Abstracts Pathologe 2010 · 31:497–514 DOI 10.1007/s00292-010-1362-6 © Springer-Verlag 2010

Abstracts

Joint Annual Meeting SGPath – ÖGP/IAP Austria .–. November  Feldkirch

Group 1: Oral Presentations (OP) part 1 and 2

OP-102. HPV in Tonsillar Carcinoma A. Hirschmann1, T.D. Collen2, R.H. Greiner2, W. Müller3, J. Diebold1 1 Institute of Pathology, 2 Dept. of Radiation Oncology 3 Clinic for Otorhinolaryngology, Lucerne Cantonal Hospital, Lucerne, Switzerland Background: HPV has been implicated as causative agent in head and neck carcinomas. The biology of HPV tumours seems to differ from alcohol and tobacco related tumours. Methods: Formalin fixed and paraffin embedded tissue of tonsillar carcinomas of 83 patients was retrieved from the archives of the Pathological Institute. 76 of 83 patients had been treated at the Dept. of Radiation Oncology between 1997 an 2008 either primarily or in an adjuvant setting. HPV status was analysed by multiplex PCR and direct sequencing. The results were entered into clinicopathological correlation. Results: HPV was found in 34/83 cases (41%), in 33 cases HPV-16, in one case HPV-33. 9 HPV-16 positive tumours harboured additional HPV-types (35, 45, 51, 59). Positive HPV status was most prevalent in basaloid carcinomas (11/13 cases, 85%) compared to keratinizing and non-keratinizing squamous cell carcinomas and lymphoepithelial carcinomas (HPV positivity in 18%, 42% and 33%). TNM-tumour stage of HPV positive cases was significantly lower. Comparison with clinical data showed an inverse correlation between HPV and alcohol consumption and cigarette smoking. In multivariate analysis positive HPV status as well as non-smoking and tumour-free resection margins were favourable prognostic factors. HPV positivity was associated with prolonged overall and disease free survival. Conclusions: HPV seems to be an important causative factor in tonsillar carcinomas. Although most prevalent in basaloid carcinomas, it can be detected in all histological types. HPV positive cases are associated with favourable prognosis which may be due to increased radiosensitivity

OP-101. 402 HPV Subtyping Results Comparing Smears, Biopsies and Conisations R. Silye1, Ch. Webersinke1, M. Winkler1, St. Doppler1, G. Palmisano1, B. Traxler1, W. Stummvoll2 1 Department of Pathology, Nerve Clinic Linz, Austria 2 Department of Gynaecology, Sisters of Mercy Hospital Linz, Austria

OP-103. Poorly Differentiated Thyroid Carcinoma – How Much Poorly is Needed? M. Dettmer1, 6, A.M. Schmitt6, H. Steinert2, A. Haldemann3, A. Meili4, H. Moch1, P. Komminoth5, A. Perren6 1 Institute of Surgical Pathology, University Hospital Zurich, Switzerland 2 Division of Nuclear medicine, University Hospital Zurich, Switzerland 3 Division of Nuclear medicine, Triemlispital, Zürich, Switzerland 4 Division of Nuclear medicine, Kantonsspital Winterthur, Switzerland 5 Institute of Surgical Pathology, Triemlispital, Zürich, Switzerland 6 Institute of Pathology, University of Bern, Switzerland

Background: The link between squamous cell cervical carcinoma and HPV is well-established and screening methodes have developed rapidly during the last decade, provoked by molecular HPV subtyping. Most commercially available tests for HPV subtyping are able to distinquish between low and high risk HPV infection, but a list of particular HPV subtypes is missing. Here we present a study on 402 cases, where a macroarray analyzes 32 HPV types. We compared cytology specimens and histological ones like biopsies, cones and loop excisions. Methods: GP5+/GP6+ and My11/9 primed PCRs were performed followed by subtyping 16 high and 16 low risk HPV types by a macroarray of Chipron. Results: HPV 16, 31 and 51 are the predominant high risk types, HPV 42 and 53 the most common low risk types. In 49, 1% there is a single HPV infection, whereas in 27, 2% a dual and in 12,8% a triple one. We observed up to 9-fold infections. In 71, 8% there is an identical subtyping result in pap smears and biopsies. In 21, 6% the smear subtpyping yielded additional subtypes, only in 5, 5% biopsies were more sensitive. Cytological controls after conisation exhibit a HPV infection in nearly one half of the cases, sometimes HPV subtypes constellation do differ from the original ones. Conclusions: Cytological sampling is the method of choice for HPV subtyping in cervical screening. HPV clearing is not achieved satisfactory by conisation. Therefore subtyping of HPV should be discussed as a standard procedure in addition to cytological controls.

Background: Poorly differentiated carcinomas of the thyroid (PD) are conceptually situated in between classic papillary and follicular carcinomas and anaplastic thyroid carcinomas. Today, it is not clear, how much of a PD area in a given tumor is required to allow such a diagnosis. Methods: We identified via the nuclear medicine departments all patients with a carcinoma of the thyroid with an adverse clinical outcome, defined as more than one relapse or tumor associated death. We estimated the area of a poor differentiation per tumor, according to the Turin criteria of PD carcinomas (solid/trabecular/insular growth pattern; lack of nuclei of papillary carcinoma and one of the following: 1. convoluted nuclei, 2. tumor necrosis, 3. three or more mitoses per ten high power fields). Results: We examined 92 Patients and correlated the results with the overall survival (OS), tumor specific survival (TSS) and relapse free survival (RFS). With a cut-off of 10%, we identified 35 PD carcinomas. 57 age, stage and gender matched classic follicular carcinomas served as controls. Even with only a small 10% of the area being poorly differentiated, the OS, TSS and RFS in a KaplanMeier analysis was significantly worse than in the control group (p<0.001). In a multivariate analysis including age, gender, tumor stage and PD area above 10% for OS, TSS and RFS, the only consistent significant factor was PD (p<0.001). Conclusions: Even very minor PD part in a thyroid carcinoma affects prognosis significant. The presence of such areas may be worth reporting in thyroid carcinomas.

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OP-104. Prognostic Role of CD68-Positive Macrophages in the Context of the Reactive Environment of Classical Hodgkin Lymphoma (HL) S. Hoeller1, M. Matter1, A. Tzankov1, S. Dirnhofer1 1 Institute of Pathology, University of Basel, Switzerland Background: HL consists of neoplastic Hodgkin and Reed-Sternberg cells (HRSC) and a micromilieu of non-neoplastic cells that greatly outnumber the HRSC. Studies on HRSC-related prognostic biomarkers were largely unsuccessful, but the composition of the microenvironment is of prognostic importance. Recently, the number of macrophages has been correlated with adverse survival in HL and there was a claim for results validation. Methods: We analyzed the prognostic importance of the CD68-positive macrophage number compared to other cellular environmental components in an unselected series of 105 HL in tissue microarrays by immunohistochemistry. Results: Applying a cut-off score of >0.82% tumor macrophages, cases with increased numbers showed worse overall survival (mean 185 months, median 192) compared to cases with lower amounts (mean 285 months, median not reached). Eleven of 62 patients with ≤0.82% macrophages died compared to 19 of 43 with >0.82% (p<0.001). Importantly eight of the latter died of a second malignancy. The number of macrophages correlated with low FOXP3/high Granzyme B/high PD-1-positive cellular background and with patient age, and was not of independent prognostic significance in our collective. However, a combination background score with all negative prognostic microenvironmental components (CD68-, PD-1- and Granzyme B-positive cells) was of independent prognostic significance (p=0.002, relative risk 2.63). Conclusions: The reactive non-neoplastic micromilieu is of decisive prognostic importance in HL. An increased number of macrophages is associated with an adverse outcome. However, the number of macrophages is probably only a part of a complex interaction network of reactive immune cells, especially T-cell subsets, in HL. OP-105. SDHB Loss Predicts Malignancy in Pheochromocytomas/ Sympathethic Paragangliomas, but not through Hypoxia Signalling A.M. Schmitt1, A. Blank2, E. Korpershoek3, F.H. van Nederveen3, T. Rudolph4, N. Weber4, R.T. Strebel5, R.R. de Krijger4, P. Komminoth6, A. Perren1 1 Institute of Pathology, University Bern, Bern, Switzerland 2 Technical University Munich, Institute of Pathology, Munich, Germany 3 Department of Pathology, Josephine Nefkens Institute, Erasmus MC, University Medical Center, Rotterdam, Netherlands 4 Institute of Surgical Pathology, Department of Pathology, Zurich, Switzerland 5 Department of Urology, University Hospital Zurich, Switzerland 6 Department of Pathology, City Hospital Triemli, Zurich, Switzerland Background: Prediction of malignant behaviour of pheochromocytomas/sympathetic paragangliomas (PCC/PGL) is very difficult if not impossible on a histopathological basis. In a familial setting, it is well known that SDHB-associated PCC/PGL very often metastasize. Recently, absence of SDHB expression as measured via immunohistochemistry was shown to be an excellent indicator of the presence of an SDH germline mutation in PCC/PGL. SDHB loss is believed to lead to tumour formation by activation of hypoxia signals. Methods: To clarify the potential use of SDHB immunohistochemistry as a marker of malignancy in PCC/PGL and its association with classic hypoxia signalling we examined SDHB, Hif-1alpha and its targets CA-9 and GLUT-1 expression on protein level using immunohistochemistry on a tissue micro array on a series of familial and sporadic tumours of 115 patients. Survival data was available for 66 patients. Results: SDHB protein expression was lost in the tumour tissue of 12 of 99 patients. Of those 12 patients 5 had an SDHB germline mutation, in 5 patients no germline mutation was detected and mutational status remained unknown in parts in 2 patients. Loss of SDHB expression was not associated with increased classic hypoxia signalling as detected by HIF-1alpha, CA-9 or GLUT-1 staining. Loss of SDHB expression was associated with an adverse outcome. Conclusions: The lack of correlation of SDHB loss with classic hypoxia signals argues against the current hypoxia hypothesis in malignant PCC/PGL. We suggest SDHB protein loss as a marker of adverse outcome both in sporadic and in familial PCC/PGL.

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OP-106. Absence of Extramural Venous Invasion is an Excellent Predictor of Metastasis-free Survival in Colorectal Carcinoma Stage II – A Study using Tangential Tissue Sectioning K. Dirschmid1, W. Sterlacci1, F. Oellig2, M. Edlinger3, M. Rhomberg1, Z. Jasarevic1, H. Dirschmid1, F.A. Offner 1 Institute of Pathology, University Teaching Hospital Feldkirch, Austria 2 Institute of Pathology, Mühlheim/Ruhr, Germany 3 Department of Medical Statistics, Informatics and Health Economics, Innsbruck, Austria Background: Patients with colorectal carcinoma (CRC) stage II pose a significant therapeutic management problem, since considerable controversy exists regarding the role of adjuvant chemotherapy. Patients with a high risk profile may benefit from adjuvant chemotherapy but the recognition of these patients is difficult. Extramural venous invasion (EVI) is an established risk factor of hematogenous metastasis. However, published incidence rates of EVI of 8.7–33% indicate major technical inconsistencies in the assessment of this important parameter. The present study applies tangential vessel preparation and correlates results with hematogenous metastasis. Methods: CRC stage II diagnosed between 1994 and 1996 were included in our study. Resection specimens were analyzed by tangential sectioning of the tumor periphery. Confirmation of haematogenous metastasis was assessed by computer tomography, ultrasound and biopsy. The median follow-up period for patients in stage II without metachronous hematogenous metastasis was 49 months. Results: EVI was detected in 50/79 (63%) CRC stage II. 13/50 (26%) of these patients developed metachronous hematogenous metastasis. The rate of hematogenous metastasis for patients with venous invasion was virtually independent of adjuvant chemotherapy. Only 1/29 patients without EVI (3.5%) progressed to hematogenous metastasis. Absence of extramural venous invasion is significantly associated with metastasis-free survival (p<0.011). Conclusion: EVI is an excellent predictor of tumor progression in CRC stage II. In particular absence of extramural venous invasion is a highly specific (93%) negative predictor of metastasis. However, this is only true if the incidence of EVI is assessed properly by tangential sectioning of the tumor periphery. OP-207. VEGFA Gene Amplification and Protein Expression in Breast Cancer M. Andreozzi1, S. Schneider1, I. Zlobec1, L. Tornillo1, S. Eppenberger1, L. Terracciano1, C. Ruiz1 1 Institute for Pathology, University Hospital Basel, Basel, Switzerland Background: The vascular endothelial growth factor A (VEGFA) protein is a chemical signaling molecule that is known to be a major factor in the induction of angiogenesis during tumor initiation and progression, and is also a target of anti-angiogenic therapies. Recently, we discovered the genomic amplification of the VEGFA gene in a small subset of colorectal cancers. Aim of this study was to investigate the presence of VEGFA gene amplification in breast cancer and to determine its potential impact on VEGFA protein expression. Methods: VEGFA gene amplification was evaluated by FISH on a multitumor tissue microarray (MTMA) comprising 132 different tumor types. Further, a small tissue microarray was constructed from breast carcinoma samples whose VEGFA protein concentration had been previously quantified by chemiluminescence (check!). In order to interrogate tissue heterogeneity, VEGFA gene amplification was also analyzed on large tissue sections from 70 primary breast cancers. Results: We detected VEGFA gene amplification in 2% of the breast cancer samples from the MTMA and in 5% of the breast cancer samples with known VEGFA protein concentration. In addition, 8% of the samples (5 out of 65) were characterized by a high polysomy. Interestingly, elevated VEGFA gene copy number was strongly correlated with higher VEGFA protein levels (p<0.0001, check!). Conclusions: VEGFA gene amplification defines a small subset of breast carcinomas with elevated VEGFA protein expression. Our data suggest that FISH analysis of VEGFA could represent an additional evaluation system for the identification of breast cancer patients who might benefit from anti-VEGFA therapies.

Abstracts OP-208. Genomic Alterations and Allelic Imbalances are Strong Prognostic Predictors in Osteosarcoma D. Baumhoer1–3, J. Smida1,4, M. Rosemann5–6, A. Walch5, S. Bielack7, C. Poremba8, K. Remberger9, E. Korsching10, W. Scheurlen11, C. Dierkes12, S. Burdach4, G. Jundt2–3, MJ. Atkinson ��������5–6, M. Nathrath1,4   1 Clinical Cooperation Group Osteosarcoma, Helmholtz Center Munich, German Research Center for Environmental Health, Germany  2 Institute of Pathology and   3 Bone Tumor Reference Center at the Institute of Pathology, University Hospital Basel, Switzerland   4 Department of Pediatrics, Technical University Munich and Pediatric Oncology Center, Germany  5 Institute of Pathology and   6 Institute of Radiation Biology, Helmholtz Center Munich, German Research Center for Environmental Health, Germany   7 Klinikum Stuttgart Olgahospital, Pediatrics 5 - Oncology, Hematology, Immunology, Germany   8 Institute of Pathology, Heinrich-Heine-University, Duesseldorf, Germany and Center of Histopathology, Cytology and Molecular Diagnostics (CHCMD), Research Park Trier, Germany  9 Institute of Pathology, University of the Saarland, Homburg-Saar, Germany 10 Institute of Bioinformatics, University of Muenster, Germany 11 Cnopfsche Kinderklinik Nuremberg Children’s Hospital, Germany 12 Institute of Pathology, Justus-Liebig-University, Giessen, Germany Background: Osteosarcoma, the most common primary malignant tumor of bone, is characterized by complex karyotypes with abundant structural and numerical alterations. Despite attempts to establish molecular prognostic markers already at the time of initial diagnosis, histologic regression grading following neoadjuvant chemotherapy is still the gold standard concerning prognostic prediction. Methods: To detect recurrent loss of heterozygosity (LOH) and copy number variations (CNV) with potential prognostic and therapeutic impact for osteosarcoma patients we performed a genome-wide search using Affymetrix 10K2 high-density SNP arrays. 45 well characterized pretherapeutic biopsy samples were investigated and numerical aberrations and allelic imbalances were correlated with clinico-pathological data including follow-up and the histologically assessed regression grading. Results: The most frequent genomic alterations included amplifications of chromosome 6p21 (15.6%), 8q24 (15.6%, harboring MYC) and 12q14 (11.1%, harboring CDK4) as well as LOH of 10q21.1 (44.4%). All these aberrations and the total degree of heterozygosity of each tumor were significantly associated with an unfavourable clinical outcome of patients. We defined a new chromosomal alteration staging (CAS) system with a superior predictive potential compared to the conventional regression grading. Conclusions: Structural chromosomal alterations detected by SNP analysis provide a simple and reliable tool to predict the response to neoadjuvant chemotherapy. The proposed CAS system might therefore help to better anticipate the clinical course of osteosarcoma patients already at the time of initial diagnosis and to adept neoadjuvant treatment in patients resistant to the current protocols. OP-209. Her2 Amplification is Significantly more Frequent in Lymph Node Metastases from Urothelial Bladder Cancer than in the Primary Tumors Fleischmann A.1, Rotzer D.1, Seiler R.1, Thalmann G.N.2 1 Institute of Pathology 2 Department of Urology, University of Bern, Switzerland Background: Her2 might be an attractive therapeutic target in metastasizing bladder cancer. Primary tumors and their metastases may differ genetically and phenotypically. Consequently, Her2 status must be determined in both tumor components to assess the full therapeutic potential of this target. Methods: Hundred and fifty lymph node positive patients with urothelial bladder cancer underwent cystectomy and pelvic lymphadenectomy. A tissue microarray was constructed with four tumor samples per patient, two from the primary tumor and two from nodal metastases. Her2 status was determined by immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH).

Results: Her2 amplification was significantly more frequent in nodal metastases (15.3%) than in primary bladder cancers (8.8%; p=0.003). Her2 gene alterations of primary tumors were highly conserved in corresponding metastases. There was a high concordance in Her2 FISH results between both samples from the primary tumor (kappa: 0.853) and from the metastases (kappa: 0.930), IHC results were less concordant (kappa: 0.539 and 0.830). FISH and IHC results were only moderately linked in primary tumors (kappa: 0.566) and metastases (kappa: 0.673). While Her2 amplification in the primary tumor significantly predicted poor outcome (p=0.044), IHC based survival prediction was unsuccessful. Conclusion: There is a high potential for anti-Her2 therapies in a substantial subset of bladder cancer patients with metastases. For patient selection Her2 determination in the metastases is advantageous and FISH might be more useful than IHC. OP-210. KRAS Mutational Status may be Underestimated in Rectal Cancer Specimens Obtained after Neoadjuvant Chemoradiation M.Nucifora1, F.Molinari1, E.Zanellato1, S.Crippa1, A.Franzetti-Pellanda2, P.Saletti3, L.Mazzucchelli1, M.Frattini1 1 Institute of Pathology, Locarno, Switzerland 2 Radiotherapy and Tomotherapy Center, Clinica Luganese, Lugano, Switzerland 3 Oncology Institute of Southern Switzerland, Bellinzona, Switzerland Background: KRAS testing represents a prerequisite before EGFR-targeted therapies administration to metastatic colorectal cancer patients. The choice of representative tumor samples for KRAS testing is fundamental. In patients with locally advanced rectal cancer (LARC), neoadjuvant chemoradiation is offered in most protocols. Therefore, due to tumor regression, residual tumor in resection specimens may be scarce. We tested KRAS mutations in diagnostic biopsies and resection specimens to evaluate potential differences of KRAS detection rates in rectal cancer before or after neoadjuvant treatment. Methods: Tumors samples obtained before (diagnostic biopsy) and post (resection specimen) neoadjuvant chemoradiation from 61 LARC patients were analyzed. Tumor microdissection was performed. KRAS status was evaluated by direct sequencing (DS, sensitivity: 10–20%) and by mutant-enriched PCR (ME-PCR, sensitivity: 0.01%). Results: In diagnostic-biopsies, DS revealed KRAS mutations in 24=39% cases and ME-PCR in 28=46%. After chemoradiation, DS found KRAS mutations in 13=21% cases and ME-PCR in 24=39%. Eight cases showed a discordant pattern in pre- and post-therapy tissues: 6 patients showed the mutation limited to the pre-treatment biopsy (in 4 cases detected by DS and ME-PCR, in 2 cases only by ME-PCR) while 2 patients showed the mutation limited to the posttreatment tissue (detected only by ME-PCR). Conclusions: The KRAS mutational status in rectal cancer after neoadjuvant treatments may be underestimated. We suggest that DS of pre-treatment biopsies is more reliable than analysis of resection specimens. Although carefully microdissected, post-treatment specimens must be assessed only if diagnostic biopsies are not available, and, in case, by high sensitive methodologies (e.g. ME-PCR). OP-211. Morphological Criteria of Nuclear Grading: a Critical Re-Appraisal H.A. Lehr1, B. Mora1, D. Bombari2, S. Schaefer3, F. Mast2 1 Institute of Pathology, University Hospital Laussanne, Laussanne, Switzerland 2 Department of Cgnitive Psychology, University of Bern, Bern, Switzerland 3 Department of Pathology, University of Bern, Bern, Switzerland Background: Medical students are taught in pathology class that nuclei of malignant tumors are big (enlarged), dark (hyperchromatic), irregular (heterochromatic, with irregular chromatin texture), unround (showing irregular contours), and nucleolated. However, are these truly the criteria that we use in grading tumor nuclei in our day to day diagnostic practise as diagnostic pathologists? Methods: 20 Pathologists in various stages of formation/experience were asked to grade nuclei in each of 40 high power fields of prostate carcinomas projected on a computer screen. Eye tracking allowed to identify which nuclei the pathologists fixed during the 8 second period during which each HPF was shown and Der Pathologe 6 · 2010 

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the nuclear grades that they assigned were compared with various morphological measures to the selected nuclei (Photosho-based image analysis). Results: Linear regression analyses showed that 14 of the 20 pathologists bases his/her nucelar grade assignment on a sole morphological feature, rather than a combination of the various features listed above: 13 used solely nuclear size and one used solely hyperchromasia. For one pathologist, a combination of nuclear size and unroundness was relevant, and for five pathologists, not a single one of these morphological criteria was statistically linked to their grade assigment. Conclusion: In contrast to what we teach medical students, in our daily practise we do not assign nucelar grades on a variety of morphological features. Without being aware of this, we tend to base our grade assignment on only a single morphological feature. OP-212. PIM Serine/Threonine Kinases in Diffuse Large B-Cell Lymphoma T. Menter1, L. Brault2, S. Knapp3, S. Thommen2, J. Schwaller2, A. Tzankov1, E. Obermann1 1 Institute of Pathology, 2 Department of Biomedicine, University Hospital Basel, Switzerland, 3 SGC, Oxford, UK Background: PIM1–3 are a family of constitutive active serine/threonine kinases regulating cellular proliferation and survival that have been reported to be over-expressed in several human cancers. Their proto-oncogenic role has been demonstrated in several experimental lymphoma models but never been systematically analyzed in primary lymphoma biopsies. Methods: We have studied the expression of PIM1–3 by immunohistochemistry in a large cohort of well-characterized diffuse-large B-cell lymphoma (DLBCL) cases (n=101, stages I-IV, GC-DLBCL=29 as defined by the Chi algorithm), using tissue microarrays. Functional studies were performed in a panel cell lines derived from GC-type and ABC-type human DLBCLs applying two previously characterized small molecule PIM inhibitors. Results: In most (91%) cases cytoplasmic expression of PIM1 was observed, only 12 cases showed also nuclear PIM1 staining in >50% of the cells. Only modest levels of PIM3 were found. PIM1 expression significantly correlated with activation of STAT3 and STAT5 as assessed by phospho-STAT staining, and with the fraction of actively proliferating cells. Nuclear expression of PIM1 expression correlated significantly with the Ann-Arbor lymphoma stage. No significant correlations between PIM expression and the status of MYC-, BCL2- and BCL6-genes were found. Treatment of DLBCL cell lines with two structurally different small molecule inhibitors significantly impaired cellular proliferation. Conclusions: Our study strongly suggests that PIM kinases are associated with JAK/STAT pathway activation in a significant fraction of human DLBCLs. Blocking proliferation of DLBCL cells by small molecule inhibitors implies that PIM kinases might represent rational therapeutic targets.

Group 2: Posters with Brief Oral Presentation Part 1 and 2 PO-113. Putative Cancer Stem Cells and Epithelial-to-Mesenchymal Transition in Malignant Pleural Mesothelioma S. Thies1, I. Opitz2, A. Schramm2, E. Felley-Bosco3, W. Weder2, H. Moch1, A. Soltermann1 1 Institute for Surgical Pathology, 2 Clinic of Thoracic Surgery and 3 Clinic and Policlinic of Oncology, University Hospital Zurich, Rämistrasse 100, CH-8091 Zurich, Switzerland Background: Malignant pleural mesothelioma (MPM) is derived from cells having undergone epithelial-mesenchymal transition (EMT). EMT confers

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stem cell traits to tumor cells. We aimed for investigating the protein expression of putative cancer stem cell (CSC) markers SOX10, podoplanin, nestin and beta-catenin, together with the recognized EMT marker periostin in MPM. Patients and Methods: Tumor tissue of a retrospective cohort of 352 MPM patients was analysed by immunohistochemistry of a tissue microarray with antibodies against CSC markers. Protein expression data of CSC markers was correlated with clinico-pathologic parameters including histotype, asbestos exposure and survival as well as with the diagnostic qualifier calretinin. Results: Of the 352 MPM, 117 were of epithelioid, 45 of sarcomatoid and 190 of biphasic histotype. Expression of SOX10 was found in 58.3%, nestin in 62.7%, beta-catenin in 58.6%, podoplanin in 51.3% and periostin in 98% of the tumor cells (calretinin in 86.9%). The CSC marker SOX10 correlated with the sarcomatoid and biphasic HT and with the EMT-marker periostin, which is also correlated to the sarcomatoid HT (p-value <0.001). Calretinin, podoplanin and beta-catenin associated with the epitheloid HT. Calretinin denoted also like nestin an inverse correlation with periostin (p-value <0.001) and was associated with a better overall survival, whereas a high expression of periostin was associated with a poor survival. Conclusions: Cancer stem cell markers are expressed in malignant pleural mesothelioma and indicate a differentially pattern in the histological subtypes. Also EMT plays an important role in the development of MPM. PO-114. Deregulation of Cell Cycle Control in Non-Small Cell Lung Cancer is Associated with Unfavorable Prognosis W. Sterlacci1, M. Fiegl2, W. Hilbe2, H. Jamnig3, W. Oberaigner4, T. Schmid5, F. Augustin5, J. Auberger6, E.C. Obermann7, A. Tzankov7 1 Institute for Pathology, University Teaching Hospital Feldkirch, Feldkirch, Austria 2 Department of Internal Medicine, Division of Hematology and Oncology, Medical University Innsbruck, Innsbruck, Austria 3 Department of Pneumology, Hospital Natters, Natters, Austria 4 Institute for Clinical Epidemiology, Medical University Innsbruck, Innsbruck, Austria 5 Department of Visceral, Transplantation and Thoracic Surgery, Medical University Innsbruck, Innsbruck, Austria 6 IIIrd Medical Department for Hematology, Oncology, Hemostasiology, Infectious Diseases and Rheumatology, University Hospital Salzburg, Salzburg, Austria 7 Institute for Pathology, University Hospital Basel, Basel, Switzerland Background: A large group of interacting molecular factors are implicated in playing an important role in cancerogenesis and progression of non-small cell lung cancer (NSCLC). However, success concerning potential clinical correlations is still lacking. Combined analysis of a multitude of intertwined factors is currently a promising approach for gaining further insight. Methods: Cyclins (D1, D2, D3, and E), p21, p27, EGFR, Snail, E-cadherin, Beta-catenin, phosphatidylinositol-3’ kinase, phosphatase and tensin homologue, phosphorylated Akt, and phosphorylated signal transducer and activator of transcription-3 were analyzed by immunohistochemistry in 405 surgically resected NSCLC, using a standardized tissue microarray platform. The gene status of EGFR and cyclin D1 (CCND1) was examined by fluorescence in-situ hybridization (FISH). Extensive clinical data was acquired, enabling detailed clinicopathological correlation over a postoperative follow-up period of fifteen years. Results: Protein overexpression of nuclear p27, cyclin D3, E-cadherin and EGFR were all associated with a significant reduction of overall survival time. FISH analysis revealed frequent amplifications of CCND1 and EGFR, which correlated with protein overexpression. Cyclin D1 proved to be especially important, being the only molecular tumor-related factor with prognostic significance by multivariable analysis (hazard ratio: 1.577, 95% confidence interval: 1.090–2.282, p=0.016). Conclusions: The results emphasize that deregulation of controlling factors of the early G1 phase is of significant oncogenic relevance and may represent a potential treatment target in NSCLC.

Abstracts PO-115. Differentiated Dysplasia is a Frequent Precursor or Associated Lesion in Invasive Squamous Cell Carcinoma (SCC) of the Oropharynx R. Arsenic1, M.O. Kurrer2 1 Pathologisches Institut, Kantonsspital Aarau, Switzerland 2 Pathologie Institut Enge, Zurich, Switzerland

PO-117. Impact of CpG Island Methylator Phenotype (CIMP), O-6-Methylguanine-DNA Methyltransferase (MGMT) and Microsatellite Instability on Tumor Budding in Colorectal Cancer I. Zlobec1, M. Bihl1, A. Foerster1, A. Rufle1, L. Terracciano1, A. Lugli1 1 Institute for Pathology, University Hospital Basel, Basel, Switzerland

Background: Vulvar cancer ����������������������������������������������������������� precursor lesions are – more so than in uterine cervical cancer – related to either HPV or chronic inflammation and can manifest under the VIN paradigm or as differentiated dysplasia. Oral cavity cancer precusor lesions are etiologically more variable, related to smoking, HPV or chronic inflammation, but the spectrum of histological types of precursor lesions has so far not well been characterized. Methods: We reviewed the histological slides of all patients with oropharyngeal biopsies and subsequent resections specimens on file in the archives of the Department of Pathology of the Cantonal Hospital Aarau from 1988 until 2008. Results: Different basic and combined patterns of precursor lesions or SSC associated lesions were identified: Pleomorphic similar to severe bronchial dysplasia (19/152), basaloid stratified similar to anal basaloid dysplasia of AIN III (19/152), differentiated similar to lichenoid lesions with minimal basal cell layer irregularities (49/152), mixed pleomorphic and differentiated (35/152) as well as verrucous with open raisin like nuclei (5/152). In 25/152 no precusor lesion could be identified. Progression of isolated differentiated dysplasia was documented in 10% of patients (16/152) over variable time periods ranging from months to years. Conclusions: Differentiated dysplasia is a frequent precursor or associated in situ lesion in oropharyngeal SCC. Failure to recognise differentiated dysplasia results in underdiagnosis of a sizable proportion of patients at risk for invasive carcinoma. Our cases of documented progression of differentiated dysplasia call for efforts to refine criteria for separation of differentiated dysplasia from morphologically related lichenoid lesions.

Aim: Tumor budding is an established prognostic factor in colorectal cancer patients and infrequently observed in tumors with high-level microsatellite instability (MSI-H). Because of its strong association with MSI-H but negative effect on outcome, we hypothesized that high-level CpG Island Methylator Phenotype (CIMP-high) may be involved in the process of tumor budding. The aim of this study was therefore to determine the effect of CIMP on tumor budding in microsatellite stable (MSS/MSI-L) and MSI-H colorectal cancers. Methods: 127 patients were included in this study. MSI-H was defined as instability in ≥2 Bethesda-panel markers. CIMP-high was defined as methylation in ≥4/5 of the following loci: CACNA1G, CDKN2A, CRABP1, MLH1, and Neurog1. Mutations in KRAS (codons 12/13) and BRAFV600E and MGMT methylation were evaluated. Tumor budding was scored using CK22-stained whole tissue sections and defined as high-grade (HG-TB) at a threshold of 6 buds/HPF. Results: HG-TB was more frequent in MSS/MSI-L (83%) than MSI-H (59%) cancers (p=0.005), but was not associated with any other molecular correlates. In MSI-H cancers only, CDKN2A methylation was strongly predictive of HGTB (p=0.01; AUC=0.72; PPV=81%; NPV=61%), right-sided tumor location (p=0.009), advanced pT (p=0.022), vascular invasion (p=0.013), an infiltrating growth pattern (p=0.089) and more unfavourable survival time (p=0.052). Discussion: CDKN2A methylation is a predictive factor for tumor budding in MSI-H colorectal cancers and a negative prognostic factor in this subset of patients. These results suggest that CDKN2A gene silencing by promoter methylation in MSI-H cancers may play a critical role in the activation of a tumor budding phenotype.

PO-116. Small Bowel Adenocarcinomas Share a Molecular Profile with Colorectal Adenocarcinomas A. Riva1, R. Trezzi1, E. Zanellato1, M. Nucifora1, F. Molinari1, A. Bordoni2, A. Movilia3, R. Boldorini4, V. Bertolini5, P. Saletti6, L. Mazzucchelli1, M. Frattini1 1 Institute of Pathology, Locarno, Switzerland 2 Ticino Cancer Registry, Locarno, Switzerland 3 Department of Pathology, Civil Hospital, Legnano, Italy 4 Department of Medical Sciences, University School of Medicine, Novara, Italy 5 Department of Pathology, Multimedica, Milan, Italy 6 Oncology Institute of Southern Switzerland, Bellinzona, Switzerland Background: Primary adenocarcinomas of the small intestine are rare and show several epidemiological and pathological similarities with colorectal carcinomas leading to the hypothesis that these neoplasms might share the same genetic profile. To demonstrate this hypothesis we investigated the most important molecular markers involved in colorectal carcinogenesis in a cohort of small bowel adenocarcinomas. Methods: Thirty-nine primary sporadic non-ampullary adenocarcinomas of the small intestine were analyzed by microsatellite instability (MSI), gene sequences of K-Ras and TP53, and loss of heterozygosity (LOH) of chromosome 18q. Results: MSI was found in 9 cases (23%). K-Ras mutations were detected in 18 patients (46%); in particular, 17 mutations were found in exon 2 (including codons 12 and 13) and 1 in exon 3. We detected 14 TP53 mutations (36%), the majority of which occurred in exons 5, 6 or 7 (11 cases), while 2 mutations occurred in exon 8 and 1 in exon 4. Two patients showed a double point mutation in two different exons of TP53 gene. In 30 cases with microsatellite stable status, we observed K-Ras mutations in 13 (43%), TP53 mutation in 11 (36%) and LOH of chromosome 18q in 18 out of 24 evaluable patients (75%). Conclusions: The rate of molecular markers alterations in small intestine adenocarcinoma are similar to those reported in the literature for colorectal cancer. We conclude that small intestine adenocarcinomas and colorectal adenocarcinomas have similar genetic profiles.

PO-118. KRAS Mutational Analysis for Colorectal Carcinoma: a Practical Approach Using Allele-Specific PCR and Pyrosequencing R. Stockinger1, S. Spreitzer1, A. Bösl1, Z. Jasarevic1, U. Gruber-Mösenbacher1, F.A. Offner1 1 Institute of Pathology, University Teaching Hospital Feldkirch, Feldkirch, Austria Background: Metastasizing colorectal carcinomas (CRC) harbouring mutations of the KRAS-gene are resistant to EGFR-targeted treatment. Timely analysis of the patients tumor for the presence of KRAS mutations is thus a pre-requisite for efficient treatment. Methods: KRAS testing was established in August 2008 by allele-specific PCR (ASP) (DxS, TheraScreen®). Pyroseqencing (Pyromark Q24®) was introduced in 2010. Validation of the tests was confirmed by sucessful participation in the quality assurance programes of the German and European Societies of Pathology. Analyses are performed in every new CRC stage IIA or higher. Results: 221 CRC were analysed between August 2008 and Juli 2010 by ASP. DNA quality was sufficient in 214/221 samples (96,8%). The overal mutation rate was 44% (97/221). Mutations detected in codons 12 and 13 were as follows: Gly12Asp (36), Gly12Val (31), Gly13Asp (17), Gly12Cys (5), Gly12Ser (4), Gly12Ala (2) and Gly12Arg (1). Two separate mutations (Gly12Val) and (Gly12Asp) were detected in one case. 36 tumor samples were analyzed, both by ASP and pyrosequencing. Concordant results were obtained in 33/36 cases (91,6%). One case showed a mutation in codon 61 by pyroseqencing. In 1 case a mutant tumor cell clone was missed by pyroseqencing but detected by ASP. One Gly12Val mutation determined by ASP proved to be a GGT>TTT mutation by pyroseqencing. Conclusions: Pyrosequencing is our method of choice for samples with a tumor cell content (area/microdissected tissue) of >5%. It detects a broader range of mutations and is cheaper than ASP. ASP is reserved for samples with low tumor cell content.

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PO-119. Comparison of the Results Obtained Using Different Methods for the Analysis of Codons G12 and G13 of the KRAS Gene M.P. Bihl1, M.C. Andreozzi1, S. Hoeller1, A. Foerster1, A. Rufle1, L. Tornillo1, L. Terracciano1 1 Institute of Pathology, University of Basel, Basel, Switzerland Background: Testing for KRAS mutations is routinely done in patients with metastatic colorectal carcinomas, since studies have shown that patients presenting with KRAS mutations do not benefit from anti-Epithelial Growth Factor Receptor (EGFR) therapy. In this study we want to analyze three different molecular testing techniques (Dideoxy-sequencing, Pyro-sequencing and INFINITY®) and their impact on the results of KRAS mutation analysis. Methods: Hundred different samples from formalin fixed and paraffin embedded colorectal cancer tissues were selected. This cohort contained a wide spectrum of mutations (61 mutated samples with 8 different mutations and 39 wild type). DNA was extracted from each samples (concentration from 15 to 60 ng/mcl) and distributed to each of the three different methods. Results: The diagnostic for KRAS mutation status was very similar in all three methods (96–97% concordant results). Only the INFINITY® method was able to detect a mutation in one case. Unclear diagnostic results were observed in 4%, 3% and 1% of cases with Pyrosequencing, Dideoxysequencing and INFINITY®, respectively. A wrong diagnostic result was obtained by the INFINITY® and Dideoxysequencing method. Conclusions: All three methods are very well applicable for KRAS mutation status evaluation and the amount of non informative or wrong diagnostic results was very low. INFINITY® technology seems to be slightly more sensitive in samples with high amounts of non mutated cells. Further studies are needed to elucidate the power of each method in samples with low DNA concentration like cytology specimens or material gained from micro laser capture. PO-120. Loss of E-Cadherin Independently Predicts the Lymph Node Status in Colorectal Cancer E. Diamantis-Karamitopoulou1, I. Zlobec2, E.S. Patsouris1, G. Peros3, A. Lugli2 1 Department of Pathology, University of Athens, Athens, Greece 2 Institute of Pathology, University Hospital Basel, Basel, Switzerland 3 4th Department of Surgery, University of Athens, Athens, Greece Background: The identification of biomarkers that improve risk stratification in patients with colorectal cancer (CRC) is still a challenge. The objective of our study was to indentify independent protein markers as predictors of lymph node stage (N) in CRC. Methods: Tumor specimens from 221 CRC patients were mounted onto a multiple-punch tissue microarray and evaluated for 21 tumor-related and 1 hostrelated factors involved in CRC carcinogenesis, namely β-Catenin, E-Cadherin, EGFR, pERK, RHAMM, pAKT, TGF-b, pSMAD2, p21, p16, Bcl-2, Ki67, APAF-1, MST1, RKIP, VEGF, EphB2, MMP7, Laminin5γ2, MUC1, CDX2 as well as intra-tumoral and stromal CD8+ tumor infiltrating lymphocytes (iTILs and sTILs). Results: Node-positive cancers showed significant losses for p21 (p=0.026), Bcl-2 (p=0.027), APAF-1 (p=0.033), EphB2 (p=0.006), E-cadherin (p<0.001), RKIP (p=0.019), CD8+ iTILs and sTILs (p<0.001; p=0.008, respectively) and cytoplasmic MST1 (p=0.014). Based on the area under the receiver operating characteristic curve (AUC) EphB2, E-cadherin, iTILs and sTILS were indentified as potential predictors of N stage (AUC values >0.6), but only loss of Ecadherin was an independent predictor in multivariate analysis. Conclusions: E-Cadherin appears to be a strong predictor of N stage in CRC and should be considered in preoperative and postoperative management of colon and rectal cancer patients. PO-221. Merkel Cell Polyomavirus Infection in Non-Melanoma Skin Cancer K.D. Mertz1, A. Arnold2, M. Baumann1, N. Willi1, G. Cathomas1 1 Kantonales Institut für Pathologie, Liestal, Switzerland 2 Department of Dermatology, University Hospital Basel, Basel, Switzerland Background: Merkel cell polyomavirus (MCPyV) has been shown to be strongly associated with Merkel cell carcinoma. The analysis of other epidermal skin

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cancers, however, produced inconsistent results. We therefore analyzed the prevalence of MCPyV in squamous (SCC) and basal cell (BCC) carcinomas in immunocompetent and immunosuppressed patients. Methods: Formalin fixed, paraffin embedded tumor tissue was analyzed by MCPyV specific PCR and immunohistochemistry using antibodies against the viral large T antigen. Results: In normal skin samples, MCPyV DNA was detected by PCR in 13 (23.6%) of 55 samples. In BCC, MCPyV DNA was detected significantly more frequently (36 [41.0%] of 88; p<0.05). However, the detection rate did not reach significance if BCCs were stratified according to immunocompetence (28 [39.4%] of 71 immunocompetent BCC patients, 8 [47.0%] of 17 immunosuppressed BCC patients). No difference in the detection rate was observed in SSCs (21 [41.0%] of 75 SCC patients, including 4 [26.7%] out of 15 immunosuppressed patients). Immunohistochemistry showed a weak reactivity in 3 (5.3%) out of 57 PCR positive tumors (1 SCC and 2 BCCs), but it none of the PCR negative tumor samples. Conclusions: A positive PCR for MCPyV is common in epidermal skin cancers, however, only in BCC a significant difference to normal skin is observed. Using immunohistochemistry, expression of viral proteins can be detected only in a minority of tumors, arguing against an essential pathogenetic role in skin cancer development. PO-222. Triploidy in Abortion and Results in Fetal Autopsies A. Reiner-Concin1, R. Arnhold1, S. Sladek1, P. Hofmeister1, M. Mollik1, H. Farfeleder1 1 Pathologisch-Bakteriologisches Institut, Donauspital, Vienna, Austria Background: Triploidy is characterized by three sets of a haploid genome. Rarely such pregnancies reach the second trimester. Methods: Our genetic laboratory found triploidy in 107 cases (88 abortions, 13 chorionic villi, 6 amniotic fluids). Karyotype was 69,XXY in 74 and 69,XXX in 33 cases. Four pregnancies survived to second trimester. Results: Three fetuses with electively terminated pregnancy presented each with severe growth retardation, macrocephaly, hypertelorism, syndactyly between the third and fourth fingers, variably microgeny, micrognathia, rocker bottom feet and syndactyly of toes. Two showed complex cardiac malformations with atrial and ventricular septal defects, overriding aorta and truncus arteriosus communis. Variable other malformations affected intestine, kidney and lungs. The fourth fetus showed severe autolysis due to intrauterine death. But syndactyly between the third and fourth fingers and cleft palate could be proven. Placentas were hypoplastic in three cases and autolytic in one. None showed cystic changes. Umbilical cord was hypoplastic in one and had a solitary artery in two cases. FISH for X and Y was performed on all placentas and triploidy could be demonstrated. Karyotype twice each was 69,XXY and 69,XXX. Conclusions: We report autopsies of four cases with triploid karyotype. Besides a complex pattern of malformations all cases were related to type II phenotype (intrauterine growth retardation, relative macrocephaly and a small, noncystic placenta) according to McFadden. This type is described to be associated with digynic triploidy and may survive to later stages in pregnancy while diandric triploid pregnancies usually are aborted in the first trimester. PO-223. Assessment of Proliferation Fraction in Breast Cancer: Core Needle Biopsy Versus Excision Biopsy E. Obermann1, S. Eppenberger1, L. Bubendorf1, C. Tapia1 1 Institute for Pathology, University Hospital Basel, Basel, Switzerland Background: Proliferation fraction (PF) in breast cancer is important for the choice of treatment. However, its assessment is challenging since PF analysis lacks international standardizations. In this work we investigated PF on core needle biopsies (CNB) and on paired excision biopsies (EB) to answer the controversial question of the best tissue sample to analyze PF in a routine diagnostic setting. Methods: PF was analyzed prospectively by immunohistochemistry in matched CNB and EB of 50 patients with breast cancer. Two pathologists estimated the PF independently on the same slides. Results were grouped in three PF categories: low PF≤15%, moderate PF 16–35%, and high PF ≥36%.

Abstracts Results: Exact corresponding results were achieved by the two pathologists in 21 (42%) CNB and in 22 (44%) EB. Identical PF was detected in 24 (48%) CNB and their matched EB. Equal proliferation category were highly correlated (Chisquare = 47; P<0.001) with same results in 39 (78%) CNB and matched EB. Discrepant proliferation categories were mainly observed in CNB with low PF since 4 cases were up-graded from low PF in CNB to moderate PF in EB. Conclusions: For treatment considerations proliferation categories are important, however, there is a considerable inter-observer variability. If we categorize three PF groups the results from CNB and EB are significantly comparable (78%) indicating that CNB and EB are suitable to validate PF. However, if PF is low in CNB up-grade can occur in the matched EB. Therefore, low PF in CNB should be confirmed in EB. PO-224. Stem Cell Like Phenotype (Cd44+/Cd24-) in Breast Cancer P. H������ ä����� uptle1, A. Eppenberger1, U. G��� ü�� th2, K. Glatz1, C. Tapia1 1 Institut f������������������������������������������������ ü����������������������������������������������� r Pathologie, Universit������������������������ ä����������������������� tsspital Basel, Schweiz 2 Departement f������������������������������������������������������������������ ü����������������������������������������������������������������� r Gyn������������������������������������������������������������ ä����������������������������������������������������������� kologie und Geburtshilfe, Universit������������������������ ä����������������������� tsspital Basel, Schweiz Background: CD44+/CD24- tumor cells are considered cancer stem cells (CSC) being able of self-renewal and tumorigenicity. Therefore, CSC phenotype and especially CD44 is discussed as a potential new molecule for targeted therapy. We investigated the prevalence of CSC phenotype in a large series of different breast cancers and correlated it with the clinical-pathological parameters and survival data. Methods: We evaluated 1580 formalin fixed and paraffin embedded breast cancers using a tissue microarray (TMA). Analysis of protein expression of CD44, CD24, CK5/6 and EGFR was done by immunohistochemistry. Clinical data (e.g. survival, recurrence, therapies) as well as hormone receptor- and HER2status were available from previous studies. Results: Evaluable tissue spots showed the following distribution: 250/1079 (23%) breast cancers were CD44+, while 819/946 (87%) were CD24-, and the CSC phenotype (CD44+/CD24-) was seen in 173/1075 (11%) tumors. No correlation was found between the CSC and the histological subtypes, or with the tumor grading or nodal status. Breast cancer with CSC phenotype were significantly associated with the triple negative (p=0.002; ER-, PR-and her2-) and the basal-like (p=0.009; ER-/PR-/her2- but CK5/6 and/or EGFR+) phenotype. Kaplan-Mayer overall survival curves displayed better survival (p=0.007) for patients with CSC phenotype and worst survival for patients with triple negative (p<0.001) and basal-like (p=0.07) tumors. Conclusions: CSC phenotype was found to be present in 11% of the analyzed breast cancers and occurred mainly within the triple negative tumors. The survival data for the CSC patients must be confirmed with a larger CSC breast cancer patient’s cohort. PO-225. The Role of CANT1 and HNF3alpha in Prostate Carcinogenesis J. Gerhardt1, M. Montani1, C. Steinbrech1, F. Fritzsche1, V. Tischler1, T. Sulser2, C. Stephan3, K. Jung3, H. Moch1, G. Kristiansen1 1 Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland 2 Department of Urology, University Hospital Zurich, Zurich, Switzerland 3 Department of Urology, Charité Berlin, Berlin, Germany Background: The aim of this project was to characterize the tumorbiological function of three newly identified prostate cancer biomarkers and thus to contribute to the elucidation of molecular processes of prostate cancer progression. Methods: The overexpression of GOLPH2, HNF3alpha and CANT1 in tumor tissue compared to adjacent normal tissue was confirmed by immunohistochemistry in a large prostate cancer cohort (n=640). Proliferation and migration assays based on RNA interference were conducted to assess the influence of expression of the candidate proteins on in vitro tumourigenicity of two different prostate cancer cell lines, each expressing two or three of the target proteins endogenously. Results: GOLPH2 and CANT1 are significantly overexpressed in malignant glands, suggesting a diagnostic usage, HNF3alpha is increasingly upregulated during prostate cancer progression. Functionally, the proliferation and migration of LNCaP and PC-3 cells was reduced considerably by knockdown of

CANT1 as well as HNF3alpha, as shown in two independent assays for both tumour related cellular processes. GOLPH2 knockdown neither influences proliferation nor migration behaviour of LNCaP cells. Conclusions: These data clearly show a correlation between CANT1 and HNF3alpha knockdown and reduced in vitro tumourigenicity. Thus, we suggest a pro-tumorigenic function for CANT1 and HNF3alpha in prostate cancer. In future experiments the mechanism how CANT1 and HNF3alpha exert their effects on proliferation and migration will be elucidated to better understand their role during prostatic carcinogenesis. PO-226. Amplification and Overexpression of Vinculin are Associated with Increased Tumor Cell Proliferation and Progression in Advanced Prostate Cancer C. Ruic1, D.R. Holz2, M. Oeggerli1, S. Schneider1, I.M. Gonzales2, J. Kiefer2, T. Zell­ weger3, A. Bachmann4, S. Mousses2, M.T. Barrett2, D.O. Azorsa2, L. Bubendorf1 1 Institute for Pathology, University Hospital Basel, Basel, Switzerland 2 Pharmaceutical Genomics Division, Translational Genomics Research Institute, Scottsdale, USA 3 Division of Urology, St. Claraspital, Basel, Switzerland 4 Department of Urology, University Hospital Basel, Basel, Switzerland Background: Androgen withdrawal is the standard treatment for advanced prostate cancer. Although this therapy is initially effective, nearly all prostate cancers become refractory to it. Approximately 15% of these castration-resistant prostate cancers harbor a genomic amplification at 10q22. Methods: We applied array-CGH to cell lines harboring 10q22 amplification and subjected the genes located in this region to an RNAi screen. We selected genes with a significant growth reduction in the 10q22 amplified cell line PC-3 but not in the non-amplified 22rv1 cells as putative target genes of this amplicon. Candidate genes were further investigated by functional assays and on prostate cancer tissue microarrays. Results: We defined the common amplified region to a region of 5.8 Mb. The RNAi screen revealed vinculin as the most promising candidate gene. Immunohistochemical and FISH analysis revealed a strong correlation between 10q22 amplification and increased vinculin protein expression (p<0.001). Further analysis of 443 specimens from across all stages of prostate cancer progression showed that vinculin expression was highest in castration-resistant prostate cancers, but negative or very low in benign prostatic hyperplasia (p<0.0001). Additionally, high tumor cell proliferation measured by Ki67 expression was significantly associated with high vinculin expression (p<0.0001). Conclusions: Although there are countless reports on vinculin as a cytoskeletal protein, its protein expression or functional role in prostate cancer has previously not been investigated. Our data strongly suggest that vinculin is a major driving gene of the 10q22 amplification and that its overexpression might contribute to prostate cancer progression by enhancing tumor cell proliferation. PO-227. Immunohistochemical Expression of Interferon-Stimulated Genes in Hepatitis C: a Possible Predictive Role? L. Tornillo1, R. Brand1, M. Sarasin-Filippowicz2, M. Dill2, D. Baumhoer1, M. Heim2, L.M. Terracciano1 1 Institute of Pathology, University of Basel, Basel, Switzerland 2 Department of Biomedicine, University of Basel, Basel, Switzerland Background: The standard therapy for chronic hepatitis C is a combination of interferon (IFN) and Ribavirin. 40% of treated patients are nonresponders. IFN achieves its effects through the regulation of hundreds of IFN-stimulated genes (ISGs). Dysregulation of the expression of 29 genes, mainly ISGs, may correctly predict the response to therapy. Methods: 116 liver biopsies of 80 HCV-patients were taken at different times after the beginning of IFN-therapy (4, 16, 48, 96, 144 hours, 4, 8, 12 and 96 weeks). Immunohistochemistry for glypican 3 (GPC3), pSTAT1 and SOCS3 (ISGs), CD3, CD20, CD56 and CD68 was performed. The positive cells for each marker were counted and correlated with the response to IFN. Results: There was a significant difference in the number of GPC3+ cells (p<0.001) between responders and not-responders at 4, 12 and 96 weeks. No significant difference (p=0.619) was found before and after the therapy. By a cut-off of 60 GPC3+ cells, the response to therapy could be predicted in 84.1% Der Pathologe 6 · 2010 

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of the cases. An association between pSTAT1-positivity and activation of macrophages was seen (p<0.001). Conclusions: GPC3 IHC positivity was a strong predictor of the response to therapy. It is not clear how GPC3 may influence the response to IFN. It may be involved in the control of the receptor-ligand interactions or play a role in macrophage recruiting, as suggested by the dramatic increase of CD68+ cells in biopsies with high pSTAT1 count. It is possible that the determination of ISGs may help to identify responders to IFN therapy. PO-228. Expression of eIF3a in Gallbladder Carcinomas J. Haybaeck1, A. Matak1, R. Spilka2, T. Brunhuber2, C. Lackner1, P. Obrist2 1 Institute of Pathology, Medical University of Graz, Austria 2 Laboratory of Pathology, Hospital Zams, Austria Background: Gallbladder carcinoma (GBC) is the most common type of biliary tree carcinomas. GBC is an aggressive and often lethal cancer. It is usually diagnosed at advanced stages, with a 5-year survival rate <10%. eIF3a is the largest subunit of eukaryotic initiation complex eiF3, which regulates a subset of mRNA involved in regulation of cell cycle. eIF3a is up-regulated in many cancer types and suppression of eIF3a expression leads to regression of tumour cells in vitro.We investigated the expression of eIF3a in GBC with the intention to improve currently used prognostic markers of GBC. Methods: eIF3a expression was analyzed by immunohistochemistry and scored using paraffin embedded tissue from 74 GBC and compared with patient survival. In addition, expression of eIF3a mRNA and protein levels was analysed in three GBC cell lines using immunocytochemistry, western blotting and quantitative real-time PCR (qPCR). Results: High expression of eIF3a as assessed by immunohistochemistry was associated with poor prognosis as compared to GBC with lower expression. Elevated expression of eIF3a mRNA and protein was observed in GBC cell lines as compared to normal gallbladder mucosa. Conclusions: Overexpression of eIF3a in GBC indicates more aggressive tumor biology and is associated with lower 5-year survival. The mechanistic role of eIF3a in tumour progression can be studied using GBC cell lines.

Group 3: Posters on Display PP-129. Giant Hemosiderotic Dermatofibroma, a Case Report M. Pusztaszeri1, P.Y. Jaquet2, C. VVilliamson3 1 Department of Pathology, Geneva University Hospital, Geneva, Switzerland 2 General Practitioner, Bassins, Switzerland 3 Pathology and Cytology Laboratory, Unilabs-Cytopath, Geneva, Switzerland. Background: Dermatofibroma (DF) is a common benign fibrohistiocytic lesion that usually appears as a slow-growing firm dermal nodule with predilection for the legs of middle-aged women. They are usually smaller than 2 cm. Many histological variants have been described. Generally, the clinical and histological diagnosis is straightforward, but it can be difficult in atypical cases and rare variants such as giant DF. Methods: We report the case of a giant hemosiderotic DF. Results: A 54-year-old patient presented with a 5,1 cm left foot mass which was surgically excised. He recalled a previous pea-sized lesion which was present for 20 years and grew in size over the last eighteen months. Macroscopically, it was pedunculated, ulcerated, solid and heterogeneous with white and brown areas. Microscopically, it contained a dermal, ill-defined proliferation of elongated fibrohistiocytic cells. Hypocellular and collagenized areas alternated with more cellular and focally storiform areas. No significant nuclear atypia, mitotic activity or necrosis were found. There were giant multinucleated (Touton-like) cells and numerous.

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PP-130. Teleangiectasia Macularis Eruptiva Perstans – a Potential Pointer to the Diagnosis of Systemic Mastocytosis S. Spreitzer1, S. Dertinger1, N.S. Häring2, R. Strohal2, F.A. Offner1 1 Institute of Pathology, University Teaching Hospital Feldkirch, Feldkirch, Austria 2 Department of Dermatology, University Teaching Hospital Feldkirch, Feldkirch, Austria Background: Teleangiectasia macularis eruptiva perstans is a rare form of cutaneous mastocytosis and accounts for less than one percent of patients with mastocytosis. Methods: We report the case of a 59-year-old male patient who presented with a diffuse exanthema on the trunk and extremities with a duration of one year. He denied pruritus or flush symptoms. The primary clinical differential diagnosis were mycosis fungoides, lichen ruber and urticaria pigmentosa. Skin biopsies revealed mildly dilated thin-walled blood vessels in the superficial dermis and a minimal increase in the number of mast cells. This was confirmed by immunohistochemistry (IHC) using anti-CD117 antibodies. Consequent analyses of serum tryptase revealed a remarkable elevated level of >20 μg/l (normal value 0–11,4 μg/l). A subsequent bone marrow biopsy showed a compact infiltration by spindel-shaped CD117-expressing mast cells. The percentage was estimated to be 15–20% of the cellular bone marrow. A systemic mastocytosis was diagnosed. Molecular analysis showed a c-KIT D816 V mutation. Conclusions: Minimal increased mast cells around ectatic vessels, the typical findings of Teleangiectasia macularis eruptiva perstans, should alert to think of the possibility of an underlying systemic mastocytosis. PP-131. Her2 FISH: Beyond Guidelines V. Martin 1, U. Perriard1, A. Spitale2, L. Mazzucchelli1 1 Institute of Pathology, Locarno, Switzerland 2 Ticino Cancer Registry, Locarno, Switzerland Background: IHC is used as frontline assay to determine Her2 status in breast cancer (BC) patients. Most guidelines recommend that cases showing Her2 score 2+ should be analyzed by FISH to confirm eligibility for Trastuzumab therapy. The importance of external quality assurance in diagnostic testing for Her2 has been emphasized in many publications. By contrast, the clinical impact of an internal quality control system has been poorly investigated. Methods: We examined Her2 status by IHC (clone CB11, Novocastra) and FISH (PathVysion, Abbott) in 516 consecutive BC collected monthly on TMA (control series/conBC) and in 198 BC patients selected for diagnostic purpose (whole tissue sections/diaBC). Patients were stratified depending on staining (score 0,1+,2+,3+) or gene status (amplified/A, not amplified/notA or borderline/bor) according to the well established criteria. Results: Overall, by FISH, in the conBC series, 22% were A, 76% were notA and 2% were bor. In the diaBC cohort, 29% were A, 60% were not A, and 11% were bor. In both series Her2 status detected by FISH and IHC was significantly correlated (P<0.0001, Fisher’s exact Test), see tables. DiaBC and conBC FISH patterns distribution were overlapping, and revealed that a considerable subgroup of score 1+ patients exhibited Her2 gene amplification (14% and 19%, respectively). Conclusion: In our institution we recommend FISH analysis in score 1+ and score 2+ BC patients. In our experience, an internal quality control system is able to improve patients selection to targeted therapy, is cost effective, and have more clinical impact than acritical adherence to guidelines.

Tab. 1  zu PP-131 conBC (n=516)

FISH

IHC 0 1+ 2+ 3+ tot

347   34   22    1 404

notA

borA 97 % 79 % 69 %   1 %

5 1 2 0 8

A 1 % 2 % 6 % 0 %

   6    8    8   82 104

tot   2 % 19 % 25 % 99 %

358   43   32   83

Abstracts Tab. 2  zu PP-131 diabC (n=198)

FISH

IHC 0 1+ 2+ 3+ tot

  17   26   75    2 120

notA

borA 85 % 74 % 62 % 10 %

 2  4 16  0 22

A 10 % 12 % 13 %   0 %

 1  5 31 19 56

tot   5 % 14 % 25 % 90 %

  20   35 122   21

PP-132. Determination of the Her-2/Neu Gene Amplification Status in Cytologic Breast Cancer Specimens Using Automated Sish F.R. Fritzsche1, P.K. Bode1, H. Moch1, G. Kristiansen1, Z. Varga1, B. Bode1 1 Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland Background: Silver enhanced in situ hybridization (SISH) is a new emerging tool for the determination of the Her-2 status in breast cancer. SISH is technically comparable to Fluorescence in situ hybridization (FISH) but does not require a fluorescence microscope for its interpretation. Methods: While histological evaluations of SISH are promising, we aimed to evaluate its use for cytologic specimens. The Her-2 status as routinely determined by FISH was available for all 71 cases. Results: SISH signals in cytologic cell blocks and smear specimen were easy to evaluate. The Her-2 status, as determined by the Her-2/Chr17 SISH, was basically identical to the results of the Her-2 FISH. Interobserverconsistency was high (kappa = 0.972). Limits included the lack of tumor cells in cell blocks or smears and the distinction of in situ and invasive carcinomas. Conclusions: Her-2/Chr17 SISH is a useful and accurate method for the evaluation of the Her-2 gene status in cytologic breast cancer specimens. Advantages comprise signal permanency and result interpretation with conventional bright field microscopes. PP-133. So Called Pleomorphic Adenoma of the Breast; a Case Report M. Pusztaszeri1, C. Williamson2, J.M. Wenger3, M.F. Pelte1 1 Division of Clinical Pathology, Geneva University Hospital, Geneva, Switzerland 2 Pathology and Cytology Laboratory, Unilabs-Cytopath, Geneva, Switzerland 3 Gynecologist, Geneva, Switzerland Background: Breast pleomorphic adenoma (BPA) is a very rare benign lesion morphologically similar to salivary glands PA. Some authors consider BPA to be a form of intraductal papilloma with extensive cartilaginous metaplasia or a ductal adenoma variant, while others recognize it as a specific entity. Methods: Case report of a “so-called” BPA in a 56-year-old female with a retroareolar partially calcified nodule, which was assessed immunohistochemically. Results: Histology revealed a 1 cm intraductal papillomatous lesion, composed of mixed epithelial and stromal elements with various proportions and morphology. Epithelial cells (EC) formed papillary structures or glands associated with myoepithelial cells (MC). Different types (spindle, plasmocytoid and stellate) of MC were present with fibrous, chondro-myxoid and osseous areas, admixed with EC. There were transition zones between these elements, morphologically and immunohistologically. The immunohistochemical profile of MC was heterogeneous. They reacted positively for S-100 and smooth muscle alpha actin (SMAA), except plasmocytoid MC (SMAA negative and multikeratins positive). P63 was detected only in MC associated with EC. Estrogen and progesterone receptors were expressed partially in EC and in rare MC. Conclusions: Most BPA have been reported in the retroarealoar region and are thought to arise from the large ducts like central papillomas. This and the fact that adenomyoepithelioma, PA-like and ductal-like aspects can be seen in association with intraductal papillomas suggest that they are related entities. A gradual transition between morphologically different areas in BPA supports the hypothesis that it arises from a single cell type capable of divergent differentiation, probably an altered MC.

PP-134. A Rare Case of Photosensitive Trichothiodystrophy with Complex Cerebral Abnormalities S. Dertinger1, H. Schober2, J. L������ ü����� tschg2, F.A. Offner1 1� Department of Pathology, University Teaching Hospital, Feldkirch, Austria 2 Department of Pediatrics, University Teaching Hospital, Feldkirch, Austria Background: Trichothiodystrophy (TTD) is a rare ectodermal disorder with congenital hair dysplasia and autosomal recessive transmission. Tigerstripe pattern of hair under polarized light plus trichoschisis and low sulfur content of hair define the disorder. Most mutations are found in the ERCC2/XPD and ERCC/XPB gene, the first more common. In the case of photosensitivity the disease is typically associated with additional DNA repair defects which can be classified by distinct mutations. Methods: We performed an autopsy on a male baby in the age of 5 weeks, 5th child of non consanguineous parents from Chechenia. Index child died in 2004 with the age of 3 month (no autopsy) presenting identical phenotype: severe congenital dystrophy, ichthyosiform erythrodermia (collodion baby), microcephaly, cataract, puffy cheeks, Typical sparse and brittle hair as well as onychodystrophy led to clinical diagnosis of TTD. The child died after recurrent apnoe and bradykardic events with secondary aspiration. A DNRDNI procedure was agreed by the parents. Results: Autopsy affirmed cerebral abnormalities with pachygyria, hypoplasia of corpus callosum, cerebellum, vermis and showed histologically a cortical migration disorder. Death was due to bilateral bronchopneumonia. The diagnosis TTD was confirmed by the characteristic tiger-stripe pattern of hair under polarized light and reduced sulfur content of hair. The karyotype was normal (46,XY) and additional molecular analysis were made. Conclusions: TTD is a rare ectodermal disorder with a broad phenotype and several additional mutations, which led to a new classification of this disorder based on different gene mutations, which supports the need of genetic analysis in these cases even if diagnosis seems clear. PP-135. VACTERL-Association with Pulmonary Isomerism, Airway Stenosis and Anisosplenia: Overlap with Heterotaxy? T. Menter1, J. Schneider2, J. Benzing3, J. Hammer5, R. Glanzmann3, P. Miny4, I. Filges4, R. Röthlisberger6, A. Huber7, E. Bruder1 1 Department of Pathology, Hospital of the University of Basel, Basel, Switzerland 2 Department of Pediatric Radiology, University Children’s Hospital, UKBB, Basel, Switzerland 3 Division of Neonatology, University Children’s Hospital Basel, UKBB, Basel, Switzerland 4 Division of Medical Genetics and Department of Biomedicine, University Children’s Hospital Basel, UKBB, Basel, Switzerland 5 Division of Intensive Care and Pulmonology, University Children’s Hospital Basel, UKBB, Basel, Switzerland 6 Centre for Oncology and Hematology, Kantonsspital Aarau, Switzerland 7 Divison of laboratory medicine, Kantonsspital Aarau, Switzerland Background: VACTERL is a well-recognized association with major anomalies involving the vertebral bodies, anus, heart, trachea and/or esophagus, kidneys and the limbs. Anisosplenia and lung isomerism are elements of part of laterality defects. However, recently, over the past 13 years, 17 patients have been described with features overlapping between VACTERL association and X-linked heterotaxy. Methods: An autopsy was performed including extensive macroscopic and microscopic processing. The ZIC3 coding region was sequenced. A medline research on the key words VACTERL and heterotaxy was done. Results: We now report on an infant with multiple vertebral defects, atrial septal defect type II, tracheoesophageal fistula type C, tracheal and bronchial stenosis, alobar pulmonary isomerism, left pelvic kidney and anisosplenia suggesting an overlapping phenotype of malformations part of the VACTERL association and heterotaxy. Conclusions: To the best of our knowledge there are only 17 previous patients linking elements of VACTERL association and heterotaxy. Recent studies showing the implication of FOX transcription factor gene cluster also in lung lobation defects, the polyalanine expansion in the ZIC3 gene in a patient with symptoms of X-linked heterotaxy and VACTERL as well as studies of embryonic pathways challenge the view of VACTERL association as a distinct malforDer Pathologe 6 · 2010 

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mation entity. Our findings support a related or even common origin of clinical manifestations in heterotaxy and VACTERL association. PP-136. Growth Differentiation Factor 15 in Trophoblast Regulation K. Ornig1, F. Rimser1, R. Silye1 1 Department of Pathology, Nerve Clinic Linz, Austria Background: Growth differentiation factor 15 (GDF 15) is a member of the type ß-transforming growth factor (TGF-ß) superfamily. Members of this family are involved in regulating cellular functions like proliferation, apoptosis, extracellular matrix secretion, adhesion, terminal differentiation and development. GDF 15 was identified 1977 in macrophages called Macrophage inhibitory cytokine 1 (MIC 1) and simultaneously in the placenta – named placental transforming growth factor-ß. Serum GDF 15 concentrations are measurable through ELISA routinely, the marker has become attractive in several clinical settings, like sickle cell anaemia, prostatic cancer and during pregnancy, were MIC 1 serum concentrations were low weeks before miscarriage and discussed as a possible predictor for miscarriage. In 50 miscarriage specimens we analysed GDF 15 distribution in trophoblast cells, using a commercial available antibody. Methods: Miscarriage specimens were fixed in formalin for at least 12 hours. After routine pathological proceeding immunhistochemical staining was performed using a Histochom autostainer and GDF-15 antibody (Sigma Lifescience, HPA 011191). Results: GDF-15 is expressed in the syncytiotrophoblast in all specimens. The cytotrophoblast is negative in early villi. Cytotrophoblast proliferation, like in moles exhibit no GDF-15, this is high lightened by Ki 67 positivity. Conclusions: GDF-15 plays an essential role in trophoblast cell differentiation. Due to its apoptotic function trophoblast cell proliferation like in moles or in polar proliferations lack GDF-15, in contrast to the positive syncytiotrphoblast – a useful staining pattern in routine pathology. Factors influencing GDF-15 expression like hypoxia, inflammation are discussed. PP-137. An Unusual Uterine Tumor with Signet Ring Cell R. Sarro1, M. Fiche1, N. Ketterer2, J. Benhattar1, C. Achtari3, L. de Leval1 1 Institute of Pathology, 2 Multidisciplinary Oncology Centre 3 Department of Obstetrics and Gynecology, University Hospital, Lausanne, Switzerland Background: Lymphomas with signet ring cell features are rare, as is uterine dissemination of lymphomas. We report an exceptional case of a uterine tumor combining these two characteristics. Case and Methods: A 56-year-old female was diagnosed in 2004 with localized nodal grade 2 follicular lymphoma (Stage IA). She received local radiation therapy, experienced total remission, and did well until 2009 when a systematic CT scan evidenced a pelvic anterior-lateral mass. Total enlarged hysterectomy was performed. Results: The anterior uterine wall contained a 4.8 cm fish-flesh well delineated mass corresponding to a mostly diffuse and focally nodular proliferation of medium to large cells with extensive signet ring cell changes. Tumor cells were CD20, CD10, Bcl2, and Bcl6 positive with a low proliferation rate (<10–15%), CD21 underlined a focal follicular architecture. The vacuoles were PAS-negative and did not stain for immunoglobulin; ultrastructural analysis revealed non-specific degenerative vacuole. No lymph nodes were identified isolated from the surgical specimen. The tumor was considered as a secondary localization of the systemic follicular lymphoma though no signet ring cells were evidenced in the cervical lymph node biopsy (reviewed). Follow- up showed retroperitoneal tissue infiltration (PET-CT) and medullar normal biopsy. She recently started R-CHOP chemotherapy. Conclusion: This case illustrates both an unusual site of dissemination and challenging cytological characteristics in a follicular lymphoma. The signet ring cell changes challenged the adequate classification of this lymphoma as either a large B cell or a follicular B cell lymphoma.

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PP-138. Sclerosing Stromal Tumor of the Ovary: a Case Report J.C. Tille1, F. Sciotto1, P. Heymans2, M.F. Pelte1 1 Division of Clinical Pathology, 2 Department of Gynecology, University Hospital of Geneva, Geneva, Switzerland Background: Sclerosing stromal tumor (SST) is a rare benign typically unilateral sex cord-stromal ovarian tumor occurring in more than 80% in the second and third decades. Steröid hormones are usually not produced by SST. Methods: Pelvic ultrasound and MRI were performed in a 30 years-old woman with deep dyspareuny revealing a unilateral right ovarian mass of 6 cm with a prominent peripheral vascularization. Serum levels of CA-125, β-HCG, LDH and AFP were normal. Results: During laparoscopy the abdominal cavity, the uterus and the left ovary were normal. A right ovarectomy was performed showing a lobulated ovary with two gelatinous cysts and a white-yellow nodule. The tumor was pseudolobulated with hypocellular, oedematous or collagenous foci, as well as the prominent vasculature with hemangiopericytoma-like pattern. Cellular areas were heterogenous with vacuolated luteinized theca-like and spindle-shaped cells. Mitoses were not found. The tumor was diffusely immunostained with vimentin. Spindle-shaped cells reacted positively with smooth muscle actin and desmin, and theca-like cells with inhibin and calretinin. Conclusions: SST was first described in 1973 as a distinct subgroup within the thecoma-fibroma family of sex cord-stromal tumors. Accounting for less than 5% of the latter, SST caracteristically differentiates histologically and clinically (age, hormonally inactive) from both thecoma and fibroma. The differential diagnosis for SST includes sex cord-tumors (Sertoli cell tumor, granulosa cell tumor) and epithelial ovarian tumors. Clinical information (patient’s age, hormonal dosage), uni or bilateral involvement, histological pattern and immunohistochemistry are important for the diagnosis of an ovarian mass. PP-139. Ovarian Mucinous Borderline Tumor and Zollinger-Ellison Syndrome: a Case Report J.C. Tille1, O. Zimmer1, T. Jauch Klein2, P. Petignat2, M.F. Pelte1 1 Division of Clinical Pathology, 2 Department of Gynecology, University Hospital of Geneva, Geneva, Switzerland Background: Ovarian tumors may rarely cause a paraneoplastic syndrome. We report the case of a mucinous borderline ovarian tumor responsible of Zollinger-Ellison syndrome. Methods: Woman of 50 years-old consulted for secretory diarrhea up to 30 watery stools per day persisting for 6 months. The abdominal CT-scan discovered a large complex left adnexal mass with no other abnormalities in the gastroenteropancreatic tract. Blood test showed elevated gastrin to 687 pg/ml (norm <115) and a high chromogranin 800 ug/L (norm <96) compatible with Zollinger-Ellison syndrome. A left adnexectomy was performed. Results: Macroscopically the adnexectomy comprised an ovary of 20 cm with a smooth surface. The ovary was multicystic, with mucinous material and solid areas. Microscopically cysts and glands were lined by a stratified epithelium of intestinal type with goblet cells. Focally nuclear atypia were marked with a glandular architectural complexity. No stromal invasion was found. Gastrin was immunohistochemically detected in some cells of the borderline component. The staging was completed by a hysterectomy, right adnexectomy, appendectomy and biopsies. The blood test showed a postoperative normalization of gastrin and chromogranin. Conclusions: In general, the gastrin-secreting tumors are located in the gastroenteropancreatic tract. Argyrophil cells have been identified in ovarian mucinous, serous, endometriöid and Brenner tumors. Many hormone peptides can be localized in these cells as gastrin, ACTH, somatostatin but only a small number had clinical manifestations. The reported cases of ovarian tumors responsible of Zollinger-Ellison syndrome are rare and only described in mucinous tumors comprising benign, borderline and carcinoma.

Abstracts PP-140. ADAM12 is Overexpressed in Prostate Cancer and Correlates with Clinicopathologic Parameters V. Tischler1, M. Kveiborg2, P.J. Wild1, M. Beer1, K. Ikenberg1, A. Mortezavi3, I. Hofmann1, T. Hermanns3, H. Moch1, G. Kristiansen1 1 Institute of Surgical Pathology, University Hospital Zurich, Zurich 2 Department of Biomedical Sciences & Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark 3 Department of Urology, University Hospital Zurich, Zurich, Switzerland Background: In a mouse model of prostate cancer, the disintegrin and metalloprotease ADAM12 is upregulated in carcinoma-associated stroma and is involved in tumour progression. In this study, we analyzed the expression of ADAM12 in a large cohort of human prostate cancers. Methods: Tissue microarray with 502 clinically characterized prostate tissues, semiquantitative scoring of ADAM12 immunohistochemical staining, correlation with clinicopathological parameters including relapse-free survival. Results: High cytoplasmic ADAM12 expression was observed in 92% of prostate cancer cases whereas benign prostate glands showed high ADAM12 in 46%. High prostate carcinoma-associated stroma expression of ADAM12 was found in 54% cases versus 6% high ADAM12 expression in stroma surrounding normal prostate glands. A high cytoplasmic ADAM12 expression was correlated with higher pT stage, larger tumour size, higher pre-operative PSA and high stromal ADAM12 expression. High ADAM12 was associated with shortened relapse-free survival (p<0.05). Conclusions: ADAM12 is overexpressed in prostate cancer and might play a role in tumour progression. PP 141. High Cytoplasmic Cyclin D1 Expression in Lymph Node Metastases from Prostate Cancer Independently Predicts Early Biochemical Failure and Death in Surgically Treated Patients C. Rocha1, N. Saxer-Sekulik1, I. Zlobec2, G. Sauter3 , G. Thalmann4, A. Fleischmann1 1 Institute of Pathology, University of Bern, Switzerland 2 Institute of Pathology, University of Basel, Switzerland 3 Institute of Pathology, University of Hamburg, Germany 4 Department of Urology, University of Bern, Switzerland Background: Prognostic factors in nodal positive prostate cancer are poorly defined. Cyclin D1 is expressed in prostate cancer cells and might be prognostically relevant. Methods: Nuclear and cytoplasmic Cyclin D1 expression was evaluated in 119 nodal positive prostate cancer patients undergoing radical prostatectomy and extended lymphadenectomy. Cyclin D1 was correlated with various tumor features and biochemical recurrence-free (bRFS), disease-specific (DSS) and overall (OS) survival. Results: In the metastases, high cytoplasmic Cyclin D1 expression predicted independently poor outcome (5-year bRFS: 12,5% vs. 26.4% p=0.006; 5-year DSS: 56.3% vs. 80.7%, p=0.007; 5-year OS: 56.3% vs. 78.7%, p=0.011). These patients had a 2.62 fold elevated risk of dying compared to patients with low cytoplasmic Cyclin D1 expression (p=0.031). All other sub-cellular compartments of Cyclin D1 expression in primary tumors and metastases were prognostically insignificant. Conclusions: The sub-cellular location of Cyclin D1 expression in prostate cancer is linked to specific clinical courses. Survival stratification according to biomarker expression in metastases indicates an important role for tumor sampling from these tissues.

PP-142. Androgen Receptors are Differentially Expressed in Gleason Patterns of Prostate Cancer and Down-Regulated in Matched Lymph Node Metastases A. Fleischmann1, S. Schobinger1, R. Seiler1, G.N. Thalmann2, C. Rocha 1 Institute of Pathology and 2 Department of Urology, University of Bern, Switzerland Background: Androgen receptor (AR) expression profile in the different Gleason patterns (GP) of primary prostate cancers and nodal metastases is unknown. More information about AR distribution is needed to optimize evaluation methods and to better understand the role of AR in development and progression of prostate cancer. Methods: A tissue microarray (TMA) was constructed from 119 hormone-naïve nodal positive, surgically treated prostate cancers containing tissues from all GP present in every primary tumor and the matched metastases. ARs were evaluated immunohistochemically and an expression score (intensity x percentage of positive cells) was assigned for each tissue spot. Results: ARs were up-regulated in primary tumors compared to normal glands and significantly different expressed in the GP (mean AR scores: GP3=128.7, GP4=159.1, GP5=123.5; p=0.016). A similar expression profile was observed in metastases, however, on significantly (p<0.001) lower level (mean AR scores: GP3=70.5, GP4=90.4, GP5=71.7; p=0.114). High AR expression in metastases was associated with larger total size of metastases (p=0.008). All other correlations of AR expression in primary tumors and metastases with quantitative (age, prostate cancer volume, number of metastases) or categorical (tumor stage, Gleason score of the primary tumor and metastases) tumor characteristics or with survival were insignificant. Conclusion: ARs are differentially expressed in GP what should be considered in prognostic models which include AR. In nodal metastases, ARs are significantly down-regulated suggesting decreased dependence on androgens already under hormone-naïve conditions. AR expression level is not prognostic in nodal positive disease. PP-143. Protein-1 Helps to Identify Transplant Glomerulopathy in Kidney Transplant Biopsies J. Radke1, S. Schaub2, U. D�������� ü������� rm����� ü���� ller1, M.J. Mihatsch1, H. Hopfer1 1 Pathology, 2 Clinic of Transplantation Immunology and Nephrology, University Hospital Basel, Switzerland Background: Transplantat glomerulopathy (TG) is a progressive form of chronic, antibody-mediated kidney transplantat rejection that leads to dysfunction, graft loss, and reduced survival. Early phases of TG are often difficult to diagnose because of their focal and segmental distribution. The glomerular expression of Plasmalemmal Vesicle-Associated Protein-1 (PV-1) has been suggested as an ancillary tool in the diagnosis of TG. Methods: Double immunofluorescence staining of PV-1 and CD34 was performed on cryo sections of transplant biopsies with TG and compared to scheduled 6 months control biopsies without relevant pathological findings and late indication biopsies without glomerular pathology. Photographs of all glomeruli were scored as negative, segmentally or globally positive. Image analysis of glomeruli was performed to obtain the area of each glomerular cross section, the PV-1 and CD34 positive area. Results: Using a cut off of 0.01 PV-1+ area/glomerular area 20/24 (83.3%) cases with TG were identified. 18 of these cases had at least one globally affected glomerulus. In contrast, only 1/18 (5,6%) protocol biopsies showed PV-1 positivity above the cut off. 6/32 (18.8%) late indication biopsies without glomerular pathology were PV-1 positive. Interestingly, 1 of these cases developed TG and additional 3 graft failure within 6–45 month of follow-up compared to only 1/26 graft losses in the PV-1 negative group. Conclusions: PV-1 sensitively indicates TG and may be helpful in the detection of early phases of TG. Furthermore, PV-1+ without established TG may have the potential to serve as a prognostic marker.

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PP-144. Endoplasmic Reticulum Stress-Induced Enteritis in Xbp1-Deficient Mice is Dependent on NfκB Signaling L. Niederreiter1*, T. Adolph1*, F.A. Offner2, T. Fritz1, A.R. Moschen1, B. Enrich1, E. Sarcevic2, N.C. Kaneider3, A.H. Lee4, L.H. Glimcher4, H. Tilg1,3, R.S. Blumberg5†, A. Kaser1† 1 Dept of Medicine II, Innsbruck Medical University, Innsbruck, Austria 2 Dept of Pathology, Academic Teaching Hospital Feldkirch, Feldkirch, Austria 3 Christian-Doppler Research Laboratory for Gut Inflammation, Innsbruck Medical University, Innsbruck, Austria 4 Dept of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA 5 Div of Gastroenterology, Hepatology, and Endoscopy, Dept of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA * contributed equally; † shared senior authorship Background: The IRE1/XBP1 pathway allows cells to cope with endoplasmic reticulum (ER) stress. Conditional deletion of Xbp1 in intestinal epithelial cells (IECs) results in murine enteritis, and XBP1 is a genetic risk factor for inflammatory bowel disease (IBD). Xbp1/ epithelia deplete Paneth cells and exhibit a pro-inflammatory phenotype via JNK. We hypothesized that hypomorphic XBP1 might overactivate NFκB via IRE1. Methods: The IEC line MODE-K was silenced for XBP1 or additionally for IRE1α and stimulated with TNFα. Phosphorylation status of IKKs, IκBα, and NFκB were analyzed, as well as DNA binding activity to the NFκB consensus sequence and IκBα mRNA levels by qPCR. Xbp1flox/flox.Villin-Cre-ERT2 mice were treated with the IKK2 inhibitor BAY11–7082 or vehicle, enteritis prevalence determined and IHC for several targets performed. Results: TNFα-stimulated XBP1-silenced MODE-Ks exhibited increased phosphorylation of IKKs, IkBα, and nuclear NFκB p65, along with increased NFκB DNA binding activity and IκBα mRNA expression, which upon co-silencing for IRE1α was restored to normal. Treatment of Xbp1flox/flox.Villin-Cre-ERT2 mice with BAY11–7082 reduced enteritis prevalence from 83% to 25% (p=0.0801). Hyperproliferation (BrdU) of the epithelium, increased apoptosis (Tunel) and loss of Paneth cells (Lysozyme) in XBP1-hypomorphic mice could be prevented by BAY11-7082. Conclusions: Unresolved ER stress in IECs due to XBP1 hypofunction overactivates the NFκB pathway, secondary to hyperactivated IRE1. Enteritis development appears dependent on NFκB overactivation and can be prevented by IKK2 blockade, which may open novel avenues for the treatment of IBD interfering immediately downstream of a genetic risk factor. PP-145. RAS, BRAF and PIK3CA are Rarely Mutated in Hepatocellular Carcinoma D. Romanelli1, M. Nucifora1, M. Bolli2, A. Riva1, E. Zanellato1, J. Barizzi1, P. Saletti3, L. Mazzucchelli1, M. Frattini1 1 Institute of Pathology, Locarno, Switzerland 2 Department of Surgery, San Giovanni Hospital, Bellinzona, Switzerland 3 Oncology Institute of Southern Switzerland, Bellinzona, Switzerland Background: Despite major efforts to improve outcome of patients with advanced hepatocellular carcinoma (HCC), therapeutic options remain limited. Recently, sorafenib, a multikinase inhibitor targeting the RAS-RAF pathway, has proven to increase overall survival in this setting as compared with best supportive care. We investigated deregulations of both, RAS-RAF-MAPK and the cross-linked PI3K pathways, to clarify the putative role of these alterations in carcinogenesis, possibly helping in defining predictive markers of response to sorafenib. Methods: Fifty-one primary resected HCC were analyzed. By using direct sequencing, we searched for KRAS, HRAS and NRAS mutations in exons 2 and 3, for BRAF mutations in exon 15 and for PIK3CA mutations in exons 9 and 20, which include hotspot codons where the large majority of oncogenic mutations occur. Results: All patients were analysable for all the molecular markers. No mutations in KRAS, HRAS, BRAF and PIK3CA were observed. We found a point mutation in codon 27 of NRAS gene, which involved the first base of the codon leading to the H27Y change (CAC-tAC, His-Tyr). Conclusions: Consistently with data reported in the literature, in our series we confirm that RAS, BRAF and PIK3CA genes are rarely mutated in HCC, sug-

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gesting therefore that these genes do not directly play a role in carcinogenesis. It remains to be clarified whether these pathways can be deregulated through other mechanisms, involving for example receptor tyrosine kinases upstream MAPK and PI3K axes. PP-146. Comparative Study of Immunopathological Mechanisms Operative in Patients with Collagenous Colitis, Ulcerative Colitis and Non-Inflamed, Normal Colon V. Genitsch1, D. Kassahn1, A. Johner1, C. Mueller1 1 Institute of Pathology, University of Bern, Bern, Switzerland Background: The pathogenetic relationship of collagenous colitis (CC) and inflammatory bowel diseases, particularly, with ulcerative colitis (UC) continues to be debated. Methods: We compared the expression of genes related to macrophages, T-lymphocytes and intestinal epithelial cells from CC, UC and healthy control (HC) patients (N=10, each), using RNA isolated from tissue sections of formalinfixed, paraffin-embedded colonic biopsies for subsequent qRT-PCR. Results: In UC the expression of the T-cell lineage-specific transcription factors t-bet and GATA3 is increased compared to HC and CC, indicating a higher frequency of effector CD4+ T-cells in UC. Furthermore, the expression of TGFβ1 and TGFβ3 is exclusively increased in UC and is associated with elevated FoxP3 expression, suggesting a counter-regulation to chronic inflammation through an increased local accumulation of Treg’s in UC, but not in CC. TREM1 mRNA is markedly increased in UC, but almost completely absent in CC and HC, whereas TREM2 mRNA expression is comparably low in UC and CC. This may indicate differential macrophage differentiation (M1vs.M2). Transcription of the cytokine TSLP, which is preferentially expressed in intestinal epithelium, is reduced in CC in comparison to HC. Conclusions: The established methodology is suitable to determine distinct gene expression profiles using paraffin embedded tissues. Preliminary data indicate differences in the gene expression patterns in the myeloid, epithelial and T-cell compartment in the colonic mucosa of UC, CC, and HC. Hence, the analysis on colonic tissue sections by laser capture microdissection may allow to define distinct, cell type-specific pathogenetic pathways operative in CC, UC and HC. PP-147. Intravascular Methylene Blue Injection Improves Lymph Node Detection in Neoadjuvant Treated Rectal Cancers S. Stadlmann1, M. Trippel1, D. Lenggenhager1, A. Keerl2, T. Kocher2, G. Singer1 1 Institute of Pathology, 2 Department of Surgery, Kantonsspital Baden AG, Switzerland Background: Lymph node assessment is an essential part of staging in colorectal cancer in general and crucial for prognosis and therapy. Lymph nodes smaller than 5 mm are especially important as they carry the majority of metastases. We performed a study comparing the lymph node harvest in neoadjuvant treated rectal cancers with and without methylene blue staining. Methods: The study cohort included 14 patients with rectal cancers treated by neoadjuvant radiochemotherapy. Methylene blue injection into the superior rectal artery was performed in 7 cases. 10/14 (71.4%) patients were male and 4/10 (28.6%) were female. Median age was 63.5 years (range 46–76 years). 5/13 (38.5%) had already hepatic metastasis at time of diagnosis (1 had no clinical staging information). Results: Lymph node harvest in the methylene blue-stained and unstained resections was 51.9±12.2 and 40.4±17.3, respectively. Most examined lymph nodes measured <5 mm, with a significantly higher number of small lymph nodes detected in the methylene blue-stained group (38.9±10.7 vs 27.7±12.1; P=0.027). Lymph node metastases were found in 7 cases (2 in the methylene blue-stained group and 5 in the unstained group). Isolated tumor cells were not detected. Conclusions: Methylene blue injection technique improves harvest of in particular smaller lymph nodes in neoadjuvant treated rectal cancers. A study including a higher number of cases may improve lymph node staging.

Abstracts PP-148. Clofazimine Induced Peritonitis – a Confusing Finding for Surgeons and Pathologists B.M. Aberer1, S. Dertinger1, E. Wenzl2, F.A. Offner1 1 Department of Pathology, University Teaching Hospital, Feldkirch, Austria 2 Department of General and Thoracic Surgery, University Teaching Hospital, Feldkirch, Austria Background: Clofazimine is a highly lipophilic riminophenazine dye used for the treatment of mycobacterial infections as well as Melkerson-Rosenthal syndrome. Methods: We report a 69-year old female patient with a previous history of tuberculosis and a Melkerson-Rosenthal syndrome who was treated with clofazimine. Treatment was started at a dose of 100 mg/day in 1998 and continued intermittently. 11 years after initiation of treatment the patient presented with acute abdominal pain. Explorative laparatomy did not confirm cholecystitis as suspected by MRI, but revealed unexpected black discolorations of the peritoneum. Results: Frozen sections of the peritoneum revealed accumulations of foamy macrophages under the surface mesothelium. Malignancy was excluded. Under polarized light the frozen tissue revealed numerous bright-red crystals in the cytoplasms of the macrophages. The lipophilic crystals became invisible after dehydration. The patient developed pneumonia and died one week after laparotomy due to sepsis. Work-up of autopsy tissue and previous biopsy specimens revealed depositions of clofazimine in the intestinal wall. Conclusion: Fifteen cases of clofazimine enteropathy and two cases of clofazimine peritonitis have been reported in the world literature – mainly in Asia and South America. Over 70% of these patients underwent exploratory laparotomy for acute abdomen, suspected subacute intestinal obstruction or perforated duodenal ulcer. A proper pre-operative diagnosis may be extremely difficult and depends on the respective patient history and the detailed analysis of frozen tissue. PP-149. Extensive Characterization of EGFR Pathways May Help in Integrating the Use of EGFR-Targeted Therapies in Patients with Squamous Cell Anal Cancer E. Zanellato1, V. Martin1, F. Molinari1, S. Crippa1, S. De Dosso2, A. Franzetti-Pellanda2, A. Mobilia3, A. Assi3, R. Boldorini4, A. Paganotti4, L. Deantonio5, L. Mazzucchelli1, P. Saletti2, M. Frattini1 1 Institute of Pathology, Locarno, Switzerland 2 Oncology Institute of Southern Switzerland, Bellinzona, Switzerland 3 Department of Pathology, Civil Hospital, Legnano, Italy 4 Department of Medical Sciences, University School of Medicine Novara, Italy 5 Radiotherapy Unit, University School of Medicine Novara, Italy Background: Chemo-radiation is the standard treatment for local squamous cell anal cancer (SCAC). In locally advanced head and neck squamous cell carcinomas, monoclonal antibodies (MoAbs) targeting EGFR have proven to improve survival in combination with radiotherapy. In colorectal cancer, patients benefit from anti-EGFR MoAbs in presence of EGFR gene copy number gain (CNG) and absence of mutations in EGFR downstream members (except for PIK3CA exon 9 mutations). We investigated the EGFR pathways in SCAC, to evaluate whether anti-EGFR MoAbs can be integrated in the management of this disease. Methods: Thirty-five SCAC biopsies were collected in the Departments of Pathology in Locarno, Legnano and Novara. EGFR gene status was assessed by fluorescent in-situ hybridization, whilst K-Ras, BRAF and PIK3CA mutations by direct sequencing. Results: EGFR CNG was observed in 5 (20%) cases. No BRAF mutations were detected. K-Ras was mutated in 1 (3%) case, PIK3CA in 8 (23%); 6 mutations in exon 9, 2 in exon 20). EGFR CNG was concomitant either to K-Ras or PIK3CA exon 9 mutations (in 1 case each). The 3 remaining EGFR CNG cases did not show other EGFR downstream signal alterations. Overall 4/36 (11%) SCAC patients might have a proficient pattern for anti-EGFR MoAbs treatment. Conclusions: EGFR gene deregulation, K-Ras and PIK3CA mutations are involved in SCAC carcinogenesis.. Clinical trials involving anti-EGFR MoAbs in this disease should consider analysis of predictive factors to identify patients who might benefit from targeted therapies.

PP-150. T-PTLD in a 73 Years Old Patient 21 Years after Heart Trans­ plantation Ph. Niederberger1, E. Mueller-Garamv����� ö���� lgyi1, A.M. Schmitt1 1 Institute of Pathology, University of Bern, Bern, Switzerland Background: A 73 years old patient who had undergone heart transplantation 21 years before was hospitalized due to worsening of the general condition and apathy. On the emergency department the patient was found in an acute respiratory insufficiency and tachycardy. The blood test results revealed leucopenia and thrombopenia which had not been known a month before and the reason of which remained unclear. The patient died 8 days after hospital admission in respiratory insufficiency. Methods: An autopsy with histological examination of the internal organs including bone marrow was performed. Tissue sections of the bone marrow were stained with monoclonal antibodies directed against B and T cell antigens. In situ hybridization for EBV in the lungs and bone marrow was performed. Clonal rearrangement of the gamma chain of the T cell receptor was examined by PCR. Results: The bone marrow showed a replacement of normal hematopoiesis by a peripheral T cell lymphoma, NOS. Clonal rearrangement of the gamma chain of the T cell receptor was proven by PCR. A subset of the neoplastic cells were positive for EBV in an EBER in situ hybridization. Sparse lymphoma infiltrates were also found in the lungs. Due to thrombocytopenia bilateral subdural hematoma had developed. Conclusions: 10–15% of PTLD are of T cell lineage, around 30% of which are associated with EBV. In adult recipients of heart trans­plantation, PTLD has been reported to occur in 1–6.3%. Immunosuppression during 21 years due to heart transplantation in this patient led to a peripheral T cell lymphoma, NOS with lethal outcome. P-151. No Evidence for C-MAF Gene Alterations In Angioimmunoblastic T-Cell Lymphoma: a FISH-Based Study B. Bisig1,2, C. Thielen2, S. Gofflot2, C. Herens3, J. Boniver1,2, P. Gaulard4, L. de Leval1,2,5 1 Department of Pathology, CHU Sart Tilman, University of Liège, Liège, Belgium 2 Laboratory of Experimental Pathology, Groupe Interdisciplinaire de Génoprotéomique Appliquée (GIGA) Research, University of Liège, Liège, Belgium 3 Department of Human Genetics, CHU Sart Tilman, University of Liège, Liège, Belgium 4 Department of Pathology and Inserm U955, Hôpital Henri Mondor, Créteil, France 5 Institute of Pathology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland Background: The molecular alterations underlying angioimmunoblastic T-cell lymphoma (AITL), one of the most common peripheral T-cell lymphomas (PTCLs), are largely unknown. The c-Maf transcription factor is expressed in follicular helper T cells, the normal counterpart of AITL, and reportedly mice transgenic for c-MAF develop T-cell lymphomas. Thus, we hypothesized that cMAF could be deregulated in AITL as a consequence of gene rearrangements. Methods: Fluorescence in situ hybridization (FISH) assays were conducted on tissue microarrays comprising 48 AITLs and 45 PTCLs not otherwise specified (NOS), using a home-made break-apart probe for c-MAF @ 16q and centromeric probes. FISH results were correlated to c-Maf expression studied by immunohistochemistry. Results: A normal hybridization pattern for c-MAF was observed in all 48 AITLs. Extra copies (up to five) of non-rearranged c-MAF were identified in 13/45 PTCLs NOS (29%), owing to polysomy 16 or polyploidy. Immunohistochemistry demonstrated c-Maf expression in 29/35 AITLs (83%) and 6/27 PTCLs NOS (22%). Among PTCLs NOS, no significant association was identified between c-Maf expression and extra copies of c-MAF locus (p=0.29). Conclusions: Nuclear expression of c-Maf is detected in the majority of AITLs, while it is absent in most PTCLs NOS. In AITLs, no structural or numerical aberrations are observed at the c-MAF locus. Extra copies of c-MAF, identified in a subset of PTCLs NOS, are due to polysomy 16 or polyploidy and do not correlate with c-Maf expression.

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PP-152. EBV+ Cutaneous Diffuse Large B Cell Lymphoma of the Leg in an Elderly Patient with Mycosis Fungoides and Methotrexate Treatment T. Rausch1, A. Cairoli2, J. Benhattar1, D. Hohl3, L. de Leval1 1 Institut Universitaire de Pathologie, 2 Service et laboratoire central d’Hématologie, 3 Service de Dermatologie, Centre Hospitalier Universitaire Vaudois (CHUV) et Université de Lausanne, Switzerland Background: Diffuse large B cell lymphoma, the most frequent lymphoma in adults, is a heterogeneous disease comprising several subtypes and entities defined by a combination of clinical and pathological features. EBV infection of the neoplastic cells is a typical feature of several entities known to occur in a context of immune suppression, and of a new entity, namely “EBV+ DLBCL of the elderly”. Case Report: We present a case of a 77-year-old man who had been diagnosed with Mycosis Fungoides since 2006 and had received several lines of therapy including courses of IV methotrexate (MTX) for the past two years. In November 2009, he developed an ulcerated nodule on the leg. Biopsy revealed diffuse sheets of EBV+ large B-cells (CD20+ CD30 +/- CD10- MUM1+ IgM Lambda), with an angiocentric distribution. Monoclonal IGH gene rearrangement was demonstrated by PCR. No evidence of MF was found. Discussion: Although the pathological features were clearly diagnostic for an EBV-positive DLBCL, several possibilities could be considered for assignment to a specific subtype/entity: EBV-positive DLBCL of the elderly, methotrexateinduced lymphoproliferative disorder (LPD), Lymphomatoid Granulomatosis. The occurrence of EBV+ LPD has been reported in three other patients with MF under MTX, the leg is one of these and the current case may question the relatedness to so-called “Primary cutaneous DLCBL leg type”. PP-153. Comparison Between the SIAPEC-IAP and the Bethesda Systems for Reporting Thyroid Cytopathology: Experience in Two Hospitals S. Crippa1, M. Bongiovanni1 1 Institute of Pathology, Locarno, Switzerland Background: The 5-tiered SIAPEC-IAP thyroid FNA System and the new 6tiered Bethesda thyroid FNA System offer two approaches to the problem of reporting thyroid FNA results. In this study, we present the combined experience from our institutions for reporting thyroid FNAs using these two different systems and evaluate their efficacy based upon surgical follow up. Methods: Data on thyroid FNAs and their corresponding surgical specimens were collected at our two institutions over a two-year period. We compared the sensitivity and specificity for each of the component groups within the 2 systems where a diagnosis lead to surgery (CAT III, IV, V, VI and TIR 3, 4, 5 versus benign). Results:

Tab.  zu PP-153 SIAPEC-IAP system Categor y TIR 1 TIR 2

FNA

%

Malignant

Benign

34 306

8.8 79.3

2 2

4 24

TIR 3 TIR 4 TIR 5 TOTAL

24 6 16 386

6.2 1.6 4.1

3 5 14 26 (37 %)

14 1 1 44 (63 %)

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Bethesda system Categor FNA y CAT I 31 CAT II 156 CAT III 4 CAT IV 79 CAT V 13 CAT VI 11 TOTAL 294

%

Malignant

Benign

10.5 53 1.4 27 4.4 3.7

0 2 / 5 4 9 20 (28 %)

4 15 / 28 4 0 51 (72 %)

The sensitivity for TIR 3, 4, 5 and for CAT III, IV, V, VI were almost equal in the two reporting systems, SIAPEC-IAP and Bethesda (91,6% vs 90%), while specificity was higher in the British (60% vs 31,9%). Interestingly, the number of cases in the TIR 3 and CAT IV categories (indeterminate/suspicious) differed significantly between the two reporting systems (6,2 and 27%). For these two categories, the rate of malignancy on surgical excision is similar (21% and 18%), with a PPV of 17% and 15% respectively. Conclusions: The two reporting systems, the SIAPEC-IAP and the Bethesda, show similar sensitivities but the SIAPEC system exhibited better specificity, possibly related to differences in the use of the so called “grey zone” (TIR 3 and CAT IV) categories. Larger studies will be useful to further confirm these data. PP-154. Adenomatoid Tumor of the Adrenal Gland – a Case Report M. Dumser1, Z. Redzic2, H. Feichtinger1 1 Dept. of Pathology and Microbiology, 2 Dept. of Radiology, KA Rudolfstiftung, Vienna Background: Adenomatoid tumors are benign neoplasms of mesothelial origin, most commonly occurring in the male and female genital tract. Exceptionally, they can be found at extragenital locations, the adrenal gland being exceedingly rare. They may impose diagnostic difficulties in a specific setting. Methods: Case report of a 70 year old man clinically presenting with aldosteronism, a unilateral nodular enlargement of the right adrenal gland and a left sided colorectal carcinoma. Synchronous surgery of the adrenal and the colon was performed. Results: Intraoperative frozen section of the adrenal gland revealed nodular cortical hyperplasia and an unclassifiable, microcystic tumor, probably benign; however, a partially pseudomucinous appearance warranted no final diagnosis and definite exclusion of metastasis. Permanent sections showed a poorly demarcated, intradrenal, partially largely cystic tumor with predominant areas of microcystic spaces and anastomosing channels. Interspersed residual adrenocortical tissue and lymphocytic infiltrates were seen. By immunohistochemistry, the tumor cells showed co-expression of vimentin with several cytokeratin subtypes except CK 20, were negative for endothelial markers, but exhibited immunoreactivity with antibodies against calretinin and podoplanin confirming their mesothelial origin. Conclusions: Due to the rarity of these lesions they are a diagnostic challenge, especially in the setting of frozen section and a concurrent malignant neoplasm. Associated reactive changes of the adrenal cortex may cause clinically manifest endocrine symptoms.

Abstracts PP-155. Cribriform-Morular Variant of Papillary Thyroid Carcinoma in a Man with Gardner’s Syndrome: Pathological Features and Clinical Implications S. Crippa1, L. Giovanella2, S. Suriano2, P. Saletti3, M. Frattini1, V Nosé4, L. Mazzucchelli1 1 Institute of Pathology, Locarno, Switzerland 2 Nuclear Medicine and PET/CT centre, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland 3 Medical Oncology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland 4 Department of Pathology, Brigham and Women’s Hospital, Boston MA, USA Background: Papillary thyroid carcinoma (PTC) is an extra-intestinal manifestation of patients with familial adenomatous polyposis (FAP), mostly occurring young women. Recent publications highlight that FAP-associated PTC represents a distinct type of follicular cell neoplasm histologically characterized by cribriform-morular aspects, of which the incidence probably has been underestimated so far. We report a case history of PTC occurring in 55-year-old man with Gardner’s syndrome. Clinical History: Screening thyroid ultrasonography revealed a solid and well-circumscribed nodule in the left lobe of 3,5 cm, hard in clinical examination. Fine needle aspiration (3 passes) was not representative (Tir1 according to “Consensus citologico” SIAPEC-IAP 2007). The patient underwent left hemithyroidectomy. The thyroid lesion consisted of neoplastic cells in several small nests dispersed in an encapsulated nodule characterized by highly hyalinized fibrous stroma with calcifications and bone metaplasia. Immunohistochemical staining revealed focal but strong nuclear beta-catenin expression restricted to nests with morular aspects but not in adjacent normal appearing follicular structures. Conclusions: PTC is a rare extraintestinal manifestation of Gardner’syndrome that should not be disregarded. Thyroid screening ultrasound examination is appropriate in women as well as in men. We suggest that any thyroid nodule in FAP individuals should be considered as suspicious until proved otherwise. A “negative” fine needle aspiration should be interpreted with caution, due to the extensive fibrosis and lack of characteristic nuclear changes of the cribriform-morular variant of PTC. Finally, we suggest that morular nests with focal beta-catenin expression within a thyroid nodule represents a putative precursor lesion of PTC. PP-156. Aberrant Expression of the HER-2 Oncogene is not a Common Feature in Osteosarcoma D. Baumhoer1–3, J. Smida3–4, K. Specht5, K. Bink6, L. Quintanilla-Martinez6, M. Rosemann6‑7, H. Siggelkow8, W.B. Nathrath9, M.J. Atkinson6–7, S. Bielack10, G. Jundt1–2, M. Nathrath3–4  1 Institute of Pathology and   2 Bone Tumour Reference Center at the Institute of Pathology, University Hospital Basel, Switzerland   3 Clinical Cooperation Group Osteosarcoma, Helmholtz Center Munich, German Research Center for Environmental Health, Germany   4 Department of Pediatrics, Technical University Munich and Pediatric Oncology Center, Germany  5 Institute of Pathology, Technical University Munich, Germany  6 Institute of Pathology and  7 Institute of Radiation Biology, Helmholtz Center Munich, German Research Center for Environmental Health, Germany   8 University Medical Center Goettingen, Department for Gastroenterology and Endocrinology and Endokrinologikum Goettingen, Germany   9 Institute of Pathology, Klinikum München-Harlachingen, Munich, Germany 10  Klinikum Stuttgart Olgahospital, Pediatrics 5 - Oncology, Hematology, Immunology, Germany Background: HER-2 expression in osteosarcoma has been correlated ambiguously with both favorable and poor prognosis in several studies, most of them relying on immunohistochemical analyses only. Due to the decisive need of prognostic markers and effective new treatment options for osteosarcoma patients we evaluated the role of HER-2 in two well characterized sets of pretherapeutic osteosarcoma samples (46 paraffin embedded and 46 fresh frozen biopsy samples).

Methods: Immunohistochemistry with two different antibodies (DAKO A0485 and Novocastra CB11) as well as FISH, qRT-PCR, and SNP array analyses were performed. The results were correlated with clinico-pathological parameters. Results: No unequivocal evidence of HER-2 gene amplification or overexpression of HER‑2 mRNA or protein in any of the investigated samples was detected. Only in a small subset of tumors, a moderate increase in mRNA levels (13.6%) or focal membranous immunoreactivity (8.7%, A0485) was identified but did not correlate with survival or response to chemotherapy. Cytoplasmic staining was recognized more frequently (63%, CB11) but again did not show any association with clinico-pathological parameters. Conclusions: Our study does not support a role for HER-2 as a prognostic marker or as a therapeutic target in osteosarcoma. Due to the abundance of studies with conflicting results it is crucial to apply generally accepted techniques and scoring schemes and to also report negative studies to avoid publication bias. PP-157. Crystal Precipitations and Granulomatous Inflammation in Multiple Organs Following Foscarnet Therapy in a Lung Transplant Recipient V. Tischler1, M. Schuurmans2, A. Boehler2, A. Gaspert1 1 Institute for Surgical Pathology, 2 Clinic of Pulmonary Medicine, University Hospital Zurich, Zurich, Switzerland Background: Cytomegalovirus infection or reactivation is a serious complication after organ transplantion leading to potentially life threatening decrease of organ function. Irreversible renal insufficiency has been described after systemic therapy with foscarnet for cytomegalovirus infection. Several reports on foscarnet nephrotoxicity describe acute tubular necrosis, glomerular crystal deposits and tubulo-interstitial nephritis. We present the case of a 42 yo male lung transplant recipient with disseminated granulomatous reaction to crystal precipitation after therapy with foscarnet for recurring ganciclovir resistant cytomegalovirus reactivation. Methods: Autopsy, macroscopic and histopathologic evaluation. Results: We found chronic airway rejection with constrictive bronchiolitis obliterans (ISHLT C1) and mild acute rejection (ISHLT A2), without evidence of CMV infection. Multiple disseminated small birefringent crystal structures surrounded by granulomatous inflammation were found in both lungs. The kidneys showed disseminated deposits of birefringent short angular crystals in glomeruli with concomitant rupture of glomerular capillary loops and Bowman’s capsule, associated with multinucleated giant cells and focal granulomatous reaction. The tubules showed acute tubular necrosis in regeneration and multiple intratubular crystals. We found a pronounced fibrinous inflammation of epi- and pericardium with widespread birefringent crystal structures and granulomatous inflammatory reaction. Same crystal deposits with granulomatous reaction were found in the myocardium and esophageal muscularis propria. Conclusions: This is the first description of a systemic granulomatous inflammation with crystal precipitation after foscarnet therapy with involvement of the heart, kidneys and lung. Administration of foscarnet for ganciclovir resistant CMV infection calls for careful evaluation due to its potential for complications. PP158. Solitary Fibrous Tumors of the Pleura: is Immuohistochemical Analysis Helpful in Prediction of Malignant Behavior? D. Krenbek1, S.B. Watzka2, 3, H. Prosch4, U. Setinek1, F. Lintner1 1 Division of Pathology, Otto Wagner Hospital, Vienna, Austria 2 Division of Thoracic Surgery, Otto Wagner Hospital, Vienna, Austria 3 Karl Landsteiner Institute for Thoracic Oncology, Otto Wagner Hospital, Vienna, Austria 4 Division of Radiology, Medical University of Vienna, Vienna, Austria Background: Solitary fibrous tumours of the pleura (SFP) are rare tumors of mesenchymal origin that are characterized by their distinct morphological appearance. Occurrence of necrosis, frequent mitoses and cell atypia lead to the diagnosis of malignant SFP. Most SFPs are benign and can be cured by complete surgical excision, however in some case of seemingly benign SFPs local recurrence and metastases occur after total resection of the primary tumor, whereas a large number of SFPs with features of malignancy do not metastasize. Therefore the finding of reliable markers to predict biological behavior of SFPs Der Pathologe 6 · 2010 

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is important. Some promising markers were described in literature showing contradicting results. Methods: In this study we performed immunohistochemical staining to analyze the expression of CD34, bcl-2 and CD99 as well as ER, PR, p53 and p16 in 17 cases of solitary fibrous tumors (5 malignant and 12 benign). Results: All tumors stained positive for at least two of the standard markers CD34, Bcl2 or CD99. Expression of p53, ER and PR was similar in malignant and benign tumors. Convincing p16 intensity was observed in only a small amount of tumor cells in benign SFPs, in contrast to a strong staining pattern in malignant tumors. Conclusions: CD34, Bcl-2 and CD99 are reliable markers in the diagnosis of benign and malignant SFPs. ER, PR and p53 are not useful in prognosticating tumor dignity whereas p16 might be a valuable marker in predicting biological behavior of SFPs. PP-159. Histologic Assessment of Drug-Eluting Vascular Prosthesis According to Implantation Site: Subcutaneous vs. Aortic J.C. Tille1, S. de Valence2, D. Mugnai3, W. Mrowczynski3, R. Gurny2, M. Moeller2, B.H. Walpoth3 1 Division of Clinical Pathology and 2 Department of Pharmaceutics and Biopharmaceutics, EPGL, University of Geneva, Switzerland, 3 Department of Cardiovascular Surgery, University Hospital of Geneva Background: Drug-eluting vascular prostheses represent a new direction in vascular surgery to improve early thrombosis and late intimal hyperplasia for small calibre grafts. Subcutaneous implantation of such prostheses is a rapid and cost-effective screening model to assess the drug-elution effect and to compare the results with the vascular implantation site. Methods: Polycaprolactone (PCL), paclitaxel-PCL (PTX-PCL) and dexamethason-PCL (DXM-PCL) grafts were implanted subcutaneously and in an infrarenal abdominal aortic replacement model for 1, 3 and 12 weeks. At conclusion of the study histological analysis was performed in all groups and models. Results: Cellular graft invasion was analyzed by morphometry in the subcutaneous and aortic group revealing time differences of infiltration between PTXPCL and PCL/DXM-PCL groups in both models. Cell infiltration was continuously increasing with time in the aortic model compared to the subcutaneous model for all groups. Morphological cell counting revealed major differences in fibroblast, macrophage and giant cell graft colonisation in all groups and models with time. Macrophages and giant cells increased in the PCL aortic group; whereas in the subcutaneous model these cell types increased only slightly after 3 weeks or even decreased in the drug-eluting PCL groups. Other major findings were observed only in the aortic replacement such as neo-angiogenesis and chondroid metaplasia. Conclusions: The subcutaneous polymer implant model can be used for screening, especially when drug-eluting effects are studied. However major histological differences in cell type reaction, depth of cell penetration compared to the aortic replacement model indicate some limitation and the need to perform vascular implantations. PP-160. What does a Diagnostic Repository Require to Become a Useful Biobank for Translational Research? S. Eppenberger1, I. Zlobec1, U. D��������� ü�������� rrm����� ü���� ller1, L. Terracciano1 1 Institut f������������������������������������������������ ü����������������������������������������������� r Pathologie, Universit������������������������ ä����������������������� tsspital Basel, Schweiz Background: An institute of pathology is itself a biorepository: human specimens are received, analyzed and stored for diagnostic quality control. The tissue bank of the Institute for Pathology (IPB) is implementing quality management rules in order to provide optimal material for translational research. Methods: Since 1985 approximately 23’000 cryopreserved tissues and FFPE specimens of more than 600’000 patients have been collected. All pathological, molecular and clinical parameters are in the process of being extracted from previous documents and registered in single fields of a new relational database, a modified version of CAISIS®. Our tissue bank works with a general (2008) and a specific authorization (1996) of the SAMW and corresponding patient’s consents. Banking activities are regulated by workflows and SOPs.

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Results: The registered specimens with related data allow the identification of pathologically and molecularly well classified cohorts. We successfully compared and optimized cryopreservation procedures, extracted RNAs of good quality in 80% of 2’200 selected frozen samples, compared several methods of protein analyses acquiring relevant information on limits and strengths of each procedure. 30’000 well characterized FFPE samples have been spotted on TMAs for new biomarker investigations. Furthermore, miRNA and DNA studies as well as comparative studies at different expression levels are being conducted. Conclusions: Well structured work flows, SOPs, clinical annotations acquired with respect of patient rights and ethical laws, storage of data in relational database is what renders a biobank valuable for translational research projects. PP-161. Congenital Cytomegalovirus Infection – an Unusual Autopsy Case A. Korntheuer1, S. Dertinger1, P. Schw������ ä����� rzler2, F.A. Offner1 1 Department of Pathology, University Teaching Hospital, Feldkirch, Austria 2 Department of Obstetrics & Gynaecology, University Teaching Hospital, Feldkirch, Austria Background: Human cytomegalovirus (CMV) is one of the most common causes of congenital infection. It occurs in 0,2%–2,4% of all live births. However, only 5–15% of infants with congenital CMV infection have clinical evidence of disease at birth. Methods: A 22-female patient presented at a general practitioner with acute abdominal pain. Ultrasonography revealed an unsuspected pregnancy and a cerebral abnormality of the fetus. The patient had not been seen by an obstetrician because she had denied her pregnancy. She was then referred to the Department of Obstetrics & Gynaecology of the University Teaching Hospital Feldkirch. An obstetric ultrasonography was performed and a holoprosencephaly and Dandy Walker malformation was diagnosed. A syndromal disorder with severe neurological disability was suspected and a feticide was recommended and agreed by the mother. Results: The autopsy of a male growth restricted fetus exposed a generalized cytomegalic inclusion disease (CID) with extensive necroses, intracranial calcifications, secondary microcephalus, splenomegaly and petechiae. The diagnosis was confirmed by histology and immunohistochemistry. Virus inclusion bodies were detected in multiple organs and the placenta showed leukemic infiltrates. The complex pathology of the brain was interpreted to be the result of the severe CMV infection and not to be due to a classic primary malformation. Conclusions: An autopsy should always be preformed in cases of feticides to verify the clinical diagnoses. If complex cerebral deformities are discovered apart from syndromal or genetic disorders severe congenital CMV infection should be considered as a possible differential diagnosis. PP-162. The Neuropathology of Neurocysticercosis: the Geneva Experience K. Burkhardt1 1 Neuropathology Unit, Surgical Pathology Service, Geneva University Hospitals, Geneva, Switzerland Background: Cysticercosis, caused by Taenia solium, is the most frequent parasitic disease affecting the human CNS worldwide. Morbidity and mortality related to neuro-cysticercosis represent a major public health issue in Asia, Africa and Latin America. In rural zones of endemic areas, conditions favouring disease transmission prevail. The disease was eradicated in much of the Western world in the first half of the twentieth century, but industrialised nations have seen a resurgence due to intensified tourism and migratory flows. This study should enable the pathologist, when faced with a histological image in a given clinical context, to recognise neurocysticercosis and not overlook a potentially curable condition. Methods: Cases collected retrospectively span the period from 1954 to 2007, including both autopsy and biopsy material. Diagnosis was based on histology, in combination with serological, clinical and radiological data when available. Results: 42 cases of neurocysticercosis were identified: 31 at autopsy, 11 in biopsies. In 38, a parasitic remnant was present. 27 contained a fibrous capsule, 25 calcifications, and 15 cystic components. In 21, inflammation was predominantly lymphocytic, whereas in 13 giant cells were prominent. A significant eo-

Abstracts sinophilic component was seen in only 6. In 19, gliosis was marked, leading to an erroneous preliminary diagnosis of glioma in 2. Conclusions: Though no longer endemic in the West, physicians here will no doubt be confronted with neurocysticercosis through a traveller or immigrant. It is essential that pathologists be familiar with its many forms, due to the parasite’s complex life cycle and protean host responses. PP-163. Low Host-Dwarf Tapeworm Reaction in Human J. Hench1, M. Dettmer1,G. Cathomas1 1 Kantonales Institut f����������������������������������� ü���������������������������������� r Pathologie, Liestal, Switzerland Background: Based on epidemiology and animal experiments, helminth colonization has been described to be protective against chronic colitis. In mice, infection with the dwarf tapeworm Hymenolepis diminuta – a rat specific cestode – provokes an anti-parasite response and significantly reduces the intensity of chemically induced colitis. In human, however, very little is known about the natural reaction against Hymenolepis sp. We report the type of inflammatory response in rare human dwarf tapeworm infection. Methods: In a 33 year old male who underwent resection of an asymtomatic Meckel’s diverticulum during unrelated surgery, histologically a dwarf tapeworm infection was observed and the inflammatory response was analyzed by immunohistochemistry. Results: In comparison to normal intestinal mucosa, no increase of CD3+, CD4+ or CD8+ T-cells was observed in the mucosa close to the adult worm as well as in the vicinity of the intramucosal larva which was surrounded by a very sparse CD68+ cell infiltrate. In addition, no granulomatous reaction, no overall increase in CD68+ histiocytes and no eosinophilia was present. Conclusions: To our knowledge, this is the first description of the inflammatory reaction against a dwarf tapeworm in human. The lack of a significant cellular inflammatory response against the intraluminal and intramucosal parasite match the findings from Hymenolepis diminuta infection in rats. Our result supports the concept that these helminths suppress the host reaction and may further down-regulate the inflammation in intestinal disorders including inflammatory bowel disease. PP-164. Efforts to Standardise Ki-67 Reporting of Breast Cancer? A Pilot Study of the Swiss Working Group of Gyneco- and Breast Pathologists Z. Varga1, J. Diebold2, C. Domman-Scherrer3, H. Frick4, D. Kaup5, A. Noske1, E. Obermann5, C. Öhlschlegel6, C. Rakozy7, O.S. Sancho8, S. Schobinger-Clement8, H. Schreiber9, G. Singer10, C. Tapia5, U. Wagner11, H.A. Lehr12  1 Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland  2 Institute of Pathology, County Hospital Luzern, Luzern, Switzerland  3 Institute of Pathology, County Hospital Winterthur, Winterthur, Switzerland  4 Institute of Pathology, County Hospital Graubünden, Chur, Switzerland  5 Institute of Pathology, University Hospital Basel, Basel, Switzerland  6 Institute of Pathology, County Hospital St. Gallen, St. Gallen, Switzerland  7 Pathology Institute for Biopsy Diagnostics, Zurich, Switzerland  8 Medical Laboratory, Pathology, Promed SA, Marly, Switzerland  9 Institute of Pathology, Clinic Konstanz, Konstanz, Germany 10 Institute of Pathology, County Hospital Baden, Baden, Switzerland 11 Pathology, Unilabs Mittelland, Bern, Switzerland 12 Institute of Pathology, University Hospital Laussanne, Laussanne, Switzerland Background: Adjuvant chemotherapy decisions in breast cancer are increasingly based on the pathologist’s assessment of the proliferation fraction in the tumor. Yet, exactly how to count and in which areas to count within a tumor remains inadequately standardised. The Swiss Working Group of Gyneco- and Breast Pathologists has tried to better define the magnitude of this dilemma and to propose ways to obtain more reproducible results. Methods: In a first phase, 5 senior pathologists evaluated Ki-67 (MIB-1) counts in 10 breast cancers by exact counting (500 cells) and by eyeballing. Pathologists were free to select the region in which Ki-67 was evaluated. In a second phase 16 pathologists evaluated Ki-67 counts in 3 breast cancers also by exact counting and eyeballing, but in predefined fields of evaluation. In both phases, Ki-67 was assessed in centrally immunostained slides (ZH) and on slides immunostained in the 11 participating laboratories.

Results: Discordance of Ki-67 assessment was due to each of the following 4 factors: (i) pathologists’ divergent definitions of what counts as a positive nucleus (ii) the mode of assessment (counting vs. eyeballing), (iii) immunostaining technique/protocol/antibody, and (iv) the selection of the area in which to count. Conclusion: Defining the field of evaluation (representative field in the tumor periphery and omitting hot spots) reduces the discordance rates of Ki-67 readings between laboratories/ pathologists (counting better than eyeballing), but discordance rates are still unacceptably high, notably since chemotherapy decisions are increasingly based on this parameter. PP-165. Can an Involuntary Selection of Nuclei Explain the Cognitive Bias in Nuclear Grading of Malignant Tumors B. Mora1, D. Bombari2, S. Schaefer3, F. Mast2, H.A. Lehr1 1 Institute of Pathology, University Hospital Laussanne, Laussanne, Switzerland 2 Department of Cognitive Psychology, University of Bern, Bern, Switzerland 3 Department of Pathology, University of Bern, Bern, Switzerland Background: We have previously shown that nuclear grade assignment of prostate carcinomas depends on a cognitive bias induced by the architectural organization of the tumor (Fandel et al., J Pathology, 2008). We asked the question whether this bias is “transported” by the non-conscious selection of nuclei that “match the expectation”. Methods: 20 Pathologists in various stages of formation/experience were asked to grade nuclei in high power fields of prostate carcinomas projected on a computer screen. HPF images were projected in a circle before a background of low power architectural images of the carcinomas. Unknown to the subjects, each HPF was shown twice, once before a background of a Gleason 2–3 carcinoma and once before the background of Gleason 4–5 carcinoma. Eye tracking experiments allowed to identify which nuclei the pathologists focused on during the 8 second projection period. Results: For each of the 20 pathologists, nuclear grade assignment was significantly biased by the architectural differentiation of the background image. All pathologists tended to focus on bigger, darker, and more irregular nuclei when HPFs were projected before a Gleason 4–5 carcinoma (and vice versa), but this involuntary nuclear selection accounted for only about 10% of the bias in nuclear grading induced by the architecture. Conclusion: The confirmational bias in nuclear grade assignment induced by the tumor architecture can only in part be explained by an involuntary focus on the nuclei that “match the expectation”. PP-166. A Proposal for a New Cervical Cancer Screening Strategy in Vorarlberg (Austria) – Primary HPV DNA Testing with Cytology Triage R. Stockinger1, G. Hartmann1, W. Widder1, H. Dirschmid1, A. B��� ö�� sl1, H. Amann1, F.A. Offner1 1 Department of Pathology, University Teaching Hospital Feldkirch, Feldkirch, AUSTRIA Background: Cervical cytology is the current standard test for cervical cancer (CC) screening and has contributed to a decrease in the incidence of CC in Vorarlberg from 34 in 1982 to 6,8 in 2007. The PAP smear as a primary screening tool has recently been challenged. Several reports indicate that primary human papillomavirus (HPV) DNA testing might be a superior technology to screen for CC. Methods: We performed a Medline search regarding HPV DNA testing in CC prevention and identified studies, reviews and meta-analyses where this test method was used in conjunction with cervical cytology or as the primary mode of screening. We tried to identify a screening strategy that best suits our setting and exploits the greater sensitivity of HPV DNA Testing while minimizing unnecessary colposcopy referrals and overtreatment. Results: HPV-testing combined with cytology triage seems to be more sensitive and more effective in preventing invasive CC. Screening has to be implemented in an organized fashion to be most effective. Cytology should not be omitted but used as the primary diagnostic tool to detect neoplasia after a positive HPVtest. Screening intervals might be extended to 5 years. Conclusions: From our point of view there are now several studies that justify a change in the mode of screening for CC. Primary HPV DNA testing would alDer Pathologe 6 · 2010 

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low for a more sensitive, objective screening and positive cases would be triaged by cytology to minimize unnecessary treatment. This new screening approach should be cost effective. PP-167. Targeted Reviewing for Quality Assurance in Cervical Cytology H. Ammann1, B. Böckle1, J. Brunner1, E. Karnowsky1, J. Kohler1, M. Linortner1, V. Seifert1, K. Sutter1, C. Nemes1, F.A. Offner1, W. Widder1 1 Institute of Pathology, University Teaching Hospital Feldkirch, Austria Background: Quality assurance in cervical cytology is an important but labourintensive and costly task. We describe the implementation of targeted reviewing (TR) as a method of quality management at the Institute of Pathology of the University Teaching Hospital Feldkirch and report our experience gathered in 7 years. Methods: The work load of the cytology laboratory is 65.000 PAP smears per year. All patients with cervical intraepithelial neoplasia (squamous and glandular) and all patients with invasive cervical carcinoma proved by histological analysis of resection specimens (cone biopsy or hysterectomy) are subjected to TR. The last negative smears of these patients taken before surgery are rescreened in a blinded fashion bi-monthly by physicians and technicians. A negative and positive control smear is included. A special schedule of classification was designed to exclude ambiguous cases. Finally all slides are discussed at a multi-headed microscope within the guidelines of a certified “Quality Circle” approved by the General Medical Council of Austria. Results: Out of 446.033 smears 428 (0,1%) cases were submitted to RTR. Thereof 122 (28,5%) cases had to be reclassified because of screening or interpretation errors. Conclusions: TR contributes to integrated and continuous training of the laboratory personal as well as pathologists. Furthermore an improvement of error management and positive dynamics of the team could be achieved. The method is much less labour-intensive and also cheaper than other methods of quality assurance and has recently been adopted as an appropriate quality management method by the Austrian Society of Cytology. PP-168. Use of MALDI Imaging for the in situ Detection, Identification and Validation of Breast Cancer Biomarkers in Tissue S. Rauser1, D. Suckau3, S.O. Deininger1, C. Albers1, E. Belau1, K. Specht4, M.P. Ebert2, M. Schmitt5, H. Höfler1,4, A. Walch1 1 Institute of Pathology, Helmholtz Zentrum München - German Research Center for Environmental Health, Neuherberg, Germany 2 Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, Munich, Germany 3 Bruker Daltonik GmbH, Bremen, Germany 4 Institute of Pathology, Technische Universität München, Munich, Germany 5 Department of Obstetrics and Gynecology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany Background: MALDI Imaging analysis of tissue has established itself as new promising approach in a number of biomedical applications: from drug distribution and metabolism testing to molecular histopathology and clinical research. In this work we used high definition MALDI imaging (HDMI) as a molecular dimension to accelerate the clinical decision making process and to obtain prognostic information for the treatment of breast cancer. Health state assignments of tissue specimens (HER2 positive and negative breast cancer samples) were based on the distribution of either individual proteins or of protein populations and attempts were undertaken to actually identify proteins that were of clinical relevance. Methods: Breast cancer tissues were subjected to MALDI-IMS and protein profiles were obtained through direct analysis of tissue sections. Multivariate statistical methods (SVM, ANN) were applied to obtain classification of the HER2 (+/-) cancer tissue with 50–100 µm class image resolution. Protein identification was performed by tissue micro-extraction and fractionation followed by top-down tandem mass spectrometry on an ion trap with ETD. Results: Specific protein expression changes strongly correlated with the HER2 over expression in the 4–8 kDa range achieving an overall accuracy of 89%. Among these, we identified m/z 8404 as Cysteine-rich intestinal protein 1 (CRIP1). CRIP1 is a newly detected protein biomarker presumably involved in

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cell proliferation. Initial IHC experiments with anti-CRIP1 polyclonal antibodies confirmed its co localization with the HER2-positive phenotype. Conclusion: Our results underscore the potential of MALDI-IMS proteomic algorithms for morphology-driven tissue diagnostics such as HER2 testing and show that MALDI-IMS can reveal biologically significant molecular details from tissues which are not limited to traditional high-abundance proteins. CRIP1 is a cytosolic protein that is potentially useful for serum based diagnostics of HER2 if tissue leakage can be demonstrated. PP-169.High Tenascin-C Expression Marks Vital Residual Breast Cancer Tissue after Neoadjuvant Treatment C. Hauser-Kronberger1*, S. Bastelberger1*, S. Swierczynski1*, E. Hinterseer1., L. Pradel1, J. Beil1, G. Rendl2, O.Dietze1 1 Department of Pathology and 2Department of Nuclear Medicine, Paracelsus Medical University, Salzburg, Austria.* contributed equally Background: The extracellular matrix molecule Tenascin-C (TNC) in its many isoforms is highly expressed during tissue repair and pathological conditions such as cancer. Utilising the selective pressure of neoadjuvant chemotherapy on carcinoma cells we screen for a subgroup of breast cancer patients with elevated TNC expression and a bad therapy response, evaluated by immunopathological markers, histopathological data (pTNM staging system) and clinical data. Methods: ��������������������������������������������������������������������� Sections of paraffin embedded samples from 68 preoperatively treated breast cancer patients, are used for automated Immunohistochemical staining with appropriate antibodies targeting TNC, AE1/AE3, Ki67, CD3 and p53. In addition F-18 FDG PET-CT was performed to evaluate response. Protein- andgene expression experiments performed onto six breast cancer cell lines (MCF7, MDA-MB231, MDA-MB453, SK-BR3, SUM-159, T47-D) by qRT-PCR and IHC showed an TNC-expression of MDA 454 breast cancer cell line. Results: We show that TNC expression in breast cancer is immensely reduced after neoadjuvant chemotherapy (43% of patients lost TNC expression) and that expression profiles after treatment correlate with histopathological data, especially the grade, indicating prognostic value. However, there is a strong correlation and colocalisation of the proliferation marker Ki67 in cancer cells and TNC in the surrounding tumour-stroma of patients having a less beneficial response to chemotherapy. The evaluation of expression levels revealed a direct linear correlation of TNC and Ki67. We expect TNC to be a dependable marker for vital cancer tissue remaining after neoadjuvant treatment. Conclusions: These findings concur with the role TNC plays in tissue reorganisation, wound healing and it’s growth promoting properties in cancer.