal malformations. They concern a-/hypogonadism in a fetus with PAGOD-, with Casamassima-Morton-Nance-(CMNS), and with Splenogonadal fusion limb defect syndrome (SGFLD). Results.  In the female fetus with PAGOD-syndrome the characteristic features comprising pulmonary hypoplasia/hypoplasia of the pulmonary artery, unilateral a-/hypogonadism resp., omphalocele/diapragmatic defect and dextrocardia associated with complex cardiac malformations were confirmed. The female fetus with CMNS showed costovertebral dysplasias – as the main feature – in association with anal and renal anomalies. In the male fetus with SGFLD we detected fused spleen and left gonad, tetramelic ectro-/hemi-/amelia and severe micrognathia. Our cases represent rare syndromes and are so far the 12th, 5th and 11th observation, resp. Conclusions.  The presented syndromes show a spectrum of gonadal anomalies with associated overlapping features. The continuum in the phenotypic spectrum of gonadal anomalies ranges from ’pure’ agonadism in the PAGOD to streak- and fused gonads in the other syndromes. Interestingly, in contrast to the 11 cases of PAGOD syndrome reported so far, our case had unilateral remains of gonads. The overlapping features in all three syndromes might indicate a common pathogenetic pathway. Molecular genetic studies are still in progress. In all three cases the gonadal anomalies were not detected by prenatal ultrasound scan. Detailed investigation by the fetal pathologist may thus enabling clear syndromic assignment.

P10.05 The embryo-placental CD15-positive „vasculogenic zones“ as a source of propranolol-sensitive pediatric vascular tumors L. Seidmann*, P. Stenzel, W. Roth Institut für Pathologie, Universitätsmedizin, Mainz, Deutschland Background.  Propranolol-induced involution is a unique biological feature of some pediatric vascular tumors, for instance infantile haemangioma (IH), cerebral cavernoma or chorioangioma. We tested the hypothesis that propranolol-responsive pediatric vascular tumors are derived from common vessel-forming CD15+ progenitor cells which occur in an early stage of embryo-placental vascular development. The aim of this study was to identify the tumor-relevant CD15+ progenitor cells at the early stages of normal embryo-placental development. Methods.  Human embryo-placental units of 4–8 weeks gestation and pediatric vascular tumors were tested for expression of the tumor-relevant markers CD15, CD31 and CD34. Results.  Vessels of physiologically immature early placentas (4–8 weeks of gestation) were characterized by immunostaining for CD15, CD31, and CD34. In embryonic tissue, a discontinuous CD15+/CD31+/CD34+ cell population was detected in immature vessels of the skin, neural tube, spinal and cerebral meninges. Similarly, vessels in infantile haemangioma and chorioangioma exhibited a co-expression of CD15, CD31, and CD34. In contrast, the majority of embryonic and umbilical vessels presented a CD31+/CD34+, but CD15-negative immunophenotypic pattern. Conclusions.  Our results suggest the existence of a CD15+ “vasculogenic zones” in the normal embryo-placental unit as well as in IH and chorioangioma. A site-specific correlation between normal embryo-placental and tumoral vessel-forming CD15+ progenitors was demonstrated. Hence, normal vessel-forming CD15+ progenitors could be considered as propronalol-sensitive targets and potential source of pre- and postnatal vascular tumors.

P10.06 Tufting enteropathy (intestinal epithelial dysplasia); a notable differential diagnosis among rare cases of pediatric congenital enteropathies S. Boral*1, W. Luck2, K. Hauptmann1 Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland, Klinik für Pädiatrie, Charité – Universitätsmedizin, Berlin, Deutschland

1

2

Background.  Congenital enteropathies are uncommon disorders presenting with early-onset intractable diarrheas and malabsorption, and resulting in failure to thrive. Having significant clinical consequences, early and accurate diagnosis is crucial to direct clinical treatment and prevent severe complications. With increase in knowledge on their genetic origin the number of these diseases have increased contributing to a broad differential diagnosis. Tufting enteropathy (TE) is one of the rare congenital enteropathies and should be excluded in any case of intractable diarrheas. Methods.  2 cases of patients with intractable diarrheas and malabsorption will be presented. H&E stained duodenal sections were assessed for abnormalities, particularly for the presence of villous blunting/atrophy, crypt hyperplasia, intraepithelial lymphocytosis, lamina propria inflammatory cell infiltrates and surface enterocyte tufts. EpCAM antibody staining was performed. One case of an infant without any anamnestic or histomorphological features served as control for EpCAM staining. The findings were correlated with ultrastructural findings (electron microscopy). Information regarding clinical presentation/outcome was gathered from treating gastroenterologists. Results.  Clinical presentation: The patients were both males and 5 (A) and 3 (B) months old at the time of the initial duodenal biopsie and normotroph at birth. They presented with early-onset untractable diarrhea and failure to thrive.

H&E morphology: biospsie of patient A = partial villous blunting, focal subtle enterocyte tufts, preserved brush border and physiological inflammatory changes. No intraepithelial lymphocytosis (IEL). initial biopsy of patient B = subtotal villous atrophy, inflammatory cell infiltrates with lymphocytes, histiocytes, eosinophiles in the lamina propria, no IEL. IHC: Complete loss of membranous EpCAM expression on duodenal epithelial cells in both cases. Electron microscopy (patient B): nonspecific ultrastructural findings. Conclusions.  Congenital enteropathies are rare diseases and still poorly understood. Their pathologic features may be subtle, focal, or inapparent on routine stains. A definitive diagnosis based on morphologic assessment of biopsies is often very difficult or impossible. The consideration of TE as a differential diagnosis is decisive in patients meeting specific age/clinical criteria for rare congenital enteropathies. The use of specific IHC staining may be essential for the determination of the correct diagnosis.

P10.07 An 18 months old girl with recurring strokes due to fibromuscular dysplasia P. Stenzel*, L. Seidmann, W. Roth Institut für Pathologie, Universitätsmedizin, Mainz, Deutschland Background.  Fibromuscular dysplasia (FMD) is an idiopathic non-atherosclerotic and non-inflammatory disease of the arteries. Accurately labelling pediatric stroke patients as ’fibromuscular dysplasia’ based purely on clinical-radiological features is not possible. Histopathological examination remains the only means by which FMD can be confirmed. Methods.  We report the case of an 18 months old girl with a past medical history of several strokes. MR-angiography revealed highly altered internal carotid arteries with long-segment beaded stenosis and short-segment stenosis of the vertebral and hepatic artery. FMD was clinically suspected and amongst others STA-MCA-bypass surgical procedure was carried out. The patient exhibited progressive therapy-refractory strokes and died of multi-organ failure. Results.  Postmortal examination showed multifocal arteriopathy with socalled “fatty streaks”, fibroplasia of arterial intima and media with irregular proliferation of smooth muscle cells and destruction of the internal elastic lamina and multiple micro-aneurysms with incipient dissection of the vascular wall. The arteriopathy was predominantly localized in the basilar artery, internal and external carotid artery, aorta, renal artery and hepatic artery. There was no inflammation or calcification of the arteries. Neuropathological examination of the brain showed strokes in different stadiums. Conclusions.  Recurring strokes in pediatric patients are very rare. The prevalence of FMD associated strokes in the literature varies between < 1 % up to 7 %. In this case FMD was clinically suspected, but could only be confirmed post mortem by histopathological examination. Especially the intimal affection is typical for young children, whereas other entities (medial, perimedial or adventitial FMD) appear more often in adults. In cases of clinically suspected pediatric stenosing arteriopathy inflammatory, neoplastic, metabolic, dysplastic or genetic etiologies must be differential diagnostically considered. Fibromuscular dysplasia is an important morphological differential diagnosis in pediatric patients with recurring strokes.

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Abstracts P10.08 Schistosomiasis of the urinary bladder in a 12 year old boy E. Gradhand*1, H. Rees2, M. Woodward3, S. Planas1 Histology Department, Pädiatrische und Perinatalpathologie, Bristol, Vereinigtes Königreich, 2Bristol Children’s Hospital, Pädiatrische Onkologie, Bristol, Vereinigtes Königreich, 3Bristol Children’s Hospital, Pädiatrische Urologie, Bristol, Vereinigtes Königreich 1

Background.  A twelve year old boy residing in UK was referred from a small hospital for investigation of a bladder mass. The boy had a five week history of penile pain on micturation and one week history of haematuria. In the previous 3 years he was vacationing abroad for periods of time, and he swam in Lake Malawi on two occasions. The bladder ultrasound and MRI showed abnormal wall bladder thickening with a localised mass and enlarged lymph nodes. The boy underwent cystoscopy and bladder biopsies were taken. Methods.  We received four biopsies; each measuring 2 × 1 × 1 mm. Routine H&E stains were performed. Results.  The microscopic examination revealed bladder biopsies which were covered by transitional epithelium. The subepithelial tissue showed lymphocytes, eosinophils and abundant granulomas containing calcified Schistosoma eggs with a typical spike. The transitional epithelium showed no squamous metaplasia or an intraepithelial neoplasia. There was no evidence of a botryoid embryonal rhabdomyosarcoma or any other paediatric malignancy. The diagnosis was that of a Schistosomiasis of the urinary bladder. The histological diagnosis was as well confirmed by serology. The boy was treated with praziquantel and he will be under surveillance. Conclusions.  Bladder Schistosomiasis is still a rare differential diagnosis in paediatric bladder tumours in Western Europe. However, this entity will potentially occur more often with the increase of travelling and patients with a migration history from countries in which Schistosomiasis is endemic. The Schistosoma eggs are relatively easy to recognise on a routine H&E stain, but one has to know their typical morphology. In countries where Schistosomiasis is endemic, such as Egypt, symptomatic children are usually treated empirically with praziquantel without histological confirmation. Untreated, Schistosomiasis can result in hydronephrosis and renal failure or in Schistosoma associated bladder cancer.

P10.09 VACTER association with tracheal agenesis A. M. Müller*1, B. Wiebe2 Zentrum für Kinderpathologie und Pathologie, MVZ Uniklinik, Bonn, Deutschland, 2Asklepios Klinik, Abteilung Neonatologie/Päd. Intensivmedizin, St. Augustin, Deutschland 1

Background.  The diagnosis VACTER (vertebral defects, anal atresia, tracheoesophageal fistula with esophageal atresia, radial and renal dysplasia) is made when at least three of the defects are present. Although esophageal atresia is a key sign for the diagnosis VACTER with patent oesophagus but tracheal atresia is not published yet. Methods.  Case: Delivery of a male newborn at 34 weeks of gestation with prenatal diagnosis of d-TGA, hydronephrosis, left-renal agenesis, ovarial cyst and ascites. At birth presentation with severe respiratory distress syndrome. Intubations failed but tracheotomy seemed to result in lung ventilation. As no sufficient oxygenation could be reached and with view of the numerous malformations parents were counseled regarding the poor outcome and decided for withdrawal at day 2 of life. Results.  Post mortem findings: Tracheal atresia Typ II (Floyd’s classification). Patent esophagus without obvious fistula from the oesophagus resp stomach to the bronchial bifurcation. Histologically ventilated lungs. Vitium cordis (large VSD, pulmonary atresia). Sinistral renal aplasia, anal atre-

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sia, cloacal malformation. Radiology: No os sacrum. Wedged vertebrae. Horizontal vertebral clefts. Normal radius. Conclusions.  Synopsis of clinical and post mortem findings allowed the diagnosis of a VACTER-association with the up to now not reported tracheal agenesis with patent eosophagus, only detected on postmortem. The inflated lungs support the strong theory of a fistula between distal esophagus resp. stomach and trachea, too thin to be found on preparation (as not longer airfilled). Hence this case emphasizes the importance not only of profound clinical informations but of a detailed analysis and adopted techniques of this region as soon as this differential diagnosis is discussed.

P10.10 Autopsy of a 18 month old, male toddler with measles infection and cadiomyopathy I. Klempert*, K. Hauptmann, M. Dietel Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland Background.  Measles are a highly contagious viral disease, with serious or even fatal consequences for infants, children, juveniles as well as adults. We investigated the case of a male, 18 month old, unvaccinated Toddler, who was hospitalized because of fever and loss of consciousness. During the inpatient stay a cardiomyopthy was diagnose and an exanthema, typically for a measles infection, evolved. Extensive laboratory tests revealed measles specific IgM antibodies, due to a recent measles infection. According to medical history, an infection with an enterovirus and parvovirus B 19 3 and 2 month prior were known. The boy died after 5 days in hospital. Methods.  A complete clinicopathological autopsy was performed, including histological, immunohistochemical and molecular pathological processing. Additionally nucleic acids isolated from spleen tissue were genotyped at the Referenzzentrum MMR. Results.  After clinicopathological autopsy cause of death of the 18 month old, male toddler was a combination of a cerebral edema and an acute (global) decompensation of the by an inflammatory cardiomyopathy impaired, hypertrophied heart. The measles infection caused the cerebral edema and also enhanced the decompensation of the already impaired heart and thereby influenced the time of death. The inflammatory cardiomyopathy suggests a not recent, previously existing process, which led to the myocardial changes. Most likely the parvovirus B 19 infection is causative for these changes. The genotyping analysis resulted in the variant “D8 Rostov on Don”. Conclusions.  The boy would have benefited from vaccination – despite the known heart disease and the recurrent infections –, but also from a passive immunization (“Herdimmunität”). Parents should be inforemd more detailed about teh benefit of vaccination of children with preexisting or recurrent diseases.

Postersitzung AG Kopf-Hals-Pathologie P11.01 Analyses of ERCC1 expression and other biomarkers in salivary gland carcinomas G. Keller*, K. Hussein Institut für Pathologie, Medizinische Hochschule Hannover, Hannover, Deutschland Background.  High expression of excision repair cross-complementation group 1 (ERCC1) in adenocarcinomas of the lung is associated with treatment failure after platinum-based chemotherapy. Among head and neck cancers, squamous carcinomas have been evaluated for ERCC1 but, until

now, salivary gland carcinomas (SGC) have not been analysed. Furthermore, there is currently no established algorithm for the molecular profiling of therapy-relevant defects in SGC. Therefore, we established receptor and cell signalling profiles of therapy-relevant factors and propose a molecular diagnostic algorithm for SGC. Methods.  Formalin-fixed and paraffin-embedded tissue samples from SGC (n = 38) were analysed with immunohistochemistry (ERCC1 protein expression was verified by qPCR) and flourescence in situ hybridization (FISH). Results.  ERCC1: Nuclear ERCC1 expression was detectable in 15/38 carcinomas (9/15 adenoid cystic carcinomas) and in all cases the intensity of protein expression was weak (1+); none of the SGC showed overexpression. Corresponding to weak protein expression, low levels of transcript expression was found. The ERCC1-encoding chromosomal segment 19q13 was not amlified or deleted. Receptor and cell signalling profils: Two or more expressed receptors and/or receptor gene amplification were detectable in 8/38 tumours: HER2 3+/AR 1+, HER3 gene amplification/AR 1+/EGFR 1+, ER 3+/AR 1+, EGFR 2+/PR 1+ and EGFR 2+/PR 1+/AR 1+. No FGFR1-3, MET, ALK1, ROS1, RET, BRAF or VEGFA defects were detectable. Conclusions.  Personalized therapy of patients with SGC should include HER2 and EGFR signalling testing and, in negative cases, evaluation of rare potential target molecules. ERCC1 is not a reliable markers for the decision for or against chemotherapy in SGC.

P11.02 Retrospective Analysis of BRAF Status in Papillary Thyroid Carcinoma S. Scheil-Bertram*1, M. Demes2, S. Saalabian3, J. Schirren4, O. Kollmar4, A. Fisseler-Eckhoff1 HELIOS Dr. Horst Schmidt Kliniken Wiesbaden, Institut für Pathologie und Zytologie, Wiesbaden, Deutschland, 2Gemeinschaftspraxis für Pathologie Wiesbaden, Molekularpathologie, Wiesbaden, Deutschland, 3 DKD HELIOS Kliniken Wiesbaden, Endokrine Chirurgie für Schilddrüse und Nebenschilddrüse, Wiesbaden, Deutschland, 4HELIOS Dr. Horst Schmidt Kliniken Wiesbaden, Klinik für Thoraxchirurgie, Wiesbaden, Deutschland 1

Background.  Papillary thyroid carcinoma (PTC) is the most common thyroid malignoma. The majority of PTCs demonstrate a transversion from T to A at nucleotid 1799, leading to a valine to glutamic acid change in codon 600 (up to 90 %). The prevalence of other genetic abnormalities like RET rearrangements is much lower (5–30 %). The prognostic relevance of a BRAF mutation in codon 600 is still discussed controversely. In the current study, we analyzed BRAF mutations of PTCs retrospectively. Methods.  We analyzed 52 PTC samples (86 % papillary and its subtypes: 8 % follicular, 2 % tall cell, 4 % oncocytic; gender: 18 male, 34 female; median age at diagnosis: 45.5 years, range 16 to 78 years) by conventional staining methods. Using SNaPshot and Sanger sequencing or pyrosequencing technique we analyzed the BRAF status of codon 600 (n = 52), and codon 464, 466 or 469 (n = 42). TNM classification, margins, tumor size, multifocality, lymphovascular invasion, calicification and tumor caspule were evaluated. In 35 cases 413 lymph nodes were prepared (N-: mean of lymph nodes: 4.3 +/– 5.7; N+: mean of lymph nodes: 19.6 +/– 13.7). Results.  None of the tumors demonstrated a BRAF mutation in codon 464, 466 or 469. Nearly 50 % of cases were microcarcinomas (pT1a; median size 0.6 +/– 0.2 cm). 79 % of all PTCs and 81 % of pT1a/pT1b tumors demonstrated BRAFV600E mutation (BRAFmut). Additional activating mutations in V600, such as V600K and V600D mutations are not found. BRAFmut was found more frequently in women (82 %, 28/34; median age at diagnosis 46 years) than in male patients with PTCs (72 %, 13/18; median age at diagnosis 41 years). 89 % of PTCs with capsule were BRAF mutated (9 of these were bilateral). All PTCs with calcification demonstrated a BRAFV600E mutation (n = 12). We did not find a correlation of lateral cervical lymphnode infiltration and BRAF status (N-: 21/22 BRAFmut, N+: 10/14 BRAFmut).

Conclusions.  In the present retrospective study, we confirmed that BRAFmut is more frequent in women than in men. BRAFmut was found in all PTCs with calcification. In the future, it will be important to determine if calcification in PTC might predict the BRAFmut status.

P11.03 If incidential diagnosis-finding of a papillary microcarcinoma requires completion lymphadenectomy: the tall-cell variant of this malignant tumor lesion I. Bartella*1, F. Meyer1, K. Frauenschläger2, K. Reschke3, T. Wallbaum4, B. Buth1, C. Bruns1, C. Chiapponi1 1 Klinik für Allgemein-, Viszeral- und Gefäßchirurgie, Universitätsklinikum, Magdeburg, Deutschland, 2Institut für Pathologie, Universitätsklinikum, Otto-von-Guericke-Universität, Magdeburg, Deutschland, 3 Universitätsklinikum Magdeburg A. ö. R., Klinik für Nieren- und Hochdruckkrankheiten, Diabetologie und Endokrinologie, Magdeburg, Deutschland, 4Universitätsklinikum Magdeburg A. ö. R., Klinik für Radiologie und Nuklearmedizin, Magdeburg, Deutschland

Background.  Papillary thyroid carcinoma is the most common neoplasia of the thyroid gland. It is usually associated with a very good prognosis. Methods.  Report of a rare and extraordinary case based on selected scientific references, own clinical experiences and interdisciplinary management. Results.  A 46-year-old patient’s thyroid had a sonographic volume of approximately 80 ml and the right thyroid lobe was more enlarged than the left. Scintigraphy showed a cold nodule of 4 cm in size, filling the complete right thyroid lobe. Because of intraoperatively detected multiple small hard nodules in the left thyroid lobe, a complete thyroidectomy was performed. The histopathologic examination revealed a follicular adenoma without any malignancy in the right thyroid lobe and a “tall-cell” variant of a papillary thyroid microcarcinoma with capsular invasion, parathyorideal infiltration of the connecting tissue and a diameter of 0.6 cm in the left lobe. The “tall-cell” variant of the papillary thyroid carcinoma is a rare subtype and it is known for its aggressive oncobiologic behaviour as well as the high risk for lymphogenic metastases. For this reason, a lymphadenectomy of the central compartments was performed. This revealed 30 tumour-free lymph nodes. The postoperative course was uneventful and was followed by initiation of a radioiodine therapy. Conclusions.  The tall-cell variant is a rare and more aggressive subtype of the papillary thyroid carcinoma, which is characterised by a worse 5-year-survival and a higher recurrence rate. This diagnosis requires a more aggressive surgical treatment and a continuous follow-up. The aim of this case report is the presentation of the course of a rare tumour entity.

Postersitzung AG Zytopathologie P12.01 An enzymatic antigen retrieval method for Ep-CAM immunocytochemistry on air-dried, May-Grünwald-Giemsa-stained smears of serous effusions B. E. Silva de Araujo, S. Biesterfeld, N. Pomjanski, B. Buckstegge, I. Esposito, M. Schramm* Institut für Pathologie, Funktionsbereich Cytopathologie, Heinrich Heine Universität, Düsseldorf, Deutschland Background.  Diagnostic immunocytochemistry on unstained, air-dried smears is commonly used. However, in cases with sparse material, all Der Pathologe Suppl 1 · 2016

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Abstracts smears are stained according to May-Grünwald-Giemsa for cytological diagnosis. In this case, subsequent immunocytochemistry is not possible with current methodology. To overcome this limitation, we analysed the effects of treatment with proteinase K as the antigen retrieval method for the immunocytochemical detection of Ep-CAM with the monoclonal antibodies Ber-EP4 and MOC-31 on air-dried, May-Grünwald-Giemsastained smears of serous effusions. Methods.  Residual material of 100 malignant and 100 cellular reactive serous effusions, sent to the department of Cytopathology from November 2014 to June 2015 was included in this retrospective case/control study. New air-dried smears, stained according to May-Grünwald-Giemsa were prepared. The number of relevant cells was estimated semi-quantitatively under the microscope. After removal of the coverslip the smears were incubated with proteinase K at room temperature for 10 minutes. Then, immunocytochemistry was performed according to the avidin-biotin-complex method with Ber-EP4 and MOC-31. The expression of Ep-CAM was compared with the cytological diagnosis. Results.  Sensitivity and specificity for the detection of malignant epithelial cells with Ber-EP4 immunocytochemistry was 96 % and 99 %, respectively. Using MOC-31, the sensitivity was 95 % and the specificity was 99 %. Conclusions.  Introduction of proteinase K pre-treatment in the immunocytochemical procedure is suitable for the detection of malignant epithelial cells on air-dried smears of serous effusions, stained according to May-Grünwald-Giemsa. The diagnostic accuracy is similar to the published evidence on alcohol-fixed smears, stained according to Papanicolaou. This possibly enables the application of immunocytochemistry on limited samples with few, air-dried smears submitted, when pre-staining with May-Grünwald-Giemsa of all available smears is necessary for the cytological diagnosis.

P12.02 Diagnostic value of cytology in ophthalomogy – a clinical follow-up study S. Sarim1, M. Schramm1, N. Pomjanski1, R. Guthoff2, S. Biesterfeld*1, 3 Heinrich Heine-Universität, Schwerpunkt Cytopathologie, Düsseldorf, Deutschland, 2Heinrich Heine-Universität, Augenklinik, Düsseldorf, Deutschland, 3Institut für Pathologie, Koblenz, Deutschland

1

Background.  In this study, the diagnostic value of brush cytology and fine needle aspiration cytology from 191 consecutive ophthalmologic patients, diagnosed between 2002 and 2014, was evaluated by analysing the clinical and histological follow-up. Methods.  The majority of the 191 specimens were brush biopsies from the cornea or the conjunctiva, respectively. Some specimens were prepared from cystic processes, and a few number resulted from fine needle biopsy of the vitreous body. The original cytologic smears had been stained according to Papanicolaou. In 38 cases, adjuvant methods (DNA-cytomety or immunocytochemistry) had been applied. Clinical follow-up data was collected from the original files of the patients and using a questionnaire. Additionally, results from histology were considered. Results.  52 % of the patients were male, 48 % were female; the mean age at diagnosis was 50 years. Clinically, the investigations were made due to inflammation (n = 68, 36 %), pigmented lesions (n = 58, 30 %), squamous epithelial lesions (n = 42, 22 %), ulcer (n = 14, 7 %) or degenerative changes (n = 9, 5 %), respectively. Cytologically, 167 specimens were classified as benign (87 %), 17 as suspicious (9 %) and 7 seven as malignant (4 %). If a lesion had been clinically classified as benign, malignancy was found only very rarely (1 %). On the other hand, clinically suspicious lesions were also benign in the majority of cases (~ 80 %). Histology was performed in 47 cases (24.6 %). All seven diagnoses of malignancy were confirmed. 28 lesions were diagnosed as benign, 12 as suspicious or dysplastic, respectively. Conclusions.  The application of cytology in ophthalmology is a useful diagnostic tool especially if squamous epithelial lesions of the cornea or the conjunctiva have been diagnosed. With the exception of vitreous body aspiration,

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cytology in ophthalmology represents a non-invasive method that is complication-free and can be performed easily also under outpatient conditions.

P12.03 Fluorescent in situ hybridization in endoscopic ultrasound-guided fine needle aspiration cytology of the pancreatobiliary system A. Heywinkel, S. Biesterfeld, N. Pomjanski, M. Kazimirek, I. Esposito, M. Schramm* Institut für Pathologie, Funktionsbereich Cytopathologie, Heinrich Heine Universität, Düsseldorf, Deutschland Background.  Endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) cytology is an established diagnostic procedure for cystic, solid and mixed pancreatobiliary lesions with moderate sensitivity and high specificity. Conventional cytology is equivocal regarding malignancy in a substantial number of solid or mixed solid/cystic lesions due to low number of suspicious cells or artefacts. The most frequent malignant pancreatobiliary lesions, ductal adenocarcinoma and bile duct carcinoma usually display aneusomy, which can be analysed by multicolour fluorescent in situ hybridization (FISH). The aim of this study is to determine if the addition of FISH could enhance the diagnostic accuracy of conventional EUS-guided FNA-cytology, especially regarding equivocal cytological diagnoses. Methods.  A cohort of 273 EUS-guided FNAs from solid and mixed solid/cystic pancreatobiliary lesions sent to the Department of Cytopathology from January 2011 to December 2012 was included in the study. All specimens were alcohol-fixed and stained according to Papanicolaou for cytological diagnosis. Aneusomy was analysed with the UroVysion™ FISH multiprobe. A histological and/or clinical follow-up according to a predefined reference-standard, 6 months after EUS-guided FNA at the earliest, was available for 218 lesions, including 125 pancreatobiliary carcinomas, 75 benign lesions, 11 metastases and 7 neuroendocrine neoplasms. Results.  In the 200 cases with pancreatobiliary carcinoma and benign lesions, cytology had both a sensitivity and specificity of 87 %, if all equivocal specimens were considered positive for statistics. FISH achieved 74 % sensitivity and 96 % specificity. If a positive and a negative cytology was accepted as reliable and FISH was evaluated in cytologically equivocal specimens only, sensitivity, specificity and positive predictive value of combined cytology/FISH were 76 %, 96 % and 97 %, respectively. 75 % of the neuroendocrine neoplasms and all metastases with cytologically detectable abnormal cells showed aneusomy. Conclusions.  Analysis of aneusomy with FISH on EUS-guided FNA cytology specimens of solid and mixed solid/cystic pancreatobiliary lesions, is specific and predicts malignancy with high accuracy, especially in equivocal cases.

P12.04 HPV (Human Papillomavirus) vaccinated and still dysplasia U. Hahlbohm*, G. Richter Institut für Pathologie Dr. Richter, Hameln, Deutschland Background.  It should be the aim to protect women from a carcinoma of the cervix and its preliminary stages with a type specific vaccination. Half of all women form barely to no antibodies against HPV. A possible advancement of the HPV vaccination with antibody-titer-identification could be a sensible support for women with HPV infection. Furthermore the continuing care, education and invitation for vaccinated women to take part in gynecological screening with morphological diagnostics apply. Methods.  During microscopic inspection of gynecological cells of the cervix of vaccinated women using the known malignancy criteria, mild, moderate and severs dysplasia as classified in the Münchner Nomenklatur III in German-speaking areas, can be detected.

Results.  From January 2015 to June 2015 20 cases of dysplasia of the cervix in Pap smears of vaccinated women were diagnosed. 65 % of these cases were vaccinated with Garadasil, in 35 % the vaccine wasn’t specified. The dysplasia show the following distribution: 65 % of cases show a mild dysplasia (low SIL). In 35 % a high SIL lesion can be verified. There of 30 % have a moderate dysplasia and 5 % a severe dysplasia. Development and progress of the dysplasia: in 75 % of cases the year of vaccination is unknown. In 10 % of cases a dysplasia shows after 1 year, in 5 % after 4 years and in 10 % after 5 years of HPV vaccination. There are 65 % of cases in dysplasia follow-up examinations, 20 % of these are healed up within 2 years. In 15 % of high SIL cases a histological clarification followed, which confirmed the high SIL lesion 100 %. Conclusions.  The actual rate before the formation of a dysplasia in vaccinated women to the rate of formation of a dysplasia in non-vaccinated women, cannot be illustrated at the moment. At this particular time there is no valid specific marginal limit for a HPV-antibody-titer-identification and also a subtype determination of HPV antibodies isn’t possible, that’s why a reliable statement about the length of immunity and the actual raising of specific antibodies against HPV is not known. The HPV test doesn’t indicate dormant HPV infections reliably. The effect of the HPV vaccination into a dormant HPV infection to the actual effect on the formation of a dysplasia is therefore unclear. Thus the importance of gynecological screening with morphological diagnostics becomes apparent.

P12.05 Numbered agar bodies to individualize needle biopsy specimens – a paradigm shift in specimen identification

protein (e. g. fine minced meat). After solidification, the numbered agar bodies were stored in formalin 4 % until the slots were filled with needle biopsy specimens.To firmly attach the specimens to the surrounding agar body liquid agar was poured onto the specimens. The agar block was routinely paraffinized, cut and stained. Results.  Agar bodies could be successfully individualized by filling the figure slots with a colored agar-protein mixture. At all steps of processing, the number was clearly visible to the naked eye (. Fig. 1). This was especially true for the unstained and stained sections. The number remained on the slide even at immunohistochemistry and fluorescence in situ hybridization. The needle biopsy specimens could be securely fixed to the surrounding agar by using liquid agar. This liquid agar (55°C) did not harm the immunoreactivity of the unfixed tissue specimens. Conclusions.  By using numbered agar bodies with firmly attached needle biopsy specimens it is possible to individualize the specimens theirselves and not only the specimen containers. Therefore, the specimens can be clearly identified at all steps of processing and evaluation making specimen mix-ups extremely unlikely.

Postersitzung AG Molekularpathologie P13.01 The miR-183 cluster is activated in glioblastomas carrying EGFR amplification B. Schneider*1, D. William2, N. Lamp1, C. F. Classen2, A. Erbersdobler1

U. F. Vogel*

1

Institut für Pathologie, Universitätsklinik, Eberhard-Karls-Universität, Tübingen, Deutschland

2

Background.  Specimen mix-up in the histology laboratory is a fortunately rare, but potentially devastating source of medical error concerning biopsy specimens of different organs. The main reason why it is difficult to detect the accidental switching of tissue specimens is the fact that normally not the tissue itself but the sample container (e. g. transportation containers, embedding cassettes, slides) is marked. To achieve a real individualization of the specimens, I propose the use of numbered agar bodies with slots into which needle biopsy specimens can be inserted and secured with liquid agar. Methods.  Standard steel embedding molds for pouring paraffin blocks were filled with liquid agar. Before solidification of the agar spacer bars and a random set of spacer figures (base area: 3 mm2/figure) were put into the mold. After solidification of the agar the spacers were removed. The figure slots were filled with a mixture of liquid agar, dye of different colors and

Background.  Genomic amplification of the EGFR gene is a hallmark of glioblastoma (GBM) and occurs in ~50 % of these tumours, associated with a poor prognosis. The accompanying expression of the EGFRvIII deletion variant lacking the extracellular domain leads to ligand-independent aberrant EGFR signalling activating additional pathways. MicroRNAs (miRs) are short regulatory non-coding RNAs which are known to be deregulated in different tumours and can show oncogenic or tumour suppressive activity. MiRs are also considered as targets for specific therapies. This study wants to elucidate the miR expression profile of glioblastomas carrying EGFR amplification compared to others. Methods.  24 samples were chosen for analysis, 12 samples each with or without EGFR amplification, determined by qPCR. MicroRNA was extracted from FFPE material and miR-expression profiling was performed with the Nanostring nCounter miRNA assay. The expression levels of miRs

Universitätsmedizin Rostock, Institut für Pathologie, Rostock, Deutschland, Universitätsmedizin Rostock, Kinder- und Jugendklinik, Rostock, Deutschland

Fig. 1 I P12.05 8 a–g: Different specimens Der Pathologe Suppl 1 · 2016

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Abstracts of interest were verified by qPCR. Potential target genes were identified by different databases and expression was analysed by immunohistochemistry (IHC). Results.  Array analysis comparing the two groups revealed multiple differentially expressed miRs, but three of them, miR-183, miR-96 and miR182, all arranged in the miR-183 cluster, were highlighted by a 6–9 fold higher expression in EGFR amplified GBM, whereas all other upregulated miRs showed only a factor of up to 3. Database analysis revealed the pro-apoptotic factor FOXO1 as an important target gene for the miR-183 cluster members. IHC showed a higher FOXO1 expression in GBMs with normal EGFR compared to EGFR-amplified tumours. Conclusions.  All three mostly upregulated miRs belong to the miR-183 cluster, which has been shown to play a role in multiple tumours. Dependent on tumour entity and study, its members are considered both as oncogenes and tumour suppressors. Our current data indicate, that in EGFR amplified glioblastomas the miR-183 cluster has an oncogenic function, as a pro-apoptotic target gene, FOXO1, is downregulated. This mechanism could be a contribution to higher treatment resistance, therefore the members of the miR-183 cluster can be potential targets for specific therapy.

P13.02 Prognostic relevance of Aldehyde dehydrogenase expression in Glioblastoma multiforme F. Lämmer*1, 2, 3, C. Delbridge2, S. Baur2, C. Grubmüller2, J. Gempt4, F. Ringel4, K. Matiasek3, J. Schlegel2 1 Klinik und Poliklinik für RadioOnkologie und Strahlentherapie, Klinikum rechts der Isar, Technische Universität München, München, Deutschland, 2 Institut für Allgemeine Pathologie und Pathologische Anatomie der Technischen Universität München, Neuropathologie, München, Deutschland, 3Institut für Tierpathologie, Ludwig-Maximilians-Universität München, München, Deutschland, 4Neurochirurgische Klinik und Poliklinik, Klinikum rechts der Isar, Technische Universität München, München, Deutschland

Background.  Glioblastoma multiforme (GBM, WHO IV) is the most common adult glioma with a medium survival of 15 months. For the choice of medication, it is essential to have prognostic markers to predict the course of the disease, or to evaluate developed medications. As a novel maker for GBM, ALDH1A1 (Aldehyde dehydrogenase 1A1) was introduced. There is also evidence that ALDH1A3 activity is increased in mesenchymal GBM but not in proneural GBM. An increased proportion of ALDH1A3 positive cells seem to correlate with malignancy of glioma and poor survival of patients. The role of the ALDH enzymes in human GBM and especially ALDH1 isoforms is re-evaluated with immunohistochemistry (IHC) on formalin fix paraffin embedded material. A clear correlation between ALDH isoform expression and survival of human GBM patients, will make the inhibition of the enzyme activity interesting for targeted therapy. Methods.  IHC analysis was performed blinded on formalin fix paraffin embedded tissue of 90 GBM patients for ALDH1, ALDH1A1, ALDH1A3. The percentage of tumour cells with stained cytoplasm was scored and the intensity of the staining was taken into account. We defined two categories of IHC reaction: none and up to 10 % positive cells counted as no IHC reactivity, more than 10 % positive cells as IHC reactivity. Results.  ALDH1A1 IHC-expression in GBM was found primarily in the tumour adjacent region, whereas ALDH1A3 positive cells were more frequently found among tumour cells. Prognostic relevance for GBM patients’ outcome was found for the ALDH1 IHC-expression. Moreover, GBM patients have prolonged survival if neither ALDH1A1 nor ALDH1A3 expression is present. Conclusions.  The presented results indicate that there is, though challenging, a necessity for isotype specific analysis of ALDH expression. The heterogeneity of GBM is a main issue in GBM studies. The investigated specimens represent only small pieces within the particular tumour. De-

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tection of reliable routine prognostic markers is usually based on FFPE material. RT-PCR analysis does not give any hint for enzyme activity or relevance after post transcriptional regulation. A combined analysis with IHC, enzyme activity measurements and RT-PCR or methylation assays would give a general overview on ALDH presence on all levels and might help to find a new therapy target of GBM tumours.

P13.03 rs849142 Is A Biomarker Predicting Skin Toxicity on Targeted anti-EGFR Therapy of Metastatic Colorectal Cancer Using Cetuximab M. Frölich*1, 2, S. Stintzing2, 3, G. Lenz1, 2, S. Schaaf2, 4, U. Mansmann2, 4, V. Heinemann2, 3, T. Kirchner1, 2, A. Jung1, 2 1 Ludwig-Maximilians-Universität München, Pathologisches Institut der Universität München, München, Deutschland, 2Deutsches Konsortium für Translationale Krebsforschung (DKTK), Heidelberg, Deutschland, 3Klinikum Grosshadern und Comprehensive Cancer Center, Ludwig-MaximiliansUniversität München, Medizinische Klinik III, München, Deutschland, 4 Institut für medizinische Informationsverarbeitung, Biometrie und Epidemiologie, Ludwig-Maximilians-Universität, München, Deutschland

Background.  Mutations in the KRAS- and NRAS (RAS) genes are negative predictive biomarkers in anti-EGFR targeted therapy of metastatic colorectal cancer (mCRC)and are part of the approval. But only about 70 % of patients with WT RAS respond to anti-EGFR targeted therapies, indicating a weak predictive value of this biomarker. Interestingly, patients developing skin toxicity (ST) under therapy respond well what is also independent of RAS mutations.[1] Due to its manifestation in tumor unrelated sites ST seems to be related to the patient’s individual genetic make-up. By an educational guess we created a panel of single nucleotide polymorphisms (SNPs) known to be related to the development of 1.) acne, 2.) systemic lupus erythematodes (SLE), or 3.) Fcγ-receptor (FcγR) affinity. Our hypothesis was that ST predicting SNPs should be present in this panel of polymorphisms. Methods.  61 SNPs were selected from public sources and specific amplicon sets containing 41 SNPs were generated. These were analyzed by Next Generation Sequencing (NGS) using DNA from formalin fixed paraffin embedded (FFPE) tissue on an Ion Torrent PGM platform. Results were obtained from a training-set of 16 patients showing high- (3), medium- (2)or absence (1) of ST (CTCAE v4.0-Common Terminology Criteria for Adverse Events) using an association analysis model applying the PLINK software. [2] An independent set of 51 patients (all grades of ST: 1–3) was used for validation. Results.  Association analysis of the training set revealed SNP rs849142 as highly associated with ST (p = 0.00395). This significant association was verified in the validation set (p = 0.04362) thus unraveling rs849142 to be a predictive biomarker for ST. As ST is also correlated with progression free survival (PFS) (Kaplan-Meier model, p < 0.001) rs849142 was used as a surrogate in this analysis but failed to reach significance (p = 0.112). Thus, rs849142 is a biomarker predicting ST but not PFS in our test-validation approach. Conclusions.  rs849142 is located in the JAZF1 gene (juxtaposed with another zinc finger protein 1). This gene has been shown to be significantly correlated to systemic lupus erythematosus and is involved in prostate cancer as well as the metabolism of lipids. If rs 849142 is only coincidentally associated with the JAZF1 gene or if it is the gene function by itself indicating ST and thus response on anti-EGFR targeted therapy has to be shown. References 1. Stintzing S et al (2013) Prognostic value of cetuximab-related skin toxicity in metastatic colorectal cancer patients and its correlation with parameters of the epidermal growth factor receptor signal transduction pathway: Results from a randomized trial of the GERMAN AIO CR, Int J Cancer, 132:236–245 2. Purcell S et al (2007) PLINK: A Tool Set for Whole-Genome Association and Population-Based Linkage Analyses, Am J Hum Genet, 81:559–575

P13.04 Qualitative Comparison of Fresh Frozen (FF) and Formalin-Fixed-Paraffin-Embedded (FFPE) Tissue Samples: Impact of tissue entity, preservation and storage duration C. Lucena-Porcel*1, 2, S. Schmitt2, P. Schirmacher1, E. Herpel1, 2 Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, NCT Gewebebank am Pathologischen Institut, Heidelberg, Deutschland

1

2

Background.  Human biomaterial is an inestimable resource for genomic and proteomic research and important in personalized medicine. Tissue biobanks meet the increasing demand by providing FF and FFPE preserved specimen. To ensure high quality samples, appropriate tissue preservation and processing is crucial for final analyses. The aim of this study is evaluating how tissue preservation and storage duration impact sample quality by analysing RIN and DIN. We focused on the comparative analysis of FF and FFPE samples as it is known that therapy alters tissue quality. Despite the increasing number of FF preserved biomaterial, FFPE samples are the main part of biomaterial collections. To efficiently use this source we hypothesize that FFPE samples are still suitable for molecular studies. We installed several methodological devices for DNA/RNA extraction to compare different FF and FFPE entities from 2005–2014. Methods.  After establishing the protocols of nucleic extraction we evaluated DNA/RNA integrity in matched pairs of FF and FFPE tissue samples of several organs of the Tissue Bank of the NCT (Heidelberg) between 2005–2014: prostate (n = 50), colon (n = 30), kidney (n = 40), breast (n = 20), lymph node (n = 40), soft tissue (n = 50). DNA/RNA was extracted with QIAcube (QIAGEN)/Siemens TPS Hamilton in 50 µl buffer and quantified by fluorescence signal with Qubit device (Agilent Technologies) and validated DNA/RNA quality by electrophoresis (2200 TapeStation, Agilent Technologies). To prove usability of extracted DNA/RNA, analyses will be performed for specific genes. Results.  DNA quality and quantity are higher in FF than in FFPE tissue. The quality of FF and FFPE specimen stored in 2009–2014 is higher than in samples from 2005–2008. DNA extracts of FF prostate samples have DIN = 7 and DNA concentration = 30.2 ng/µl than FFPE samples (DIN = 3.3; concentration = 13.6 ng/µl). Evaluating defined preservation time, FF prostate samples from 2005–2008 have a DIN of 7 and a concentration of 30.2 ng/µl while FF prostate samples from 2009–2014 show a DIN of 8.3 and a concentration of 42 ng/µl. FFPE samples of the same preservation time were comparatively analysed. Samples stored between 2005–2008 have a DIN of 3.3 and a concentration of 13.6 ng/µl, while samples of 2009–2014 display DIN = 3.5 and concentration = 14.8 ng/µl. To demonstrate quality control of isolated DNA comparative PCR analysis will be performed. Conclusions.  In common our study demonstarted higher DNA quality out of FF samples which have shorter preservation time.

P13.05 Detection of rearrangements and transcriptional up-regulation of ALK and ROS1 in lung adenocarcinomas using a novel, sensitive quantitative RT- PCR assay C. Kalla1, K. Gruber1, D. Mönch*1, M. Kohlhäufl2, G. Friedel2, G. Ott3 Dr. Margarete Fischer-Bosch – Institut für Klinische Pharmakologie, Stuttgart, Deutschland, 2Klinik Schillerhöhe, Zentrum für Pulmonologie und Thoraxchirurgie, Stuttgart, Deutschland, 3Robert-Bosch-Krankenhaus, Abt. für Klinische Pathologie, Stuttgart, Deutschland 1

Background.  Successful treatment of lung cancer patients with crizotinib depends on the accurate diagnosis of ALK and ROS1 gene rearrangements. The approved FISH tests are low-throughput assays difficult to use in daily diagnostic practice. We devised a sensitive and robust diagnostic test for the detection of rearrangements and transcriptional up-regulation of ALK and ROS1.

Methods.  We developed a qRT-PCR assay adapted to routine FFPE specimens and applied it to 652 and 695 NSCLC specimens to screen for ALK and ROS1 rearrangements, respectively. The reliability of this technique was investigated by comparison with FISH and immunohistochemistry. Results.  qRT-PCR analysis detected unbalanced ALK and ROS1 expression indicative of gene rearrangement in 24/523 (4.6 %) and 5/680 (0.7 %) interpretable tumors, respectively. Among 182 NSCLC (21 ALK-rearranged) and 127 NSCLC (five ROS1-rearranged) simultaneously analyzed by FISH and qRT-PCR, the latter accurately typed 97 % (ALK) and 99 % (ROS1) and identified ALK/ROS1 activation in three cases with insufficient FISH. In addition, up-regulated expression of non-rearranged transcripts was observed in 1.1 % (ALK) and 9.6 % (ROS1) NSCLC. Immunohistochemistry detected ALK and ROS1 protein expression in tumors with gene fusions and transcriptional up-regulation, but did not distinguish between the two. Conclusions.  Our qRT-PCR assay reliably identifies and distinguishes ALK and ROS1 rearrangements and up-regulated transcription. It is an easy-to-perform, cost-effective and high-throughput tool for the diagnosis of ALK/ROS1 activation. The expression of non-rearranged ALK and ROS1 transcripts may be relevant for NSCLC therapy.

P13.06 A comparison of molecular alterations between newly established gastrointestinal cancer cell lines and their corresponding primary tumors D. Hirsch*1, P. Téoule2, S. Seyfried2, T. Staib2, U. Aghamaliyev2, F. Rückert2, P. Kienle2, T. Ried3, T. Gaiser1 1 Pathologisches Institut, Universitätsmedizin Mannheim, Mannheim, Deutschland, 2Chirurgische Klinik, Universitätsmedizin Mannheim, Mannheim, Deutschland, 3Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, Vereinigte Staaten von Amerika

Background.  Human cancer cell lines are widely used as model systems to study molecular mechanisms and genetic changes in cancer. However, the model is criticized due to the lack of proximity to the original tumor. Therefore, understanding in what ways and to what extent cell lines cultured under artificial conditions retain the biological properties and reflect the genomic profiles of their corresponding primary tumors is crucial. Methods.  We have established cancer cell lines from four patients with esophageal cancer, gastric cancer, colon cancer and rectal cancer. The individual identity of the cell lines was confirmed by short tandem repeat fingerprinting. Results.  The newly established cell lines mimicked the morphology and closely resembled the immunohistochemical staining profile of the tumors they had been derived from. Targeted re-sequencing and microarray-based comparative genomic hybridization revealed similar genomic profiles of the cell lines and their corresponding primary tumors. The chromosomal aberration patterns of the parental tumors were largely maintained in the cell lines and the distribution of gains and losses was typical for gastroesophageal and colorectal carcinomas. Furthermore, the model allows for the analysis of the development and conservation of subclones. Conclusions.  Taken together, this comprehensive characterization of four newly established gastrointestinal cancer cell lines and their corresponding primary tumors shows that histomorphological and immunophenotypic features as well as molecular alterations of the parental tumors are generally preserved in the cell lines. Hence, this newly established panel of four gastrointestinal cancer cell lines in conjunction with patient history could help to gain valuable insight into the mechanisms of drug sensitivity or resistance of tumors.

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Abstracts P13.07 Intratumoral heterogeneity in colon cancer identified by quantitative multiregion tissue proteomics K.-F. Becker*1, K. Grundner-Culemann2, N. Dybowski2, J. Slotta-Huspenina1, C. Schott1, A. Tebbe2, C. Schaab2, 3, H. Daub2 Technische Universität München, Institut für Pathologie, München, Deutschland, 2Evotec München GmbH, Martinsried, Deutschland, 3Max Planck Institut für Biochemie, Martinsried, Deutschland 1

Background.  The implications of intratumoural heterogeneity for diagnostic and therapeutic approaches in the era of personalized medicine are still at a very early phase. The variabilities of protein abundances within tumors have not been systematically addressed on a proteome-wide level. To analyze intratumoral heterogeneity in colon cancer, we have devised a robust proteomics workflow, applying mass spectrometry and reverse phase protein arrays (RPPA) to quantify proteins in formalin-fixed and paraffin-embedded tissue samples from multiple tumor regions and adjacent non-malignant tissues. Methods.  Formalin-fixed and paraffin-embedded (FFPE) tumor tissues from 5 stage II/III colon cancer patients were analyzed. Multiple (6–10, depending on the size of the tumor) samples from different locations of each individual tumor and 2 samples from adjacent non-malignant tissues from each patient were taken. In total, 47 tissue samples (37 tumor and 10 control samples) were prepared for proteome analyses using reverse phase protein arrays (RPPA) and mass spectrometry. Results.  Using mass spectrometry, we quantitatively recorded 3500 to 4000 distinct proteins per tumor and detected considerable intratumoral heterogeneity of protein levels. Nevertheless, hierarchical clustering and principal component analysis consistently segregated tumor samples from individual patients into separate groups, whereas non-malignant tissue samples clustered together irrespective of patient origin. Thus, tumor-specific proteome characteristics were sufficiently preserved and allowed robust patient assignment despite intratumoral variability. Distinct sub-proteomes, including extracellular matrix and plasma membrane proteins, showed particularly intratumoral variability. Interestingly, adjacent tumor areas do not necessarily have similar protein profiles. Moreover, proteins showing high heterogeneity within individual tumors also exhibited pronounced expression differences amongst tumors from different patients. The analysis of intra-patient heterogeneity by RPPA analysis showed variability of some of the analyzed 34 proteins and phosphoproteins within the tumors of the patients. Low and moderate heterogeneity was seen. Conclusions.  Our study provides a proteome-wide view on intratumoral heterogeneity and identifies subsets of the proteome which are particularly prone to sampling bias. These results add relevant information for the selection of protein biomarker candidates and the design of clinical trials.

P13.08 Array-based detection of ALK fusion variants in lung cancer F. Wagner*1, A. Streubel1, V. Heiser2, T. Mairinger1, A. Roth1 HELIOS Klinikum Emil von Behring, Institut für Gewebediagnostik/MVZ, Berlin, Deutschland, 2Chipron GmbH, Berlin, Deutschland

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Background.  Detection of ALK break apart events in lung cancer is performed by a variety of methods, most commonly by in situ hybridization (either fluorescence-based or chromogenic, FISH/CISH), immunohistochemistry (IHC), or reverse-transcription PCR (RT-PCR). Other methods available are mass spectrometry, NanoString technology, and next-generation sequencing. Each of the more often used methods has its specific strengths and weaknesses: FISH detects all break apart events regardless of the fusion partner, but it can be false-negative due to complicated rearrangements of the ALK locus [1] [2]; IHC is easy to perform, but can be challenging wrt an unequivocal distinction between positive and negative cases [3]; RT-PCR precisely detects specific fusion variants, but the ever growing number of ALK fusion variants is difficult to cover by RT-PCR in the daily laboratory routine.

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Methods.  In order to overcome some of the above mentioned problems, we have designed a DNA array representing 10 variants of EML4-ALK, 3 variants of TFG-ALK, 2 variants of KLC1-ALK, and 3 variants of KIF5BALK fusions. Each variant is represented by a specific oligonucleotide derived from the ALK fusion partner; in addition, ALK expression is detected by a region derived from ALK exon 20. Results.  We have compared the performance of the ALK array with RT-PCR and FISH. In comparison with RT-PCR and subsequent LightCycler-based discrimination of variants, the array is more sensitive and requires less handson time. It also detects ALK break apart-positive cases missed by FISH. Conclusions.  In summary, array-based detection of ALK variants is a robust, sensitive, fast, and easy to perform method. References 1. Peled N, Palmer G, Hirsch FR et al (2012) Next-generation sequencing identifies and immunohistochemistry confirms a novel crizotinib-sensitive ALK rearrangement in a patient with metastatic non-small cell lung cancer, J Thorac Oncol 7:e14–16 2. Ren S, Hirsch F, Varella-Garcia M et al (2014) Atypical negative ALK break apart FISH harbouring a Crizotinib-responsive ALK rearrangement in non-small cell lung cancer, J Thorac Oncol 9:e21–3 3. Von Laffert M, Warth A, Penzel R et al (2013) Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC): results of a multi-centre ALK-testing, Lung Cancer 81:200–6

P13.09 Detection of HPV Subtypes by Mass Spectrometry: A Reliable Tool for Routine Diagnostics P. Wandernoth*1, M. Kriegsmann2, N. Arens1, S. Gemballa1, C. Mohanu1, R. Casadonte3, R. Longuespée1, J. Kriegsmann1 1 Molekularpathologie Trier, Trier, Deutschland, 2Institut für Pathologie, Ruprecht-Karls Universität Heidelberg, Heidelberg, Deutschland, 3 Proteopath GbR, Trier, Deutschland

Background.  Recently the U:S Food and Drug Administration has approved high-risk Human Papilloma Virus (HPV) tests as a primary screening tool for cervical cancer in women aged 25–65 years, without a simultaneous Pap smear. Thus, it is tempting to speculate that pathologists and molecular biologist will be faced with a rising number of HPV testings in the near future. Therefore test systems are required that detect high- and low-risk HPV subtypes with high specificity and sensitivity in a time and cost effective manner. Methods.  We developed a custom designed assay for routine mass-spectrometric (MS) (MassARRAY, Agena Bioscience) analysis to test for the presence/absence of 19 specific HPV infections and compared the results to hybridization assays (COBAS and Chipron). In total 45 Formalin fixed paraffin embedded (FFPE) tissue samples and 10 Pap smear liquid samples were analyzed. Moreover we tested three different isolation techniques with variable hands-on-time (from 5–45 min) and compared the results. Results.  All high risk HPV subtypes detected by the COBAS or the Chipron system were also detected by MS. MS outperformed the number of HPV subtypes detected when a time consuming, but very effective DNA purification step had been applied. In all patients HPV infections were found. Conclusions.  MS is a valuable method to detect HPV subtypes in a high throughput setting in FFPE samples and in Pap smears.

P13.10 Fluorescence in-situ hybridization as an aid in the diagnosis of Whipple’s disease P. Braubach*1, T. Lippmann1, M. Kühnel1, I. Anagnostopoulos2, H. H. Kreipe1, D. Jonigk1 Institut für Pathologie, Medizinische Hochschule Hannover, Hannover, Deutschland, 2Institut für Pathologie, Charité – Universitätsmedizin, Berlin, Deutschland

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Background.  Whipple’s disease is a systemic chronic infection with the bacterium Tropheryma whipplei (T. w.). It is a rare disease with an approximate incidence of 1/1,000,000. The diagnosis of Whipple’s disease is challenging and relies on the detection of characteristic but unspecific histopathological changes: foamy macrophages with PAS positive and diastase resistant inclusions. PCR based amplification of T. w. specific sequences is used as a confirmatory test, but often lacks sensitivity. In recent years the introduction of a polyclonal T. w. specific antibody has improved the diagnostic toolkit for establishing the diagnosis of Whipple’s disease. Methods.  Fluorescence in-situ hybridization (FISH) with species specific probes targeting ribosomal RNA of bacteria is an advanced molecular method enabling the detection and localization of bacteria in formalin fixed and paraffin embedded (FFPE) tissue. It has been suggested as an aid in the diagnosis of Whipple’s disease, but until present there are no published studies on the value of FISH in the diagnosis of Whipple’s disease. We present a study evaluating 20 cases of confirmed Whipple’s disease and appropriate controls with standard stains (PAS, PAS-D), T. w. specific immunohistochemistry and T. w. specific bacterial FISH. Specific FISH signals of T. w. were detectable in 50 % of the investigated cases and none of the control cases. Results.  In conclusion bacterial FISH is a valuable aid in the diagnosis of Whipple’s disease and can add confirmatory support to the pathological diagnosis. FISH negative cases are probably due to treatment effects inactivating and/or killing of vital bacteria. Conclusions.  We will further investigate the properties of T. w. specific FISH in combination with T. w. specific immunostains to highlight the differences between the diagnostic methods.

P13.11 Experiences of the Comprehensive Cancer Center Freiburg Molecular Tumorboard B. Kraczyk*1, L. Lutz1, C. Kayser1, R. Claus2, H. Busch3, M. Börries3, N. von Bubnoff2, M. Werner1, 3, S. Lassmann1, 3 Universitätsklinikum, Institut für Klinische Pathologie, Freiburg, Deutschland, 2Universitätsklinikum, Klinik für Innere Medizin I, Freiburg, Deutschland, 3Deutsches Konsortium Translationale Krebsforschung (DKTK) und Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Deutschland 1

Background.  Integration of advanced molecular diagnostics into the management of the broad spectrum of cancer patients beyond guideline treatment regimens requires multidisciplinary approaches. This need is increasingly matched by development of molecular tumorboards (MTB) in university medical centers. Here we report on the experiences of the MTB at the Comprehensive Cancer Center Freiburg (CCCF). Methods.  Since March 2015, the MTB at the CCCF is scheduled biweekly at 7:30 am. It accommodates requests for advice of all CCCF member departments beyond standard molecular pathology testing in organ-specific tumorboards. Hence, the MTB sees cancer patients with rare tumor entities, CUPs or patients with tumors under progress receiving standard or targeted therapy. The MTB approach is clinico-pathologically focused by interdisciplinary pre-selection of molecular analyses according to histology and patient-specific clinical possibilities (e. g. off-label drug, inclusion into ongoing clinical studies). In few MTB cases, explorative molecular analyses of whole genome or exome sequencing and/or RNA transcriptome analyses are performed in a research setting. Diagnostic reports are jointly discussed and a recommendation is provided to the treating physician and/or to the organ-specific tumorboards. Results.  So far, in 20 MTB sessions 68 patients with e. g. CUPs, soft tissue or metastatic GI and breast carcinomas were evaluated. At least 59 % (40/68) patients were re-discussed for optimal follow requirement of the molecular data. So far a total of 223 analyses were performed in the Institute of Surgical Pathology including 49 sequencing analyses via targeted Next Generation Sequencing, 145 immunohistochemically stainings, 22 in-situ hybridizations and 7 microsatellite analyses. Moreover, in 13 cases,

DNA and/or RNA were submitted to WES/RNA transcriptome analyses. Until now, 37 % (25/68) tumor patients received a therapy recommendation and among 20 % (5/25) patients were included into clinical trials. For breast cancer and lung cancer advanced diagnostic pathways were established and played back to the organ-specific tumorboards. Conclusions.  The CCCF MTB is a valuable supportive interdisciplinary structure to optimize cancer patient management. Moreover, it contributes to the development and integration of novel molecular markers into routine diagnostic workflows and patient pathways as well as education of professionals working in the medical field.

P13.12 Comparison of different methods for the detection of gene fusions in formalin-fixed and paraffin-embedded tissue samples E. Moskalev*, A. Agaimy, A. Hartmann, F. Haller Institut für Pathologie, Friedrich-Alexander Universität, Erlangen, Deutschland Background.  The detection of specific gene fusions is of increasing diagnostic and predictive value in non-small cell lung cancers (NSCLC) and soft tissue sarcomas. While fluorescence in situ hybridization using dual color break apart probes is the current gold standard for the detection of gene fusions in FFPE tissue samples, it is expensive, laborious and consumes valuable tissue if only small biopsies are available. Detection of gene fusions on RNA level using next-generation sequencing or the amplification-free nCounter Analysis System provide promising methods for detection of gene fusions in FFPE tissue. For the current study, we evaluated the two different methods for their usefulness in daily routine diagnostic work-up of NSCLC and soft tissue sarcomas. Methods.  A cohort of 8 NSCLC samples with known gene fusions including ALK and ROS1 as well as 8 soft tissue sarcomas were simultaneously analyzed using the NGS approach and the nCounter Analysis System. For the NGS analysis, a commercially available library preparation using a multiplex PCR based approach was employed (Archer FusionPlex Panels). For the nCounter Analysis System, modified versions of the published Lung Fusion and Sarcoma Fusion panels were generated. Results.  Sequencing libraries were generated from FFPE-derived RNA from a total of 16 cases, and were analysed simultaneously by NGS and the nCounter Analysis System. All previously known gene fusions were successfully detected. The usefulness of the two different detection methods for their integration in daily routine diagnostic work-up were compared in regards of sensitivity, specificity, time, costs and applicability. Conclusions.  Both tested methods are applicable for detection of gene fusions in FFPE tissue material.

P13.13 Proteomic deciphering of echinococcosis using laser microdissection coupled to LC-MS/MS analyses and MALDI Imaging R. Longuespée*1, R. Casadonte1, M. Kriegsmann2, G. Mazzucchelli3, J. Kriegsmann1, 4 Molekular Pathologie Trier, Trier, Deutschland, 2University of Heidelberg, Institute of Pathology, Heidelberg, Deutschland, 3University of Liège, Mass Spectrometry Laboratory, Systems Biology and Chemical Biology, Liège, Belgien, 4MVZ for Histology, Cytology and Molecular Diagnostics Trier, Trier, Deutschland 1

Background.  Echinococcosis is a neglected tropical disease caused by the parasitic tapeworm Echinococcus. Commonly infected animals are dogs and foxes that represent hosts for human infection, after spreading of eggs in food and water. The human manifestation mostly occurs in the liver and the symptoms which are amongst others include abdominal pain and fever, which can be very weak until the cyst by the worm reaches a big size. The treatment generally consists in surgical removal of infected organs but Der Pathologe Suppl 1 · 2016

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Abstracts ing liver tissue by mass spectrometry. Thus, mass spectrometry may serve as diagnostic tool for the identification of an echinococcal infection. Further analyses may allow to distinguish between different species of Echinococcus.

P13.14 A novel Insulin like growth factor-1 (IGF-1) and Insulin Receptor (IR) RNA in situ Hybridization assay: Potential application in Precision Medicine V. Sailer*1, T. MacDonald1, L. C. Cantley2, M. A. Rubin2, J. M. Mosquera1 Weill Cornell Universität für Medizin, Englander Institut für Präzisionsmedizin, New York, Vereinigte Staaten von Amerika, 2Weill Medical College of Cornell University, Meyer Cancer Center, New York, Vereinigte Staaten von Amerika 1

Fig. 1 I P13.14 8 High expression of IR in prostatic adenocarcinoma (x40)

Fig. 2 I P13.14 8 Quantitative analysis of IR expression in a prostatic adenocarcinoma this can be impossible if the cysts are presents in multiples sites. In these cases, chemotherapeutic agents or antiparasitic drugs such as albendazole are administered. Finding new biomarkers of different echinococcal species may help to treat patients more effectively. Methods.  Sections of a liver infected by Echinococcus granulosus were processed using a recently developed method for laser microdissection based microproteomics. The sampling step consisted in isolating the worm itself and his secretions from the cyst and the liver. Digested tissues were analyzed by LC-MS/MS using a Nanoacquity UPLC (Waters) coupled to a Q-exactive Orbitrap mass spectrometer (Thermo) and the data processing was performed using MaxQuant. Subsequent imaging mass spectrometry was also performed to map ion peptides related to Echinococcus infection. Results.  The proteomic analysis was performed using two databank interrogations. Since the proteome of Echinococcus granulosus is poorly described and this worm develops in human, human sequences were also added for identification interrogations. Each of the tissue regions allowed us to retrieve more than 1000 proteins. As expected most of the worm proteins were found in the tissue region occupied by the worm. Interestingly, the three different compartments (liver, echinococcal cyst and the worm) could clearly be distinguished at the molecular level. Conclusions.  In the present study we show differences in the protein pattern comparing the worm Echinocuccus, the echinococcal cyst and the surround-

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Background.  Novel kinase inhibitors (PI3Ki) like the pan-Class I PI3K inhibitor BKM120 target the PI3K pathway and are currently under clinical investigation in a variety of malignant diseases. A common side effect is hyperglycemia but a subsequent treatment with insulin might counter antitumor effects. Evaluating the expression of IGF-1 and IR in formalin fixed paraffin embedded (FFPE) tissue could unmask tumors that are more likely to be resistant to PI3Ki due to reactivation of the PI3Ki/AKT signaling axis when exposed to insulin. For this purpose we developed a chromogenic RNA in situ hybridization (RNAish) assay to evaluate IGF1 and IR expression in FFPE tissue. Methods.  The Affymetrix QuantiGene® ViewRNAish Tissue 1-Plex Assay was optimized in HELA cells. PC3 knock down cells (IGF-1 or IR) were used as control (western blot). The assay was validated in a prostate cancer TMA with 39 cases and corresponding prostate biopsies. Results.  This pilot study of IGF-1 and IR RNAish showed that our newly developed assay is robust and reproducible. Using imaging analysis software, over-expression was detected in a subset of advanced prostate cancers. Conclusions.  This newly developed IGF-1 and IR RNAish assay can be employed to test for high expression of IGF-1 and IR in malignant tumors. We confirmed its quality in control cell lines as well as in a small prostate cancer cohort. In the latter high expression was seen in advanced prostate tumors (. Figs. 1, 2). This assay could be employed as a biomarker in metastatic tumors that are likely to respond with reactivation of the PI3Ki pathway when a treatment-induced hyperglycemia is medicated with insulin. Our findings warrant further investigation in the context of clinical trials. Quantitative analysis was done using Definiens® software.

P13.15 Establishment of a pyrosequencing assay for determination of MLH1 methylation status J. Seitz*1, E. Moskalev1, A. Hartmann1, G. Seitz2, F. Haller1 Institut für Pathologie, Friedrich-Alexander Universität, Erlangen-Nürnberg, Deutschland, 2Institut für Pathologie, Bamberg, Deutschland

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Background.  A subset of colorectal cancers (CRC) is characterized by deficiency of the DNA mismatch repair (MMR) system, e. g. microsatellite instability. This deficiency is either caused by germline mutations of any of the DNA MMR proteins MLH1, MSH2, MSH6 or PMS2 in patients with hereditary non-polyposis colon cancer (Lynch syndrome), or can occur sporadically via aberrant MLH1 hypermethylation with subsequent loss of the protein. Additionally, up to 25 % of endometrial cancers display MMR deficiency, among which the majority occurs via somatic hypermethylation of MLH1. In colorectal cancer, BRAF mutation analysis is currently used as a surrogate parameter for aberrant MLH1 hypermethylation, as the presence of the BRAF V600E mutation is significantly correlated with MLH1 hypermethylation. In endometrial cancer, the frequency of the BRAF V600E is so low (<2 %) that a similar correlation is unlikely. For the

current study, our aim was to develop a pyrosequencing assay for quantification of MLH1 methylation in DNA from formalin-fixed and paraffin-embedded (FFPE) tumor tissue, and to evaluate thresholds for aberrant MLH1 hypermethylation in colorectal cancer and endometrial cancer. Methods.  The MLH1 promoter region was analyzed in silico for the presence of CpG sites and compared to published data on aberrant methylation status. Accordingly, a pyrosequencing assay interrogating the methylation level at 5 individual CpG sites was designed and tested using CRC tissue samples and cell lines (HCT-116 and HT-29) with known microsatellite status. BRAF mutation analysis was performed using a self-designed pyrosequencing assay, and the samples harboring the V600E mutation were regarded as sporadic MSI-H carcinomas. Genomic DNA from FFPE tissue samples was extracted using the QIAamp DNA FFPE Tissue Kit, while bisulfite conversion was carried out using the EZ DNA Methylation-Gold Kit. Additionally, FFPE samples of endometrial cancer were analyzed for MLH1 methylation status and BRAF V600E mutation status. Results.  Using CRC samples harboring the V600E mutation as positive controls, MLH1 methylation levels of >50 % were regarded as hypermethylated. In the samples with BRAF wildtype status, the MLH1 methylation was observed in the range between 5 and 30 %. In the endometrial cancer samples, similar methylation levels could be observed. Conclusions.  Direct measurement of MLH1 methylation levels may be superior to BRAF mutation analysis in MSI-H cancers with a low frequency of BRAF mutations (e. g. endometrial cancer).

P13.16 BRCA1/BRCA2 analysis by next-generation sequencing: the Aachen-Heidelberg experience N. Ortiz Brüchle*1, V. Endris2, A. Maurer1, R. Penzel2, T. Eggermann3, P. Schirmacher2, R. Knüchel-Clarke1, E. Dahl1 Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland, Institut für Pathologie, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Deutschland, 3Institut für Humangenetik, Uniklinik RWTH Aachen, Aachen, Deutschland 1

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Background.  The identification of deleterious BRCA1/BRCA2 tumor mutations has recently been approved as an indication for PARP inhibitor (Olaparib) therapy in women with platinum-sensitive relapsed ovarian cancer. The analysis of both genes is complicated by their size (BRCA1-cDNA: 5592bp; BRCA2-cDNA: 10257bp). To address the need of a fast analysis we established a next-generation sequencing (NGS) based BRCA1/ BRCA2 analysis. In order to compare our workflow with another method as a part of our validation process we cooperated with the Institute of Pathology in Heidelberg. The performance differences will be presented. Methods.  DNA was isolated using the Maxwell System (Promega) from FFPE material of 20 patients (tumor cell content >20 %). For library generation in Aachen we used the Human BRCA1/BRCA2 Panel (GeneRead V2; Qiagen) according to manufacturer’s protocol. The libraries were subsequently sequenced on the MiSeq system (Illumina). Data analysis was performed with the SeqNext module (SEQUENCE Pilot software, JSI medical systems). In Heidelberg a customized BRCA1&2 Research Panel (Ion AmpliSeq Community Panel, ThermoFisher) was used for library preparation. Samples were sequenced on the Ion PGM System (ThermoFisher). Data analysis was performed with an individual “homemade” pipeline. Variants with a frequency of >5 % (>500x coverage) in the coding sequence and 10bp of the adjacent introns were taken into account. Results.  Coverage was usually >98 % >500x in the ROIs in both labs. 71 frequent polymorphisms with an allele frequency of 17–100 % were equally detected at both sites. Allele frequencies differed between the labs with a maximal difference of 50 % (e. g. 30 % vs. 80 %). Additionally in 13 cases a clearly deleterious mutation was confirmed in both labs. However, two samples showed divergent results. A large duplication of about 40bp was missed in one lab at first and one frequent polymorphism could not be detected in the

other lab. Further analyses regarding a possible drop-out are ongoing. Overall 84/86 changes in 20 patients were called identically within both workflows. Conclusions.  The detection of larg rearrangements is challenging using the methods mentioned which is well known in the NGS community. Furthermore allelic drop-out is a commonly described problem of PCR-based sequencing methods. Both limitations should be kept in mind. Nevertheless, our data show that the described two different mulitplex-PCR based next-generation sequencing BRCA1/BRCA2 analysis workflows perform almost alike.

P13.17 Rapid and accurate ALK detection in lung adenocarcinoma using a microfluidic tissue processor S. Brajkovic*1, G. Repond1, D. G. Dupouy1, A. Soltermann2 Lunaphore Technologies, Lausanne, Schweiz, 2Universitätsspital Zürich, Institut für Klinische Pathologie, Zurich, Schweiz

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Background.  Anaplastic lymphoma kinase (ALK) is a key oncogenic driver in lung adenocarcinoma. The oncologic treatment decision is based on the respective molecular pathologic analysis of patient’s tumor cells, which includes immunohistochemistry/immunofluorescence (IHC/IF). Lunaphore Technologies SA developed a medical device called “microfluidic tissue processor” (MTP). It is based on a chip-confined low-volume technology and allows for rapid IHC/IF staining of formalin-fixed paraffin-embedded (FFPE) tissue samples. In comparison to standard IHC, MTP-based IF demonstrated better separation of HER2 positive versus negative breast carcinoma samples (Ciftlik AT et al., PNAS 2013). In this study we have used the MTP platform for rapid and accurate determination of ALK protein expression level in lung adenocarcinoma by IF. Methods.  FFPE whole tumor sections of surgically resected lung adenocarcinoma patients of the University Hospital Zurich were used. The primary mouse anti-human ALK antibody (clone 5A4, Novocastra) was used with dilutions of 1/10, 1/50, 1/100 and 1/200. Next to MTP-based IF, automated Leica Bond IHC as well as manual IF were performed as controls. The reagents further included: secondary antibody AlexaFluor647 (Life technologies), horseradish peroxidase (HRP) secondary antibody system from ImmPRESS (Vector Laboratories), TSA kit (Life technologies, TSA dilutions 1/100, 1/200, and 1/500), BSA 1 % (Life Technologies), 2.5 % horse serum (Vector Laboratories), Top Block (Lubioscience), and Sudan Black B (Sigma-Aldrich). An ImageJ plug-in was developed for quantification of IF intensity. Results.  All 3 methods yielded specific ALK protein detection on FFPE whole sections of lung adenocarcinoma. The total staining time on the MTP device was 22 minutes, whereas manual IF required 2 hours 7 minutes. The ALK IHC protocol on the Leica Bond automat used for routine diagnostics needed 60 minutes. Best staining was obtained by using a blocking solution of 2.5 % horse serum, 8 minutes incubation of the primary antibody, a primary antibody dilution of 1/50, 8 minutes of the HRP-coupled secondary antibody ImmPRESS with 2 minutes of TSA, and a TSA dilution of 1/100. Conclusions.  The MTP-based protocol for IF detection of ALK protein has been optimized for different steps and it is 6 times faster than manual IF. We foresee that the increased rapidity and accuracy provided by Lunaphore’s technology will open the roads for clinical studies that would improve the efficacy of lung adenocarcinoma targeted therapy.

P13.18 FGFR1 alterations in squamous cell carcinomas M.-C. Demes*1, S. Stallmann2, S. Scheil-Bertram3, A. Fisseler-Eckhoff2, 3 Gemeinschaftspraxis für Pathologie, Molekularpathologie, Wiesbaden, Deutschland, 2Gemeinschaftspraxis für Pathologie Wiesbaden, Pathologie, Wiesbaden, Deutschland, 3Dr. Horst-Schmidt-Kliniken (HSK), Institut für Pathologie und Zytologie, Wiesbaden, Deutschland

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Abstracts Background.  Currently, no standardized molecular marker is tested for squamous cell carcinomas in routine diagnostics and only little is known about FGFR1 mutational behaviour. Genetic alterations in FGFR1 are associated with distinct malignancies. For instance, myeloproliferative malignancies are treated with ponatinib which also inhibits the FGFR family. FGFR1 amplifications in squamous histologies are currently the focus of Research. Methods.  The test slides were analyzed with the Fluorescence Microscope (Carl Zeiss MicroImaging GmbH) by using 40x or 63x objectives. If the signals of interest were homogeneously distributed, random areas were selected for cell signal counting. For FGFR1/CEN8 analysis 20 tumour cell nuclei (if possible non-overlapping cell nuclei) from three distinct random tumour areas were assessed resulting in a total of 60 nuclei. Green FGFR1 and orange centromer 8 (CEN8) signals were counted and calculated as proposed by Schildhaus and colleagues. Each FGFR1 mutation was confirmed by taking three distinct areas of the same tumour in order to ensure valid results. Results.  FGFR1 amplifications detected by FISH were observed in 11/60 (18.3 %) of squamous cell carcinoma histologies. 8 of the positive FISH cases presented a predominant pattern of microamplifications which were shown as several clusters of green signals. The remaining FGFR1 amplified cases were presented as single green dots or chain formations. This study has demonstrated no evidence of intratumoral heterogeneity with respect to FGFR1 mutations. Conclusions.  Squamous cell carcinomas of the lung are characterized by an heterogenetic pattern of the FGFR1 gene copy number. As shown in the results section, distinct patterns of FGFR1 amplification have to be kept in mind, ranging from chain formation and single dot presentation to microcluster amplification patterns. Polysomies must also be kept in mind in order to avoid false positive FGFR1 tumours. So far, there is not yet any standardized method to judge FGFR1 FISH assays. One unanswered question remains how to predict the meaning of the distinct FGFR1 patterns.

P13.19 Tumor tracking of non-small cell lung carcinoma by ultra-deep sequencing of the entire mitochondrial genome M. Odenthal*1, W. Amer1, C. Toth2, H. Eischeid1, U. Koitzsch1, A. Arens3, R. Büttner1, S. Schäfer1 Institut für Pathologie, Universitätsklinikum, Köln, Deutschland, Pathologisches Institut, Universitätsklinikum, Heidelberg, Deutschland, 3 Qiagen GmbH, Hilden, Hilden, Deutschland 1

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Background.  Mitochondrial (mt) genome analysis is a well established molecular tool for cell lineage tracing, because the mtDNA has an up to 10 fold higher mutation rate than nuclear DNA due to a high rate of reactive oxygen species generation, lack of protective histones and limited DNA repair activity. Here, we designed a unique approach for tracking the cell lineage of multiple phenotypic divergences of non-small cell lung carcinoma (NSCLC) cell clones to identify the intra-tumor clonal structure based on comprehensive ultra-deep sequencing of the entire mitochondrial genome. This approach provides an appropriate tool in understanding how tumors originate, develop and progress. Methods.  A total of 58 formalin fixed and paraffin embedded NSCLC tumors archived from 26 patients diagnosed with lung carcinoma were enrolled in this study. Matching nodules of different histological differentiation or grad were studied. Lesional areas of NSCLC were macro- or laser-microdissected. 108 primer sets spanning the whole mtDNA were designed and a multiplex PCR-based approach was established. Target enriched libraries of mtDNA were sequenced by means of the MiSeq platform and NGS data interpreted by the Biomedical Genomics Workbench 2.5.1 (www.clcbio.com). mt-variants were additionally validated by conventional Sanger sequencing. Results.  NGS data analysis revealed a good run performance with 98 % of mapped reads aligned to mt-genome, and only around 2 % of mapped reads aligned to chromosome 1, and < 0.1 % to other chromosomes, indicating the efficiency of our approach for target enrichment of the whole

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mt-genome. Whole mt-genome analysis revealed a wide spectrum of mt alterations, and high frequency of variants observed in the D-Loop region and genes of the respiratory chain complex. Interestingly, most tumor nodules harbor identical mutations indicating a monoclonal origin of multiple phenotypic divergencies of NSCLC. In particular, the homoplasmic variant status refers to clonal expansion from single cell progeny. Conclusions.  In summary, our novel approach may be considered as a standard diagnostic method for comprehensive analysis of mitochondrial genome in a simultaneous, cost effective and very specific and sensitive manner in order to address the clonal architecture of NSCLC. In addition, we have shown that the NCSLC are progeny of a single transformed cell.

Postervorstellung: Geschichte der Pathologie PO.01 About the foundation of the German Society of Pathology in 1897 K. Donhuijsen* Städt. Klinikum Braunschweig, Institut für Pathologie, Braunschweig, Deutschland Background.  The German Society of Pathology was founded the 20th. september 1897 in Braunschweig during the congress of german natural scientists. The foundation was prepared by Rudolf Virchow, Berlin and a special commission with the pathologists Chiari, Prag, Hanus, St.Gallen, Ponfick, Breslau, von Recklinghausen, Straßburg, and Ziegler, Freiburg. Previously,there was a controversial discussion about necessity, goals and designation of a new special scientific society. Virchow first being an opponent of the foundation was outvoted and he agreed finally. Details of the discussions and the local circumstances reflect the scientific field at the end of the 19th. century. Methods.  Minutes of proceedings,reports of local news paper and reviews serve for the analysis of the foundation process. Results.  The decision to establish the German Society of Pathology at 20th. september 1897 in Braunschweig was the essential action to the development of the scientific importance of the german pathology. 1898 the first proceedings of the new society were held in Düsseldorf. Conclusions.  The separation of the pathology from the society of natural scientifics was the beginning of the most important period of german pathology till the first decades of the 20th.century.

PO.02 Development of university professorships for pathology in Germany S. Hübler*, R. Meyer Deutsches Herzzentrum Berlin, Berlin, Deutschland Background.  The aim was to examine the historical development of pathology at universities in Germany since 1845. Methods.  Using a standardized form, 18 pieces of information on all professors of pathology appointed between 1845 and the present were recorded in databases. The information was taken from: 55obituaries appearing in the annual proceedings volumes of the Deutsche Gesellschaft für Pathologie (DGP, German Society for Pathology) 55announcements in the professional journals (English titles: Journal for General Pathology and Pathological Anatomy; The Pathologist) 55material made publicly available by the universities and academies 55the internet. Data concerning different questions were recorded in different databases and analyzed. Results.  The first professorship for pathology was created in 1845 at the University of Würzburg and was held by Bernhard Mohr (1809–1848). Be-

tween then and the present day, 37 university professorships for pathology came into being. At present there are still 35. Almost half of these professorships (46 %) were in existence when the DGP was founded in 1897; a further seven were created before 1950 and the remaining 12 subsequently. By 2015 a total of 219 pathologists had been appointed to these professorships or had exercised the function temporarily. Conclusions.  The information that has been gathered is a suitable basis for further investigations concerning the historical development of university professorships in Germany.

PO.03 The Institute of Pathology in Mainz – 100 years interwoven with the fate of the region C. Brochhausen*1, P. Hochgesand2, W. Roth3, C. J. Kirkpatrick4 REPAIR-Lab, Institut für Pathologie, Universität Regensburg, Regensburg, Deutschland, 2Augenarztpraxis Dr. Hochgesand, Mainz, Deutschland, 3 Institut für Pathologie, Universitätsmedizin, Mainz, Deutschland, 4REPAIRLab, Johann Wolfgang von Goethe Universität, Frankfurt, Deutschland 1

Background.  The development of institutions is deeply embedded in its historical context. The region of the left bank of the Rhine river was the scene of significant changes and eruptions during the past centuries. Methods.  We illustrate the development of pathology and that of the Institute of Pathology in Mainz with a view to the historical context of the region. Results.  In the 18th century the elector Karl Joseph von Erthal promoted the development of the university. The medical faculty earned high recognition since the appointment of Thomas Soemmering to the chair of anatomy. His plan for an anatomical theatre failed due to the chaos of the revolutionary war, following by the closing of the university. In the 19th century the “Society of Medical Physicians” was founded to promote the performance of autopsies. The Institute of Pathology was founded 1914 by a donation of Jacob Hochgesand. The first Director Dibbelt died in the battle of Verdun before being active in the newly formed institute. In 1917 Gruber became Director of the institute. He was followed by Müller until 1946. With the re-opening of the university in 1946 the Institute of Pathology became integrated. The first chair of Pathology was occupied by Klinge, a pioneer in the field of rheumatopathology. He was followed by Bredt in 1954. His research focus was given by cardio-pathology, arteriosclerosis, paidopathology and the pathology of aging. In 1974 Thoenes became chair, and had significant impact to renal pathology. Under his chairmanship further groups became very active regarding uro-, gastrointestinal-, hepatopathological pathology and amyloidosis. Under his leadership electron microscopy became a highly active diagnostic tool. Kirkpatrick took the chair of pathology from 1994–2015 and continued the non-oncological research tradition of Klinge and Bredt, namely within the fields musculo-skeletal and cardio-vascular-pathology with the heritage of the prominent ultrastructural approach established by Thoenes. During his chairmanship the research laboratory, named the REPAIR-lab, was a founding member of The European Institute of Excellence for Tissue Engineering and Regenerative Medicine. Since October 2015 Wilfried Roth took the chair of Pathology and will further establish cancer research. Conclusions.  The development of pathology in Mainz provides a good example of the dependence of the development and the research focus of an institution on the historical context.

PO.04 Overview of clinical autopsies in the last decade – an analysis of two University centers F. Erlmeier*1, W. Weichert1, R. Knüchel2, J. Andruszkow2 Institut für Pathologie, Technische Universität München, München, Deutschland, 2Institut für Pathologie, RWTH Universitätsklinikum, Aachen, Deutschland 1

Background.  In the last decade, numbers of clinical autopsies performed by pathologists are declining in most institutes of pathology in Germany. Nevertheless, autopsies still represent an integral part in training during residency and a fundamental asset of learning pathophysiology via morphology. Therefore, it was the aim of our study to analyze differences in patients’ characteristics due to decreasing autopsy numbers. Methods.  Autopsy reports between 2005 and 2014 of two university institutes of pathology were reviewed. Age and sex of the patients were documented as well as the specialty department and the hospital that was registering the patient for autopsy. Then, cause of death was determined and categorized in organ systems for better evaluation. Results.  The number of clinical autopsies performed per year was virtually continuously decreasing: While in 2005, 2007 and 2008 240 autopsies were performed annually, only 167 autopsies were performed in 2014 (−30 %). Since 2010, the number of external hospitals that admitted patients for autopsies was declining from 26.3 to 12.6 % per year (p = 0.001). Among the 2183 autopsies that were collectively performed in both institutes, the median age of patients was 64.5 years without significant changes during the decade. In each year, the quantity of male patients was higher (58.3–66.5 %) than the quantity of female patients (33.5–41.7 %). The main specialty registering patients for autopsy was internal medicine, although the proportion of these patients was decreasing from 63.4 % (2005) to 51.9 % (2014) (p = 0.02). At the same time registration of surgical patients has slightly increased from 26.8 (2005) to 33.3 % (2014). The main causes of death during the decade were cardiovascular and circulatory diseases (45.5–55.7 %). This cause of death is relatively declining in the last years, since systemic diseases like sepsis are increasing (9.1 to 26.3 %). Conclusions.  Although numbers of autopsies were declining in the last years, there were no differences in patients’ demographics like age or sex. Changes in the registering clinic and hospital result in different patient morbidities and a different spectrum of causes of death. The pathologist should exchange findings thoroughly with the clinician, and will certainly provide additional information for the clinical situation.

PO.05 Military autopsies in Rostock 1939–1946 A. Erbersdobler* Universitätsmedizin Rostock, Institut für Pathologie, Rostock, Deutschland Background.  882 military autopsies were performed at the Institute of Pathology, University of Rostock, between 1939 and 1946, the protocols of which were archived separately. Methods.  Autopsy protocols were revisited and collected in a table. Causal chains of death were classified according contemporary criteria and coded with respect to the ICD-10. Results.  Median age of the deceased was 27 years. 48 % of cases of death were classified as “not natural” according to contemporary criteria. The three main causes of death were special infectious diseases, air plane accidents, and deadly injuries during the act of war (. Fig. 1). War prisoners, and Russian soldiers and civilians which came to autopsy after the end of war, were subgroups with different frequencies according to the special causes of death. Conclusions.  Autopsy protocols from the time of World War II are historical documents which allow a special view on the state of things of that time.

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Abstracts

Fig. 1 I PO.05 7 Causes of Death in 882 military autopsies

PO.06 Analysis of the incidence of death at Benedictine monks V. Chupina*1, R. Meyer2, J. Götze3 1 Klinikum Frankfurt(Oder), Medizinische Klinik III, Frankfurt(Oder), Deutschland, 2Deutsches Herzzentrum Berlin, Kardiovaskuläre Gewebebank, Berlin, Deutschland, 3Berlin, Deutschland

Background.  An analysis of mortality and morbidity delivers an important information about population health state as well as quality of medical care. The epidemiological data before the establishment of statistical bureaus are rare and incomplete. Methods.  A retrospective analysis of data from the members’ registers of Kremsmuenster and Admont monasteries in the 17th-19th centuries is performed. Results.  There are no significant differences between both monasteries determined. Age at death in the monasteries of Kremsmuenster und Admont between 1600 and 1899 averaged 58,1 y. o. The most common causes of death were cardiovascular diseases (37.5 %), infections (18.0 %) and neoplasms (12.0 %). Among the cardiovascular pathology there prevailed apoplexy (47 %) and heart failure (26 %). Among infections the tuberculosis played the key role (54 %). The comparison of the Benedictines with the males of Ulm in the 17th century shows a similar age at death in both populations (54.2/55.0 y. o.). The most common causes of death in both groups were cardiovascular (37.2 %/34.7 %) and infectious diseases(18.4 %/26.3 %). The 3rd frequent cause of death among the monks were neoplasms, which occurred among the Ulm’s males seldom (12.3 %/6.3 %). An average life expectancy at the age of 20 years was in Kremsmuenster and Admont higher than in the Bavarian monasteries. An average life expectancy of the Austrian monks was comparable with the Common Mortality Table (CMT) of German Empire 1871/81. The life expectancy of the resident population(Imhof ’s sources)was in Hartum, Ortenau and Saarland significantly lower, however in Herrenberg, Ostfriesland, Schwalm/Eder and Hamburg it was similar or significantly higher than in the CMT. An average life expectancy at the age of 21 years among monks in 1600–1899 was considerably lower, than among the Austrian males in 2007 (38.8/56.9 y. o.). The quote of cardiovascular diseases in both groups was the highest one (37.2 %/38.5 %). The rate of neoplasms in modern Austria was more than doubled (28.7 %/12.3 %). The 3rd place in 2007 the violent death (7.9 %/1.0 %) achieved. The infections were among Benedictines more often(18.4 %/0.8 %). The incidence of coronary artery disease in current Austria increased (50 % vs 7 %); the rate of stroke decreased (18 % vs 47 %).

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The tuberculosis rate decreased from 54 to 8 %. In 2007 some new pathogens appeared. Conclusions.  The monks age of death was comparable with a resident population. The most common causes of death were cardiovascular diseases, infections and tumors.

PO.07 Back to Future – epistemological concepts for the definition of pathological entities – the unique example of „the disease of the right lower quadrant“ C. Brochhausen*1, 2, C. J. Kirkpatrick3 REPAIR-Lab, Institut für Pathologie, Universität Regensburg, Regensburg, Deutschland, 2Zentrum für Rheumapathologie, Universitätsmedizin Mainz, Mainz, Deutschland, 3REPAIR-Lab, Institut für Pathologie, Universitätsmedizin, Mainz, Deutschland 1

Background.  In Germany Virchow introduced the concept of cellular pathology in clinical practice. Untill today this concept represents the essential basis of understanding of disease. The critical aspect consists in the correlation between structure and function, which is represented on the molecular level by the receptor-model. Aim was to prove the applicability of the theoretical concept from Ludwig Fleck on the evolution of the nosological term of appendicitis. Methods.  Fleck’s relativistic view on scientific knowledge based on the association of history and the theory of science. We correlated the history of the nosology of appendicitis with Fleck’s model of creating and changing of scientific facts, which he thought is critically influenced by its social and cultural context. We included modern data and knowledge on the pathomechanisms of inflammation. Results.  In the concept of humoralpatholgy, which represent the major concept of health and disease until the 19th century, no basis for the causal link between inflammation of the appendix vermiformis, perforation and peritonitis exists. In this period perforation of the appendix was interpreted as a secondary process caused by an abscess in the right lower quadrant, leading to the term “disease of the right lower quadrant”. Systematical analyses of histomorphological changes was firstly introduced with cellular pathology. In this concept the sequence of inflammation, perforation and peritonitis was described. The molecular basis for this process in leucocyte trafficing was demonstrated later wtihn the 20th century. Conclusions.  The historical overview showed clearly that the changes of the concepts of the “disease in the right lower quadrant” was not the result of rational scientific debate, but was critically embedded in social and cultural developments.