Abstracts DGP Wuppertal 2005

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Pathology – Research and Practice 201 (2005) 153–300 www.elsevier.de/prp

Local Welcome

Symposium: Most Outstanding Research Communications 2005

Pathology in Wuppertal

1 Abberant methylation of the Wnt antagonist SFRP1 in breast cancer

S. STO¨RKEL Institut fu¨r Pathologie, Universita¨t Witten/Herdecke th

In 2005 the Institute of Pathology celebrates the 100 anniversary. Prof. M. Koch from Berlin, an assistant of R. Virchow, became the first pathologist in the Elberfeld hospital. He was followed by Dr. B. Funccius from Erlangen, who managed the Institute for 42 years including the years of both World Wars. At the Barmen Hospital Prof. J. Wa¨tjen from Freiburg started in 1922, he was later chairman at Halle University. His successor in 1925 was Prof. J. Miller from Tu¨bingen, who wrote the famous Handbuch der Speziellen Pathologie ‘‘Krankheiten des Eierstocks’’ edited bei Henke-Lubarsch. In 1950 the Elberfeld and Barmen Institutes of Pathology merged and Prof. O. Koch from Du¨sseldorf was the first head of the united department in the Elberfeld hospital. He was killed as a pedestrian by a car accident in 1951. His successor was Prof. G. Liebegott from Freiburg, who focussed on endocrine pathology and cardiovascular research. He became member of the managing board of the DGP for 14 years and was DGP president in 1973. He died in an aircraft crash 1975, when he visited the Congress of the International Academy of Pathology in Australia. In 1976 Prof. G. Schubert from Tu¨bingen became Director of the Institute. He constructed the new Institute at the Barmen hospital and in 1984 received the chair of Pathology of the first private University in Germany Witten/Herdecke. His research interests concentrated on Genitourinary Pathology. Besides Clinical Pathology Experimental Pathology played an important role in Wuppertal too. Prof. G. Domagk, leading pathologist of the Research Center of Bayer Pharmaceutical Company, developed the sulfonamides (Prontosil) and received the Nobel Prize for Medicine in 1939. Even Prof. E. Grundmann run the Department of Experimental Pathology at Bayer Pharmaceutical Company before he became Chairman of Pathology at the University of Mu¨nster. 0344-0338/$ - see front matter doi:10.1016/j.prp.2005.03.001

HX. AN, J. VEECK, I. GERMANTA, G. KRISTIANSEN1, I. LOSEN, R. KNU¨CHEL-CLARKE, E. DAHL Institut fu¨r Pathologie, Universita¨t Aachen 1 Institut fu¨r Pathologie, Charite´ Berlin Aims: The Wnt signalling pathway plays a key role in embryonic development and defects in this pathway have also been implicated in the carcinogenesis of various types of tumors. The SFRP1 gene on chromosome 8p12 encodes a Wnt signalling antagonist and was recently found to be frequently inactivated by promoter methylation in colon cancer. Allelic deletion of 8p12 is frequently observed in breast cancer. Our previous study has demonstrated loss of SFRP1 protein expression in 44% of breast cancers and suggested a putative tumor suppressor gene function of SFRP1. To reveal the cause of SFRP1 deregulation in breast cancer, we performed a parallel expression and promoter methylation analysis of SFRP1 in cell lines and primary tumors of the breast. Methods: Expression and hypermethylation of SFRP1 were detected by real-time PCR, immunohistochemistry and methylation-specific PCR (MSP). T-cell factor (TCF) regulated transcription activity was analyzed by reporter assay with TEC-LEF-responsive reporter constructs. Proliferation and apoptosis of cells were examined by FACS scanning. Results: Methylation-associated silencing and re-expression after treatment with 5-aza-2’-deoxycytidine were found in five of five analysed breast cancer cell lines. Promoter hypermethylation of the SFRP1 gene was frequently detected in primary breast cancer. Preliminary data indicate that restoration of SFRP1 function in breast cancer cells can reduce or block Wnt signalling. Conclusions: The results of this study suggest that epigenetic inactivation of the SFRP1 gene is the

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Symposium: Most Outstanding Research Communications 2005 / Pathology – Research and Practice 201 (2005) 153–300

predominant mechanisms of SFRP1 gene silencing in breast cancer.

2 Association of CXCR4 chemokine receptor expression with MMP and TIMP secretion in primary clear cell renal cell carcinoma K. STRUCKMANN1, K. MERTZ1, P. STALLER2, W. KREK2, H. MOCH1 1 2

Institut fu¨r Klinische Pathologie, Universita¨t Zu¨rich Institut fu¨r Zellbiologie, ETH Zu¨rich

Aims: CXCR4 chemokine receptor mediated signaling is of importance for the metastatic process in a variety of human cancers. We have recently found an association between strong CXCR4 expression and poor tumor-specific survival in clear cell renal cell carcinoma (ccRCC; Nature, 425, 2003). It has been shown in vitro that the expression of certain MMPs/ TIMPs correlates with the CXCR4 expression status in epithelial tumors indicating that the CXCR4MMP/TIMP pathway might play a role in tumor progression. To verify this hypothesis, we correlated the mRNA abundance of certain MMPs and TIMPs with the CXCR4 expression status in primary ccRCC. Methods: mRNA expression levels of MMP2, MMP3, MMP9, TIMP1, TIMP2, and TIMP3 were determined by RT-PCR in primary ccRCC and normal renal tissues. MMP/TIMP expression levels were related to the CXCR4 expression status of the given tissue samples. Results: The average increase in CXCR4 expression was 7.3-fold (range 2.3–18.8-fold) in 26 of 29 primary ccRCC compared with normal renal tissue. First experiments revealed a strong association between MMP9 and CXCR4 expression and a weaker but also clearly detectable correlation of MMP2 and CXCR4 expression levels. Conclusions: CXCR4 upregulation in ccRCC may influence expression of at least MMP2 and MMP9. These preliminary data indicate that the CXCR4-MMP/ TIMP pathway might play a role in renal cancer progression.

3 Stem cells normalize hyperglycaemia in diabetic mice R. HUSS, X. XIANGWEI1, H. HEIMBERG1 Pathologisches Institut, Ludwig-Maximilians-Universita¨t Mu¨nchen 1 Diabetes Research Center, Vrjie Universitat Bru¨ssel (VUB)

Aims: The potential role of adult stem cells in the regeneration of beta cells in diabetes is still controversial. Although islet cell transplantation is currently the most pursued field of research, we have investigated the capacity of multipotent adult stem cells to correct hyperglycaemia in an experimental murine diabetes model. Methods: Cloned stem cells were labelled with eGFP or transfected with a pTie2-RFP construct to show endothelial differentiation in-vivo. The beta cell toxin alloxan was injected intravenously and all mice became hyperglycaemic (4400 mg/dl) within 2 days and lost more than 90% of their beta cell mass. Stem cells were then injected either directly into the pancreas or given systemically. Results: Mice that received stem cell transplantation reached normal blood glucose levels within 14 days and the beta cell mass fully recovered within 1 month after treatment, regaining normal body weight soon after stem cell infusion. The host pancreas was then dissociated and further analyzed. The eGFP+ donor cells did not express insulin or other endocrine markers, but showed a red fluorescence (RFP+) and CD31 expression instead, characteristics of endothelial cells after pTie2 activation. It was further shown that remaining (eGFP-) beta cells showed increased cell cycle activity. Conclusions: Endothelial differentiation from transplanted stem cells, induced by the environment of an injured pancreas, allows the regeneration of insulin production either through proliferation of still existing and residual beta cells in the islet or the recruitment and differentiation of beta cell progenitors mostly from the duct region via enhanced vasculogenesis and microcirculation.

4 Nuclear localization of heterochromatic regions varies in hyperplastic and preneoplastic lesions of the breast N. ARENS, M. STILLER, M. BIERBAUM, R. HILDENBRAND Pathologisches Institut, Universita¨tsklinikum Mannheim Aims: Previous molecular cytogenetic studies in breast cancer revealed numerous chromosomal changes and identified alterations involving the chromosomes 1 and 16 as early incidents in mammary carcinogenesis. Since both chromosomes reveal pericentromeric heterochromatic areas, these chromosomal alterations might result from instable heterochromatin caused by DNA hypomethylation. In the present study, we investigated whether hyperplastic and neoplastic lesions of the breast differ regarding the distance between the heterochromatic areas of chromosomes 1 and 16 within the nuclei.

ARTICLE IN PRESS Symposium: Most Outstanding Research Communications 2005 / Pathology – Research and Practice 201 (2005) 153–300

Methods: We hybridized fluorescence-labeled DNA samples specific for the heterochromatic regions of chromosomes 1 and 16 to formalin-fixed tissue sections. Histological classification of the lesions was supported by immunohistochemical staining using cyto keratinspecific antibodies. The methylation state of the hetero chromatic regions was tested by staining with an antibody specific for methylated cytidin. Results: Our results revealed an increased frequency of paired intranuclear signals specific for chromosomes 1 and 16 in neoplastic lesions (atypical ductal hyperplasia, ductal carcinoma in situ) compared to ductal hyperplasia and normal glandular epithelium. Staining with the methylation-specific antibody reavealed a weaker staining in neoplastic lesions compared to hyperplastic lesions and normal cells. Conclusions: We conclude that atypic ductal hyperplasia represents the histomorphological equivalent for the beginning of tumor genome evolution that progresses in ductal carcinoma in situ and infiltrating carcinoma.

5 Detection of high-risk human papillomavirus (HPV) E6 and E7 oncogene transcripts increases the specificity of the detection of a cervical intraepithelial neoplasia (CIN) K. SOTLAR, D. DIEMER, A. STUBNER, S. MENTON1, M. MENTON1, K. DIETZ2, D. WALLWIENER1 B. BU¨LTMANN Institut fu¨r Pathologie 1 Universita¨ts-Frauenklinik 2 Institut fu¨r Medizinische Biometrie, Universita¨t Tu¨bingen Aims: The oncogenic potential of the high-risk human papillomavirus (HR-HPV) genotypes depends on the expression of the viral oncogenes E6 and E7. Thus, the detection of these transcripts could serve as a factor in the evaluation of a woman0 s risk of development of cervical intraepithelial neoplasia (CIN). Methods: A nested RT-PCR assay for the detection of E6/E7 oncogene transcripts of all known HR-HPV genotypes was established. Cervical scrapes of 779 HRHPV-DNA-positive women exhibiting all grades of CIN were examined. Results: Spliced E6/E7 oncogene transcripts of all the HR-HPVs were detected in numerous samples. In 459 cases with agreement between the cytologic and histologic findings, the prevalence increased with lesion severity: CIN 0, 18%; CIN I, 58%; CIN II, 77%; CIN III, 84%. Multiple transcriptionally active HR-HPVs were detected in 12% (33/279) of cases. While sensitivity and negative predictive value of HR-HPV DNApositivity for the detection of a CIN lesion were

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significantly (po0:0001) higher than those of E6/E7 mRNA positivity, the opposite was true for the specificity and positive predictive value (po0:0001). Conclusions: Besides the detection of HPV DNA, the detection of HR-HPV E6/E7 oncogene transcripts may serve as a valuable tool in increasing the specificity of HPV testing. A prospective follow-up study is currently been undertaken to evaluate its prognostic relevance for the persistence of a HR-HPV infection, and the possible evolution and further development of a CIN lesion.

6 Expression of early placenta insulin-like growth factor (EPIL) in breast cancer cells provides an autocrine loop with enhancement of predominantly Her2-related Invasivity H. BU¨RGER, D. KEMMING, M. HELMS, U. FELDMANN, A. MATUSCHEK, W. BO¨CKER, B. BRANDT Institut fu¨r Pathologie 1 Institut fu¨r Klinische Chemie, Universita¨t Mu¨nster Aims: Recently, we were able to show that the expression of early placenta insulin like growth factor (EPIL) is expressed by highly motile Her2-positive breast cancer cells in vitro (Brandt et al, Cancer Res. 2002) in Paget cells in vivo and indicates a poor clinical prognosis, irrespectively of other prognostic factors. Results: Epil overexpression in SKBR3 cells resulted in fast and frequent protrusion formation of the cells shown by laser scan microscopy. The cells were further characterized by a significantly increased invasiveness, which could be reversed by Epil specific siRNA treatment. Increased invasiveness and morphological changes were associated with a decreased erk1/2 phosphorylation. Methods: In order to demonstrate the interplay between HER2 and Epil we established a cellular model for high simultaneous Epil and Her2 expression. The Her2positive breast cancer cell line SKBR3 was modified with an EPIL expression vector. In addition, an assay for the knockdown of EPIL-expression via siRNA was established. Erk1/2 expression was measured via Western Blot. The phenotype of the viable cells was determined by laser scan microscopy. Conclusions: These data further supports the assumption that EPIL might provide an autocrine loop in Her2positive breast cancer cells that enforce metastasis, conceivably escape from adjuvant therapy and in consequence poor clinical outcome. A tight interaction between HER2 and EPIL in invasive breast cancer cells is therefore likely. The exact mechanims remain to be elucidated.

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State-of-the-art Lecture / Pathology – Research and Practice 201 (2005) 153–300

State-of-the-art Lecture

9 Classification and grading of invasive breast carcinoma C.W. ELSTON

7 Tumor stem cells. A new concept in oncology

Department of Histopathology, Nottingham City Hospital, Nottingham NG5 1PB

O. WIESTLER Deutsches Krebsforschungszentrum Heidelberg

Symposium: Breast Carcinoma I 8

Molecular genetics of sporadic and hereditary breast cancer R.K. SCHMUTZLER

Abteilung Molekuare Gyna¨ko-Onkologie, Universita¨tsFrauenklinik Ko¨ln Breast cancer is a heterogenous disease caused by mutations in tumor associated genes (TAG). The identification of TAGs can (1) improve risk prediction, (2) tailor therapy and (3) may lead to the development of new therapeutic targets. To (1) Risk prediction: While the vast majority of breast carcinomas occur spontaneously, a small subset of 5% is caused by an autosomal dominant transmission of mutations in the genes BRCA1 or BRCA2 conferring an increased risk of up to 80% for breast cancer and up to 40% for ovarian cancer. Molecular genetic diagnosis allows identification of women at high risk that can then be offered predicitve meassures. Results of the Hereditary German Breast and Ovarian Cancer Consortium supported by the German Cancer Aid will be presented. To (2) Tailored therapy by new prognostic markers: Due to a lack of reliable prognostic factors, a lot of women are presently overtreated by adjuvant therapy. The identification of gene signatures by genomic profiling or new prognostic markers like uPA/PAI or the detection of isolated tumor cells in the bone marrow may improve prognostic predictions and avoid over- or undertreatment. To (3) New therapeutic targets: Elucidation of the crucial importance of growth factor stimulation via membrane receptors has led to the design of molecular targets interacting with these signalling-pathways. Substances like the antibody against Her2/neu have already been implemented successfully in first-line therapies. These combined strategies not only improve mortality rates but also quality of life for women suffering from breast carcinoma.

Invasive carcinomas may be sub-divided morphologically according to their degree of differentiation. This is achieved in two ways, by assessing histological type and histological grade. A wide range of histological patterns is recognised in invasive carcinoma of the breast and four broad prognostic groups are recognised: the excellent prognosis group comprises the special types (tubular, cribriform, mucinous carcinomas); the good group tubular mixed, mixed ductal NST/special type and classical lobular carcinoma; the average group mixed lobular, medullary and atypical medullary carcinoma and the poor group is composed of ductal NST, mixed ductal and lobular and solid lobular carcinoma. Histological typing of breast cancer also adds to our understanding of the biology of breast cancer. For example, tumours with a medullary phenotype which express basal cytokeratins and are p53 positive and ER and c-erbB2 negative are strongly predictive of the BRCA-1 carrier state. Histological grading refers to the semi-quantitative evaluation of the morphological structure of breast carcinomas. In the Nottingham method three characteristics of the tumour are evaluated, tubule formation as an expression of glandular differentiation, nuclear pleomorphism and mitotic counts. A numerical scoring system on a scale of 1–3 is used to ensure that each factor is assessed individually. Overall grade is assigned as follows: Grade 1 3–5 points, Grade 2 6–7 points, Grade 3 8–9 points. There is a highly significant relationship between histological grade and prognosis; survival worsens with increasing grade. Histological grading has been shown to have good reproducibility and has been adopted for use in Europe, Australasia and the United States.

10 Ductal carcinoma in situ: The progenitor cell concept as a new model in proliferative breast pathology W. BO¨CKER Institut fu¨r Pathologie, Universita¨t Mu¨nster Although numerous experimental data suggest the existence of a self-renewing mammary stem cell, the question of its characteristics has never been satisfactorily resolved. Using double immunofluorescence experiments for simultaneous demonstration of basal

ARTICLE IN PRESS Symposium: Advances in Molecular Pathology I / Pathology – Research and Practice 201 (2005) 153–300

cytokeratin subgroups 5/14, the glandular cytokeratins 8/18 and the myoepithelial differentiation marker smooth muscle (sm) actin, we provided direct evidence of the existence of Ck5/14+ cells that have the potential to differentiate to either glandular (Ck8/18+) or myoepithelial cells (sm-actin+) through intermediary cells (Ck5/14+ and Ck8/18+ or sm-actin+). Based on these data we postulate that the Ck5/14+ cells are phenotypically and behaviourally progenitor (committed adult stem) cells of the human breast epithelium (Boecker et al, 2002). This hypothesis has recently been confirmed by in vitro studies of Dontu et al (2003). Based on our own data on proliferative breast disease lesions we conclude that usual ductal hyperplasia is a progenitor cell-derived lesion, whereas most breast cancers seem to evolve from differentiated glandular cells. Western blotting experiments which show large amounts of Ck5 in benign proliferative breast lesions, but not in ductal carcinoma in situ confirm the immunofluorescence data. Double-fluorescence experiments provide a new tool to phenotypically characterize adult stem cells and their progenies. Our results pave the way towards a new cell biological model of proliferative breast disease and breast cancer.

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malignancy, are classified as BI-RADS 4. Minimal invasive biopsy should be considered in patients with these lesions. BI-RADS 5 lesions are highly suggestive of malignancy. It is recommended that appropriate action should be taken for these most suspicious lesions. The accuracy of the mammography as the primary diagnostic tool can be increased by the use of ultrasound and physical examination. In some situations, MRI is helpful for further evaluation. However, classifying the lesions with precision is not trivial since overlap exists between malignant and benign features.

Symposium: Advances in Molecular Pathology I—Liver and upper Gastrointestinal Tract 12 Characterisation of subtypes of hepatocellular carcinoma on the basis of gene-expression profiles J.H. NEUMANN1,2, C. GUELLY3, C.R. BUCK3, C. LACKNER1, H. DENK1, K. ZATLOUKAL1 1

11 Radiological features of breast carcinoma

Institut fu¨r Pathologie, Universita¨t Graz Pathologisches Institut, Universita¨t Freiburg 3 Oridis-Biomed GmbH, Graz 2

U. KETTRITZ Institut fu¨r Radiologische Diagnostik, MammaZentrum, HELIOS Klinikum Berlin When considering typical features of malignant lesions, the radiologist must differentiate between invasive cancers consisting of mass lesions and ductal carcinoma in situ, typically appearing as microcalcifications. Common malignant features of invasive cancers include irregular shape and indistinct or spiculated margins. In microcalcifications, segmental distribution and pleomorphic shape are the features with the highest predictive value of malignancy. However, there is a broad spectrum of findings that confound the reliable differentiation between benign and malignant lesions. The American College of Radiology has established the Breast Imaging Reporting and Data System (BI-RADS) for standardizing radiological terms and reports in mammography screening. The Breast Imaging Reporting and Data System provides diagnostic categories that have implications for guidance regarding follow-up or biopsy of mammographic breast lesions. BI-RADS 3 lesions are considered probably benign with a malignancy risk o2%. These findings can be followed up at predetermined intervals according to current recommendations. Suspicious lesions with a substantial probability, but without the classic appearance of

Aims: Hepatocellular Carcinoma (HCC) is one of the most common human malignant neoplasms causing worldwide more than 400,000 deaths per year. Mallory Bodies (MB) and Intracellular Hyaline Bodies (IHB) are cytoplasmatic inclusions in hepatocytes associated with a variety of neoplastic and non-neoplastic liver diseases. MB and IHB were found in 10-30% of investigated HCC. Methods: We generated subtractive suppressive hybridization (SSH) cDNA libraries, highly enriched with clones representing genes both up- and down-regulated in HCC. Inserts from SSH cDNA libraries were amplified and printed onto glass slides to produce a focused HCC cDNA microarray. cDNA from 8 HCC without inclusions, 2 HCC showing MB and 2 HCC showing IHB was compared with cDNA from normal liver by competitive hybridization. Results: About 410 genes consistently deregulated in these liver diseases were identified. Clustering analysis led to the identification of 27 genes differentially expressed between HCC with and without inclusion bodies. Thus, HCC showing distinct morphological features such as inclusion bodies could be distinguished as a separate molecular entity on the basis of gene expression profiles. Conclusions: We could demonstrate that the presence of inclusion bodies in HCC is associated with various changes mainly in liver cell metabolism and detoxification.

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It can be concluded that glucocorticoids may play a role in carcinogenesis of HCC with inclusion bodies. Moreover, our data indicate that these patients may show a diverse response to drugs metabolized by members of the CYP3A subfamily. These results suggest that particularly in the context of planning and evaluation of clinical trials a difference in drug metabolism in HCC with inclusion bodies should be considered.

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Telomere shortening is correlated to increased chromosomal instability and dedifferentiation of hepatocellular carcinoma

R.R. PLENTZ1, P. FLEMMING2, H. KREIPE2, B. SCHLEGELBERGER3, K.L. RUDPOLPH1, L. WILKENS2,3 1

13 Promoter methylation profile of hepatocellular carcinomas I. TISCHOFF1, N. KRAUSE1, A. MARKWARTH1, H. WITZIGMANN2, J. HAUSS2, CH. WITTEKIND1, A. TANNAPFEL1 1

Institut fu¨r Pathologie, Universita¨t Leipzig Chirurgische Universita¨tsklinik, Universita¨t Leipzig

2

Aims: To determine the methylation profile of multiple tumor-related genes during hepatocarcinogenesis, promotor methylation of 14 newly described tumor related genes in hepatocellular carcinomas (HCC) was examined. Methods: Fresh frozen tissue of 50 HCC and corresponding non-neoplastic liver was anylsed by methylation-specific PCR (MSP) for p16, E-cadherin, SOCS1, Pax5NPax5b BLU, SEMA3B, RASSF1A, GSTP1, DAPK, CASP8, TMS1, APAF1 and TFPI-2. The corresponding mRNA was analysed using Light Cyclers PCR. Results: Several tumor related genes were frequently methylated in HCC: Pax5 (50/50 HCC [100%]), E-cadherin (45/50 HCC, [91%]), SEMA3B (42/50 HCC, [84%]), Pax5b (41/50 HCC, [82%]), SOCS1 (33/50 HCC, [66%]), RASSF1A (25/50 HCC, [50%]), TMS1 (25/50 HCC, [50%]), CASP8 (24/50 HCC, [47%]), GSTP1 (24/50 HCC, [47%]), p16 (24/50 HCC, [47%]), BLU (10/50 HCC, [20%]). Methylation of APAF1 (4/50, 7%), and TFPI-2 (2/50, 3%) were rare events. DAPK was unmethylated in all cases examined. In the corresponding non-neoplastic liver tissue, methylation of Pax5a was found in all tissues. Promoter methylation in normal liver was neither detected for APAF1, nor for TFPI-2 or DAPK. Downregulation of mRNA to various extents was observed in all genes with methylated promoters. Conclusions: Our results indicate that promoter methylation of tumor related genes is a frequent event in the carcinogenesis of hepatocellular carcinomas. The occurrence of methylation in cirrhosis points to the premalignant potential of this condition. The high prevalence of Pax5a methylation indicate, that this gene may be irrelavant in hepatocarcinogenesis.

Abteilung Gastroenterologie, Hepatologie und Endokrinologie 2 Institut fu¨r Pathologie 3 Institut fu¨r Zell- und Molekularpathologie der Medizinischen Hochschule Hannover Aims: In a previous study significant shortening of telomeres was seen in cells of hepatocellular carcinoma (HCC) compared to non-neoplastic liver cells. It was also shown, that chromosomal instability increases with morphological dedifferentiation. To test whether there is a correlation of telomere shortening and the grade of chromosomal instability we compared telomere length and the number of chromosomes 8, a chromosome often involved in HCC and an indicator for chromosomal instability. Methods: Cytological specimens of HCC (n ¼ 15) and controls (n ¼ 5) were analysed by simultaneous fluorescence in situ hybridisation (FISH) using telomer specific probes and a differently labelled probe for centromere of chromosome 8. Telomer length of all chromosomes in a cell was determined with a special software and compared to the signal constellation for chromosome 8. Results: Telomere length in carcinoma cells with more than 2 copies of chromosome 8 were significantly shorter than in non-neoplastic cells with two copies of chromosome 8 (po0:001). Moreover, telomeres in cells with 5 or more signals were also shorter than in cells with 3 or 4 signals for chromosome 8 (po0:001). However, telomere length did not differ significantly for cells with 5–10 chromosomes 8, respectively. Conclusions: Our data suggest that telomere shortening and CIN are closely correlated in HCC. However, with increasing CIN telomere shortening is limited to a basic value indicating a minimal level essential for cell survival.

15 Epigenetic inactivation of four proapoptotic genes in malignant epithelial liver tumors I. TISCHOFF1, H. WITZIGMANN2, J. HAUSS2, CH. WITTEKIND1, A. TANNPAFEL1 1

Institut fu¨r Pathologie, Universita¨t Leipzig Chirurgische Universita¨tsklinik, Universita¨t Leipzig

2

Aims: Recent studies indicate that intracellular signaling molecules with proposed roles in the regulation of

ARTICLE IN PRESS Symposium: Advances in Molecular Pathology I / Pathology – Research and Practice 201 (2005) 153–300

apoptosis, nuclear factor-kB activation and cytokine maturation may be inactivated via epigenetic mechanisms in epithelial tumors. To elucidate the role of of these molecules in hepatocellular carcinomas (HCC) and cholangiocarcinomas (CC), DNA methylation and mRNA expression of CASP8, TMS1, APAF1 and DAPK was analysed. Methods: DNA methylation of fresh frozen tissue HCC (n ¼ 50) and CC (n ¼ 20) and corresponding non nonneoplastic liver were analysed by methylation specific PCR (MSP). The presence of mRNA transcripts was evaluated by semiquantitive real time RT-PCR using the LigtCyclerssystem. Results: CASP8 DNA methylation was found in 24/50 (47%) of HCC and in 17/20 (87%) of CC. TMS1 promoter methylation was detected in 25/50 (50%) of HCC and in two corresponding normal liver tissues. APAF1 was methylated in 4/50 (7%) in HCC. TMS1. APAF1 and DAPK were neither methylated in HCC nor in CC. The promoter methylation correlated with the downregulation of the transcript expression in these tumors. Conclusions: Our study shows that DNA methylation mediated silencing and subsequent transcript downregulation of the proapoptotic genes CASP8 and TMS1 are involved in malignant liver tumors. These data indicate a possible role of epigenetic silencing of CASP8 as well as TMS1 in the carcinogenesis of malignant epithelial liver tumors.

16 Nuclear localization and differential expression of Snail, an E-cadherin repressor, in adenocarcinomas of the upper gastrointestinal tract K.-F. BECKER1, E. ROSIVATZ1,2, E. KREMMER3, H. HO¨FLER1, M. SARBIA1 1

Institut fu¨r Pathologie, Technische Universita¨t Mu¨nchen 2 Present address: Department of Biological Sciences and Chemical Consortium (CBC), Imperial College London 3 GSF-National Research Center for Environment and Health, Institute of Molecular Immunology, Mu¨nchen Aim: Mechanisms for transcriptional downregulation of the homophilic cell adhesion molecule E-cadherin in human tumors are not fully understood. The aim of our study was to analyse Snail, a direct repressor of Ecadherin transcription, in adenocarcinomas from the upper gastrointestinal tract. Methods: We analyzed a series of 239 tumors from esophagus (n ¼ 53), cardia (n ¼ 102), and stomach (n ¼ 84) arranged in tissue microarrays for Snail expression using an in-house generated novel monoclonal antibody. Results: Specificity of the new antibody was demonstrated by Western blot, immunofluorescence, and immunohisto-

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chemistry of transfected cells and primary human tissues. Nuclear Snail immunoreactivity was seen in 20 (8%) tumors and was significantly more frequent in esophageal adenocarcinomas (18.9%) than in cardiac (6.7%) or in gastric carcinomas (3.6%; p ¼ 0:0054). In 7 (35%) out of the 20 Snail positive cases E-cadherin immunoreactivity was lost. No correlation was found for nuclear Snail expression and tumor grade, Laure´n0 s classification, WHO classification, tumor stage and tumor size. Conclusion: We generated a novel monoclonal antibody against human Snail. Downregulation of E-cadherin is correlated with Snail expression in a subset of cases.

17 Hypermethylation of HPP1 and hMLH1 in esophageal adenocarcinoma and its precursor lesions H. GEDDERT, E. ISKENDER, A. FLORL1, C. FRANKE2, H. E. GABBERT, M. SARBIA3 Institut fu¨r Pathologie 1 Urologische Klinik 2 Chirurgische Klinik, Universita¨t Du¨sseldorf 3 Institut fu¨r Pathologie, Technische Universita¨t Mu¨nchen Aims: We determined the prevalence of aberrant methylation of the HPP1 and hMLH1 genes in 12 esophageal adenocarcinomas and their related morphological precursor lesions. Both genes are supposed to play a crucial role in carcinogenesis. Methods: After microdissection of 10 specimens with intestinal metaplasia, 5 with low-grade intraepithelial neoplasia and 6 with high-grade intraepithelial neoplasia a methylation-specific real-time PCR was performed including the 12 carcinomas. Normal esophageal tissue served as control in each case. Results: Hypermethylation of HPP1 was found in 4 of 10 (40%) intestinal metaplasias, in 2 of 5 (40%) lowgrade intraepithelial neoplasias, in 5 of 6 (83%) highgrade intraepithelial neoplasias, and in 5 of 12 (42%) adenocarcinomas. Hypermethylation of hMLH1 was neither detected in intestinal metaplasias nor in the low-grade intraepithelial neoplasias, but in 1 of 6 (17%) high-grade intraepithelial neoplasias, and in 2 of 12 (17%) adenocarcinomas. Hypermethylation of HPP1 or hMLH1 did not occur in normal esophageal tissue. Conclusions: Our data indicate that aberrant methylation of HPP1 and hMLH1 is restricted to pathological cellular changes. Hypermethylation of HPP1 is a frequent and early finding in carcinogenesis of esophageal adenocarcinoma. In contrast, hypermethylation of hMLH1 occurs less frequent and in later stages of carcinogenesis.

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Symposium: Advances in Molecular Pathology I / Pathology – Research and Practice 201 (2005) 153–300

18 Expression analysis of drug associated genes in Barrett0 s carcinomas before and after neoadjuvant chemotherapy R. LANGER, K. SPECHT, K. OTT1, H.J. STEIN1, J.R. SIEWERT1, M. SARBIA, H. HO¨FLER Institut fu¨r Pathologie, TU Mu¨nchen 1 Chirurgische Klinik und Poliklinik, Klinikum Rechts der Isar, TU Mu¨nchen Aims: To determine whether chemotherapeutic agents may influence the expression of drug associated genes during therapy in locally advanced Barrett0 s carcinoma. Methods: Biopsy and surgical resection tissue of 18 patients with locally advanced Barrett0 s carcinoma was examined. All patients received at least one cycle of a cisplatin and 5-FU based chemotherapy (CTX). We investigated pretherapeutic RNA expression levels of 5FU associated genes (TS, TP, DPD, MTHFR) and platinum-associated genes (Caldesmon, MRP1, MDR1) in untreated biopsy material using real time RT—PCR. The results were compared with RNA levels of these genes that were determined in neoadjuvant CTX treated surgically resected esophagectomy specimen of the same patients. Results: Expression levels of the majority of the analyzed genes did not significantly change after CTX treatment. Interestingly, however, a subgroup of patients with unfavourable clinical course (i.e. therapy abort due to disease progress) showed markedly and in part significantly higher both pre- and posttherapeutic levels of MTHFR, Caldesmon, MRP1 and MDR1 compared to patients with favourable clinical course. Moreover, tumors of the abovementioned subgroup of patients showed higher TS levels after CTX. Conclusions: CTX itself does not significantly influence expression of a number of CTX-associated genes. Pretherapeutic determination of gene expression levels for response prediction may therefore be representative during the whole course of therapy.

Aims: Previous cytogenetic studies indicate that loss of the Y chromosome is among the most frequent genetic alterations in esophageal (Barrett’s) adenocarcinomas (AC). In the current study, we determined whether alterations of the Y chromosome are more prevalent in esophageal than in cardiac or gastric AC and whether they are correlated with clinico-pathologic characteristics of the tumors. Methods: Using a Biotin-labelled centromeric probe and chromogenic in situ hybridization (CISH), 88 esophageal (Barrett’s), 23 cardiac and 15 gastric AC arranged in tissue microarrays were investigated for Y chromosome alterations. Results: Overall, loss of the Y chromosome was found in 78 cases (61.9%), polyploidy of the Y chromosome was found in 10 cases (7.9%) and normal Y chromosome number in 38 cases (30.2%). Prevalence of Y chromosome loss was not significantly different between esophageal (64.8%), cardiac (47.8%) and gastric AC (66.7%). Furthermore, no correlation was found between Y chromosome alterations and tumor stage, histological subtype (Lauren and WHO classification), tumor grade and patient’s age. Polyploidy of Y chromosome tended to be more frequent in tumors larger than 5 cm (13.7%) than in smaller tumors (4.2%). Conclusions: Loss of the Y chromosome is a very frequent genetic alteration in upper gastrointestinal AC. It may be an early event in upper gastrointestinal carcinogenesis since it is equally prevalent in early and in advanced tumor stages. Therefore, CISH-based detection of Y chromosome loss may become a molecular tool for the diagnosis of early malignant or premalignant lesions in the gastrointestinal tract.

20 Gene amplification in gastrointestinal stromal tumors (GIST) L. TORNILLO1, G. DUCHINI1, V. CARAFA1, A. LUGLI1, S. DIRNHOFER1, D. D VIZIO2, A. BOSCAINO3, G. SAUTER1, L. INSABATO2, L.M. TERRACCIANO1 1

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Alterations of the Y chromosome are frequent in upper gastrointestinal adenocarcinomas M. SARBIA1, F. PU¨HRINGER1, H. GEDDERT2, R. LANGER1, B.V. RAHDEN3, M. FEITH3, J.R. SIEWERT3, H.J. STEIN3 1

Institut fu¨r Pathologie Institut fu¨r Pathologie, Universita¨t Du¨sseldorf 3 Chirurgische Klinik und Poliklinik, Technische Universita¨t Mu¨nchen 2

Institut fu¨r Pathologie, Universita¨t Basel Institute of Pathology, University ‘‘Federico II’’ of Naples (I) 3 Department of Pathology, Hospital ‘‘Antonio Cardarelli’’, Naples (I) 2

Aims: Gastrointestinal stromal tumors (GISTs) are the most frequent gastrointestinal mesenchymal tumors. They are characterised by immunohistochemical positivity for CD-117 (KIT) and by activating mutation in different RTK genes. No single morphologic parameter can predict reliably their behavior. We have investigated

ARTICLE IN PRESS Symposium: Breast and Ovarial Carcinoma-Oral Presentations / Pathology – Research and Practice 201 (2005) 153–300

the potential significance of amplifications of 5 known oncogenes in GISTs. Methods: c-myc, ccnd1, her-2, egfr, mdm2 were analyzed by fluorescent in situ hybridization on a tissue microarray containing 100 GISTs (94 primary and 6 metastasis), 42 other mesenchymal tumors and 5 samples of normal tissues. Follow-up information was available for 59 GISTs. Amplification was defined as a gene probe/centromere probe ratio of more than 2.0. Results: Amplification was found for c-myc in 3 of 90 (3.3%), for mdm2 in 5 of 94 (5.3%), for egfr in 5 of 94 (5.3%), and for ccnd1 in 7 of 79 (8.9%) evaluable cases. No amplifications were seen for her-2. Amplifications of mdm2 and ccnd1 were associated with clinical malignancy and high risk of malignant behavior. Conclusions: mdm2 and ccnd1 amplification, both occuring in nearly 10% of the high risk/malignant samples, may have prognostic relevance in GIST. egfr, amplified in 5 of 94 cases, may have a role in determining the aggressivity of the tumor and perhaps may represent another potential therapeutic target gene in GISTs.

Symposium: Breast and Ovarial Carcinoma -Oral Presentations

21 Quality assurance in breast pathology: application of guidelines A. LEBEAU Pathologisches Institut der LMU Mu¨nchen Aims: Within the multidisciplinary process of clarifying palpable or non-palpable abnormalities, it’s the role of the pathologist to provide that information crucial for the adequate management of the patient. To achieve the aim of therapy guiding pathological assessment, the application of guidelines is recommended, that are targeted at the determination of necessary and relevant criteria. Methods: At present a practice guideline for breast pathology is available, that is part of the German S3guidelines for the early detection of breast cancer (www.senologie.org) and for the diagnosis and therapy of breast cancer in women (www.krebsgesellschaft.de). Results and Conclusions: The tissue specimens must comply with certain requirements to enable standardised handling of the tissue specimens. This includes that the resection specimens are unequivocally marked by the surgeon in order to obtain proper orientation and sent in without prior cutting or removal of tissue.

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The quality-assured pathological assessment of the following criteria is relevant for the local and/or systemic treatment of breast cancer: (a) histological type (WHO, 2003); (b) histological grade (DCIS: nuclear grade and presence or absence of comedo necrosis acc. to WHO, 2003; invasive carcinoma: acc. to Elston and Ellis, 1991); (c) tumour size; (d) pTNM classification (UICC, 2002); (e) peritumoral vascular invasion; (f) presence of additional tumour foci (multifocality/multicentricity acc. to Faverly et al., 1994); (g) residual tumour (R) classification (UICC, 2002) and distance to resection margins (for DCIS and invasive carcinomas); (h) hormone receptor status (invasive carcinomas: that is specification of the percentage of tumour cell nuclei positive for the oestrogen and progesterone receptor); (i) other special stains, if required (e. g. HER-2/neu, etc.). The standardised assessment of these performance indicators is illustrated in the lecture.

22 Prognostic significance of histopathology, tumor staging and genomic aberrations in breast cancer of young women H.P. SINN, A. HORLACHER, S. WEBER-MANGAL1, P. SCHIRMACHER Pathologisches Institut, Universita¨tsklinikum Heidelberg 1 Institut fu¨r Humangenetik, Universita¨tsklinikum Heidelberg Aims: There is a strong evidence that breast cancer in young women may genetically be different from others, but the prognostic relevance of genetic alterations compared with conventional factors in this age group are largely unknown so far. Methods: We have retrospectivly analyzed 250 young womens with primary breast cancer (age of onset: 21–35 years) for conventional tumor stage and clinical data including follow-up. Comparative genomic hybridization (CGH) was obtained from a subset of 88 patients with sufficient follow-up data. Results: Overall survival rates (OAS) at 10-years were 44.4% in patients of 21–29 years at diagnosis and 48.3% in the 30–35 years age group. Lymph node status and and Elston-Ellis grading were the most significant prognostic factors with a 10y OAS of 75.5%, 45.5%, 18.2%, and 9.7% for pN0, 1a, 2a, and 3a respectively (TNM 2003). Other significant prognosticators included hormone receptor status (10y OAS 80.9% vs. 35.9% for ER/PgR positive and negative tumors) and bilaterality. The analysis of CGH results revealed a strong association of grade 3 tumors with gains on the long arm of chromosome 8 (po0:001) and positive family history

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with gains on 12p than those without (po0:01). The multivariate analysis showed an independently adverse significance for patients with tumors exhibiting losses on 8p and gains on 18q. Conclusions: Various chromosomal alterations and specific clinico-pathologic features are associated with adverse prognosis in very young breast cancer patients.

23 Comparison of chromogenic in-situ-hybridisation (CISH) with FISH and IHC for assessment of HER2 status and prediction of response to therapy: analysis from a study of trastuzumab monotherapy M. HOFMANN, T. HENKEL, J. RU¨SCHOFF Institut fu¨r Pathologie, Klinikum Kassel Aims: Accurate determination of HER2 status is essential for identification of patients who are likely to benefit from trastuzumab (Herceptins). Current methods (IHC and FISH) are reliable, robust and reproducible, but have certein limitations, e.g. IHC 2+ cases; costs and equipment for FISH. Aim of this study was to compare an alternative method, CISH, with the established techniques. Methods: Tumor samples from monotherapy trial WO16229 of 86 patients were tested by IHC, FISH and CISH. Results: Overall concordance was 82.6%. FISH/CISH concordance was 90%, IHC/CISH: 87%. IHC/ FISH:88%. CISH, FISH, IHC and polysomy data were available for 23 patients. Sensitivity in correlation to response was 90% for CISH, 84% for FISH and 100% for IHC. Three responders were IHC 3+ but FISH negative, 2 were CISH positive and highly polysomic. Conclusions: CISH displays high concordance with FISH and ICH. This supports use of CISH for assessing HER2 gene amplification. CISH is also highly sensitive for predicting response to trastuzumab therapy. Thus CISH may have a place for determining HER2 status in laboratories where FISH is not available or in borderline cases as an additional testing alternative.

24 Determination of the HER2 status before and after primary chemotherapy in advanced breast cancer Z. VARGA1, R. CADUFF1, B. ODERMATT1, B. PESTALOZZI2 1

Department of Pathology, Institute of Clinical Pathology 2 Department of Internal Medicine, Clinic of Oncology, University Hospital Zurich, Switzerland

Aims: Encouraging results have been obtained with neoadjuvant chemotherapy in patients with clinically advanced breast carcinoma. Predictive factors are frequently determined on core needle biopsies before neoadjuvant chemotherapy. Currently, the reproducibility of the determination of the hormone receptor- and the HER2 status before and after primary chemotherapy is unclear. The aim of this study was to compare hormone receptor expression and HER2 status on core needle biopsies before chemotherapy with surgical samples after primary chemotherapy. Methods: We investigated the hormone receptor expression and HER2 prospectively in 23 patients with stage II, III or IV breast carcinomas. All patients were treated with 2–6 cycles of Fluorouracil-Epirubicin and/or Cyclophos-phamid/Epidocetaxel. The HER2 protein and gene were assessed both on core needle biopsies before and on surgical specimens after completing chemotherapy by using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) methods. Estrogen and progesterone receptors (ER/PR) were also determined on both samples by IHC. Results: The HER2 status was modified in eight patients on IHC (35%) and in three patients by FISH (13%). Changes in ER/PR expression were detected in seven patients (30%). Conclusions: Our data suggest that a different HER2 and hormone receptor status can be obtained from surgical specimens after primary or neoadjuvant chemotherapy. Whereas only minor discrepancies occurred by using FISH, more frequent changes are seen by immunohistochemical detection of HER2 and ER/PR with the potential of choosing different therapeutic options. Intratumoral heterogenity as well as sampling variations may also contribute to modification of the hormone receptor- and HER2.

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Bootstrapping approach reveals inherent regulatory pattern in 550 invasive breast cancer cases: CK5/6/CK 14 and CK8/18/CK 19 builds an antagonistic set is among the most outstanding research communications 2005 E. KORSCHING1, J. PACKEISEN2, R. VOSS3,4, W. BO¨CKER1, H. BU¨RGER1 1

Gerhard-Domagk-Institut fu¨r Pathologie, Universita¨t Mu¨nster 2 Institut fu¨r Pathologie, Klinikum Osnabru¨ck 3 Institut fu¨r Arterioskleroseforschungan der Universita¨t Mu¨nster 4 Institut fu¨r Klinische Chemie und Laboratoriums medizin, Universita¨t Mu¨nster Aims: All the preliminary observations on a lot of marker sets defining different stages in the tumor

ARTICLE IN PRESS Symposium: Breast and Ovarial Carcinoma-Oral Presentations / Pathology – Research and Practice 201 (2005) 153–300

development are building a framework of work hypothesis which can be verified in characterising large pools of histological uniform rated paraffin probes. Methods: We developed a bootstrapping algorithm based on correlation measures to uncover regulatory patterns of immunohistochemical characterized tissue arrays with 550 invasive breast cancer cases. The algorithm is implemented in ‘S’ a computer language used to model mathematical solutions. Results: Focussing on the cytokeratins versus a set of prominent markers in breast cancer differentiation it will be obvious that markers which are known to appear in early (progenitor) forms conform to CK5/6 and CK 14 while others associated with late stages conform to CK 8/18 and CK 19. Markers examined are among others EGFR, EMA, erb-B2, Vimentin, p53, ER and PR. Conclusions: The developed approach is an elegant and complete procedure to reveal the real regulatory patterns which are enclosed in a certain experimental design. The statistical significance of the results calculated by our algorithm is generally high and in the presented experimental design smaller than 0.6 * 10E-6.

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reactivity was observed in 35/106 (33%) mammary cancers. In 20 (57%) out of the 35 Snail nuclear positive cases E-cadherin immunoreactivity was reduced or lost; estrogen receptor (ER) was weekly expressed in 10/35 (29%) cases. Conclusion: Using a novel antibody, we found for the first time frequent nuclear Snail immunoreactivity in mammary cancers with concomitant reduction of Ecadherin expression in more than half of the cases. Although gaps in our knowledge remain, our results are in line with a potential role for ER in Snail repression, as suggested by others.

27 Study of efficiancy of teleconsultation: The telepathology consultation service of the professional assoziation of german pathologists for the screening program of breast carcinoma is among the most outstanding research communications 2005 T. SCHRADER1, P. HUFNAGL1, W. SCHLAKE2, M. DIETEL1 1

26 Cellular localization of the E-cadherin repressor Snail

Institut fu¨r Pathologie, Charite´, Universita¨tsmedizin Berlin 2 Institut fu¨r Pathologie, Gelsenkirchen

in breast cancer K.-F. BECKER1, E. KREMMER2, E.. ROSIVATZ1,3, K. BLECHSCHMIDT1, H. HO¨FLER1, J. NA¨HRIG1 1

Institut fu¨r Pathologie, TU Mu¨nchen GSF-Forschungszentrum fu¨r Umwelt und GesundheitInstitut fu¨r Molekulare Immunologie, Mu¨nchen 3 Present address: Department of Biological Sciences and Chemical Consortium (CBC), Imperial College London 2

Aim: Snail, a master regulator of epithelial mesenchymal transition and direct repressor of E-cadherin transcription, has been implicated in breast cancer. However, nuclear Snail immunoreactivity, indicative for the active form, has not been demonstrated in human cancers due to the absence of specific antibodies. Methods: We analyzed a series of 106 breast cancers arranged in tissue microarrays for Snail and E-cadherin immuno-reactivity using an in-house generated novel monoclonal rat antibody. Results: Using transfected cells and archival human tissues, we evaluated the specificity of the new anti-Snail antibody, Sn9H2, by Western blot and immunohistochemistry, respectively. After transfection of a Snail cDNA in MCF7 mammary cells, E-cadherin was found to be reduced by 95%, indicating that Sn9H2 reacts with the functional Snail protein. Nuclear Snail immuno-

Aims: In the autumn a screening program will start for detecting breast cancer in the population of women 50 and above. For the first time in this program, quality assurance rules were established: All statements of the radiologists and pathologists have to be confirmed by a second opinion. Under the current institutional arrangements, these new quality assurance rules impose significant costs in terms of time and resources. A new Telepathology Consultation Service was developed based on the experiences of the Telepathology Consultation Center of the UICC. Methods and material: The service is completely webbased and a client-server model is the basic communication model. The operating system is MS Windows 2003 Server. The Internet Information Server is used as the web-server, the SQL-Server (both Microsoft) as the database. The websites, forms and control mechanism were created by ASP scripts and JavaScript. A study to evaluate the effectiveness of telepathological consultation against the conventional consultation has been carried out. Pathologists of the Professional Association token part and were at the same time requesting pathologists and consultants for other participants. Results and conclusion: The Telepathology Consultation Service of the Professional Association of German Pathologists is a fast and effective Germanlanguage, internet-based service for obtaining a second opinion.

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28 Patterns of p53 mutations separate ovarian serous borderline tumors and low and high-grade carcinomas and provide support for a new model of ovarian carcinogenesis A. HARTMANN, R. STO¨HR1, R.J. KURMAN2, I.M. SHIH2, G. SINGER3 Institut fu¨r Pathologie 1 Klinik fu¨r Urologie, Universita¨t Regensburg 2 Dept. of Pathology, Johns Hopkins University School of Medicine, Baltimore/MD 3 Institut fu¨r Pathologie, Universita¨t Basel Aim: The infrequent association of serous borderline tumors (SBTs) with invasive serous carcinoma (SC) has led to the view that SBTs are unrelated to invasive SC. The aim of the present study was to evaluate p53 mutations and expression in a morphologically well defined series of SBTs and invasive low-grade and high grade SC. Methods: A total of 96 sporadic serous tumors including 25 SBTs (11 atypical proliferative serous tumors and 14 intraepithelial low-grade SC [non-invasive micropapillary serous carcinomas (MPSCs)]), 12 low-grade SC (invasive MPSCs) and 59 high-grade SC were analyzed for their p53 mutational status of exons 5-9 by direct sequencing and for p53 expression by immunohistochemistry. Results: p53 mutations were found in 30 of 59 (50.8%) high-grade SC and one (8%) of 12 low-grade invasive SC compared to 2 (8%) of 25 SBTs, both non-invasive MPSCs. Although p53 immunoreactivity was generally higher in p53 mutant tumors, p53 mutations and overexpression were not significantly associated. Conclusions: The similar frequency of p53 mutations in SBTs and low-grade invasive SC in contrast to the significantly higher frequency in high grade SC (po0:0005) suggests a common lineage for SBTs and low-grade invasive SC and supports the view that SBTs are unrelated to the usual type of high grade invasive SC. These findings confirm our hypothesis of dual pathways of serous carcinogenesis based on previous studies showing KRAS and BRAF mutations in 60% of SBTs and low-grade SC but not in high grade SC. We propose a model of serous carcinogenesis in which SBTs are the precursors of low-grade SC whereas high-grade SCs develop ‘‘de novo’’ from in situ alterations in epithelial inclusion cysts.

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Comparison of three different grading systems for ovarian carcinoma: prognostic significance, concordance rates, and reproducibility D. SCHMIDT, S. KOMMOSS, F. KOMMOSS, M. TRUNK1, J. PFISTERER2, A. DU BOIS3

Institut fu¨r Pathologie, A2,2, Mannheim 1 MTM Laboratories, Heidelberg 2 Frauenklinik, Universita¨t Kiel 3 Gyna¨kologie & Gyna¨kologische Onkologie, HSK, Wiesbaden Aims: To assess the three most widely used grading systems for ovarian carcinoma in terms of prognostic significance, concordance rates and reproducibility. Methods: Representative H&E slides from 334 cases of stage IIb-IV ovarian carcinoma (prospective randomised, multi-center, phase III study). First round: grading of all cases according to FIGO, GOG and Silverberg by one author. Second round (after 1 year): 30 randomly selected cases graded by three authors (FK, DS, MT). Survival analysis by Kaplan-Meier method. Measurement of intra- and interobserver reproducibility by kappa statistics. Results: None of the three grading systems was prognostically significant (FIGO: p ¼ 0:292; GOG: p ¼ 0:624; Silverberg: p ¼ 0:934). The concordance rates between the three systems were as follows: FIGO/GOG: 95.5%, k ¼ 0:929; Silverberg/FIGO: 69.9%, k ¼ 0:533; Silverberg/GOG: 66v8%, k ¼ 0:481: Intraobserver reproducibility was: FIGO 100%, GOG 96.7% (k ¼ 0:953), Silverberg 80% (k ¼ 0:692). Interobserver reproducibility for the three investigators using all three grading systems was similar: FIGO: 80% (k ¼ 0:716), GOG: 80% (k ¼ 0:691), Silverberg: 77.8% (k ¼ 0:651). Conclusions: In cases of advanced-stage ovarian carcinoma histopathological grading has no prognostic significance after standardized chemotherapy. In our study, the most complicated system (Silverberg) proved to be the least reproducible.

Symposium: Advances in Molecular Pathology II —Colon and Miscellaneous 30

b-catenin driven expression of hTERT (human telomerase RT-component) indicates tumor stell cells in colorectal adenocarcinomas E. HIENDELMEYER, T. BRABLETZ, A. HAYNL, H. HERBST1, T. KIRCHNER, A. JUNG

Pathologisches Institut, Universita¨t Erlangen-Nu¨rnberg 1 Gerhard-Domagk-Institut fu¨r Pathologie, Universita¨t Mu¨nster Aims: Tumor stem cells are the active elements of tumors and are thus causative for the outcome of tumor diseases. The existence of tumor stem cells has already

ARTICLE IN PRESS Symposium: Advances in Molecular Pathology II / Pathology – Research and Practice 201 (2005) 153–300

been shown in leukemias and mamma carcinomas. Thus, we wanted to investigate if colorectal carcinomas (CRC) also contain cells with stemness and where these tumor stem cells are localized. Methods: Immunohistochemistry (IHC), Electric Mobility Shift Assay (EMSA), luciferase-reporter assay, knockdown using siRNA, Western blotting, chromatin immune precipitation (ChIP). Results: hTERT (human telomerase RT-component) is a marker for adult human epithelial stem cells. Thus, high expression of hTERT should be indicative for tumor stem cells. CRC display high amounts of hTERT in tumor cells expressing nuclear b-catenin which are not evenly distributed but instead are found zonally concentrated at the invasion front. Moreover, using several molecular biologic methods we show that the expression of the hTERT gene is driven by b-catenin which was shown to be essential for the maintanance of normal colorectal stem cells. Conclusions: CRC contain tumor stem cells which are found zonally concentrated at the invasion front. The stemness of these cells is charcterized by the nuclear localization of b-catenin and the high expression of the stem cell marker hTERT. Finally, nuclear b-catenin actively induces the state of stemness in colorectal tumor cells as hTERT is a b-catenin target gene.

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Nuclear b-catenin expressing tumor cells at the invasive front of colorectal carcinomas: Migrating tumor stem cells? S. SPADERNA, F. HLUBEK, O. SCHMALHOFER, A. JUNG, T. KIRCHNER, T. BRABLETZ Pathologisches Institut, Universita¨t Erlangen-Nu¨rnberg Aims: Most colorectal adenocarcinomas have mutations in the APC tumor suppressor gene, leading to nuclear overexpression of the Wnt-pathway effector b-catenin. The aim is to further assess oncogenic role of b-catenin. Methods: Reporter assays, expression arrays, EMSA, siRNA techniques, in situ hybridisation, immunohistochemistry and animal models. Results: We have shown, that nuclear b-catenin predominantly accumulates in de-differentiated tumor cells at the invasive front. In contrast tumor cells in central tumor areas of primary tumors and metastases are differentiated and often lack b-catenin. Nuclear bcatenin is involved in two fundamental processes in embryonic development: epithelial to mesenchymal transition (EMT) and generation of stem cells. Based on these facts we proposed, that the de-differentiated tumor cells at the invasive front of CRCs act as ‘‘migrating tumor stem cells’’. In support of this hypothesis, we could define two groups of genes,

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overexpressed in invading tumor cells: (1) b-catenin target genes like MMP-7, MT1-MMP and the g2 chain of laminin-5, synergizing to exert EMT and a strong promigratory activity. (2) Stem cell factors, like survivin. Conclusions: We suggest that primary tumors and metastases are derived from a pool of de-differentiated ‘‘migrating tumor stem cells’’, defined by strong nuclear b-catenin accumulation. Nuclear b-catenin induces an unusual combinations of two features in these cells: (1) The abilitiy to disseminate (after EMT) and (2) to simultanously act as a tumor stem cell, thereby driving malignant tumor progression including metastasis.

32 Maspin expression in colorectal cancer correlates with microsatellite instability and is a predictor of survival benefit from adjuvant 5-FU treatment in stage III colon cancer M. BETTSTETTER, P. WILD, M. WOENCKHAUS, P. RU¨MMELE, A. PAUER1, G. RO¨CKELEIN2, F. HOFSTA¨DTER, W. DIETMAIER Institut fu¨r Pathologie, Universita¨t Regensburg 1 Tumorzentrum Regensburg 2 Pathologisch-Anatomisches Institut, Regensburg Aims: Overexpression of maspin was reported to suppress metastasis and angiogenesis in breast and prostate cancers and was associated with adverse prognostic features in several other tumors. The aim of our study was to analyze maspin expression in CRC and its role as a 5-FU prediction marker. Methods: Maspin expression and subcellular localization was analyzed in 41 colorectal carcinomas with high frequency microsatellite instability (MSI-H) and 159 microsatellite stable (MSS) colorectal cancers by immunohistochemistry (IHC) and partly by relative quantitative real-time RT-PCR and Western Blot analyses. Expression and subcellular localization was correlated with microsatellite status and survival. Results: We found significantly upregulated maspin expression in MSI-H tumors compared to both MSS tumors and matched benign colonic mucosa. Increased maspin expression was also found in three MSI-H colon cancer cell lines, but not in three MSS colon cancer cell lines. We found that maspin expression was regulated by promoter methylation both in cell lines and tumors. An intense nuclear maspin immunostaining was seen specifically in MSI-H tumors (P ¼ 0:013), dedifferentiated tumors (P ¼ 0:006) and at the tumor invasion front. Nuclear maspin expression correlated with poor survival but was shown to be a positive predictor of 5-FU chemotherapy response. Conclusions: Nuclear maspin expression correlates with microsatellite instability, high tumor grade, poor

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survival but predicts survival benefit from 5-FU treatment in colon stage III tumor patients.

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p14ARF and p16INK4a promoter methylation in ulcerative colitis related carcinogenesis D.E. AUST, L.I. DOBRYDEN, A. KLAWITTER, M.G. HAASE, G.B. BARETTON Institut fu¨r Pathologie, Universita¨tsklinikum Carl Gustav Carus der TU Dresden

Aims: Promoter hypermethylation is thought to be a mechanism for gene silencing. Hypermethylation of tumor suppressor genes have been reported in a variety of solid tumors. p14ARF and p16INK4a promoter methylation was shown in precancerous lesions associated with chronic inflammation, such as Barrett’s esophagus. Little is known about the role of p14ARF and p16INK4a promoter methylation in ulcerative colitis (UC)-related carcinogenesis. Methods: Methylation specific PCR was applied to colectomy specimens of 32 UC-patients: 29 samples of nondysplastic mucosa, nine dysplasias, one adenoma and 36 cancers. Thirty-three sporadic colorectal cancers, matched for tumor stage, grade and location were use as a control group. Results: p14ARF methylation was observed in 3 of 36 (8.3%) UC-related cancers, 2 of 9 (22.2%) dysplasias and 5 of 29 (17.2%) nondysplastic UC mucosa specimens. In contrast, 16 of 33 (48.4%) of sporadic cancers showed p14ARF methylation. p16INK4a promoter hypermethylation was found in 26 of 36 (72.2%) UC-related cancers, 1 of 9 (11.2%) dysplasias and 14 of 29 (48.2%) nonneoplastic UC mucosa. In sporadic colorectal cancers, p16INK4a methylation was found in 16 of 33 (48.4%) samples. Conclusions: Our data suggest that methylation of the p14ARF and p16INK4a promoter is a relatively early event in UC-related carcinogenesis. p14ARF promoter methylation occurs more frequently in sporadic than in UC-related colorectal cancers, while p16INK4a promoter methylation seems to be more frequent in UC-related than in sporadic colorectal cancers.

1

Pathologisches Institut, Universita¨tsklinikum Freiburg IMSE 3 Chirurgische Klinik 4 Institut fu¨r Pathologie, Klinikum rechts der Isar, Mu¨nchen 5 Roche Diagnostics GmbH, Penzberg, Germany 2

Aim: Evaluation of the prognostic and/or predictive value of quantitative analysis of thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) mRNA expression in primary colorectal tumors. Materials and methods: Total RNA was isolated from microdissected, formalin-fixed and paraffin-embedded tissues and subjected to quantitative RT-PCR (QRTPCR) in the LightCyclers system. QRT-PCR data was correlated to histology and the clinical follow-up of 102 cases (n ¼ 40 without CTX; n ¼ 52 with CTX; n ¼ 10 with CTX and radiotherapy). Results: Significant correlations were found for tumor (1) T and N category and UICC stage (TP mRNA, TP:DPD ratio), (2) T category (TS:DPD ratio), and (3) differentiation grade (TS mRNA and TS:DPD ratio). In cases without CTX, overall survival was significantly associated with the TS:DPD ratio (p ¼ 0:032). Finally, in cases with CTX, a high TP/DPD ratio (o8.1; p ¼ 0:002) and, marginally, low DPD (o8.2; p ¼ 0:05) mRNA levels were significantly correlated to diseasefree survival. Conclusion: We present a novel, standardized approach for TP, DPD and TS mRNA quantification in archival tissue specimens. Application of this approach to 102 cases of colorectal cancers revealed tumor-specific TP, DPD and TS mRNA expression patterns. Moreover, novel prognostic (TS:DPD ratio) and predictive (TP:DPD ratio, DPD mRNA levels) markers for patients with colorectal cancer were identified.

35 Downregulation of connexin 26 in human lung cancer is related to promoter methylation Y. CHEN, D. HU¨HN, T. KNO¨SEL, M. PACYNA-GENGELBACH, N. DEUTSCHMANN, I. PETERSEN Institut fu¨r Pathologie, Charite´-Campus Mitte, Berlin

34 Predicting the outcome of 5-FU treated colorectal cancer patients by quantitative TP, DPD and TS mRNA expression analysis S. LASSMANN1, M. HENNIG2, R. ROSENBERG3, J. NA¨HRIG4, J. SCHREGLMANN4, F. KRAUSE5, M. POIGNEE-HEGER5, H. NEKARDA3, H. HO¨FLER4, M. WERNER1

Aims: Cell-Cell communication via gap junctions plays a key role in carcinogenesis. One of the gap junction proteins, Connexin 26 (Cx26) was considered as tumor suppressor in various cancers. The objectives of this study were: (1) to analyze the expression patterns of Cx26 in lung cancer. (2) to explore the mechanism responsible for the downregulation of Cx26 in lung cancer.

ARTICLE IN PRESS Symposium: Advances in Molecular Pathology II / Pathology – Research and Practice 201 (2005) 153–300

Methods: mRNA expression of Cx26 was analyzed in 17 lung cancer cell lines by Northern blot analysis and RTPCR. Tissue microarrays comprising 138 primary lung carcinomas were constructed to evaluate the protein expression of Cx26. Additionally, demethylation test was performed and methylation status of two lung cancer cell lines H2170 and H226 was investigated by PCR amplification of bisulfite modified DNA and sequencing. Results: Cx26 was found downregulated at mRNA level in 17 lung cancer cells compared to normal control. In 138 primary carcinomas analyzed by immunohistochemistry, 85 cases (62%) exhibited no expression of Cx26, while in other 53 cases, the Cx26 staining was positive (38%). An association between Cx26 expression and higher grading of tumors was detected in the tumor collective (p ¼ 0:028). In squamous cell carcinoma, tumors with higher stage and grading were linked to higher expression of Cx26 (p ¼ 0:015 and 0.017, respectively). Demethylation test restored the expression of Cx26 in lung cancer cell lines H2170 and H226, and sequencing of PCR products from bisulfite modified DNA revealed a positive methylation status in these two cell lines. Conclusions: Cx26 was downregulated in lung cancer and hypermethylation in promoter region could be responsible for the inactivation of the gene.

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ß-catenin pathway in sarcomas of the pulmonary artery(SPA) A. GAUMANN1, B. BODE-LESNIEWSKA2, R. ZIMMERMANN2, F. HOFSTA¨DTER1, W. DIETMAIER1

1

Institut fu¨r Pathologie, Universita¨t Regensburg Institute of Pathology University of Zu¨rich

2

Aims: The Wnt signaling pathway plays an important role in cell fate determination mediated by stabilization of the ß-catenin complex through APC. Thus, ß-catenin activation is an important step in the tumorigenesis through induction of transcription and proliferation via c-myc and cyclin D1. Since activation mutation of ßcatenin/APC has been shown to occur in a variety of tumors we thought to investigate the expression and mutation of the involved molecules in SPA. Methods: Expression of ß-catenin, hMLH, hMSH2, hMSH6 were examined by immunohistochemistry in 18 SPA as well as microsatellite instability (MSI) using the recommended reference panel for colorectal cancer. In addition, sequencing of Exon 3 of the ß-catenin was performed. Results: In 10 cases a membrane staining and in 3 cases additional cytoplasmic staining of ß-catenin was ob-

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served. One case showed some cells with nuclear staining and 3 cases were negative. Mutation analysis of ßcatenin revealed wildtype sequence of Exon 3 in all examined cases. MSI could only be detected in 2 cases of the SPA. Interestingly, one of these cases showed nuclear staining for ß-catenin. In addition, 4 cases revealed a LOH at the APC gene locus. Nuclear expression of hMLH1, hMSH2 and hMSH6 was detected 18 cases. Discussion: LOH of APC was the prominent observation in our series supporting the view that misfunction of this tumor supressor gene may play a role in the tumorigenesis of SPA. However, ß-catenin showed only nuclear translocation in 1 case, which was the only patient who had also MSI. Thus, alteration of the ßcatenin pathway and MSI seem to play a minor role in the proliferative activity of SPA in our series.

37 Comparison of preamplification techniques with respect to their validity in DNA-microarray experiments J. WILHELM, J.P. MUYAL1, M.M. STEIN1, W. SEEGER, R.M. BOHLE1, L. FINK1 Medizinische Klinik II, Universita¨t Giessen 1 Institut fu¨r Pathologie, Universita¨t Giessen Aims: Linear amplification by in vitro transcription (IVT) and PCR based amplification (SMART) were applied to conduct DNA-microarray experiments from 50ng total RNA to reveal which method yield most valid results. Methods: As reference, 50 mg total RNA of rat kidney and liver were directly labeled. 50ng of the same RNA were amplified and labeled by IVT (2 rounds) as well as by SMART (12 cycles). Factors of amplification were determined with real-time PCR for different target sequences. The labeled products were hybridized on 10K 60mer oligonucleotide microarrays. Results: The cDNA of the unamplified and the 1mg SMART-dscDNA sample contained 40pmol incorporated dyes. IVT yielded 50mg aRNA with 2000pmol dye. Both methods amplified the tested mRNA targets about 300-1000-fold. The factor using IVT was considerably lower (10-fold) for sequences with a distance of 41kb from the poly-A-tail. From the top-200 selected genes found to be differentially expressed between liver and kidney, 105 (58) were common between the reference and SMART (IVT). The intraassay correlations were R2 ¼ 0.92 (unamplified), 0.90 (SMART), and 0.69 (IVT). The R2 for unamplified vs. SMART (IVT) were 0.84 (0.68). Conclusions: Although the IVT protocol seems to have a higher yield than the SMART protocol, many IVT products are likely to be to short. Additionally, the

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reproducibility of two rounds of IVT is very poor. The results obtained after SMART are more valid than the results obtained after IVT. Thus, for 50ng total RNA as starting material, SMART is superior to IVT for preamplification.

38 Identification of novel pro-apoptotic genes in CD95mediated apoptosis by a functional genetic approach C. MAHOTKA, M. HASSAN, C.D. GERHARZ1, H.E. GABBERT Institut fu¨r Pathologie, Universita¨t Du¨sseldorf 1 Institut fu¨r Pathologie, Evangelisches Krankenhaus Bethesda zu Duisburg Aims: Methods are needed for rapid and efficient identification of functional genes for diagnostic and therapeutic applications. Renal cell carcinoma (RCC) exhibit increased resistance to CD95-mediated cell death, although the CD95-signalling pathway is functional. In order to identify novel pro-apoptotic genes that might determine the response of RCC to CD95mediated apoptosis, we describe here the multi-cycle technical knock out (TKO) strategy for the rapid and efficient isolation of genes that function in CD95mediated apoptosis, using antisense cDNA libraries. Methods: Cloning of antisense cDNA libraries, transfection procedures, DNA sequencing, PCR, cell culture and blotting techniques. Results: (1) Representative antisense cDNA libraries were generated by a forced cloning strategy. (2) A multicycle TKO method was successfully established. (3) Known (n ¼ 11) and unknown (n ¼ 10) functional proapoptotic genes were isolated. (4) The function of the isolated genes were confirmed by directed re-transfection of antisense cDNAs. (5) The death signal specific induction of these transcripts were determined by Northern blotting. Conclusions: The successful isolation and identification of known and novel pro-apoptotic genes convincingly confirms that the multi-cycle TKO strategy provides a powerful tool for rapid and efficient identification of functional genes. These factors are functionally involved in cytokine and/or drug induced apoptosis in renal cell carcinoma and proved to be candidates for diagnostic and therapeutic targets.

Posters: Breast, Ovary

39 Genetic classification of breast cancer by tree model analysis of CGH data

K. FRIEDRICH, A.V. HEYDEBRECK1, G. HAROSKE2, J. SCHEITHAUER, W. MEYER, K.D. KUNZE, G. BARETTON Institut fu¨r Pathologie, Technische Universita¨t Dresden 1 Global Technologies, Merck KGaA , Darmstadt 2 Institut fu¨r Pathologie, Sta¨dtisches Krankenhaus Dresden-Friedrichstadt Aims: Breast cancer progression is associated with the accumu-lation of genetic alterations. The purpose of this study was to form a tree model of genetic alteration in the progression of breast cancer and test it for its prognostic relevance. Methods: The CGH data of 106 invasive breast cancers with known clinical follow-up were analyzed by a maximum likelihood based tree model. Results: The tree model including the 14 most frequently changed chromosomal arms revealed a tree with three subtrees. The first subtree was characterized by 4q, 5q, 6q, 9p, 13q and +17q, the second subtree by +1q, +8q, +16p and +20q and the third subtree by 8p, 11q, 16q and 18q. The three different subtrees did not show any correlation with the clinicopathological parameters of the tumors. The cases associated with the first and second subtree, arising from the same node of the tree, showed a similar course of disease, whereas the cases associated with the third subtree, arising from a separate node, showed a different clinical behaviour (po0:05). Cox regression analyses revealed a loss at 10q23 as prognostically relevant when all 106 cases were considered. Analysis of the cases associated with the first two subtrees, however, revealed a gain at 1cen-q23 and losses at 5q21-qter and at 9cen-q22 as prognostically relevant. Conclusions: The application of tree models to CGH data allows insights in genetic breast cancer progression and may improve the prediction of breast cancer prognosis by detection of subsets with different course of disease. The results emphasize the role of mathematical models in predicting progression and prognosis in breast cancer.

40 Analysis of DNA repair genes in tissue microarrays of breast carcinomas M. KORABIOWSKA, U. BRINCK, T. LU¨TKE, C. CORDON-CARDO1, H. SCHULZ, P. BORTKIEWICZ, G. FISCHER Institut fu¨r Pathologie, Reinhard-Nieter Krankenhaus, Wilhelmshaven 1 Division of Molecular Pathology, Memorial Sloan Kettering Cancer Center, New York, USA Aims: Alterations of DNA repair genes have not yet been fully investigated in breast carcinomas. The main

ARTICLE IN PRESS Posters: Breast, Ovary / Pathology – Research and Practice 201 (2005) 153–300

objective of this study was to analyse the expression of DNA mismatch repair genes (MLH1, MSH2, PMS1, PMS2), DNA double strand repair genes (Ku70, Ku80) in tissue microarrays of breast cancer. Methods: Expression of DNA repair genes was investigated immunohistochemically and means in situ hybridization in 160 breast carcinomas with known follow up. Results: Multivariate analysis including tumor stage, grade, cerbB2 status and DNA repair gene expression demonstrated an independent role of Ku70 and Ku80 gene expression. Tumor grade, stage and cerB2 status demonstrated also statistically significant influence on patients’ prognosis. Conclusions: Alterations of DNA double strand repair play an important role in the progression and in the prediction of prognosis of patients with breast carcinomas. DNA mismatch repair genes seem not to have an influence on patients‘prognosis.

41 Tissue microarrays for comparing molecular features

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All expected associations between Ki67 and the analyzed markers could be reproduced with high statistical significance. Conclusions: We conclude that Ki67 LI can be reliably analyzed in a TMA format for research purposes.

42 The expression of phospho(Serin 166)-murine double minute 2 correlates with Akt-activation and is associated with a shorter overall survival in node-negative breast cancer K.J. SCHMITZ1, F. GRABELLUS1, R. CALLIES2, F. OTTERBACH1, B. LEVKAU3, R. KIMMIG2,4 K.W. SCHMID4, H.A. BABA1 1

Institut fu¨r Pathologie Klinik fu¨r Frauenheilkunde und Geburtshilfe 3 Institut fu¨r Pathophysiologie, Universita¨t EssenDuisburg 4 Westdeutsches Tumorzentrum 2

with proliferation activity in breast cancer C. RUIZ1, S. SEIBT1, K. AL-KURAYA2, A.K. SIRAJ2, M. MIRLACHER1, P. SCHRAML1, R. MAURER3, J. TORHORST1, S. POPOVSKA4, M.J. MIHATSCH1, R. SIMON1, G. SAUTER1 1

Institut fu¨r Pathologie, Universita¨t Basel (CH) King Faisal Hospital and Research Center, Riad (KSA) 3 Institut fu¨r Pathologie, Stadtspital Triemli Zu¨rich (CH) 4 Dept. of General and Clinical Pathology, Universita¨t Pleven (BG) 2

Aims: Ki67 immunohistochemistry is widely used for measurement of cell proliferation. Enhanced cell proliferation is believed to be related to poor patient prognosis in many different tumor types. Ki67 expression patterns often shows considerable variability in different areas of a tumor. It is unclear if Tissue Microarrays (TMAs) are suitable for the analysis of potentially heterogeneous markers because of the small size of TMA spots. The aim of this study was to evaluate whether Ki67 immunohistochemistry can be reliably analyzed in a TMA format. Methods: We analyzed a breast cancer TMA comprising 2200 tissue samples for Ki67 Labeling Index (LI) and additional markers with a known relationship to Ki67 expression, including ER, PR, Bcl-2, Egfr, p16 and p53, by immunohistochemistry, and amplification of HER2, MDM2, CCND1, and MYC by Fluorescence in situ hybridization (FISH). Results: Ki67 expression was significantly linked to tumor grade, stage, nodal stage and patient prognosis.

Aims: Murine double minute 2 (MDM2) is an oncoprotein that inhibits the function of p53 tumorsuppressor protein. MDM2 overexpression can be both a positive and negative predictor of outcome in different tumors. The activation of the Akt signaling pathway contributes to cancer progression by inhibiting tumor apoptosis. Akt related phosphorylation of MDM2 at serine 166 allows MDM2 to gain nuclear entry and fullfill its p53 regulating function. The aim of this study was to determine the prognostic relevance of phosphorylated MDM2 (Serin 166) in node-negative breast cancer and to analyze its relationship with Akt-activation and p53 protein expression. Methods: Phospho-MDM2 (Serin 166), phospho-Akt (Serin 473) and p53 protein expression status was analyzed in 123 paraffin-embedded breast cancers by immunohistochemistry. Mean Follow up was 7.5 years. Results: The expression of pMDM2 exhibited a direct correlation with Akt-Activation. Moreover univariate analysis revealed that the expression of pMDM2 is a prognostic factor in patients with p53-negative tumors. Conclusions: This data argues strongly that MDM2phosphorylation (Serin166) is mediated by the Akt kinase in vivo. Phosphorylated MDM2 is thought to form a complex with (wild type) p53, which initiates its degradation and thereby inactivates (wild-type) p53 function. Our findings indicate that pMDM2 immunohistochemical analysis may provide useful information concerning the prognosis in node-negative breast cancer patients.

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43 C-myc oncogene amplifications in primary breast carcinomas and their local recurrences S. AULMANN, N. ADLER, J. ROM1, B. HELMCHEN, H.P. SINN, P. SCHIRMACHER Institut fu¨r Pathologie, Universita¨t Heidelberg 1 Frauenklinik der Universita¨t Heidelberg Aims: The c-myc oncogene has previously been shown to be amplified in a subset of invasive breast carcinomas and has been associated with progression from preinvasive to invasive breast cancer. We examined c-myc amplifications in a series of 48 primary invasive breast carcinomas and their local recurrences. Methods: C-myc copy number was determined using fluorescence in situ hybridisation. In addition, clinicopathological and histomorphological data as well as hormone receptor expression and overexpression of HER2 and p53 were included in the analyses. Results: Eleven (22%) of the primary carcinomas harboured c-myc amplifications, 6 additional tumours had aquired the genetic alteration at the time of relapse. The mean recurrence free survival (rfs) was 23.6 months, c-myc amplified tumours relapsed earlier than carcinomas without amplification (17.9 vs. 26.7 months, p ¼ 0:017) and were associated with a worsened overall survival (p ¼ 0:033). C-myc oncogene amplification was not associated with tumour type, grading, hormone receptor positivity or overexpression of HER2 and p53. However, patients with primary tumours containing c-myc amplifications were younger at the time of diagnosis (45 vs. 52 years,p ¼ 0:023). Conclusions: Our results indicate that while amplifications of the c-myc oncogene can be observed in early stage breast cancer, especially in younger patients, they often occur later in tumour development and thus appear to be associated with disease progression.

ing the clinical and prognostic relevance of MUC1 expression in breast cancer. Therefore, we tried to evaluate the role of MUC1 and MUC2 as predictors of the clinical course and prognosis in mammary carcinomas. Methods: The expression of various MUC1 epitopes and MUC2 was studied immunohistochemically applying numerous monoclonal antibodies (mabs) in a series of 140 patients with breast cancer. The staining was evaluated semiquantitatively and the results were correlated statistically with clinicopathological variables as well as overall survival. Results: Generally, more than 90% of the mammary cancers were stained with various MUC1 specific mabs. Ductal and lobular carcinomas were strongly MUC1 positive, whereas MUC2 binding was significantly elevated in mucinous neoplasms. Associations between any of the antigens under study and pTNM staging could not be observed. However, a strong reactivity of the sialylated MUC1 epitope detected by mab MY1. E12 revealed as a favourable independent prognostic factor indicating a longer survival. Conclusions: Our results confirm that MUC1 is generally strongly expressed in mammary carcinomas. As an exception, mucinous carcinomas contain significantly less MUC1, but more MUC2. Our data suggest that only the overexpression of a sialylated short-chain MUC1 glycoform is associated with a better prognosis, whereas other epitopes did not show such correlations.

45

C-kit expression in ductal carcinoma in situ of the breast: co-expression with HER-2/neu R. DIALLO1, A. RODY2, M. KAUFMANN2, H.E. GABBERT1, C. POREMBA1, C. JACKISCH3 1

Institut fu¨r Pathologie, Universita¨t Du¨sseldorf Frauenklinik, Universita¨t Frankfurt 3 Frauenklinik, Universita¨t Marburg 2

44 Expression of MUC1 and MUC2 in mammary carcinomas: Prognostic significance of a sialylated MUC1 epitope S.E. BALDUS, J.R. WIENAND, J.P. WERNER, S. LANDSBERG, F.-G. HANISCH1, H.P. DIENES Institut fu¨r Pathologie, Universita¨t Ko¨ln 1 Institut fu¨r Biochemie II, Universita¨t Ko¨ln Aims: MUC1 represents the characteristic mucin molecule involved in mammary carcinogenesis. However, due to the structural complexity of the MUC1 glycoprotein, various different epitopes can be detected by monoclonal antibodies. This fact may be responsible for the contradictory results of previous investigations regard-

Aims: The proto-oncogene c-kit (CD 117) is highly expressed in normal breast epithelium and is decreased in invasive breast cancer during malignant transformation. The aim of this study was to determine c-kit expression in ductal carcinoma in situ (DCIS) of the breast and to investigate the relationship of c-kit expression with nuclear grading and with the expression of HER-2/neu, estrogen receptor-a (ER) and progesterone receptor (PR). Methods: C-kit, HER-2/neu, ER and PR expression were analyzed immunohistochemically on paraffin sections of 59 cases of DCIS (25 cases were classified low grade, 8 cases as intermediate grade and 26 cases as high grade. Results: A total of 64.2% of all DCIS were positive for c-kit and c-kit expression was significantly associated

ARTICLE IN PRESS Posters: Breast, Ovary / Pathology – Research and Practice 201 (2005) 153–300

with HER-2/neu positivity (p ¼ 0:022). Furthermore, ckit expression was significantly lower in ER-positive DCIS (p ¼ 0:037). There was a trend of positive correlation of c-kit expression with high grade differentiation (76.9% of high grade and 75% of intermediate DCIS Versus 45.4% of low grade DCIS, p ¼ 0:061). Conclusions: Our findings suggest that co-expression of c-kit and HER-2/neu may define a high risk subgroup of DCIS with reduced expression of the estrogen receptor and a trend to poorer differentiation. The exact biochemical link between c-kit and HER-2/neu in this subgroup of DCIS and the influence on breast carcinogenesis have to be further evaluated.

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Origin of vimentin expression in invasive breast cancer: Epithelial-mesenchymal transition, myoepithelial histogenesis or histogenesis from progenitor cells with bilinear differentiation potential? H. BU¨RGER, E. KORSCHING, J. PACKEISEN1, D. HUNGERMANN, C. LIEDTKE, T. DECKER, W. BO¨CKER Institut fu¨r Pathologie, Universita¨t Mu¨nster 1 Institut fu¨r Pathologie, Klinikum Osnabru¨ck

Aims: Vimentin expression in invasive breast cancers currently explained by two different biological theories: a direct histogenetic derivation from myoepithelial cells and epithelial-mesenchymal transition (EMT), reflecting the end stage of breast cancer dedifferentiation.In this study we aimed to obtain further insights into the biological hallmarks of these vimentin expressing breast cancers. Methods: We applied immunohistochemistry for vimentin and 15 other differentiation markers on a series of 366 invasive breast cancer cases using the tissue microarray technology. 144 cases have been characterized by CGH before. Results: A total of 7.7% of all tumours expressed vimentin. Almost all these cases (19/21) were ductal invasive Grade 3 carcinomas, and the majority (13/21) of these were associated with an ductal in situ component. Vimentin expression was also seen in the respective in situ components and positively correlated with the expression of SMA, CD10, Ck5, p53, Mib-1 and EGFR. An inverse significant correlation was seen for the expression of Ck8/18 and the estrogene receptor. Conclusions: Our present results demonstrate that a myoepithelial histogenesis as well as EMT can not exclusively explain the biology of these distinct tumours. We therefore propose the alternative hypothesis that vimentin-expressing breast carcinomas may derive from breast progenitor cells with a bilinear (glandular and myoepithelial) differentiation potential.

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47 Expression of epoxide hydrolase predicts tamoxifen treatment failures in tamoxifen treated primary breast cancer patients P. FRITZ1, U. ZANGE2, T. MU¨RDTER2, J. DIPPON3, M. EICHELBAUM2 1

Institut fu¨r Pathologie Robert Bosch Krankenhaus, Stuttgart 2 Institut fu¨r Klinische Pharmakologie Robert Bosch Krankenhaus, Stuttgart 3 Lehrstuhl fu¨r Mathematik Universita¨t Stuttgart Aims: Microsomal epoxide hydrolase (EC3.3.2.3) is a enzyme (m-EH) of phase I drug metabolism. It’s function in tamoxifen metabolism is unknown. Recently, m-EH has been described as part of the ABS (anti-estrogen binding site) in rat. We have demonstrated in a first hypothesis generating retrospective study that high expression of m-EH in primary breast cancer is related to tamoxifen treatment failures in breast cancer patients (Fritz et al. 2001). Methods: M-EH was iimmunostained in primary breast cancer patients with known long time follow-up and known treatment including 206 cases of tamoxifen treated patients. Results: m-EH expression in primary breast cancer was related to disease outcome and treatment failure only in those patients, treated by tamoxifen in estrogen receptor positive cases. M-EH was found to be a predictor of treatment failure either in univariate and multivariate analysis only in tamoxifen treated patients. Conclusions: We present now – following the rules of evidence based medicine- a second independent long time retrospective cohort study which identifies m-EH as a treatment predictor in breast cancer patients. Supported by the Robert Bosch Foundation Stuttgart and BMF Germany

48 Low cost low amount fluorescence in situ hybridization for the evaluation of the HER2-status of mammary carcinoma U.F. VOGEL, B.D. BU¨LTMANN Institut fu¨r Pathologie, Universita¨t Tu¨bingen Aims: For the evaluation of the HER2-status of mammary carcinoma with fluorescence in situ hybridization (FISH) commercially available DNA probes like the PathVysion –kit (Vysis-Abbott) are recommended. However, these kits are very expensive. To reduce the costs, the probe mixture can be diluted or the area of hybridization reduced. In order to use the original probe we minimized the target area.

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Methods: Since 1998, about 2000 hybridizations of formalin-fixed paraffin-embedded mammary carcinoma specimens were performed mostly using the HER2-DNA probes from Vysis. Instead of the provided 10 ml of the probe mixture to hybridize a 22  22 mm area of each section, only 1–1.2 ml of the probe mixture was applied and covered by a 7  7 mm glass coverslip. To prevent evaporation, the coverslip was sealed using rubber cement. Furthermore, the hybridization chamber was humidified. In addition, the probe and the specimen were denatured separately. The best preserved tumor area in HER2-immunohistochemistry (A0485 Dako polyclonal) was chosen as target area for FISH. If a tumor displayed areas with a different expression of HER2 in immunohistochemistry, more than one area was hybridized. Results: No drawbacks were seen by the use of the 7  7 mm glass coverslips. The hybridized area was more than large enough to evaluate the required number of 60 tumor cells per slide. Problems of evaporation occurred only if the measures described above were not carried out. Conclusions: It is possible to reduce the costs of a FISH evaluation to a tenth without loosing the quality of staining by minimizing the hybridized area using small 7  7 mm coverslips.

of at least 3 levels were stained with H&E and CK-IHC. Results: Frozen sections of the SLNs contained metastases in 22/ 87 (25.3%) patients. Final histology revealed positivity in 34/87 (39%) cases by H&E stain, which means an overall accuracy of intra-operative assessment of 86.2%(false-negative rate 12/34 ¼ 35%). The H&E stains of the SLNs predicted the state of the axilla in 79/ 80 pat. (false-negative rate: 3%). There were no additional MI detected through CK-IHC in SLNs or NSLNs. With CK-IHC ITC were found in the SLNs of 13/90 (14.4%) patients and in the NSLNs of 9/80 (11.3%) patients. In one case we found ITC only in CKIHC of SLN, but 19 of 24 NSLN contained macrometastases (42 mm). Conclusions: Our results suggest that intra-operative assessment of SLNs allows a one-step procedure for most of the patients. Our data do not support the routine use of immunohistochemistry on SLNs, because the detection of ITC in SLNs did not result in a significantly increased yield of NSLN metastases.

50 Bone metastases in breast cancer W. POKIESER1, G. FISCHER2, W. ULRICH1 1

49

Sentinel lymph node biopsy in breast cancer: significance of intra-operative examination and value of immunohistochemistry J. WAGNER, A. LEBEAU, U. LO¨HRS Pathologisches Institut der Universita¨t Mu¨nchen

Aims: Sentinel lymph node (SLN) biopsy might spare patients axil-lary lymph node dissection in case no metastases are detected. Because the pathological workup is a matter of debate, we examined the accuracy of the intra-operative assessment of SLNs. Additionally immunohistochemical staining for cytokeratin (CKIHC) was performed on paraffin step-sections to evaluate the detection rate of micrometastases (MI) and isolated tumor cells (ITC) in SLN and its predictive value for non-sentinel lymph node (NSLN) metastases. Methods: SLN biopsy was performed on 90 patients with clinically node-negative breast cancer. Eighty of them underwent confirmatory axillary lymph node dissection. Intra-operative frozen sectioning was performed on one halve of the bisected SLNs in 87 patients. The residual tissue was formalinfixed and paraffin-embedded. SLNs and NSLNs were step-sectioned (200mm intervals) and the sections

Jakob Erdheim Institut fu¨r Pathologie und Klinische Bakteriologie, KH Lainz, Wien 2 Ludwig Boltzmann Institut fu¨r klinische Onkologie und photodynamische Therapie, Wien Aims: Distant metastases in breast cancer are associated with a poor survival. We investigated the role of the tumor grading and hormone receptor status of invasive breast cancer in developing bone metastases. Methods: A total of 3856 women with a history of invasive breast cancer between 1961 and 1994 were analyzed in a retrospective study. Ninety one patients (group A) developed bone metastases as the first visible clinical sign of distant metastasis. A control group of 91 patients (group B) without bone metastases were matched in terms of patients age, tumor staging and tumor typing. The two groups were compared in respect of the tumor grading and the hormone receptor status. Results: There was no difference in histological grading of the tumors in both groups. Hormone receptor expression: ER+ and PgR+ : group A (n ¼ 41=45:1%); group B (n ¼ 52=57%) ER- and PgR-: group A (n ¼ 14=15:4%); group B (n ¼ 16=17:6%) ER- and PgR+: group A (n ¼ 5=5:5%); group B (n ¼ 4=4:4%)

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ER+ and PgR- : group A (n ¼ 31=34;1%); group B (n ¼ 19=20; 9%) Conclusions: Breast carcinomas with positive estrogen and negative progesterone receptor expression were associated with a higher rate of bone metastases as the first visible clinical sign of distant metastasis. This type of metastases does not depend on the histological tumor grade in our patient collection.

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Methodological aspects of a standardized clinical evaluation of mitotic activity in breast cancer tissue S. BIESTERFELD, M. REITMAIER Institut fu¨r Pathologie, Universita¨t Mainz 1 Frauenklinik der Universita¨t Go¨ttingen Aims: The mitotic activity index can only be used as a prognostic factor in breast cancer as far as it is evaluated in a standardized manner. In this paper methodological aspects that merit special consideration are summarized. Methods and results: (1) Values attained by calculating the mitotic cells after fixation are lower than those obtained in vivo due to complex factors associated mainly with hypoxia. This fact has been demonstrated in several experimental studies and has to be considered if results from frozen sections and from paraffin sections are compared. (2) Mitotic activity should be calculated only in areas of sections where there are numerous mitotic tumour cells, following the methodological proposals of Baak. (3) The specification of the final magnification (‘‘400  ’’ high power field) is not a sufficient methodological basis, since the viewing field area of standard light microscopes differed up to 2.6-fold in own investigations. Thus, it is necessary to find correction factors for a comparison of values obtained with two different microscopes. As a standard, a viewing field area of 0.159 mm2 (400  magnification at visual field value 18) appears to be appropriate. (4) In two independent studies on each4100 breast cancer patients we could demonstrate a high prognostic significance of the mitotic activity on long-term survival. Additionally, mitotic activity was of higher clinical relevance than conventional tumour grading. (5) The calculation of the interspecific reproducibility makes this method reliable for clinical application (r ¼ 0:90). Conclusion: The calculation of the mitotic activity is a valid and reliable method for breast cancer prognosis. For clinical purposes the two most important methodological aspects are experience in identifiying mitotic figures correctly and to use a correction factor to base the interpretation of their number on a standardized area.

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52 Needle core biopsies: B-Classification, text and meaning K. LEHMANN, E. HODEK1, M. WOLF2, V. LOY1 1

Fachbereich Pathologie Vivantes Berlin Zentrum fu¨r Brusterkrankungen Vivantes Berlin

2

Aims: Needle core biopsies (NCB) are widely used as diagnostic procedure in breast screening. Standardized diagnostic and reporting guidelines for pathologists were first developed in GB (NHSBSP) and updated in 2004 by the ECWGBSP. Without special training the categories B1/B2/B3 might be confused. Methods: Since 2003 we examined 1087 NCB. 790 were classified according to the NHSBSP literally. 297 were classified according to to the meaning the forthcoming EU-Guidelines. Results: NHSBSP: B1 7%, B2 40%, B3 1%, B4 2%, B5 50%. ECWGBSP: B1 19%, B2 29%, B3 6%, B4 1%, B5 45%. Conclusions: The B1 category is defined as normal ’’ tissue‘‘, which is rarely seen in NCB. NCB mostly show minor pathological changes and therefore often are erroneously classified as B2 (‘‘benign’’). Pathologists ought to keep in mind that B1 means that the NCB is probably not appropriate and has to be reevaluated with the radiologist. As we changed our classification schema from the mere text of the NHSBSP to the meaning of the EU-Guidelines the percentage of the B2 category dropped from 40% to 29%, the B1 category rose from 7% to 19%. The category B2 does not mean all benign breast lesions, but only a small group of neoplasias which are clearly defined and have to be identified in close accordance between radiology and pathology. B3 often is misunderstood as well. This category comprises well defined lesions with uncertain malignant potential, e.g. papillary lesions, which should not be classified as B2 ‘‘benign’’, because in the NCB a DCIS could be missed and a diagnostic excision might be mandatory.

53 Sonographical breast biopsy: An analysis of clinical, radiologic and pathologic findings in 427 cases H.-J. TERPE, H. ROTT1, L. HENSCHER2, J. TERPE2, H. STEIN, B. LAMPE1 Institut fu¨r Pathologie 1 Frauenklinik, Klinikum Leverkusen 2 Mammografiezentrum der radiologischen Gemeinschaftspraxis, Leverkusen Aims: Nowadays the sonographical breast biopsy is the gold standard to examine circumscribed breast abnormalities. The purpose of this retrospective study was to

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determine the correlation between clinical, radiologic and pathologic sentences. Methods: About a period of 12 months from 427 patients were implemented 3–5 sonographical breast biopsies of a circumscribed breast lesion. We used an automated biopsy 14-gauge large core needle and a coaxial system. Core needle biopsy results were compared with clinical, radiologic and final surgical findings. For the radiology we utilized the BI-RADS classification and for the histology the B-classification. Results: (1) About 97% of the biopsies were so well done, that we found tissue of the circumscribed lesion in the biopsies. (2) (53%) (229 cases) were classified as B5 and 42% (178 cases) as B2 lesions. (3) (44%) were classified as BI-RADS V, 40% as BI-RADS IV and 16% asoBI-RADS IV. (4) In the cases of BI-RADS IV we found in 23% B5 and 67% B2 (ca. 80% fibroadenomas) lesions. (5) In the cases of BI-RADS V we detected in 91% B5 lesions. (6) There were nearly no discrepancies between the histological diagnosis of biopsies and the final histology. Conclusions: Our results confirm that sonographically guided large core breast biopsy is a safe method for the assessment of sonographically circumscribed breast abnormalities.

54 Predominance of high grade pathway in breast cancer development of saudi arabian women C. TAPIA, K. AL-KURAYA1, S. SHEIKH2, S. AMR2, P. SCHRAML, M. MIRLACHER, R. SIMON, G. SAUTER Institut fu¨r Pathologie, Universita¨t Basel 1 King Faisal Specialist Hospital and Research Centre, Riyadh, KSA 2 Pathology Services Division, Dharan, KSA Aims: To identify potential molecular differences between breast cancers in Europe and the Middle East, we analyzed consecutive breast cancer series from Switzerland (n ¼ 2197) and Saudi Arabia (n ¼ 204). Methods: Tissue microarrays were analyzed by fluorescence in situ hybridization (FISH) for HER2, CCND1, MYC, and EGFR amplification. Results: Saudi breast cancers had a markedly higher frequency of HER2 (31.3% vs. 17.3%; po0:0001) and MYC (15.8% vs. 5.3%; po0:0001) amplifications than Saudi women. This was mostly due to a much higher incidence of grade 3 cancers in the Saudi than in the Swiss population (65.2% vs. 31.9%; po0:0001). However, differences in amplification frequency hold also true within grade 3 cancers (HER2: 40.4% vs. 30.3%, po0:05; MYC: 22.1 vs. 10.8%, p ¼ 0:002). The incidence of high grade breast cancer is comparable for

Saudi and Swiss women, while the incidence of low grade breast cancers is about 14 times lower in Saudi than for Swiss women. Conclusions: These observations suggest that a difference in genetic susceptibility and/or life style results in marked phenotype/genotype differences between Saudi and Swiss breast cancers. A comparison of histologic features revealed a virtual absence of low grade tumors in the Saudi group.

55 ETV6-NTRK3 gene fusion in a secretory carcinoma of the breast of a male-to-female transsexual F. GRABELLUS1, K. WORM1, K. J. SCHMITZ1, R. KIMMIG2, K. W. SCHMID1 1

Institut fu¨r Pathologie, Universita¨tsklinikum Essen Klinik fu¨r Frauenheilkunde und Geburtshilfe, Universita¨tsklinikum Essen 2

Aims: Secretory carcinomas of the breast were first described as ‘‘juvenile carcinomas’’ by McDivitt and Stewart. This term has been replaced by ‘‘secretory breast carcinoma’’ because it can occur at any time in life. Recently it was reported that human secretory breast carcinomas express the ETV6-NTRK3 gene fusion. We present the case of a 46-years-old male-tofemale transsexual, where the diagnosis of a secretory breast carcinoma was confirmed by detecting the ETV6NTRK3 gene fusion. Methods: The initial diagnosis was made on paraffin sections with standard stains and immunohistochemistry. Snap frozen tissue as well as paraffin-embedded samples of the secretory carcinoma and three other breast tumors where investigated by RT-PCR analysis and sequencing. Results: The ETV6-NTRK3 gene fusion was detected in both, frozen and paraffin material of the secretory breast carcinoma, whereas the other investigated tumors showed a negative result. Conclusion: The diagnosis of a secretory breast carcinoma can be confirmed by the detection of the ETV6NTRK3 gene fusion. For this purpose paraffin-embedded tumor samples can be used.

56 Infiltrating lobular carcinoma of the breast in a male patient S. SONNENTAG, N. ARENS, R. KLA¨S1, R. HILDENBRAND Institut fu¨r Pathologie, Universita¨tsklinikum Mannheim 1 Institut fu¨r Humangenetik, Universita¨tsklinikum Heidelberg

ARTICLE IN PRESS Posters: Breast, Ovary / Pathology – Research and Practice 201 (2005) 153–300

Aims: We report about a case of an inflitrating lobular carcinoma in the breast of a 59-year old man. Usually, histology of the normal male breast does not reveal any acini or lobules, so the lobular tumor type is very uncommon. To our knowledge there are only 21 cases described in literature, either as lobular carcinoma or small cell carcinoma. Methods: Immunohistochemical analysis of prognostic factors and E-cadherin was performed. A combined DNA probe specific for the chromosomes X and Y was hybridized to paraffin-embedded tissue sections and to peripheral blood cells of the patient. Comparative genomic hybridization (CGH) was used to identify chromosomal copy number changes within the tumor genome. Mutation screening of all coding exons including the exon-intron boundaries of the breast cancer susceptibility genes BRCA1 and BRCA2 was performed applying DHPLC. Apparent DNA fragments were further investigated by sequence analysis. Results: Immunohistochemical analysis showed a HER2/ neu- and E-cadherin-negative phenotype. FISH and cytogenetical analysis revealed a normal XY male karyotype in the tumor and in the blood sample. CGH revealed a gain of the long arm of chromosome 1 as well as a loss of the long arm of chromosome 16 within the tumor tissue. Mutation screening of BRCA1 and BRCA2 and sequence analysis revealed a point mutation (c.5972C4T) in exon 11 of the BRCA-2 gene leading to an amino acid exchange from methionin to threonin (T1915 M). Conclusions: Our results from immunohistochemical and molecular genetic analysis of this tumor support the histological diagnosis of an infiltrating lobular carcinoma in this male patient.

57 Gynecomastia-like lesion of the female breast. A Case report F. OTTERBACH, E.A.M. HAUTH1, R. KIMMIG2, K.W. SCHMID Institut fu¨r Pathologie, Universita¨t Essen-Duisburg 1 Institut fu¨r Diagnostische und Interventionelle Radiologie, Universita¨t Essen-Duisburg 2 Klinik fu¨r Frauenheilkunde und Geburtshilfe, Universita¨t Essen-Duisburg Aims: Gynecomastia-like changes of the female breast are rarely reported in the literature. According to Umlas, these changes can occur as small gynecomastia-like areas (GLA) and gynecomastia-like lesions (GLL), the latter with tumorous appearance and no association with another tumorous lesion . We report a case of a GLL in a 39 years old fertile woman. Results: In the right upper outer quadrant of the right breast a tumor of 3 cm in diameter was palpable.

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Mammography exhibited an inhomogenous spiculate density of the same size. MRI showed a contrast enhancing mastopathic breast tissue with an 8 mm large area of fast enhancement and wash-out phenomenon, suspicious for malignancy. Gross examination of the open biopsy specimen showed an ill defined induration with greyish glossy cut surface. Microscopically this area was characterized by a complete lack of normal lobules. Tubular ducts were found in an edematous cellular onion-skin-like stroma with pseudoangiomatous features. The luminal duct epithelia showed micropapillary hyperplasia. Microcalcifications or a fibrous capsule were absent. Conclusions: Pathologists should be aware that the clinical and radiological appearance of gynecomastialike lesions can mimic invasive breast cancer. Multidisciplinary approach is vital for the interpretation of gynecomastia-like changes in needle core biopsies. The pathogenesis is obscure. GLL should be distinguished from mammary hamartomas. Data concerning the relative risk of subsequent breast carcinomas are not available.

58 Leiomyoadenoma of the female breast. A Case report F. OTTERBACH, E.A.M. HAUTH1, R. KIMMIG2, K.W. SCHMID Institut fu¨r Pathologie, Universita¨t Essen-Duisburg 1 Institut fu¨r Diagnostische und Interventionelle Radiologie, Universita¨t Essen-Duisburg 2 Klinik fu¨r Frauenheilkunde und Geburtshilfe, Universita¨t Essen-Duisburg Aims: Myogenic tumors in the female breast are rare. Exceptional cases of Leiomyoma, Rhabdomyoma, and fibroepithelial tumors with smooth muscle metaplasia have been reported. We present a case of a biphasic tumor with fibroadenomatous architecture but pure leiomyomatous differentiation of the mesenchymal component. Results: A 39 years old woman presented with a 2 cm in diameter palpable node in the lateral portion of the left breast. Mammography exhibited an area of clustered micro- and macrocalcifications in a well defined minute density. Specimen radiography revealed a lobulated border of the density. Gross examination showed a well defined firm lobulated tumor with a whorled tan cut surface. Microscopically the tumor consisted of fusiform leiomyocytes, growing in whorled, anastomosing fascicles and individually dispersed small slit-like ducts or atrophic lobules lined by attenuated epithelial cells. The presence of a myoepithelial layer could be confirmed by actin immunohistochemistry. Laminated micro- and macrocalcifications appeared in the ducts and in the

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leiomyomatous stroma. The tumor was surrounded by regular breast tissue. Conclusions: In 1901 Abramow published one case of Adenomyoma of the breast which was described as a leiomyocytic tumor with a lot of regular glands. Because of the predominant leiomyomatous component we classified our tumor as Leiomyoadenoma. No recurrence occurred 16 months after excision.

noses are papillomatosis of the nipple, papillary adenoma, tubular carcinoma or adenosquamous carcinoma.

60 Massive adenomyosis in a patient with uterus septus completus T. HANSEN, S. WULGARIS1, W. SIGGELKOW1, H. KO¨LBL1, C.J. KIRKPATRICK

59 Syringomatous adenoma of the nipple: A case report of a very rare and therefore sometimes misinterpreted lesion D. MAYR1, W. PERMANETTER2, W. STOCK3 U. LO¨HRS1 1

Pathologisches Institut der Ludwig-MaximiliansUniversita¨t Mu¨nchen 2 Pathologisches Institut Klinikum Landshut 3 Plastische Chirurgie, Chirurgische Klinik der Universita¨t Mu¨nchen Introduction: Syringomatous adenoma of the nipple is an extremely rare, locally invasive and recurrent, but non metastasizing tumour. The exact origin of the tumour is uncertain, but because of its sweet duct differentiation, derivation from eccrine structure of the nipple is supposed. In the literature less than 30 cases have been described. Case report: A 31-year-old woman with a known prolactinoma with normal prolactine in blood-serum presented herself with galactorrhoe and a painful mass in the subareolar region of her left breast. First clinical, surgical and pathological diagnoses lead to inflammation. Because persistence of the symptoms a second stay at hospital was necessary. Mammography: Indurative mastopathy, several well defined, firm and mobile nodules and a discrete, immobile mass situated behind the nipple and subareolar region. Clinical examination, including mammography and needle biopsy indicated a high possibility of carcinoma. An excision followed. Gross findings: A poorly circumscribed tumour in the nipple and subareolar region and some well-described nodules in the remaining breast tissue. Microscopic findings: Solitary, small, compressed and comma-shaped tubular structures, lined by one or more layers of epithelial cells and small keratotic cysts, imbedded in fibrous stroma. No nuclear atypia or perineural infiltration and only less mitosis. After diagnosis the patient was sent to plastic surgery for total excision and reconstruction. Conclusion: Because of its expansive nature and high recurrence ‘‘infiltrating syringomatous adenoma’’ seems to be the best term for this lesion and a wide excision should be performed. The most frequent wrong diag-

Institut fu¨r Pathologie 1 Klinik fu¨r Geburtshilfe und Frauenkrankheiten, Universita¨t Mainz Aims: Congenital uterine malformation is estimated to occur in one of 300 womens. It is classified according to the developmental defect into three groups: agenesis and aplasia; defective fusion of the mu¨llerian ducts; and defected resorption of the mu¨llerian ducts. The latter defect causes uterus septus, which may be incomplete, or – less frequently – complete. We present a case of a uterus septus completus with regard to the endometrial changes. Methods: We describe a 46-year-old female patient with a known uterus septus suffering from hypermenorrhea. Sonography demonstrated a massively enlarged uterus with several nodes. Hysterectomy was performed, and tissue specimens were routinely processed. Results: Macroscopical examination revealed a uterus septus completus weighing 1230 g. In particular, the myometrium was enlarged and exhibited a cystic cut surface with several nodes measuring up to 4.5 cm.Histologically, we found prominent adenomyosis with several leiomyomas. Conclusions: Patients with uterine malformations are known to suffer from endometrial dysfunction, most commonly due to endometriosis. However, massive adenomyosis, as presented in this case report, has not been described so far. These endometrial changes are clinically significant, since they can contribute to infertility.

61 Uterine arterial embolization with tris-acryl gelatin microspheres: A histopathologic evaluation W. WEICHERT, C. DENKERT, A. GARUDERBURMESTER1, A.-R. KURZEJA1, B. HAMM2, M. DIETEL, T.-J. KRO¨NCKE2 Institut fu¨r Pathologie, Universita¨tsklinikum Charite´ Berlin 1 Klinik fu¨r Gyna¨kologie, Universita¨tsklinikum Charite´ Berlin 2 Abteilung fu¨r Radiologie, Universita¨tsklinikum Charite´ Berlin

ARTICLE IN PRESS Posters: Breast, Ovary / Pathology – Research and Practice 201 (2005) 153–300

Aims: Uterine artery embolization (UAE) as an alternative treatment of uterine fibroids and adenomyosis uteri becomes increasingly popular. Because there is considerable uncertainty with respect to the wanted and unwanted morphological changes induced by UAE we conducted a histopatholgic study on post UAE tissue specimen. Methods: A total of 173 women were treated with UAE using tris-acryl gelatin microspheres (TGMS) for either symptomatic adenomyosis or leiomyoma. Surgical specimen of eight women who underwent subsequent myomectomy or hysterectomy were evaluated by conventional histology and immunohistochemistry. Results: TGMS were readily apparent in both macroscopy and routine histology. In patients with fibroids, TGMS accumulated preferably in medium size vessels in the direct tumor vicinity. In patients with adenomyosis, a random distribution of TGMS was noted throughout the outer myometrium. A granulomatous foreign body reaction occurred in the vicinity of particles, eventually followed by complete vessel destruction. Leiomyoma treated with UAE showed either fibrinoid necrosis, coagulative necrosis or no change at all. Foci of adenomyosis remained unaltered. Conclusions: In conclusion, after UAE with TGMS, particles were identified predominately but not exclusively at the periphery of fibroids. Pathologists must be aware of the morphologic changes induced by UAE in leiomyoma, to avoid misinterpretation of induced tissue alterations as signs of malignant tumor growth.

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Silverberg grading of ovarian carcinoma: A critical reappraisal in cases of advanced stage ovarian carcinoma after standardized chemotherapy S. KOMMOSS, D. SCHMIDT, F. KOMMOSS, M. TRUNK1, J. PFISTERER2, A. DU BOIS3

Institut fu¨r Pathologie, A2,2, Mannheim 1 MTM Laboratories, Heidelberg 2 Frauenklinik, Universita¨t Kiel 3 Gyna¨kologie & Gyna¨kologische Onkologie, HSK, Wiesbaden Aims: To assess the prognostic significance and reproducibility of the Silverberg grading in a large number of advanced stage ovarian carcinoma of all types after standardized chemotherapy. Methods: Representative H&E slides from 334 cases of stage IIb-IV ovarian carcinoma of all types (prospective randomised, multi-center, phase III study). First round: grading of all cases according to Silverberg and Shimizu by one author (F.K.). Second round (after 1 year): 30 randomly selected cases graded by three authors.

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Survival analysis by Kaplan-Meier method. Measurement of intra- and interobserver reproducibility by kappa statistics. Results: In this group of cases no prognostic significance could be found for the Silverberg grading, neither for the final score (p ¼ 0:9346) nor for one of the parameters of the grading system (architectural grade: p ¼ 0:3262; mitotic activity: p ¼ 0:3575; nuclear pleomorphism: p ¼ 0:4894). Intraobserver reproducibility was: final score 80% (k ¼ 0:692), architectural grade: 93.3% (k ¼ 0:874), mitotic activity: 76.7% (k ¼ 0:624), nuclear pleomorphism: 86.7% (k ¼ 0:767) Interobserver reproducibility was: final score 77.8% (k ¼ 0:651), architectural grade: 82.2% (k ¼ 0:690), mitotic activity: 80% (k ¼ 0:646), nuclear pleomorphism: 66.6% (k ¼ 0:381). Conclusions: In our series of advanced ovarian carcinomas after standardized chemotherapy, the Silverberg grading system was of no prognostic significance.

63 Ki—S5/Ki—S2 proliferation rate in advance stage ovarian carcinoma S. KOMMOSS, F. KOMMOSS, R. PARWARESCH1, D. SCHMIDT, M.J. TRUNK2, J. PFISTERER3, A. DU BOIS4 for AGO-OVAR Study Group Institut fu¨r Pathologie, A2,2, Mannheim 1 Pathologie, Universita¨t Kiel 2 MTM Laboratories, Heidelberg 3 Frauenklinik ,Universita¨t Kiel 4 Frauenklinik, HSK, Wiesbaden Aims: The goal of this study was to evaluate the prognostic significance of proliferation activity in cases of advanced ovarian cancer. Further stratification of patients after complete debulking would be of special interest for clinical studies. Methods: Representative H&E slides from 302 cases of stage IIb-IV ovarian carcinoma of all types (prospective randomised, multi-center, phase III study). Paraffin sections were subjected to immunohistochemistry using the monoclonal antibodies Ki-S5 to the Ki67-antigen and Ki-S2 to the S-phase specific protein repp86. For technical reasons some cases had to be excluded (Ki-S5: n ¼ 6; Ki-S2: n ¼ 21). Patients were divided into two groups (low/high) according to the proliferating rate of o/4 36% (median split). Results: Patients with a Ki-S5 proliferation rate of over 36% revealed a significantly better 5-year-survival rate (o36% Ki-S5: 26.8%;436% Ki-S5: 37.6%; p ¼ 0:03). Patients with complete debulking showed a 5-year survival rate of 43 and 68% in the low and high proliferation group, respectively. However, significance

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was failed (p ¼ 0:06), but numbers were low in this subgroup analysis. No significant results could be shown for Ki-S2. Conclusions: All results imply that advanced ovarian carcinoma with a high proliferation rate have a significantly better prognosis after chemotherapy independent of postoperative residual tumor. A better efficacy of antiproliferative chemotherapeutic agents in highly proliferative tumours might be responsible for this observation. Future studies should focus on the capability of proliferation rate as a prognosticator of a patients’ outcome in optimally debulked collectives.

Conclusions: The results imply that expression of pRb, but not of p16INK4a is of prognostic significance in advanced stage OvCa after chemotherapy.

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Protein expression of cancer testis (CT) antigens MAGE-A1, MAGE-A3, MAGE-A4, CT7 and NY-ESO1 in low stage epithelial ovarian tumors A.A. JUNGBLUTH1, D. FROSINA1, K. COPLAN1, K. IVERSEN1, E. SATO1, M. LEITAO2, R.A. SOSLOW3

1

Ludwig Institute for Cancer Research, New York Department of Surgery 3 Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 2

64 Prognostic significance of pRb- and p16INK4a-expression in advanced stage ovarian carcinoma after standardized chemotherapy M.J. TRUNK1, S. KOMMOSS2, F. KOMMOSS2, R. RIDDER1, D. SCHMIDT2, J. PFISTERER3, A. DU BOIS4 for the AGO-OVAR Study Group 1

MTM laboratories, Heidelberg Institut fu¨r Pathologie, A2,2, Mannheim 3 Frauenklinik, Universita¨t Kiel 4 Frauenklinik, HSK, Wiesbaden 2

Aims: To assess the prognostic significance of p16INK4aand pRb- expression in advanced stage ovarian carcinomas (OvCa). Methods: Representative sections from 302 pts. with FIGO stage IIb-IV OvCa (prospective randomised, multi-center, phase III study AGO-OVAR-3) were immunostained for p16INK4a and pRb. Immunostaining was evaluated using a score (ranging from 0 to 12) based on staining intensity and fraction of tumor cells stained. A cut-off for high and low expression levels was set at a score of 5 for p16INK4a and pRb, respectively. Survival analysis by Kaplan & Meier method. Results: p16INK4a and pRB-expression were detected in 94.4% and 97.3%, respectively. p16INK4a-and pRbexpression scores showed a weak negative correlation (CC – 0.108, po0:05). Low pRb-scores showed a significantly better prognosis than high pRb-scores, the 5-year survival rates were 40% and 19%, respectively (po0:0001), with corresponding median survival of 47 and 31 months (po0:0001). Furthermore, patients with complete surgical tumor debulking showed better prognosis with low pRb-expression (po0:05). p16INK4a-expression was not significantly correlated to survival: 5-year survival rate for patients was 33%, irrespective of p16INK4a-scores (p ¼ 0:2430). pRb- and p16INK4a-expression was not correlated with other established prognostic factors, such as age, tumor stage.

Aims: CT antigens (CTAs) are expressed in various malignant tumors and in normal adult tissues only in testicular germ cells. Due to this expression pattern, CTAs are potential targets for immunotherapy. RTPCR analyses of CTAs have been performed in ovarian epithelial tumors (OETs) but little is known about their protein expression. We previously generated monoclonal antibodies (mAbs) to various CTAs such as members of the MAGE family, CT7 and NY-ESO-1. In the present immunohistochemical study (IHC), we analyzed low stage OETs for CTA expression. Methods: Hundred and six low stage OTs were studied using tissue microarrays: 34 borderline tumors (BTs) & 72 carcinomas were analyzed by IHC with the following mAbs (to the following CTA): mAb MA454 (to MAGEA1), mAb M3H67 (to MAGE-A3), mAb 57B (to MAGE-A4), mAb CT7-33 (to CT7) and mAb ES121 (to NY-ESO-1). Results: The result of our IHC analysis is shown in the table below: mAb:

MA454

M3H67

57B

CT7-33

ES121

BTs 0/34(0%) 1/34(3%) 2/34(6%) 4/32(12%) 1/34(3%) Stage I 4/44(9%) 11/44(25%) 10/44(23%) 8/44(18%) 4/44(9%) Stage II 12/28(43%) 14/28(50%) 13/28(46%) 13/28(46%) 6/28(21%)

Conclusions: Among low stage OETs, little or no CTA expression is found in BTs, while carcinomas, particularly serous carcinomas, show varying degrees of expression. CTA expression appears stage related (II4I). Though little is known about the function of CTAs, our findings suggest that CTA expression in ovarian carcinoma parallels tumor progression. Therefore, CTAs appear to be attractive targets for immunotherapy in ovarian carcinoma.

ARTICLE IN PRESS Posters: Breast, Ovary / Pathology – Research and Practice 201 (2005) 153–300

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Survival benefit for patients with advanced-stage transitional cell carcinomas vs. other subtypes of ovarian carcinoma after chemotherapy with platinum and paclitaxel F. KOMMOSS, S. KOMMOSS, D. SCHMIDT, J. PFISTERER1, A. DU BOIS2

Institut fu¨r Pathologie, A2, 2 Mannheim 1 Frauenklinik, Universita¨tsklinikum SchleswigHolstein, Kiel 2 Dr.-Horst-Schmidt-Kliniken (HSK), Wiesbaden Aims: To compare incidence and outcome of patients with transitional cell carcinomas (TCCs) and other subtypes of ovarian carcinoma from a large homogeneous collective of patients with primary advancedstage ovarian carcinoma. Methods: H&E stained sections of 302 cases from a prospective randomized, multi-center, phase III study of patients with ovarian cancer, FIGO stages IIB-IV, comparing cisplatin plus paclitaxel with paclitaxel plus carboplatin were available for histological retyping applying current WHO criteria. TCC of the ovary was diagnosed if one or more of the typical transitional cell patterns (excluding malignant Brenner tumor) were present exclusively or in at least 90% of the tumor tissue present. Survival analysis was performed according to the method of Kaplan & Meier. Results: Sixteen of 302 tumors (5.3%) were diagnosed as TCC. The remaining cases were of the following histological types: 135 (44.7%) serous, 24 (7.9%) mucinous, 58 (19.2%) endometrioid, 37 (12.3%) clear cell, and 32 (10.6%) undifferentiated carcinomas. Only one of the 16 TCCs had previously been diagnosed as such by referring pathologists. 5-year survival of patients with TCC was 57% as compared to 32% for patients with ovarian carcinomas of other types (p ¼ 0:03). Conclusions: TCC of the ovary seems to be a less well recognized and possibly underdiagnosed entity. In the current series, TCC had a significantly better prognosis as compared to all other subtypes of ovarian carcinomas after standardized chemotherapy. Thorough histological typing of ovarian cancer is of clinical importance.

67 Osteopontin (OPN) expression in gestational trophoblatstic diseases (GTD) J. BRIESE , M. OBERNDO¨RFER, H.M. SCHULTE1, T. LO¨NING, A.M. BAMBERGER Institut fu¨r Pathologie, Abteilung fu¨r Gyna¨kopathologie, Universita¨tsklinikum Hamburg-Eppendorf 1 Endokrinologikum Hamburg

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Aims: The human placenta is a tissue with a unique capacity for rapid, but tightly controlled proliferation and invasion. Osteopontin (OPN) is a glycoprotein of the extracellular matrix which has been shown to mediate cellular migration and invasion and to contribute to tumorigenesis in several types of cancers. The purpose of the present study was to investigate the expression pattern of OPN in gestational trophoblastic diseases, including hydatidiform moles, placental site nodule (PSN), placental site trophobastic tumor (PSTT) and choriocarcinomas, and to compare this expression with the one observed in the normal trophoblast. Methods: Immunohistochemistry was performed on 20 cases of GTD and 50 normal placentas using a specific polyclonal antibody for OPN. The cases were additionally characterized by diagnostic markers including Ki67, HPL and CK7. Results: Osteopontin was detected in the villous cytotrophoblast and in the extravillous (invasive) trophoblast in both normal placenta and hydatidiform molar samples and also in trophoblast proliferations of molar villi. Strong OPN expression could also be found in PSN, as well as in PSTT and partially in choriocarcinomas. Conclusions: The specific localization to extravillous trophoblast and its expression pattern in GTD indicate that OPN might play a role in the pathogenesis of GTD and to be useful as an additional diagnostic marker for such lesions.

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Evaluation of the E-cadherin repressor snail in endometrial adenocarcinomas K. BLECHSCHMIDT1, K.-F. BECKER1, E. KREMMER2, I. MYLONAS3, H. HO¨FLER1, M. KREMER1 1

Institut fu¨r Pathologie, TU Mu¨nchen GSF-Forschungszentrum fu¨r Umwelt und GesundheitInstitut fu¨r Molekulare Immunologie, Mu¨nchen 3 Klinik und Poliklinik fu¨r Frauenheilkunde und Geburtshilfe, LMU Mu¨nchen 2

Aim: There is no information available on the expression pattern of Snail, a regulator of epithelial mesenchymal transition and direct repressor of E-cadherin transcription, in tumors of the female genital tract. Consequently, the aim of our study was to better understand the role of Snail in this type of tumors. Methods: So far, we have analyzed two endometrial carcinoma cell lines and a series of 42 endometrial cancers for Snail expression using an in-house generated novel monoclonal antibody by Western blot and immunohistochemistry, respectively.

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Results: A differential expression of Snail, E-cadherin and metastasis associated gene 3 (MTA3) was seen in the two cell lines expressing different levels of estrogen receptor (ER). Nuclear Snail immunoreactivity was identified in 15/42 (35%) endometrial cancers, typically observed in poorly differentiated tumors. In 4 (27%) out of the 15 Snail positive cases E-cadherin immunoreactivity was reduced or lost. ER immunoreactivity was markedly reduced or lost in 10/15 (67%) Snail positive cases. Conclusion: We found frequent nuclear Snail immunoreactivity in endometrial cancers with concomitant reduction of E-cadherin expression in some cases. Potential regulators of Snail expression include ER and MTA3. The ability of Snail to extinguish Ecadherin transcription in endometrial cancers, however, depends on additional but currently unknown factors.

Posters: Hematopathology 69

T-cell marker expression in classical Hodgkin lymphoma

A. ZIMPFER, C. BOURGAU, A. TZANKOV1, S. PILERI2, R. MAURER3, M. MIRLACHER, G. SAUTER, P.H. WENT, S. DIRNHOFER Institut fu¨r Pathologie, Universita¨t Basel/CH 1 Institut fu¨r Pathologie, Universita¨t Innsbruck/A 2 Third Chair of Pathologic Anatomy & Lymphoma Unit, University of Bologna/I 3 Institut fu¨r Pathologie, Triemli Spital/ Zu¨rich/CH Aims: Hodgkin/Reed-Sternberg cells (HRS cells) are of B-cell origin in most cases. However, expression of one or more T-cell related antigens may be present in rare cases pointing to a putative T-cell origin. We aimed to comprehensively analyze the expression of T-cell antigens in 330 cHL using the tissue microarray (TMA) technique. Methods: A TMA containing 752 cores of 330 cHL samples representing all cHL subtypes was constructed as previously reported. Immunohistochemical staining was performed using antibodies against CD2, CD3, CD4, CD5, CD7 and CD8. Results: A total of 278/330 (78%) cases were morphologically representative. Thirteen (5%) cases were positive for at least one T-cell marker. Of those, 9 (3%) cases expressed only one marker whereas 4 (1%) cases expressed two and more T-cell antigens. Eleven belonged to the nodular sclerosis subtype and 2 to the mixed cellular subtype of cHL.

Conclusions: Aberrant expression of T-cell antigens in HRS cells is present in 13 (5%) of cHL cases. This confirms that a low fraction of cHL may be of T-cell origin.

70 A rare case of a cytotoxic thymic lymphoblastic T and NK cell lymphoma W. HAEDICKE1, T. GAISER1, A. MARX2, J.L. NELLESSEN3, J. RU¨SCHOFF1 1

Institut fu¨r Pathologie, Klinikum Kassel Pathologisches Institut, Uni Wu¨rzburg 3 Kinderklinik Park Scho¨nfeld, Klinikum Kassel 2

Aims: Attempts to establish a concise classification of lymphoblastic lymphomas (LBLs) has gained momentum in the recent years, mainly due to the expanding possibilities of immunohistochemical characterization of different disease entities. But due to the relatively low number of cases on which the reports are based that propose new classifications are making it unlikely that the complete clinico – pathologic spectrum is adequately delineated. To further characterize this disease entity we present an unusual pediatric case of LBL that cannot be categorized into one of the subgroups and exhibits a positive prognosis after surgical treatment and subsequent chemotherapy. Methods: Immunohistochemistry was performed on Formalin – fixed paraffin – embedded tissue sections using various monoclonal antibodies and the indirect immunoperoxidase technique using standard methods. Results: The tumor cells reacted with TdT and had a positive cytoplasmic, but no membranous immunoreactivity for CD3. TIA-1 was strongly positive. Granzyme B was negative. T cell markers CD1a, CD4, CD8 and CD10 and the NK cell markers CD56 reacted positively, but CD57, CD16 and CD 30 (Ber H2) were all negative. The proliferation fraction (MIB-1 index) was about 80%. B cell markers (CD20, CD22, Cd79a and IgM) were all negative. No clonal B cell Ig or T cell gamma chain rearrangements were detectable. Epstein Barr virus and other Herpes Virus RNA was not detected using a sensitive RT-PCR assay Conclusions: Localization and morphology made the tumor a typical member of the LBL group. But immunophenotypically it combined the phenotype of a lymphoblast committed to the T cell lineage with the phenotype a NK cell precursor and on the other hand it combined the phenotype of a blastic/immature and a mature cytotoxic lymphoma.

ARTICLE IN PRESS Posters: Hematopathology / Pathology – Research and Practice 201 (2005) 153–300

71 ‘‘Downgrade’’ malignant non-hodgkins lymphomas – a histomorphological, molecular biological, and genetic analysis S. HO¨LLER, T. KATZENBERGER, E. HARALAMBIEVA, S. GATTENLO¨HNER, J. KALLA, M.M OTT, H.K. MU¨LLER-HERMELINK, G. OTT Institut fu¨r Pathologie, Universita¨t Wu¨rzburg Aims: ‘‘Downgrading’’ or ‘‘reverse progression’’ denotes a rare phenomenon in which a ‘‘high grade’’ lymphoma recurs as a ‘‘low grade’’ lymphoma. Methods: We studied 13 selected cases identified in a 8 year reference center based lymphoma cohort by morphology, immunohistochemistry, B cell receptor rearrangement analysis and interphase cytogenetics (fluorescence in situ hybridization). Results: All cases were initially diagnosed as a diffuse large B cell lymphoma (DLBL) and relapsed later on as a low grade lymphoma. ten tumors with a diagnosis of DLBL (four of them with a follicular lymphoma (FL) grade III component) relapsed between 1 and 5 years (mean: 2,9 a ; median: 2,5) after diagnosis as a follicular lymphoma grade I, II or IIIa). Two DLBL relapsed as a marginal zone B cell lymphoma and one as a prolymphocyte-rich chronic lymphocytic leukaemia after 1 and 3 years (mean: 2,3; median: 3). Unequivocal clarification of clonal identity by molecular rearrangement analysis was possible in only five cases (four identical clones: Two MZBCL 2 FL cases; 1 different clone: FL grade IIIa). By FISH for the t(14;18) one additional lymphoma pair was suggestive for clonal identity, while two additional cases were probably not related and six cases were not informative so far. Conclusions: Our results indicate that (1) the phenomenon of downgrading is a rare event in NHL, (2) most relapses are clonally related, but that a part obviously are not, (3) in some cases a hither to not sufficially recognized low grade component may be present already in the first biopsy.

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POEMS syndrome: A patient with an IgA lambda plasma cell disease and high expression levels of VEGF C. SCHRADER1, M. TIEMANN2, S. DIETRICH1, M. GRAMATZKI1, D. JANSSEN2, U. ZIRRGIEBEL3, R. PARWARESCH2, M. KNEBA1 1

II.Medizinische Klinik, Universita¨t Schleswig-Holstein, Campus Kiel 2 Institut fu¨r Ha¨matopathologie und Lymphknotenregister Kiel, Universita¨t Schleswig-Holstein, Campus Kiel 3 Proqinase GmbH, Freiburg

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Aims: POEMS syndrome is a rare disease characterized by polyneuropathy, organomegaly, endocrinopathy, M protein and skin changes. We present a woman with a 4 years history of a polyneuropathy caused by an IgA lambda positive plasma cell disorder. Methods: Blood investigation was done for serum electrophoresis and analysis of VEGF, bFGF and IL6. Bone marrow biopsy specimen was investigated immunohistochemically for k; l; a; g; CD20, CD56, Cyclin D1, VEGF. Results: Blood investigation revealed a monoclonal gammopathy IgA l: High levels of VEGF (1468,7 pg/ ml), bFGF (112,9 pg/ml) and IL6 could be found in the blood. Immunohistochemical investigation of the bone marrow biopsy showed a infiltration of l and a positive plasma cells (10%). Only few plasma cells expressed k: The tumor cell were negative for CD20, CD56 and Cyclin D1, but positive for VEGF in line with the high VEGF levels in the blood. Conclusion: POEMS syndrome is a rare disease caused by only few monoclonal plasma cells. Beside bone marrow investigation also VEGF and bFGF expression analysis showed be performed to confirm the diagosis.

73 Immunohistochemical detection of CD95L (Fas Ligand) in mantle cell lymphoma: a prognostic factor for clinical outcome C. SCHRADER1, P. MEUSERS2, G. BRITTINGER2, D. JANSSEN3, R. PARWARESCH4, M. KNEBA1, M. TIEMANN4 1

II.Medizinische Klinik, Universita¨t Schleswig-Holstein, Campus Kiel 2 Klinik fu¨r Ha¨matologie, Universita¨t Essen 3 Institut fu¨r Ha¨matopathologie und Lymphknotenregister Kiel, Universita¨t Schleswig-Holstein, Campus Kiel Aims: Proliferation indices are important prognostic factors in the clinical outcome in mantle cell lymphoma. We investigated immunohistochemically the expression of the apoptotic marker CD95L (Fas Ligand) in relation to the clinical course. Methods: Biopsy specimen from 69 untreated patients enrolled in two multicenter prospective trials were investigated immunohistochemically with monoclonal antibodies against CD20, CD5, CD3, CD23, cyclin D1, CD95L. The CD95L expression was analyzed in three groups: negative, weak positive and strong positive. The expression was compared with the overall survival data analysed according to Kaplan and Meier. Results: Patients with mantle cell lymphoma that had negative and weak positive CD95L expression had a median overall survival time of 32 months compared to 18.3 months for patients with a strong CD95L

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expression. The Kaplan-Meier analysis showed a significant difference in the overall survival time between the patients with strong CD95L expression and the group of patients with negative or weak positive tumor cells (po0:0038). Conclusion: Based on these findings, the immunohistochemical detection of CD95L in mantle cell lymphoma is a prognostic factor.

74 Proliferationmarker in chronic lymphocytic leukemia: prognostic factors for clinical outcome in high risk patients C. SCHRADER1, M. TIEMANN2, S. KARIMI2,3, D. JANSSEN3, S. STILGENBAUER4, H. DO¨HNER4, R. PARWARESCH2, P. DREGER5, M. KNEBA1, M. RITGEN1 1

II.Medizinische Klinik, Universita¨t Schleswig-Holstein, Campus Kiel 2 Institut fu¨r Ha¨matopathologie und Lymphknotenregister Kiel, Universita¨t Schleswig-Holstein, Campus Kiel 3 Department of Pathology, National Research Institute for Tuberculosis and Lung Disease, Darabad, Tehran 4 III Medizinische Klinik, Universita¨t Ulm 5 AK St Georg, Hamburg Aims: Ki-67 is the most used proliferation marker in lymphomas. Repp86 is a new proliferation marker expressed in cell cycle phases G2, S, M but not in G1. We investigated the Ki-67 and Repp86 expression in bone marrow biopsies of 44 patients with high risk CLL in correlation to the mutation status, ZAP 70 expression, progression free and overall survival data. Methods: Bone marrow biopsies from 44 untreated patients were investigated immunohistochemically with monoclonal antibodies against CD20, CD5, CD23, ZAP 70, Ki-67 and Repp86. Cells with clear positive staining were counted and the percentage was calculated. Molecular analysis was done from fresh peripheral blood tumor cells. Results: CLL with a low Ki-67 expression of o10% have a longer progression free survival (median undefined) than patients with a high Ki-67 expression of 410% ( median ¼ 41 months, p ¼ 0:0035). In median 1.6% (range: 0.4–4.6%) of tumor cells where Repp86 positive. Repp86 expression was independet from Binet stage, VH-mutational status, ZAP 70 expression, age and other known risk factors. Patients with CLL and more than 41% repp86 positive cells had a significant shorter progression free survival time compared to patients with less than 1% positive cells (48 moths vs. not reached; p ¼ 0:04).

Conclusion: Based on these findings the expression of Ki-67 and repp86 antigen in CLL might be prognostic factors even in this selected high risk group.

75 Onocytoid B-cell proliferation associated with an epstein-barr virus infection-an early feature of primary viral infection? I. ANAGNOSTOPOULOS1, M. HUMMEL1, B. FALINI2, H. STEIN1 1

Institut fu¨r Pathologie, Charite´, Campus Benjamin Franklin, Universita¨tsmedizin Berlin 2 Institute of Hematology, University of Perugia, Italien Aims: Monocytoid cells are a subset of B lymphocytes with characteristic morphology and immunophenotype, which proliferate in a broad variety of reactive lymph node conditions. Up to now no direct infection of these cells by a pathogenic organism has been identified. In the present study we searched our files for lymph nodes containing monocytoid B (MCB) cell proliferates for the presence of an EBV infection. Methods: Immunohistology for EBV-encoded proteins and in-situ hybridization for EBV-encoded small nuclear RNAs (EBER-1 and –2). Results: We identified 15 cases in which a number of MCB cells contained EBERS and expressed the EBVencoded nuclear antigen-2 and and in lesser extent the latent membrane protein-1. In addition, EBV-encoded transcripts and proteins were detectable in a small number of extrafollicular B-blasts and germinal center B cells in 11/15 cases. All cases had in common a prominent MCB reaction and follicular hyperplasia without sheets of extrafollicular blasts, epithelioid cells or necrosis. Clinical and serological findings indicative for an acute EBV infection were reported only in two cases. Conclusion: This is the first report showing that MCB cells are a target for an EBV infection. The findings that the presented cases lacked the morphological features of acute infectious mononucleosis and that EBNA-2 is preferentially expressed by most of the infected MCB cells imply that the presence of EBV within MCB cells represents an early sign of a primary infection.

76 The vesicular monoamine transporter 2 (VMAT2) is expressed by normal and tumorous cutaneous mast cells and Langerhans-cells of the skin, but is absent from Langerhans-cell histiocytosis M. ANLAUF1,2, M. K.-H. SCHA¨FER2, C. DEPBOYLU2,3, T. SCHWARK1, V. BRAND1, W. HARTSCHUH4, I. LEUSCHNER1, L. E. EIDEN5, R. PARWARESCH1, G. KLO¨PPEL1, E: WEIHE2

ARTICLE IN PRESS Posters: Hematopathology / Pathology – Research and Practice 201 (2005) 153–300 1

Institut fu¨r Pathologie, Universita¨t Kiel Institut fu¨r Anatomie und Zellbiologie, Universita¨t Marburg 3 Klinik fu¨r Neurologie, Universita¨t Bonn 4 Hautklinik, Universita¨t Heidelberg 5 Section on Molecular Neuroscience, National Institute of Mental Health, NIHM, Bethesda, Maryland MD, USA 2

Aims: The aim of our study was to identify monoaminehandling normal and neoplastic inflammatory cells in the skin by their expression of the vesicular monoamine transporters (VMAT1 and VMAT2). Methods: Normal skin from various parts of the body, and 21 cases of cutaneous mastocytosis and 10 cases of cutaneous Langerhans-cell histiocytosis were analyzed by means of immunohistochemistry, immunoelectronmicroscopy, radioactive in situ hybridization and double fluorescence confocal microscopy. Results: VMAT2-positive cells in the subepidermal layer were identified as mast cells by their expression of tryptase. Neoplastic mast cells in all cases of cutaneous mastocytosis retained their VMAT2 positivity. The intraepidermal VMAT2-expressing cells were identified as Langerhans-cells by their CD1a positivity. VMAT2 was absent from Langerhans-cell histiocytosis. Conclusions: VMAT2 is an excellent marker for normal and neoplastic mast cells. The presence of VMAT2 in epidermal Langerhans-cells revealed a previously unrecognized monoamine-handling phenotype of these cells and indicates possible involvement of amine storage and release associated with antigen presentation. Absence of VMAT2 in neoplastic Langerhans-cells indicates a loss of monoamine handling capacity of these cells during tumorogenesis.

77 Modulation of c-Mpl expression in Ph-negative chronic myeloproliferative diseases (CMPD) and socalled essential thrombocythemia with ringed sideroblasts (ET/RS) A. SCHMITT-GRAEFF, A. KIRN, J. THIELE1, H.M. KVASNICKA1, J. SPIVAK2 Institut fu¨r Pathologie, Universita¨t Freiburg 1 Institut fu¨r Pathologie, Universita¨t Ko¨ln 2 Hematology Division; John Hopkins University School of Medicine, Baltimore, USA Aims: An impaired posttranslational processing of the proto-oncogene cytokine receptor c-Mpl with underglycosylation has been proposed as a molecular defect of polycythemia vera (PV) and chronic idiopathic myelofibrosis (CIMF). The aim of our study was an anaylsis of

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the subcellular localization of megakaryocyte c-Mpl protein in different Ph CMPDs and so-called ET/RS. Methods: By confocal laser scanning microscopy (CLSM) we performed an imaging study of bone marrow trephines from normal bone marrow donors (n ¼ 14), patients with essential thrombocythemia (ET, n ¼ 15), PV (n ¼ 18), CIMF (n ¼ 15), and ET/RS (n ¼ 67). Common findings in all patients with ET/RS were the presence of 415% RS and a platelet count 4500  109/L. CLSM imaging was semi-quantitatively evaluated by scoring of plasmalemmal vs. cytoplasmic labeling (score 1–3). Results: In contrast to ET, c-Mpl suface expression was significantly reduced in PV and CIMF (po0:001), while cytoplasmic expression was often strongly accentuated. ET/RS c-Mpl imaging revealed a heterogeneous positivity with different degrees of plasmalemmal vs. cytoplasmic immunoreactivity in keeping either with CIMF, true ET or MDS. Conclusions: The modulation of cell surface c-Mpl expression may result in impaired receptor domains for TPO as an epigenetic abnormality and may contribute to define positive criteria for the discrimination of the distinct subtypes in the ET/RS group.

78 Anaemia of chronic disease develops in the absence of IFN-g activity and TNF activity in a mouse model of protracted septic peritonitis T. SCHUBERT1, B. ECHTENACHER2, F. HOFSTA¨DTER1, D.N. MA¨NNEL2 1 2

Institut fu¨r Pathologie, Universita¨t Regensburg Institut fu¨r Immunologie, Universita¨t Regensburg

Aims: Interferon-g (IFNg) and Tumour necrosis factor (TNF) have been considered the inflammatory cytokines mainly contributing to the generation of anaemia of chronic disease (ACD). In this work, we used a recently described murine model for ACD based on sublethal cecal ligation and puncture (CLP) with ensuing protracted peritonitis to examine the role of TNF and IFN-g in vivo. Methods: We neutralised IFNg or TNF after CLP shortly before and during the phase of most severe bone marrow depression in order to prevent anaemia. IFNgreceptor-deficient mice or TNF-deficient mice undergoing CLP were studied as well. Additionally, TNF was neutralised in IFNg-receptor deficient mice. Two weeks after CLP the animals were sacrificed and red blood cell count, haemoglobin con-centration, haematocrit, serum iron concentration, and iron stores in the spleen were determined. Results: Neither neutralisation of IFNg or TNF after CLP could prevent mice from contracting anaemia.

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Accordingly, both IFNg-receptor-deficient mice and TNF-deficient mice developed anaemia to the same extent as wild type mice. Neutralisation of TNF in IFNg-receptor deficient mice did not alter the course of ACD. Serum iron concentration was lowered both in IFNg-receptor-deficient and wild type mice. Iron stores in untreated IFNg-receptor deficient mice were elevated as compared to untreated wild type mice. Conclusion: The putative role of IFN-g and TNF in the generation of ACD is based on clinical correlations and in vitro data. Taking into account our findings in the mouse model the contribution of IFN-g and TNF to human ACD should be critically re-evaluated.

79 Assessment of bone marrow fibrosis strongly depends

80 Marrow fibrosis in imatinib treated chronic myeloid leukemia G. BU¨SCHE1, R. HEHLMANN2, A. GANSER3, M. FREUND4, B. HEINZE5, B. SCHLEGELBERGER6, C. FONATSCH7, A. GEORGII1, H.-H. KREIPE1 1

Institut fu¨r Pathologie, MH Hannover III. Medizinische Klinik, Klinikum Mannheim 3 Klinik fu¨r Ha¨matologie u. Onkologie, MH Hannover 4 Abt. fu¨r Ha¨matologie u. Onkologie, Univ. Rostock 5 Tumorzytogenetiklabor, Abt. fu¨r Innere Medizin III, Univ. Ulm 6 Institut fu¨r Zell- und Molekularpathologie, MH Hannover 7 Abt. fu¨r Humangenetik Med. Univ. Wien 2

on technical processing of biopsies G. BU¨SCHE Institut fu¨r Pathologie, Medizinische Hochschule Hannover Aims: Marrow fibrosis (MF) is a characteristic complication of chronic myeloproliferative disorders usually shortening the survival time of patients. However, the significance of biopsy processing for proof and quantification of MF has not yet been evaluated. Methods: In this study on 360 bone marrow biopsies from 23 laboratories taken from 135 patients with chronic idiopathic myelofibrosis or chronic myeloid leukemia, the results from Five approaches of proof and quantification of MF were systematically analyzed with respect to their dependence on technical processing of bone marrow biopsies. Results: Proof and quantification of MF varied markedly between the biopsies from different laboratories regardless of the method applied to quantify MF (Po0:000005). The variability was explained by differences in fixation, method of embedment, degree of tissue shrinkage during biopsy processing, and the thickness of marrow sections (Po0:000005). The relevance of these variables was explained by their influence on the volume of marrow tissue to which the fiber content was related. The Five approaches systematically compared with respect to proof and quantification of MF significantly differed in their adjustability to these technical influences (Po0:000005). Conclusions: Assessment of MF is significantly influenced by technical processing of bone marrow biopsies. Without adequate adjustment to these influences, results from different laboratories are hardly comparable. However, the adjustability to technical influences strongly depends on the method applied to prove and quantify MF.

Aims: Imatinib has been shown to reverse marrow fibrosis (MF) in chronic myeloid leukemia (CML). The prognostic relevance of MF during imatinib treatment of CML is not clear. Methods: A total of 1509 bone marrow biopsies from 605 patients with Ph+ CML were examined to determine the extent of MF. Hundred and seven CML patients were treated with imatinib, 208 with interferon-a 7 cytosine arabinoside, 154 with hydroxyurea and 136 with busulfan. Results: Imatinib and interferon-a 7 cytosine arabinoside were significantly more effective than the other types of therapy regarding reversal of initial MF (Po0:000005). However, imatinib monotherapy was not superior to interferon-a or hydroxyurea in the prevention of MF; the estimated rate of patients with extensive MF exceeded 10% and with focal MF 25% 3 years after start of treatment. In multivariate analysis, evolving MF was an early and significant independent predictor of therapy resistance and shortened survival time of patients independent of the type of treatment (Po0:00005). Conclusions: MF may be reversed by imatinib in CML. However, MF may reoccur or develop during imatinib monotherapy in a significant proportion of patients. Progressing MF is a strong and early predictor of evolving therapy failure.

81 Heterogeneity of DNA methyltransferase expression and hypermethylation in myelodysplastic syndrome F. LA¨NGER, K. BRAKENSIEK, J. DINGEMANN, H. KREIPE, U. LEHMANN Institut fu¨r Pathologie, Medizinische Hochschule Hannover Aims: Aberrant methylation is a frequent phenomenon in MDS and provides a novel target for specific

ARTICLE IN PRESS Posters: Hematopathology / Pathology – Research and Practice 201 (2005) 153–300

inhibitors. This study addresses the question whether DNA methyltransferases are overexpressed in MDS and how expression correlates to hypermethylation. Methods: Nucleic acids were extracted from a large series of bone marrow trephines from MDS patients (n ¼ 85) and a control group (n ¼ 20). DNA and RNA was also extracted from PBMC (n ¼ 12). mRNA expression levels of DNMT1, 3A, and 3B were measured using real-time PCR methodology and employing relative quantification algorithms. Hypermethylation of the cell cycle regulatory genes p21, p27, p57, and p14ARF and of the pro-apoptotic death associated protein kinase gene was assessed after bisulfite treatment of genomic DNA using qualitative and real-time PCRbased quantitative assays. Results: DNMT1, and 3A (and 3B to a much lesser extent) show a strong overexpression in refractory anemia (RA) and refractory anemia with excess of blasts (REAB). Classification according to the new WHO classification revealed distinct differences between RCMD and RARS. Whereas the p27 gene showed no hypermethylation in all samples tested, the p21 and p57 genes tended to be more frequently methylated in RAEB and AML cases. The DAP-kinase gene showed only low level methylation. Conclusions: Expression of DNMT mRNA shows a distinct pattern in different MDS subtypes and might explain the different response rates in MDS patients to methylation inhibitors. Also the hypermethylation of cell cycle regulatory genes displays gene- and MDS subtype-specific characteristics. Only quantitative detection of methylation events provides under many circumstances reliable data.

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gated the chromosomal translocation t(11;18) and the expression of Bcl-10 as an important cell cycle protein relevant for tumorigenesis in 45 cases of BALT lymphoma. Methods: Chromosomal translocation t(11;18) was detected from formalin fixed, paraffin embedded tissue using a multiplex RT-PCR approach. BCL-10 expression was visualized immunohisto-chemically. Results: Forty seven percent of the cases showed the t(11;18) chromosomal translocation. A specific nuclear reactivity for bcl-10 was seen in 67% of the t(11;18) positive cases, but also in some of the cases without the t(11;18). Conclusions: The t(11;18) is a genetic hallmark in extranodal marginal zone B-cell lymphoma of the BALT and is corralated in a majority of cases with an ectopic nuclear expression of BCL-10. Using a tissue microarray the other known genetic aberrations (t(14;18); t(1;14); trisomies 3 and 18) are subject to further analysis. By correlating the obtained results with the clinical follow-up of the patients, possible predictive factors might be established.

83 Biopsy diagnosis of hepatic lymphomas according to the new WHO classification T. LONGERICH1,3,*, C. LODDENKEMPER2,*, M. HUMMEL2, K. ERNESTUS3, I. ANAGNOSTOPOULOS2, H.P. DIENES3, P. SCHIRMACHER1,3, H. STEIN2 1

Institut fu¨r Pathologie, Universita¨t Ko¨ln Institut fu¨r Pathologie, Charite´, Universita¨t Berlin 3 Pathologisches Institut, Universita¨t Heidelberg * both authors contributed equally 2

82 Immunohistochemical and moleculargenetic characterization of extranodal marginal zone B-cell lymphoma of the bronchus associated lymphatic tissue (BALT): expression of t(11;18) and BCL-10 P. ADAM, A. METZGER, M. HARTMANN, E. HARALAMBIEVA, H.K. MU¨LLER-HERMELINK, G. OTT Institut fu¨r Pathologie, Universita¨t Wu¨rzburg Aims: Extranodal marginal zone B-cell lymphomas of mucosa associated lymphoid tissue (MALT-type) are characterized by several genetic alterations, like the t(11;18), t(14;18), t(1;14) and trisomies 3 and 18. Generally the translocations mentioned are found alternatively. Their relative prevalence depends on the anatomic site of the primary tumor. MALT lymphoma of the bronchus associated lymphatic tissue (BALT) in particular have been claimed to harbour the t(11;18) in albeit small published series. In this study we investi-

Aims and Methods: Aim of our study was to retrospectively evaluate a panel of more than 200 cases obtained by liver biopsy with diagnosis of lymphoma according to the new WHO classification. Diagnosis was based on morphology, infiltration pattern, immunohistochemistry and additional molecular pathological analyses in a few selected cases. Results: Diffuse large B-cell lymphoma (DLBCL) was the most frequent entitiy (40%). Subdivision according to the criteria of Hans et al. (2004) revealed a predominance of DLBCL of activated B-cell type (65%) as compared to the DLBCL of germinal center B-cell type (35%). Additionally, T-cell-rich B-cell lymphoma was found in 7% of B-NHL. Many B-cell neoplasms (e.g. B-CLL, marginal zone lymphoma, BALL, Burkitt lymphoma) revealed characteristic morphological pattern, which supported a selection of restricted immunohistochemical analyses. In contrast, the infiltration pattern of T-cell lymphomas was less

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informative. Even differentiation from a reactive condition sometimes required additional molecular analyses (e.g. clonal TCR-rearrangement). In general, T-cell lymphomas were more prevalent in the liver (12%) when compared with other extranodal tissues. Conclusions: The new WHO classification can be applied successfully on liver biopsies containing malignant lymphoma. Evaluation of the infiltration pattern supported the selection of restricted immunohistochemical and molecular analyses. The relative frequency of Tcell lymphomas and T-cell-rich B-cell lymphomas may reflect the important role of hepatic T-cell immune response.

proved OS. Expression of CD10 was associated with a preserved immunocompetence whereas CD20 expression was associated with an elevated lactat dehydrogenase level. However, CD20 expression was less frequent in patients who had been treated with HAART compared to patients without HAART (p ¼ 0:04). Conclusion: Lack of CD20 or CD10 expression and a post-germinal center signature are independently associated with a worse prognosis in ARL.

85 Differential expression of activation-induced cytidine 84

Immunohistochemical expression profiles in AIDSrelated lymphoma (ARL): A clinicopathological study of 89 patients C. SCHRADER1,2, D. JANSSEN2, E. WOLF3, M. VIERBUCHEN4, R. PARWARESCH2, K. ERNESTUS5, A. PLETTENBERG6, A. STOEHR6, G. FA¨TKENHEUER5, C. WYEN5, M. OETTE7, H.-A. HORST1, M. TIEMANN2, C. HOFFMANN1 1

II.Medizinische Klinik, Universita¨t Schleswig-Holstein, Campus Kiel 2 Institut fu¨r Ha¨matopathologie und Lymphknotenregister Kiel, Universita¨t Schleswig-Holstein, Campus Kiel 3 KIS-Kuratorium fu¨r Immundefizienz, Mu¨nchen 4 AK St. Georg, Hamburg 5 Universita¨t Ko¨ln 6 Institut fu¨r Interdisziplina¨re Infectiologie (ifi), Hamburg 7 Universita¨t Du¨sseldorf Aims: There is no data on immunohistochemical expression profiles in AIDS-related lymphoma (ARL) and their relation to the clinical course. Methods: Paraffin embedded sections of 89 ARL cases during 1989–2004 were stained immunohistochemically with antibodies for CD3, CD10, CD20, CD38, CD138/Syn-1, MUM1/IRF4, BCL-2, BCL-6 and Ki-67. Results: Expression of CD10 and CD20 were associated with better OS (p ¼ 0:009 and p ¼ 0:04; respectively). Expression of CD20 was associated with better DFS (p ¼ 0:03), whereas expression of CD138/Syn-1 was associated with worse DFS (p ¼ 0:03). OS and DFS were worse in patients with immunophenotypic profiles related to post-germinal center (GC) differentiation when compared to GC differentiation. When controlled for age-adjusted International Prognostic Index, prior ADI and year of ARL diagnosis, expression of CD20 and CD10 remained significantly associated with im-

deaminase (AID) in classical and nodular lymphocyte predominant Hodgkin lymphoma A. GREINER, S. TOBOLLIK1, M. BU¨TTNER, B. JUNGNICKEL1, K. HERRMANN, E. KREMMER2, G. NIEDOBITEK Pathologisches Institut, Universita¨t Erlangen-Nu¨rnberg 1 Institut fu¨r Klinische Molekularbiologie, GSF, Mu¨nchen 2 Institut fu¨r Molekulare Immunologie, GSF, Mu¨nchen Aims: AID is essential for class switching and somatic hypermutation of immunoglobulin genes. Accordingly, AID is expressed in non-Hodgkin lymphomas with a germinal centre phenotype. Study of Hodgkin lymphomas (HL) for AID expression has so far been hampered by a lack of suitable antibodies. We have analysed HL for AID expression using in situ hybridisation and immunohistochemistry with an AID-specific monoclonal antibody. Methods: In situ hybridisation was carried out using 35Slabelled RNA probes and expression of AID protein was examined using immunohistochemistry with a newly generated monoclonal antibody. twenty three nodular sclerosis, 16 mixed cellularity and six nodular lymphocyte predominant (HLnlp) Hodgkin lymphomas, as well as tonsils showing reactive hyperplasia were studied. Results: In hyperplastic tonsils, AID expression was observed in germinal centres and in scattered extrafollicular blasts. All six HLnlp cases showed labelling of tumour cells while in all classical HL cases (HLns and HLmc) the neoplastic cells were AIDnegative. Conclusions: These results are in keeping with the notion that tumour cells of HLnlp, but not of classical HL, are germinal centre-derived cells showing evidence of ongoing somatic hypermutation. Our results suggest that AID may be a useful marker in the differential diagnosis of HL.

ARTICLE IN PRESS Posters: Cardiovascular Pathology / Pathology – Research and Practice 201 (2005) 153–300

86 Interferon-inducible chemokine IP-10 (CXCL10) and CXCR3/IP-10 receptor-positive lymphocytes in Hodgkin lymphoma and nasopharyngeal carcinoma M. TEICHMANN, B. MEYER, A. BECK, G. NIEDOBITEK Pathologisches Institut, Universita¨t Erlangen-Nu¨rnberg Aims: Hodgkin lymphoma (HL) and nasopharyngeal carcinoma (NPC) are Epstein-Barr virus (EBV)-associated tumours and display an abundant infiltrate of reactive lymphoid cells. The presence of this lymphoid stroma may affect the outcome of T-cell-based immunotherapy of these tumours. The interferon-inducible chemokine IP-10 has anti-neoplastic effects in several model systems partly mediated by T-cells expressing the CXCR3 receptor. Methods: In situ hybridisation (ISH) was used to detect IP-10 expression and CXCR3-positive lymphocytes were identified by immunohistochemistry. EBV infection was detected using EBER-specific ISH. Results: IP-10 was expressed in neoplastic cells of a proportion of HL showing a statistically significant correlation with mixed cellularity histotype and with EBV infection. IP-10 expression was also detected in most EBV-positive NPC as well as in EBV-negative squamous cell carcinomas of the tongue. Thus, IP-10 expression showed no correlation with EBV infection in carcinomas. Numerous CXCR3-positive lymphocytes were detected in HL and NPC but only infrequently in squamous cell carcinoma of the tongue. Conclusions: These observations raise the possibility of a Th1-predominant immune response in HL and in NPC. In view of the proposed anti-neoplastic functions of IP10 and CXCR3-positive lymphocytes these findings are unexpected and suggest that endogenous IP-10 expression in human tumours may not exert the anti-tumour effects described in animal experiments.

87 Anaplastic large cell lymphomas lack the expression of T-cell receptor molecules or molecules of proximal Tcell receptor signaling

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still unknown. A unifying concept for both ALK+ and ALK– as well as systemic and cutaneous ALCL is still missing. Methods: We studied 24 ALK+, 15 ALK- systemic and 7 cutaneous ALCL for their T-cell receptor (TCR) rearrangements, expression of TCRs and TCRassociated molecules (CD3, ZAP-70). Frozen material was available in 19 cases. Twenty-two not otherwise specified PTCL (PTCL-NOS) and seven angioimmunoblastic T-cell lymphomas (AILT) were studied in comparison. Results: Among 19 frozen cases, TCRb and TCRg genes were clonally rearranged in 74% each. One case each was rearranged for TCRb but not TCRg and vice versa. Despite their frequent clonal rearrangement for TCRb; only 2/47 ALCL (4%) expressed TCRb protein, while TCRs were detected 20/22 PTCL-NOS and in 7/7 AILT. CD3 was lacking in all but one systemic ALK1+ ALCL and in 40% of ALK1 systemic ALCL. ZAP-70 was lacking in more than 70% of all ALCL cases studied. Interestingly, the both TCRb+ cases lacked both CD3 and ZAP-70 indicating that proximal TCR-signaling may be impaired in all ALCL investigated. Conclusion: Defective expression of TCRs is a common characteristic of all types of ALCL which may contribute to the dysregulation of intracellular signalling pathways controlling T-cell activation and survival. This molecular hallmark of ALCL is analogous to defective immunoglobulin expression distinguishing Hodgkin lymphoma from other B-cell lymphomas.

Posters: Cardiovascular Pathology 88 Autopsy is of significant value for quality management in cardiac surgery T. GRADISTANAC1, A.J. RASTAN2, N. LACHMANN2, F.W. MOHR2, CH. WITTEKIND1 1

Institut fu¨r Pathologie, Universita¨t Leipzig Herzchirurgie, Herzzentrum, Universita¨t Leipzig

2

T. RU¨DIGER, I. BONZHEIM, E. GEISSINGER, S. ROTH, A. ZETTL, A. MARX, A. ROSENWALD, H. K. MU¨LLER-HERMELINK Institut fu¨r Pathologie, Universita¨t Wu¨rzburg Aims: Anaplastic large cell lymphoma (ALCL) designates a heterogeneous group of CD30+ peripheral T-cell lymphomas (PTCL). A subgroup of systemic ALCL is transformed by anaplastic lymphoma kinase (ALK). In ALK– cases the exact mechanism of transformation is

Aims: Autopsies rates have declined throughout the world despite serving as an important tool in quality management. We wanted to assess the impact of autopsy for early postoperative quality assurance in cardiac surgery. Methods: Between 2000 and 2003 14,313 patients received cardiac surgery. Eight hundred and ninty eight patients (6.3%) died, in 468 (52.1%) autopsy was performed. Data from clinical and post-mortem examination were prospectively analyzed regarding causes of

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death, postoperative complications, concomitant diseases and surgery associated pathologic findings. Results: Mean survival (468 patients) was 13.9 postoperative days. Autopsy causes of death were cardiac in 233 patients (49.8%), respiratory in 39(8.3%), cerebral in 30(6.4%), abdominal in 22(4.7%), multiorgan failure/ sepsis in 70(14.9%), pulmonary embolism in 31(6.6%), procedure-associated in 39(8.3%) and others in 4(0.9%). Discrepancies between clinical and post-mortem cause of death were identified in 108 patients (23.1%). Clinically unrecognized postoperative complications were found in 364 patients (77.8%), unknown concomitant diseases in 464(99.1%), with potential therapeutic relevance in 90(19.2%), pre-mortem unrecognized surgery associated patholocigal findings in 96(20.5%). Conclusions: A high overall discrepancy rate between clinical and autopsy diagnoses was recognized. Autopsy revealed clinically relevant information in a significant number of patients. Autopsy remains essential for quality management in cardiac surgery.

89 Fatal shock after heat stroke – A case report CH. BROCHHAUSEN, T. SCHREINER1, A. SCHWARTING1, J. BOHL, M. GHALIBAFIAN, C.J. KIRKPATRICK Institut fu¨r Pathologie, Universita¨t Mainz 1 I. Medizinische Klinik, Universita¨t Mainz Aims: Heat stroke is a life-threatening disease with high mortality, characterized by body temperature over 40 1C and clinical symptoms of central nervous system dysfunction. However, the pathophysiological mechanisms are not fully understood. A new interesting explanation for the clinical symptoms could be a systemic inflammatory response leading to endothelial damage and a syndrome of multiorgan dysfunction (1). Methods: We describe a 37-year-old male patient who collapsed during working in a vineyard in environmental temperature of 32 1C with body temperature of 42.5 1C. Despite intensive care treatment he died with symptoms of shock and multiorgan dysfunction. Autopsy was performed followed by the histological evaluation of paraffin embedded tissue. Results: As correlates for clinical shock symptoms shock lungs, shock kidneys and shock liver could be demonstrated. Furthermore, multiple micro-thrombi were found, clinically by undetectable fibrinogen values. Finally, the patient died due to massive diffuse gastrointestinal bleeding and bleeding in pleural and pericardial cavities. There were no signs of severe edema of the central nervous system detectable.

Conclusions: This case underlines the hypothesis that in heat stroke an endothelial damage with consecutive cascade of inflammatory and coagulatory reactions may play a pathophysiological role. Reference: A. Bouchama, J.P Knochel N Engl J Med 346:(2002)1978–1988.

90 Alcohol – related death and German reunification, a retrospect, descriptive analysis of autopsy reports of a former East-Berlin hospital from 1979 to 2000 M. SCHNEIDER, R. MEYER, R. HETZER Deutsches Herzzentrum Aims: Analysis of period of German reunification, a significant milestone of recent German history. Autopsy-reports of alcohol-related death in a former East Berlin hospital were examined in relation to the significant political, economic and societal changes in the former GDR. Methods: (1) Initial examination of 284 autopsy reports of a hospital in a highly populated district of Berlin with low social index from 1979 to 1994. (2) additional data collection of same hospital 1999 & 2000, resulting in a comparison of periods 1980/81, 90/91 and 99/2000. Main research criteria: cause of death, basic underlying disease, macroscopic and histological changes of the heart, liver and coronary arteries of alcohol-related death (ARD); comparisson with control-group (CG) without alcohol anamnesis. Results: (1) Proportion of alcohol-related death to total mortality increased ; (2) Predominant ICD-groups differ for ARD and CG, (3) Fatty transformation of left ventricle with interstitial fibrosis: predominant histol. change on heart; (4) Av. age at death of male alcoholics in 1990/91 was 12 y below early 80’s and 16 y below 1999/00; 7. 41.2% of ARD through damage of gastrointestinal tract in 1990/91. Conclusions: (1) ARD show 14.6 years reduced life expectancy in comparison to total number of death; (2) This effect is stronger in female ARD; (3) Liver damage is pre-dominant cause of death; (4) In the 2 years after fall of the wall‘‘ARD in men is increased through ’’ damage of GI- tract and occurred at a younger age; (5) Socio-economic distress seems to increase alcoholrelated death amongst the population.

91 Ductus arteriosus diverticula in adults A.M. MU¨LLER, T. VOGLER, F. SCHULZ1, K.-M. MU¨LLER Institut fu¨r Pathologie, Ruhr-Universita¨t Bochum 1 Institut fu¨r Rechtsmedizin, Universita¨t Hamburg

ARTICLE IN PRESS Posters: Cardiovascular Pathology / Pathology – Research and Practice 201 (2005) 153–300

Aims: According to angiographic studies 9% to 36% of all adults show a so called diverticulum of the ductus arteriosus, which is defined as retraction of the aortic wall at the insertion of the obliterated ductus arteriosus. As up to now there are no morphological studies confirming or contradicting these data we morphologically studied the region of the ligamentum arteriosum in uncalcified thoracic aortae Methods: In 45 thoracic aortae (age: 17–50 y) the region of the ligamentum arteriosum was examined macro- and microscopically. Preparing the specimens special care was taken to include the insertion of the obliterated ligamentum arteriosum. As it is nearly impossible to evaluate or reconstruct the normal structures of aortic walls with atherosclerosis, atherosclerotic aortae were excluded. Results: Sixteen aortae showed a retraction of the aortic wall at the insertion of the ligamentum arteriosum, in depth varying between 0.5 mm and 2 mm. Interestingly a corresponding umbilication was found on the attached pulmonary artery. In addition, the aortae showed a distorsion of the elastic fibers. twenty nine aortae showed a distorsion of elastic fibers but no insertion. Conclusion: Our findings confirm radiological studies demonstrating a diverticulum of the ductus arteriosus at the insertion of the ligamentum arteriosum in about 36% of non-atherosclerotic aortae. This, and the distorsion of the aortic elastic fibers should be considered as one reason for the localisation of dissecting aortic aneurysms and traumatic aortic rupture in the proximity to the ligamentum arteriosum.

92 Histomorphometric investigations of the aortic media in cases with aortic dissection B. BOCKHOLDT1, R. MEYER2, V. SCHNEIDER1, R. HETZER2 1

Institut fu¨r Rechtsmedizin, CBF, Charite´ Berlin Deutsches Herzzentrum Berlin

2

Aims: Information concerning quantification of selected parameters of the aortic media in cases with aortic medial diseases is very sparse; in the literature no data could be found dealing with quantification of changes of the medial structural elements in cases with aortic dissection. We believe that histomorphometric investigations are an appropriate method to quantify selected parameters of the aortic media. Methods: A comprehensive post-mortem investigation (macroscopical, microscopical and histomorphometrical) of 28 cases with an aortic dissection (showing no, or only minimal macroscopical observable arteriosclerosis).

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Results: The thickness of aortic media decreased from the ascending aorta (1.4 mm) to the descending aorta (1.1 mm) and to the abdominal aorta (1.0 mm). The thickness of elastic lamellae were 3.3 mm in the ascending, 3.2 mm in the descending and 3.2 mm in the abdominal aorta. The thickness of interlamellar spaces range from 13.1 m (ascending aorta), to 12.5 mm (descending aorta) to 13.6 mm in the abdominal aorta. All three parameters do not differ from the values of our control group (normal aortas). The amount of collagen in all regions was lower than in the control group (10.2%) in ascending aorta, 8.5% descending aorta and 9.0% in the abdominal aorta. Conclusions: In aortic medial diseases (apart from arteriosclerosis) the thickness of the media, elastic lamellae and interlamellar spaces are not primarily changed, however the amount of collagene is clearly reduced.

93 Morphometric analysis of heart muscle cells in heart donors and patients with chronic ischemic heart disease D. GENOVA, R. MEYER, N.E. HIEMANN, R. HETZER Deutsches Herzzentrum Berlin, Abt. fu¨r Herz-, Thorax- und Gefa¨ßchirurgie Aims: The aim of this study was to investigate histomorphometric characteristics of heart muscle cells (HMC) in healthy heart donors and patients with chronic ischemic heart disease (CHD). Methods: Morphometric analysis of HMC was performed in 23 healthy heart donors (7 male, 16 female, mean age 43713 yrs) and 25 male patients with advanced coronary artery disease (mean age 5976 yrs). Using EasyMeasure software the diameters of 50–00 HMC per patient were evaluated by light microscopy. In total 4,809 analyses in the donor group and 8,253 analyses in the CHD group were performed. Statistics were calculated using non-parametric tests (po0:05). Results: The mean HMC diameter was 8.7371.26 mm in the healthy donor cohort and 17.0273.39 mm in the CHD group (po0:0001). In the donor group, HMC diameters in female patients were 8.6371.29 mm and in males 8.9671.26 mm (p ¼ 0:578). More than 80% of all measurements in the donor group ranked among two classes: 6.0 to 8.99 mm (48%) and 9.0 to 11.99 mm (34%). In the CHD cohort 86% of the measured diameters ranked among five classes: 9.0 to 11.99 mm (13%), 12.0 to 14.99 mm (24%), 15.0 to 17.99 mm (23%), 18.0 to 20.99 mm (16%) and 21.0 to 23.99 mm (10%). Conclusions: CHD is associated with different grades of heart muscle cell hypertrophy. The reasons for this

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phenomenon still have to be elucidated. In this study, no differences were found in the HMC diameters of healthy male and female patients.

94 Papillary fibroelastoma in cardiac surgery: pathological aspects and review of literature S. SAENGER1, R. MEYER2, O. GRAUHAN3, S. THOMANN2, J. KNOERIG1, R. HETZER3 1

Abt. fu¨r Herzchirurgie, SANA Herzzentrum, Cottbus Abt. fu¨r Herzpathologie, Deutsches Herzzentrum Berlin (DHZB) 3 Abt. fu¨r Herz-, Thorax- und Gefa¨sschirurgie, DHZB 2

Aims: Cardiac papillary fibroelastomas (CPF) represent about 7–10% of all primary cardiac tumors. Located mostly on the cardiac valves, surgical intervention is necessary because of the tumor’s high potential to embolize and causing fatal complications. We describe our echocardiographical, surgical and histopathological findings. Methods: In a six-year period (1998–2004), 25 tumors with the histological diagnosis of CPF were examined in our institution. Twenty-four CPF were surgically removed by using the heart-lung machine and requiring circulatory arrest, and one tumor was explanted of a heart-donor patient. The age of the patients (19 women, 6 men) ranged from 32 to 86 years (mean 58.5 years). CPF were preoperatively detected by echocardiography in 23 patients (92%). Results: CPF were located on the aortic valve (14 patients), left ventricular outflow tract (2 patients), mitral valve (3 patients), tricuspid valve (2 patients), right atrium (3 patients) and left atrium (1 patient). The diameter of the 25 specimen ranged from 2 mm to 5 cm, mean 12.4 mm. Immunohistochemical specimens were positive for endothelial cells (CD 31 and CD 34 markers) and negative for cytomegaly virus. Conclusions: CPF are located on all endocardial structures of the heart – with an unexplainable preference of the valves. Cardiac fibroelastomas have a high incidence of systemic embolic events. Widespreading of echocardiography has a key-role in early and time-righted detection of CPF. Surgical removal is curative and can be performed with excellent results. The pathophysiology of CPF is still unknown.

95 Size and distribution of heart-muscle-cells conducted on patients with hibernating myocardium F. LABSCHIES, R. MEYER, H. HAUSMANN, H. SINIAWSKI, M. GUTBERLET1, R. HETZER

Deutsches Herzzentrum Berlin 1 Institut fu¨r Radiologie, Charite´, Medizinische Fakulta¨t, Humboldt Universita¨t Berlin Aims: As of yet, it has not been proven how heartmuscle-cells change their size under conditions of hibernating myocardium. It was our aim to show a specific distribution chart of the dimensions of their hypertrophy. Can this distribution be a morphological characterisation of hibernating myocardium? Methods: Myocardial biopsies from 37 patients (64+/ 10 years old) with defined areas of hibernating myocardium were measured for hypertrophy with a computerized microscope. A total of 26.865 measurements were made on 5.373 cells. Afterwards these were classified, according to their grades of hypertrophy, in 16 categories of subpopulation. Results: Heart-muscle-cells in a condition of hibernating myocardium have an average cell diameter of 18.3+/ 7.0 mm, which stands for a medium grade of hypertrophy. They may be classified according to their sizes: Only two thirds of all analyzed heart cells were hypertrophied. 40% had a strong-(419 mm), 10% had a medium-(17–18.9 mm), and 10% had a small (15–16.9 mm) hypertrophy. 40% of the analyzed heart cells did not take part in the process of hypertrophy (o14.9 mm). 17% of the cells were even defined as atrophied (o10 mm). Conclusions: Heart-muscle-cells in a state of hibernating myocardium can be characterized on a morphologically basis, since they show a ‘‘specific distribution of size’’. The result of the present study reflects a thorough and comprehensive analysis of hibernating myocardium, which has not been done in this quantity before.

96 Characterization of human atherosclerotic plaques. Histological, ultrastructural and chemical investigations I. SCHMITZ1, M. EPPLE2, A. BECKER2, K.M. MU¨LLER1 1

Institut fu¨r Pathologie, BG-Kliniken Bergmannsheil, Ruhr-Universita¨t Bochum 2 Institut fu¨r Anorganische Chemie, Universita¨t Duisburg-Essen, Campus Essen Aims: Until now the actual mechanism leading to the deposition of calcium phosphate within blood vessels is still not clear. So aim of our study was to characterize atherosclerotic plaques of patients with well-documented clinical history. Methods: Histology, scanning electron microscopy and energy dispersive X-ray analysis, transmission electron microscopy, infrared spectroscopy, thermogravimetry and high resolution X-ray diffraction were used in order to examine plaques of the aorta (6 cases, autopsy).

ARTICLE IN PRESS Posters: Cardiovascular Pathology / Pathology – Research and Practice 201 (2005) 153–300

Results: Soft, lipid-rich, fibrous lesions and hard plaques with calcified areas of different extent were distinguished. Mineralised particles were detected in all cases. All samples contained 60-70wt% biological carbonated apatite (in dry state) in a nanocrystalline form with particle sizes of about 20 nm.Structurally there were strong similarities to bone mineral. Ultrastructural investigations documented typical calcospherites, mineralisation processes, starting at collagen fibrils as well as ring-shaped mineralised structures. There were no significant ultrastructural or chemical differences between the calcification of the aortas of the individual patients. Conclusions:The results point to similar mechanisms of the vascular calcifications. Bone mineralisation and pathological calcification may be related to the same mechanism.

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Induction of the matrix metalloproteinase-2 activation-system in arteries by short time tensile stress. Involvement of the MAP-kinase pathway F. GRABELLUS, K. WORM, K.J. SCHMITZ, K.W. SCHMID, H.A. BABA Institut fu¨r Pathologie, Universita¨t Duisburg-Essen Aims: Matrix metalloproteinasen (MMPs) play a role in tissue remodeling by degrading extracellular matrix components. In vascular remodeling MMPs are involved. The regulation of MMP activity can be regulated by mitogen activated protein kinases (MAPKs). In an animal organ culture model the effect of short term pressure on the activity of MMP-2 and the MAPKs was investigated in medium sized muscular arteries. Inhibition experiments of MAPKs were carried out to prove their effect on MMP-2 activation. Methods and Results: After tensile stress the activity of MMP-2 was significantly increased (po0:002) in the arteries shown by gelatinase assay. The MMP-2 activation complex showed no changes for MMP-2 and a decrease of TIMP-2 (p ¼ 0:065) or MT1-MMP protein (po0:0001) in western blot analysis. In addition p38 and ERK1/2 were activated (p38, po0:005; ERK1/2, po0:0001) by pressure. Real-time quantitative PCR revealed a minor increase in MMP-2 transcription (po0:02). p38 but not ERK1/2 inhibition had weak effects on MMP-2 activity in weaken the pressure induced activation to a non-significant level. Conclusion: Short term pressure application activates the MMP-2 system in arterial walls and may influence early vascular remodelling during periods of transient blood pressure elevation. This mechanical signal is mediated by MAPKs and can be negatively regulated by blocking p38.

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98 Prediction of atrial fibrosis by atrial volume F. GRAMLEY, C.H. KNACKSTEDT, P. SCHAUERTE, M. ZARSE, K. MISCHKE, T.H. SCHIMPF, M. SCHMID1, J. LORENZEN2 Medzinische Klinik I, Universita¨t Aachen 1 Klinik fu¨r Herz- und Gefa¨sschirurgie, Universita¨t Aachen 2 Institut fu¨r Pathologie, Universita¨t Aachen Aims: Congestive heart failure (CHF) is the single most important risk factor for atrial fibrillation (AF). AF (like CHF) induces atrial remodelling and dilation. Left echocardiographic atrial size correlates inversely to cardioversion (CV) success. This study investigates whether the development of atrial dilation correlates to the degree of atrial fibrosis during the development of CHF-AF. Methods: Six of 14 studied dogs received atrial and ventricular pacemakers for rapid pacing-induced AF and CHF (ventricles: 6 weeks of 180-240/min; atria: 4 weeks of 600/min). Atrial dimensions were determined initially, during the development, and prior to euthanasia. Eight healthy dogs were used as histological controls. Post mortem atrial tissue was obtained to measure the degree of fibrosis, nuclear size, shapefactor, and DNA-content morphometrically. Results: The ejection fraction (EF) of CHF-AF dogs decreased from 53% to 19% after rapid pacing (po0:01). In parallel, biatrial fibrosis increased significantly. (po0:01); DNA-content, nuclear size, and shape factor changed (RA: po0:01) implying atrial myocardial cellular hypertrophy. Increases of theleft and right atrial volumes (po0:01) and diameters (p ¼ 0:02) were significant. The extent of atrial fibrosis correlated significantly with atrial volumes and diameters as determined by echocardiography (r ¼ 0:8). Conclusions: During the development of CHF and AF significant biatrial fibrosis and dilation occurs. During this process echocardiographic parameters of atrial size and volume correlate with the degree of atrial fibrosis and cellular hypertrophy. This may explain why echocardiographic parameters of left atrial size are a strong predictor of CV success.

99 Calcification in coronary and aortic intima and media of uremic patients M.L. GROSS1, H.P. MEYER2, H. ZIEBART1, P. RIEGER1, K. NEUMANN1, I. BERGER1, K. AMANN3, E. RITZ4 1

Institut fu¨r Pathologie (Universita¨t Heidelberg) Institut fu¨r Mineralogie (Universita¨t Heidelberg)

2

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Posters: Cardiovascular Pathology / Pathology – Research and Practice 201 (2005) 153–300

Institut fu¨r Pathologie (Universita¨t Erlangen) Klinik fu¨r Innere Medizin (Universita¨t Heidelberg)

Aims: Coronary calcification, as assessed in vivo, has been shown to be a potent predictor to cardiac events. In renal patients the prevalence and the intensity of the calcification of coronaries and other vessels increased intensively. There is an ongoing discussion of the extent of intimal and medial calcification and whether the pattern is different in renal and non-renal patients. Methods: In autopsies coronary and aortic samples of 11 uremic patients and eight non-uremic patients were used for this study. Tensions of the coronary arteries as well as aortic samples were examined (by electron and light microscopy). Immunohistological stains using antibodies against CD 68, osteocalcin, CRP, C5b-9, TGF-ß, ET-1, collagen IV, MMP2, VEGF were analyzed. Probes of directly adjacent tissue and chemical analyses were carried out with a Leo 440 Scanning electron microscope. Results: The calcification was significantly higher in the intima and media of the aorta of uremic patients (intima: 22.577.9%, media: 11.077.9%) compared to calcification in non-uremic controls (intima: 10.475.2%, media: 4.472.2%). The media of coronaries of uremic patients (media: 16.6710.6%) were significantly more calcified than in non-uremic controls (media: 3.872.31%). The protein expression of osteocalcin, CRP, TGF-ß and collagen IV was significantly increased in the media of aorta and coronaries of uremic patients compared to non-uremic patients with equivalent calcifications in both sites. Conclusion: Calcification in the media of uremic patients generates significantly increased expression of inflammation mediators.

100 Atherosclerotic plaque formation and chondrogenic differentiation – coexistence or pathogenetic mechanism? T. AIGNER, V. CAˆMPEAN, D. NEUREITER, TH. KIRCHNER, K. AMANN Institut fu¨r Pathologie, Universita¨t Erlangen-Nu¨rnberg Aims: Recently, chondrogenic differentiation mechanisms were suggested to be important within atherosclerotic calcification. The aim of this study was to investigate in a series of atherosclerotic lesions the distribution and expression of classical marker genes of chondrogenic differentiation. Methods: Immunostaining for marker proteins S-100 protein, collagen types II and X were performed in correlation to histomorphology in atherosclerotic lesions of different grades (according to Stary; n ¼ 41). Quantitative PCR was applied on RNA isolated from

atherosclerotic arteries (n ¼ 10) for collagen types II and X expression. Results: In most samples, no expression of collagen type II and S-100 protein was found. Exceptionally, S-100 protein and type II collagen expression was observed very focally within advanced atherosclerotic plaques. Type X collagen was not detected in any of the lesions investigated. Conclusions: Overall, we found no evidence that chondrogenic differentiation pathways are generally active and relevant in atherosclerotic plaque formation and progression. In particular type X collagen, one presumably important molecule in cartilage calcification was not expressed in any of the investigated samples. However, occasionally chondrocytic differentiation occurs, but this most likely represents a metaplastic event associated, but not causative for atherosclerotic vessel degeneration and calcification.

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How efficient would routine acceptance of heart valves from donors older than 65 years be? K. GROSSE1, R. MEYER2, E. SCHMITZER2, C. WESSLAU1, R. HETZER2 1

Deutsche Stiftung Organtransplantation, Region Nord-Ost, Berlin 2 Deutsches Herzzentrum Berlin Aims: In contrast to organ donation, there is an agreed upper age limit for the acceptance of heart valves for tansplantation of 65 years, albeit without medical justification. Therefore we tested the morphological suitability for transplantation of heart valves from donors older than 65 years. Methods: Hearts of donors above this age limit with permission for heart donation were explanted, prepared and examined in accordance with the standards of the Bio Implant Service. Results: Between 01.09.1999 and 31.08.2004 there were 95 donors: 52 females and 43 males. The average age at donation was 71 years in both sexes. Of the 190 valves prepared, 98 (52%) were morphological suitable for transplantation. With increasing donor age only the number of suitable aortic grafts fell. Despite of advanced donor age the majority (60–90%) of pulmonary grafts from all age groups were suitable for transplantation. Donors: age groups 65–69 y. 70–74 y. 75–79 y. 80–84 y. Result satisfactory:

Donors: n/% 38 (40%) 33 (35%) 19 (20%) 5 (5%) 95

Total allografts 42 (55%) 36 (55%) 17 (45%) 3 (30%) 98 (52%)

Aortic allografts 8 (21%) 6 (18%) 1 (5%) none 15 (16%)

Pulmonary allografts 34 (89%) 30 (91%) 16 (84%) 3 (60%) 83 (87%)

ARTICLE IN PRESS Posters: Cardiovascular Pathology / Pathology – Research and Practice 201 (2005) 153–300

Conclusions: Our results do not justify the age limit of 65 years for routine harvesting of heart valves of multiorgan donors. Therefore we recommend that the age limit be increased to 75 years.

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Pathomorphological findings after heart and heartlung transplantation: Single center experience with 15,571 right ventricular biopsies N.E. HIEMANN, R. HETZER, R. MEYER Deutsches Herzzentrum Berlin, Klinik fu¨r Herz-, Thorax- und Gefa¨ßchirurgie

Aims: The histological evaluation of right ventricular endomyocardial biopsies (Bx) is the gold standard for diagnosis of acute cellular rejection after heart and heart-lung transplantation (HTx, HLTx). Routine Bx presuppose sufficient morphological structures to describe and evaluate humoral rejection and vascular and myocardial textural changes. Methods: A total of 15,571 Bx from 1,351 patients (age at HTx 46 7 15 yrs, m ¼ 1,074, f ¼ 277, dCMP ¼ 803, CAD ¼ 362, valvular disease ¼ 48, others ¼ 134) undergoing HTx or HLTx between 04/86 and 12/02 were evaluated by light microscopy for acute cellular rejection (ISHLT), Quilty phenomenon, microvascular textural changes, fibrosis, scars and hypertrophy. Immunohistochemical reactions directed against different intracellular and intercellular structures (immunological cells, terminal vascular system, HLA system, collagen subtypes, humoral parameters) were performed. Qualitative and quantitative analysis was done for blood vessel structure, fibrosis, scars, size and shape of heart muscle cells. Results: In 68% (N ¼ 10:544) of Bx there was no evidence of acute cellular rejection, ISHLT grade 1A-2 was present in 23% (N ¼ 3:611), 3A and 3B in 7% (N ¼ 1:076) and grade 4A und 4B in o1% (N ¼ 4) of Bx. In total, 2% (N ¼ 307) of Bx were not representative for further morphological evaluation. In 50% of Bx graded 0 according to the ISHLT scale there was positive immunohistochemical reaction for T cells, in 20% evidence for macrophages (CD68) and in 10% Quilty phenomenon. Microvascular disease was present in 10% of Bx; in 37% of Bx microvasculopathy was suspected. The amount of fibrosis in Bx (10–14%) did not change significantly during the post-transplant period. Conclusions: Routine Bx play a central role in post-HTx and HLTx management. Their major targets have to be redefined as enabling (a) description and evaluation of acute cellular and humoral rejection, (b) grading of vascular textural changes, and therefore (c) stratification of the risk for severe adverse events.

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103 Expression of PDGF and its receptors early after clinical heart transplantation (HTx) in relation to later uncomplicated or complicated postoperative course PH.A. SCHNABEL, C.I. SCHMIDT, J. SYKORA, B. FRO¨HLICH, A. KOCH1, F.-U. SACK1, T.J. DENGLER2, M. GORENFLO3, M. HAASS2, H.E. ULMER3, S. HAGL1, H.F. OTTO Pathologisches Institut, Universita¨t Heidelberg 1 Abt. Herzchirurgie, Chirurgische Universita¨tsklinik Heidelberg 2 Abt. Kardiologie und Pulmologie, Med. Univ.-Klinik Heidelberg 3 Abt. Pa¨diatrische Kardiologie, Univ.-Kinderklinik Heidelberg Aims: Infections and acute rejections mainly influence morbidity and mortality after HTx. We investigated the expression of PDGF and its receptors (-R) in the first 2 weeks after HTx before these compli-cations occurred compared to an uncomplicated later course. Methods: Right ventricular biopsies were taken before implantation, 1 and 2 weeks after HTx. The expression of PDGF A, B, PDGF-Rs a and b was determined immunohistochemically using a semiquantitatve score. During the first year after HTx 12 patients showed an uneventful course, 12 suffered from infections and 12 revealed acute rejections (ISHLT grade 4/ ¼ 3A). Results: The expression of PDGF A, B and -R b mainly in endothe-lia and interstitial mononuclear cells and the expression of PDGF-R a in cardiomyocytes, endothelia and smooth muscle cells of blood vessels was very low before implantation. It increased significantly 1 week after HTx in all groups. Two weeks after HTx it decreased, being still significantly higher than before implantation. Significant differences between the groups were found for PDGF-R a which was lower in the infection group than in both other groups in the 1st week, and for PDGF A which was lower in the uneventful group than in in both other groups in the 2nd week. Conclusions: PDGF A may serve as a prognostik marker, showing an elevated expression 2 weeks after HTx, before infections and rejections occur, compared to low expression in uneventful course.

104 The ‘‘Shelhigh’’ stentless bioprosthesis in Active Infective Endocarditis (AIE): early and mid-term results M. MUSCI, R. MEYER, R. PETZINA, O. BIRKELBACH, Ch. DETSCHADES, M. PASIC, H. SINIAWSKI, R. HETZER Deutsches Herzzentrum Berlin, Klinik fu¨r Herz-, Thorax- und Gefa¨ßchirurgie

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Aims: We retrospectively investigated early and midterm results of the biological ‘‘Shelhigh’’ stentless bioprosthesis implanted in AIE patients. Methods: ‘‘Shelhigh’’ biological prostheses were implanted over a period of 4 years in 108 consecutive AIE patients (n ¼ 82 male, 26 female) with a mean age of 55.8 (range 21–85) years. Of these patients, 78 had native AIE (72.2%) and 30 (27.8%) a prosthetic infection. Preoperatively 30 patients (27.7%) were intubated and 42 patients (38.8.%) on high doses of catecholamines with 10 of them in septic shock (9.2%). Results: Intraoperatively an intact annulus was found in 67 (62.0%), annulus abscess in 38 (35.2%) and ventriculo-aortic dehiscence in 3 (2.8%) patients. Forty-one patients received aortic valves, 13 aortic conduits, 21 mitral valves, 24 combined aortic-mitral valves, 8 tricuspid valves und 1 a pulmonary valve. The table shows early and late deaths.

Aortic valve prosthesis Aortic valve conduit Mitral valve prosthesis Aortic and mitral valve

Patients n¼

Late Early death death o30 days o1 year

Total n¼

41

6 (14.6%) 2 (4.8%)

8 (19.5%)

13

2 (15.3%) 1 (7.6%)

3 (23.0%)

21

1 (4.7%)

1 (4.7%)

24

4 (16.6%) –

–

ical prostheses require permanent anticoagulation. This prompted us to investigate a newly developed polyurethan (PU) heart valve prosthesis for mitral position. It was tested in an animal model without long-term anticoagulation with regard to mineralization, degeneration, and thromboembolism. Methods: Seven PU and seven commercial bioprosthesis were implantated into growing Jersey calves (age: 3-5 months, weight 60-97 kg) to aim 20 weeks duration. Phenprocumon was administered for 6 weeks, acetylsalicylacid during the complete study. After explantation the valves were investigated macroscopically, via X-ray and electron microscopy. The inner organs were examinated macroscopically and histologically. Results: Clinical course of animals with PU valves was uneventful. In contrast, five of seven calves with bioprosthese had to be sacrificed after 1–9 weeks, because of congestive heart failure. The PU valve prostheses showed markedly less mineralization than biological substitutes and no material degeneration. In contrast, bioprostheses showed severe mineralization, degeneration or were thrombosed. Peripheral embolism was not observed. Conclusions: The novel polyurethan heart valve prothesis for mitral position has excellent biocompatilitity. Calcification and structural changes are mild compared to bioprostheses. Clinical studies are planned.

4 (16.6%)

Conclusion: Early and mid-term results of the Shelhigh stentless bio-prosthesis in AIE appear to be acceptable and comparable to those of ho-mografts, in particular with respect to graft infection. Subcoronary AVR is suitable for infection confined to the valve. Aortic root replacement with a conduit may be successful, even in excessive abscess formation. The mitral prosthesis is the only option well suited for mitral annular infection.

105

Biocompatibility of a novel polyurethanic heart valve prosthesis B. HERMANNS-SACHWEH, J. ALFER1, B. KLOSTERHALFEN1, J. SACHWEH2

Institut fu¨r Pathologie, Universita¨tklinikum, RWTH Aachen 1 Institut fu¨r Pathologie, Krankenhaus Du¨ren 2 Kinderherzchirurgie, Universita¨tklinikum, RWTH Aachen Aims: Currently, heart valve replacement is performed using either biological or mechanical heart valve prostheses. Disadvantages of biological valve substitutes are mineralization and degeneration, whereas mechan-

106

The effect of the donor and recipient age on the heterotop transplanted rat heart, studied in the LewisFisher 344-model C. PROCH, R. BERG, R. MEYER Institut fu¨r Veterina¨r-Anatomie des Fachbereiches Veterina¨rmedizin, Freie Universita¨t Berlin Arbeitsbereich Herzpathologie, Deutsches Herzzentrum Berlin Aims: Since the day of the first successful heart transplantation, the number of heart transplantations has increased constantly, leading to an increasing demand for donor hearts. One possible solution is to accept hearts from older donors. We studied the influence of donor and recipient age on transplant rejection using a representative allograft model. Methods: Male Lewis- and Fisher 344-rats with different ages (4 weeks and 4 months, respectively) underwent heterotopic transplantation by the ONO und LINDSEY method. Four groups were formed with 10 rats in each: (1). old donor – old recipient, (2). old donor – young recipient, (3). young donor – old recipient and (4). young donor – young recipient. After 30 days the transplanted hearts were histologically and immunohistochemically stained. They were classified by the ISHLT

ARTICLE IN PRESS Posters: Cardiovascular Pathology / Pathology – Research and Practice 201 (2005) 153–300

standard and the growth of the connective tissue was analysed using a microscope imagine analyse system. The results were compared statistically. Results: Thirty days after transplantation serious rejections (ISHLT 3b or 4) were found in all four groups but there were significant differences in the nature of rejection. Transplant vasculopathy was also more frequent if the donor or the recipient is older. Conclusions: There are great differences in the development of the immune systems of the rats, which is the reason for different kinds of rejection. Therefore we cannot say whether the age factor has any influence on the rejection of transplanted hearts.

195

Smad 4 might mediate atrial replacement fibrosis and, thus, cardiac remodeling.

108 Immunohistochemical differentiation of eosinophilic heart diseases using antibodies against eosinophil activation markers F. GRABELLUS1,2, G. MALL3, P. A. SCHNABEL4, U. PFEIFER5, C. KERSTING2, K. J. SCHMITZ1, J. WOHLSCHLA¨GER1, E. BIERHOFF6, H.-H. SCHELD7, K. W. SCHMID1, H. A. BABA1,2 1

Institut fu¨r Pathologie, Universita¨tsklinikum Essen, Institut fu¨r Pathologie, Universita¨tsklinikum Mu¨nster 3 Institut fu¨r Pathologie, Sta¨dtische Kliniken Darmstadt 4 Institut fu¨r Pathologie, Universita¨tsklinikum Heidelberg 5 Institut fu¨r Pathologie, Universita¨tsklinikum Bonn 6 Institut fu¨r Pathologie, Klinikum Essen-Mitte 7 Klinik und Poliklinik fu¨r Thorax-, Herz- und Gefa¨ßchirurgie, Universita¨tsklinikum Mu¨nster 2

107

Atrial fibrillation: FIH regulates hypoxia and apoptosis F. GRAMLEY, F. PEZZELLA1, N. GEPARDT2, E. SACHARIDOU2 M. SCHMID3, K. C. GATTER1, P. SCHAUERTE, J. LORENZEN2

Medzinische Klinik I, Universita¨t Aachen 1 Nuffield Dep. of Clin. Lab. Sciences, Oxford University 2 Institut fu¨r Pathologie, Universita¨t Aachen 3 Klinik fu¨r Herz- und Gefa¨sschirurgie, Universita¨t Aachen Aims: Selective atrial ischemia may promote the development of atrial fibrillation (AF). This study aimed at clarifying the role of hypoxic signaling in atrial fibrillation. Methods: Right atrial appendages (n ¼ 74) were grouped according to heart rhythm (sinus rhythm, SR, n ¼ 35 and AF, n ¼ 39). Immuno-histochemistry for hypoxia- and apoptosis-related proteins (Hypoxia Induced Factor HIF-1a, PHD-3, BNIP-3, DEC-1, and Factor Inhibiting HIF FIH) was performed. Smad 4 was quantified by Western Blots and fibrosis determined in Sirius Red stains. Results: In AF the following proteins were upregulated: HIF-1a (po0:02), a key regulator in hypoxia; PHD-3 (po0:01), a HIF-1a stabiliser; BNIP-3 (po0:03), a hypoxia-induced proapoptotic protein; DEC-1 (po0:03), a hypoxia-induced transcription factor with unknown function in apoptosis; FIH (po0:03), an inhibitor of HIF-1. Fibrosis increased from 14.777.8% (SR) to 22.679.2% (AF)(po0:01). Smad 4, which mediates the function of hypoxia-induced TGF-b and may lead to increased apoptosis and fibrosis, was non-significantly increased. Conclusions: We found evidence that in AF compared with SR hypoxic signaling may initiate apoptosis and fibrosis. This HIF-1 dependent pathway appears to be regulated by FIH via a negative feedback mechanism.

Aims: Eosinophilic heart syndromes are rare and include endocarditis parietalis fibroplastica (EPF) and hypersensitivity myocarditis (HM). There are stricking differences in clinical courses and morphological findings. We studied a set of immunohistochemical markers in order to characterize the activation status of the infiltrating eosinophils in both entities. Methods: This study is based on the investigation of seven explanted hearts and 1 left ventricular specimen collected during implantation of a left ventricular assist device from a total of seven patients with HM and six specimens from five patients with EPF. We used antibodies against EG1, EG2, CD44, and CD69. Results: The EG1 to EG2 ratio of eosinophils and the immunoreactivity against CD44 showed no differences between the two entities. However, eosinophils in the EPF were completely negative for CD69 whereas eosinophils reacted positively within the HMgroup. Conclusion: The immunohistochemical investigation of eosinophilic heart diseases using antibodies against CD69 can be a useful marker to distinguish between hypersensitivity myocarditis and endocarditis parietalis fibroplastica.

109 Expression of VE-cadherin in human endothelial cell cultures M.C. HERWIG, M.I. HERMANNS1, C.J. KIRKPATRICK1, A.M. MU¨LLER

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Institut fu¨r Pathologie, Ruhr-Universita¨t Bochum 1 Institut fu¨r Pathologie, Universita¨t Mainz Aims: Disruption of the endothelial barrier plays a significant role in the pathogenesis of ARDS and may be driven by inflammatory cytokines or directly by bacterial endotoxins, like lipopolysaccharide (LPS). Vascular endothelial (VE)-cadherin is the major component of adherence junctions (AJ) in the endothelium. We studied whether LPS itself or cytokines influence the VE-cadherin expression in human umbilical endothelial cells (HUVEC) and human pulmonary microvascular endothelial cells (HPMEC) mimicking the disruption of endothelial cell-to-cell junctions in septic ARDS. Methods: VE-cadherin expression in HUVEC and HPMEC, unstimulated or stimulated with LPS (1mg/ ml), tumour necrosis factor-alpha (TNF-a)(300 U/ml) and interferon-gamma (INF-g)(2000 U/ml) for 4 and 24 h, was studied by immunolocalisation and enzyme immunoassay (EIA) (for TNF-a and INF-g). Results: There was a nearly identical VE-cadherin expression in unstimulated HUVEC and HPMEC. After stimulation with TNF-a/IFN-g/LPS a formation of gaps between the cells as well as a reduced staining at the cell-cell junctions could be observed in both endothelial cell types. Conclusions: The fainter VE-cadherin expression by stimulated HUVEC and HPMEC corresponds well with the reduced VE-cadherin expression in situ. The lack of any marked differences between these two cell culture models indicates a uniform reaction of macro- and microvascular EC with respect to VE-cadherin expression after stimulation with TNF-a/IFN-g or LPS, resp.

110 Increased activities of matrixmetalloproteases 2 and 9 in the myocardium correlate with impaired cardiac function during experimental early multiple organ failure in sheep J. WOHLSCHLA¨GER, H.D. STUBBE1, K.J. SCHMITZ, A. TAKEDA2, N. TAKEDA2, F. HINDER1, H.A. BABA Institut fu¨r Pathologie, Universita¨t Essen 1 Institut fu¨r Ana¨sthesiologie und operative Intensivmedizin, Universita¨t Mu¨nster 2 Department of Internal Medicine, Jikei University, Tokyo, JP Aims: Matrixmetalloproteases (MMP) are able to degrade extracellular matrix and are involved in both inflammation and heart failure. The role of MMP-2 and -9 in acute cardiac dysfunction in multiple organ failure (MOF) due to vasoconstrictor-masked hypovolemia and endotoxinemia was investigated.

Methods: Eighteen adult sheep were assigned to the following groups: (1) vasoconstrictor-masked hypovolemia plus endotoxinemia (NMH+ENDO), (2) vasoconstrictor-masked hypovolemia without endotoxinemia (NMH), (3) recurrent endotoxinemia during normovola¨mie (ENDO) and (4) controls (CON). Cardiovascular parameters were evaluated. Post-mortem cardiac tissue was investigated by gel-zymography and Western blot. Results: MMP-2 activity was significantly elevated in all experimental groups compared to controls. MMP-9 was significantly increased in NMH+ENDO. MMP-2 was elevated on the protein level, while MMP-9 was unaltered. MMP-2/-9 activities correlate positively with heart rate and negatively with cardiac output. Conclusions: Activities of MMP-2/-9 are significantly elevated in the myocardium and play an important role in the pathogenesis of acute cardiac dysfunction during early multiple organ failure due to vasoconstrictormasked hypovolemia and endotoxinemia in an ovine experimental model of MOF.

111 Essential role of survivin in cardiomyocyte numerical growth regulation in vivo A.H. BABA, J. WOHLSCHLA¨GER, M. SCHA¨FERS1, K.W. SCHMID, J. STYPMANN2, E.D. CONWAY3, B. LEVKAU4 Institut fu¨r Pathologie, Universita¨t Duisburg-Essen 1 Klinik fu¨r Nuklearmedizin Universita¨t Mu¨nster 2 Medizinische Klinik C, Universita¨t Mu¨nster 3 Center for Transgene Technology, University of Leuven, BE 4 Institut fu¨r Pathophysiologie, Universita¨t DuisburgEssen Aims: Survivin belongs to the family of inhibitors of apoptosis proteins (IAPs) and has been implicated in both protection against apoptosis and control of cell division. The aim was to study the in vivo role of survivin in the heart. Methods: We have generated mice with a cardiomyocyte-restricted deletion of survivin (MHC-survivin/) and investigated the animals by serial MRI, small animal-PET, echocardiography, and characterized the cardiac morphology. Results: These mice progressively developed heart failure due to dilative cardiomyopathy and died prematurely. Characteristic morphological features of survivin-deficient cardiomyocytes were their enlarged and bizarrely lobated nuclei containing aneuploid and polyploid DNA (60% of the survivin-deficient cells exhibited a 44n DNA content), but without signs of increased apoptosis. Interestingly, mice deficient for cardiac survivin had profoundly decreased total

ARTICLE IN PRESS Posters: Miscellaneous / Pathology – Research and Practice 201 (2005) 153–300

cardiomyocyte numbers per left ventricle: 34% less cardiomyocytes were present already at birth, and 64, 75, 84 and 81% after 1, 2, 4, and 9 months, respectively. To cope with the resulting increased hemodynamic work load per cell, survivin-deficient cardiomyocytes responded with a progressive hypertrophy and fibrosis. Conclusions: Our study mechanistically supports that a priori reduction of cardiomyocyte numbers is sufficient to promote the clinical sequelae of heart failure, and that survivin is essential for the control of cardiomyocyte numbers and function in vivo.

Posters: Miscellaneous 112 DNA copy number changes in sporadic neuroendocrine tumours of the thymus R.J. RIEKER, P. SCHNABEL, R. PENZEL, S. AULMANN, H. BLA¨KER, P. SCHIRMACHER, G. MECHTERSHEIMER Institut fu¨r Pathologie, Universita¨t Heidelberg Aims: The aim of the study was to detect chromosomal imbalances in sporadic neuroendocrine (carcinoid) tumours of the thymus. Methods: Ten cases of sporadic thymic neuroendocrine tumour (five cases of atypical carcinoid, four cases of classic carcinoid, one case of spindle cell carcinoid) were investigated using immunohistochemistry (synaptophysin and NSE) and comparative genomic hybridisation (CGH). Results: All tumours showed a diffuse expression of neuron specific enolase (NSE) and synaptophysin. One spindle cell and one atypical carcinoid did not show any chromosomal imbalance. In the remaining eight cases, the most frequent gains were seen on chromosome Xp (3/10 cases), 7p, 7q, 11q, 12q, and 20q (2/10 each), losses were most frequently detected at 6q (5/10 each), 6p and 4q (3/10 each), 3p, 10q, 11q and 13 q (2/10 each). Conclusions: The DNA copy number changes detected in neuroendocrine tumours of the thymus are in some part similar to the chromosomal imbalances commonly observed in advanced thymomas. Further studies, including small cell (poorly differentiated neuroendocrine) carcinomas, are needed, to confirm the theory of a continuous spectrum of differentiation in neuroendocrine tumours of the thymus as proposed by Suster and Moran.

M. MONTANI, A. M. SCHMITT, S. SCHMID, T. LOCHER, P. SAREMASLANI, P. KOMMINOTH1, A. PERREN Departement Pathologie, Universita¨tsspital Zu¨rich, CH 1 Institut fu¨r Pathologie, Kantonsspital Baden, CH Aims: Medullary thyroid carcinomas (MTC) share certain similarities with pheochromocytomas (PCC) regarding morphology, immunohistochemical and molecular markers. Both tumors exhibit activating RET mutations and are part of the MEN2 phenotype. Recently, germline mutations of the mitochondrial complex II subunits SDHB, SDHC and SDHD were identified in patients with familial pheochromocytomas/ paragangliomas (PGL). The genes are located on the chromosomal bands 1p35-36, 1q21 and 11q23, regions lost in a subset of MTC. Additionally, the SDHD germline variant H50R in exon 2 was described in a family with non-RET-associated C-cell-hyperplasia. We examined sporadic and familial MTC’s for mutations and deletions of SDHB, SDHC and SDHD. Methods: We examined 35 MTC (13 familial and 22 sporadic) for alterations in all SDH- genes by PCR/ DGGE, direct sequencing and LOH analysis. Results: No mutations of SDHB, SDHC and SDHD were detected. LOH was found in 30% (SDHB) and 4% (SDHD). However, coding polymorphisms of SDHB and SDHD were increased in patients with MTC and are related to either germline or somatic RET-mutations. Conclusions: In contrast to PCC/ PGL, mutations of SDH- genes are not important in MTC. MTC are associated with an increased rate of germline polymorphisms in the SDH-genes as well as the latter are associated with underlying RET-mutation. Further studies are needed to assess whether these polymorphisms have a disease modifying aspect in MTC or other RET-associated tumors.

114

Persistent hyperinsulinemic hypoglycemia in 15 adults with diffuse nesidioblastosis: Diagnostic criteria, incidence and characterization of b-cell changes M. ANLAUF1, D. WIEBEN1, A. PERREN2, A. RAFFEL3, B. SIPOS1, M.L. KRUSE4, C. FOTTNER5, W.T. KNOEFEL3, H. MO¨NIG4, P. KOMMINOTH2, P.H. U. HEITZ2, G. KLO¨PPEL1 1

Institut fu¨r Pathologie, Universita¨t Kiel Department Pathologie, Universita¨t Zu¨rich 3 Allgemeine und Visceralchirurgie, Universita¨t Du¨sseldorf 4 1. Medizinische Klinik, Universita¨t Kiel 5 1. Medizinische Klinik, Universita¨t Mainz 2

113 Mutation analysis of the succinate dehydrogenase subunits SDHB, SDHC and SDHD in sporadic and familial medullary thyroid carcinomas

197

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Aims: Persistent hyperinsulinemic hypoglycemia (PHH) in adults that is not caused by an insulinoma is a rare and not well characterized disease that has been named nesidioblastosis. In this study we defined and scrutinized criteria for its histological diagnosis and assessed its relative incidence. Methods: In pancreatic specimens from 15 adult patients with PHH in whom no insulinoma was detected the endocrine tissue was screened for islet and b-cell changes. The diagnostic reliability of the proposed criteria were checked by an inter-observer analysis. The relative frequency of the disease was assessed in a series of 232 patients with PHH. Finally, genetic analysis of the menin gene was performed. Results: Among the various indicators of islet changes, b-cell hypertrophy was the most significant and diagnostic finding in patients with PHH. The inter-observer analysis revealed 100% specificity and 87.7% sensitivity. The hyperfunctional state of the b-cells was not associated with changes in the subcellular distribution of insulin and proinsulin, proliferative activity, or germline mutations of the menin gene. Conclusions: Diffuse nesidioblastosis in adult patients with PHH resembles that is seen in neonates suffering from PHH. Most important for the diagnosis is b-cell hypertrophy. As 4% of adult patients with PHH are affected by diffuse nesidioblastosis, this disease is not as rare as it has been thought to be. Further studies are needed to elucidate the molecular defects leading to the uncontrolled insulin secretion.

115 Nestin is

not expressed in pancreata of streptozotocin treated rats K. PETERS, R. PANIENKA, R.N. WANG1, G. KLO¨PPEL

Institut fu¨r Pathologie, Universita¨t Kiel 1 Dept. of Physiology & Pharmacology, Lawson Health Research Institute, University of Western Ontario, Canada Aims: The regeneration or neogenesis of new b-cells in diabetes mellitus is of interest for the therapy of this disease. Experimental treatment with streptozotocin (STZ) induces b-cell destruction in the pancreas and is a well known animal model for diabetes mellitus type I. We wanted to characterize the dynamics of transcription factors and putative stem cell markers in this model. Methods: Pancreata of STZ treated rats were examined 3, 7, and 14 days after administration of the drug. The expression of the transcription factors pdx1, pbx1, and meis2 was evaluated by immunohistochemistry. Furthermore, the expression of c-kit and nestin was examined. Proliferation was determined by Ki-67

labeling, insulin staining was examined. For evaluation, expression of the markers was described in three different compartments of the pancreas (duct, islet, acinus). Results were compared to untreated controls. Statistical significance was determined by using a twotailed unpaired student’s t-test. Results: pdx1 and c-kit expression is enhanced after STZ treatment, the expression levels are already decreased on day 14. No expression of nestin can be seen on neither day. Conclusions: b -cell regeneration in the STZ treated pancreas is independent on nestin expression. Pdx1 expression is a prerequisite for b -cell regeneration and neogenesis. The role of c-kit for restoring of the b cell mass may be more important than previously expected.

116 The regulatory subunit Ia of protein kinase A may not act as a tumor suppressor gene in poorly differentiated and anaplastic thyroid carcinomas M. BRO¨CKER-PREUSS, S.-Y. SHEU1, M. LIPPERT, K. WORM1, K. MANN, K.W. SCHMID1 Klinik fu¨r Endokrinologie, Zentrum fu¨r Innere Medizin 1 Institut fu¨r Pathologie, Universita¨tsklinikum Essen Aims: The activity of protein kinase A (PKA) is controled by four distinct regulatory subunits. The RIa subunit was reported to be strongly expressed and associated with high proliferation rates and dedifferentiation in various tumors. In contrast, in anaplastic thyroid carcinoma and endocrine tumors in carney complex, PKA RIa was reported to act as a tumor suppressor gene (Sandrini et al., 2002). Methods: To further elucidate the function of PKA RIa in the pathogenesis of poorly differentiated and anaplastic thyroid carcinomas, we performed mutation analysis by direct sequencing of the PKA RIa gene in 10 tissues. Furthermore, PKA RIa protein expression was analyzed by immunohistochemistry. Results: We found that expression of PKA RIa in poorly differentiated and anaplastic thyroid carcinomas was stronger than in normal thyroid tissues: 5/10 carcinomas showed a predominantly moderate staining intensity, 5/10 tissues were stained weak. No PKA RIa staining was observed in normal thyroids. Staining pattern on the cellular level was diffuse cytoplasmic. Analysis of the coding region of the PKA RIa gene showed the presence of wild-type sequences in the genomic DNA of all 10 tissues examined. Conclusions: Our results indicate that the PKA RIa protein may have a role in thyroid carcinogenesis and growth regulation of thyroid carcinomas. The hypothesis of a possible function of PKA RIa as a tumor

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suppressor gene in thyroid carcinomas could not be confirmed by our data.

117 Somatostatin

receptor (sstr) subtype expression in differentiated thyroid carcinomas: possible prognostic relevance of sstr3 S.Y. SHEU, M. BROECKER-PREUSS1, K.J. SCHMITZ, K. MANN1, K.W. SCHMID, R. GOERGES2

Institut fu¨r Pathologie 1 Klinik fu¨r Endokrinologie 2 Klinik fu¨r Nuklearmedizin, Universita¨tsklinikum Duisburg-Essen Aims: Somatostatin acts as an inhibitor of secretion and proliferation in normal and malignant cells. Expression of the five somatostatin receptor (sstr) subtypes in thyroid carcinomas was reported but the significance of certain subtype expression in correlation with clinical outcome is still unknown. To elucidate further clinical implications for diagnostic and therapeutic purposes we analysed sstr1-5 expression by immunohistochemistry and correlated it to clinical data. Methods: sstr1-5 expression status was analysed in 112 paraffin embedded tissues (27 follicular (FTC), 74 papillary (PTC) and 11 poorly differentiated thyroid carcinoma (PDTC)) from 112 patients. The staining results were correlated to clinical follow-up data available in 86 cases and scintigraphy using 111InPentetreotide (Octreoscan) in 12 cases. Results: FTC showed a significantly stronger cytoplasmatic expression of all sstr subtypes compared with PTC and PDTC. Hu¨rthle cell carcinomas revealed the highest expression rates of all subtypes consistent with scintigraphic results. Univariate survival analysis of all receptor subtypes exhibit a direct correlation with tumor stage and clinical outcome. The strong expression of sstr3 in thyroid carcinomas is significantly associated with a progressive clinical state in the multivariate Cox regression analysis. Conclusions: sstr expression in advanced disease stage may enable a more targeted radiotherapy using subtype specific analogues coupled to radionuclides and chemotherapeutic agents. The determination of sstr3 expression seems to be associated with progressive disease which may be of value in identifying high-risk patients with thyroid cancer.

118 Rhabdoid gliomas: A case of a rhabdoid glioblastoma R. KLEIN, M. BENDSZUS1, G. H. VINCE2, W. ROGGENDORF

199

Pathologisches Institut, Abteilung Neuropathologie, Universita¨t Wu¨rzburg 1 Abteilung fu¨r Neuroradiologie, Universita¨t Wu¨rzburg 2 Neurochirurgische Klinik und Poliklinik, Universita¨t Wu¨rzburg Aims: Glioblastomas show besides a typical morphology also uncommon morphological variants with epitheloid and mesenchymal components. Among brain tumors, atypical teratoid/ rhabdoid tumors and rhabdoid meningiomas are meanwhile well known, while reports on rhabdoid variants of glial tumors are rare. Methods: We report a case of a glioblastoma with rhabdoid morphology in a 42-year-old woman. After a clinical history of headaches for 5 months, MRT revealed a hyperdense tumor in the deep white matter of the left frontal cerebral hemisphere without relationship to the brain surface or its meningeal coverings. The tumor was surgically removed and histopathological investigation was carried out using conventional and immunohistochemical stains. Results: The tumor was of high cellular density, displayed areas of necrosis and the tumor cells showed a rhabdoid morphology of large tumor cells with eccentric nuclei, prominent nucleoli and inclusion-like cytoplasm. The immunohistochemical profile (GFAP+, EMA+) underscored the glial origin of the tumor. Conclusion: We conclude that rhabdoid variants or components of glioblastomas exist and should be considered in differential diagnosis of primary or secondary intracranial tumors showing a rhabdoid morphology.

119 Genetic analysis of the different histological parts of glioblastomas with oligodendroglial component by interphase cytogenetic and comparative genomic hybridization (CGH) B. KLINK, E. SCHRO¨CK1, S. PATT2 Institut fu¨r Pathologie, Universita¨t Jena 1 Institut fu¨r Klinische Genetik, Universita¨t Dresden 2 Institut fu¨r Pathologie, Universita¨t Jena Aims: In oligodendrogliomas it has been demonstrated that combined loss of 1p and 19q is a positive prognostic factor for overall survival and chemosensitivity. Interestingly, a small subgroup of glioblastomas contains areas with histological features of oligodendroglial differentiation. Our objective was to genetically characterize such glioblastomas with an oligodendroglial component (GBMO) and define prognostic markers for these tumors. Methods: We analyzed the oligodendroglial and the ‘‘classic’’ glioblastoma parts of 13 GBMOs separately by

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CGH using microdissected paraffin-material and interphase fluoreszence in situ hybridization (FISH), regions 1p, 1q, 7q, 10q, 17p, 19q, cen18, 21q. Additionally we studied 10 classical‘‘ glioblasto’’ mas (GBM) by Interphase-FISH using the same markers. The genetic and histopathological features were correlated with the clinical data (including followup data). Results: The two histologically differing parts of the GBMOs showed the same general genetic profile with small variations. All 10 GBMs and 10 of 13 GBMOs exhibited a combined gain of 7q and loss of 10q. The remaining 3 GBMOs showed other aberrations, e.g. combined loss of 1p and 19q. No specific genetic marker for GBMOs could be identified. However, occurrence of an oligodendroglial component of GBMs emerged as a significant (po0:05) independent predictor of longer overall survival. Conclusions: GBMOs show the same genetic aberrations as GBM and are of monoclonal origin. Our results underline the importance of histological characterization of GBMOs for the clinical prognosis.

120

Correlations of clinico-pathological features with epidermal growth factor receptor (EGFR) overexpression in soft tissue sarcomas are antibody-dependent C. KERSTING1, J. PACKEISEN1, B. LEIDINGER2, B. BRANDT3, R.V. WASIELEWSKI4, W. WINKELMANN2, G. GOSHEGER2, W. BO¨CKER1, H. BUERGER1 1

Gerhard-Domagk-Institut fu¨r Pathologie Klinik fu¨r Orthopa¨die 3 Institut fu¨r Klinische Chemie, WWU Mu¨nster 4 Institut fu¨r Pathologie, Medizinische Hochschule Hannover 2

Aims: With the approach of new targetted cancer therapies, the need to select cancer patients for treatment with these drugs emerges. Several studies indicate that besides carcinomas, soft tissue sarcomas (STS) might be a promising target, too. However, available data on expression of EGFR in STS is sparse and inhomogeneous. Methods: Three hundred and three specimens of STS were examined using the tissue microarray technique. Epidermal growth factor receptor expression frequencies were obtained with immunohistochemistry using five different commercially available primary antibodies. Amplification status was measured by fluorescence in situ hybridisation. These parameters were correlated with clinical follow up data available for 163 cases.

Results: EGFR expression frequency ranged between 0.3% and 52.9% depending on the antibody and scoring method used. Significant correlation existed between all of the stains. A total of 3.5% of the STS exhibited amplification in FISH. A significant correlation was observed for the expression results of two antibodies with tumour grade, and for one of the antibodies with unfavourable clinical outcome and occurrence of metastases. egfr amplification status revealed a good correlation with EGFR expression for three antibodies, while no correlation with clinical features was evident. Conclusions: The choice of the primary antibody and scoring system has a tremendous impact on the determination of EGFR overexpression in STS. A correlation between EGFR overexpression and clinical outcome could be seen with only one of five tested antibodies.

121 The use of archived tissue – comparison of german and british regulations CH. BROCHHAUSEN, N. ROSSRICKER, G. WOLFSLAST, C. WEINRICH1, C.J. KIRKPATRICK Institut fu¨r Pathologie, Universita¨t Mainz 1 Fachbereich Rechtswissenschaft, Universita¨t Gießen Aims: In daily practice of any institute of pathology human organs and tissues are archived for documentation and possible re-evaluation. From the diagnostic point of view these archives play an important role for patient safety, because further diagnostic tools could be applied if necessary. On the other hand these archives represent a tremendous resource for biomedical research. But the juridical and ethical legalisations for this purpose remain difficult. Aim of the present study was to compare the frameworks of two different juridical traditions. Methods: In the present study we analysed and compared German and British frameworks for ethicolegal regarding the use of archived tissue for biomedical research. Results: The use of archived material for research and museums or collections is regulated in Germany by statements of the central ethical board of the ‘‘Bundesa¨rztekammer’’ and in Great Britain by guidelines of The Royal College of Pathologists. Even if in both systems informed consent is required, in Great Britain a well differentiated consent process is established. Conclusions: Further investigations are needed to establish whether a ‘‘British consent process’’ could be helpful to delineate the feasibilities of further use of archived tissue in Germany.

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122 Tissue microarray for quality control in immunohistochemistry—experience and possibilities J. PACKEISEN, U. THEBING-BARRIER, H. BU¨RGER1, W. BO¨CKER1, R.-H. KRECH Institut fu¨r Pathologie, Klinikum Osnabru¨ck Institut fu¨r Pathologie, Universita¨t Mu¨nster

1

Aims: Since immunohistochemistry is used to recognize are targets of distinct adjuvant therapy approaches, it is demanded that these investigations are done with the highest reproducibility and quality assurance. To face this problem we started using tissue microarrays in 2002. This method has been introduced in different laboratories in an individual setting. In the present study we aim to give an overview investigated the objectively usability of this method. Methods: One thousand and two hundred slides of routinely stained for 24 different immunohistochemistry markers were harvested form our archive. The slides were tested for staining results and completeness of the control microarray. This was compared to the results in the diagnostic sample. Results: Completeness of the control tissue microarray was seen in 62% of all cases. The relevant loss in all 1200 control microarrays in relation to the diagnostic samples was only 2.85%. Conclusions: Using a tissue microarray for quality control in immunohistochemistry obtained the possibility of a reliable method in routine pathology. The loss of 2.85% control microarray tissue revealed in our study should be reduced by the modification of the control microarray setting.

123 Erdheim-Chester disease: case report with multisystemic manifestations including testes, thyroid, and lymph nodes S.Y. SHEU, R.R. WENZEL1, C. KERSTING2, R. MERTEN3, F. OTTERBACH, K.W. SCHMID Institut fu¨r Pathologie, Universita¨t Duisburg-Essen, 1 Internal Department, GPH Zell am See, A 2 Institut fu¨r Pathologie, Universita¨t Mu¨nster 3 Klinik und Poliklinik fu¨r Strahlentherapie und Radioonkologie, Universita¨t Hamburg Aims: Erdheim-Chester disease is a rare nonLangerhans0 cell histiocytosis with characteristic radiological and histological, but uncharacteristic clinical features. This entity is defined by a mononuclear infiltrate consisting of lipid laden, foamy histiocytes positive for CD68. We report a case of a 50 year old man who initially presented with hypogonadism and

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diabetes insipidus and died two years later due to atrial and ventricular arrhythmias. Methods: A necroscopy was done and tissue specimen were obtained from almost every organ. Conventional histological and immunohistochemical stains (CD68, CD1a, S100) were performed. Results: Macroscopically we found several soft and fatlike yellow masses in the heart and a retroperitoneal fibrosis. Almost every tissue specimen revealed an extensive mononuclear infiltrate stained positive for CD68, negative for CD1a and S100 including rare organ manifestations such as testes, thyroid and lymph nodes. Conclusions: The rare diagnosis of Erdheim-Chester disease was missed during lifetime according to uncharacteristic clinical symptoms in our patient. The diagnosis can be confirmed by verification of extensive organ involvement with CD68 positive foamy histiocytes within a broad inflammation.

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Unusual non-Langerhans-histiocytosis type Erdheim-Chester: Case report and review of the literature M.R. DOMULA1, D.E. AUST1, M.G. HAASE1, C.H. KIRSCH2, C.H. ZIETZ1, G.B. BARETTON1 1

Institut fu¨r Pathologie Medizinische Klinik I, Universita¨tsklinikum Carl Gustav Carus der TU Dresden 2

Erdheim-Chester disease (ECD) is a rare CD68 (+) and CD1a () non-Langerhanscell histiocytosis defined by characteristic infiltration of skeleton and viscera by lipid laden histiocytes leading to fibrosis and osteosclerosis. The majority of patients are middle aged males presenting with mild bone pain. Initial diagnosis of osteosclerosis is usually made by radiology. In the present case a 63 years old man complained of fever, nightsweats, exhaustion, and a growing abdominal tumor 6 months before death. Inflammatory serum parameters (CRP, IL-2R) were significantly increased. Repeated peritoneal biopsies showed chronic histiocytic and fibrosing inflammatory reaction without any signs for malignancy or dysplasia as seen in so called idiopathic peritonitis. The patient subsequently developed signs of sepsis without detection of any specific pathogen and passed away. Autopsy yielded massive disseminated fibroblastic and histiocytic proliferation in the nasal sinuses, lungs, pericard, epicard, pancreas and the peripancreatic, mesenterial and retroperitoneal tissue as well as in the bone marrow of femur and spine. Liver and spleen were not infiltrated but enlarged due to cardiac involvement. Furthermore, there was massive fibrosis in examination of the lungs. Additional histo- and immunohistochemical reactions showed lipidladen histiocytes positive for CD68 and negative for

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CD1a typical for ECD. As no major septic focus could be revealed by autopsy, clinical signs of sepsis might be related primarily to massive proliferation of activated histiocytes with high level cytokine production. ECD should be considered as rare differential diagnosis in cases of etiologically unclear histiocytic/fibrosing inflammatory reactions.

125 EGFR-expression in synovial sarcoma C. KUHNEN, D. ROSENBERGER, K.M. MU¨LLER Institut fu¨r Pathologie, Ruhr-Universita¨t Bochum Aims: The Epidermal Growth Factor Receptor (EGFR) has been analysed in various tumors, especially carcinomas with therapeutical implications. Aim of this study was the characterization of EGFR in synovial sarcoma as a sarcoma entity showing an epithelial line of differentiation. Methods: Twenty two synovial sarcomas were included. For immunohistochemistry, the DAKO-kit for EGFR was used. The immunoreaction was evaluated semiquantitatively. Results: Ninety six percent of all synovial sarcomas exhibited a positive EGFR-expression. In 59%, the immunoreaction was estimated as strong showing a membraneous distribution. A striking reaction pattern was detected in biphasic tumors: in nearly all cases, the fibrous component exhibited a strong immunoreactivity in contrast to the epithelial component, which was mostly negative. Conclusions: The presented results indicate a considerable expression of EGFR in synovial sarcoma as a sarcoma with an epithelial, albeit uncertain line of differentiation. This immunoreaction pattern might offer an additional possibility for future therapeutic trials using signal transduction inhibitors.

126 Morphological reaction

patterns of malignant soft tissue tumors to prosthetic devices – 2 case reports

M. HELWING, D. DRU¨CKE1, K.-M. MU¨LLER, C. KUHNEN Institut fu¨r Pathologie, Ruhr-Universita¨t Bochum 1 Klinik fu¨r Plastische Chirurgie, Ruhr-Universita¨t Bochum Aims: Prosthetic devices are used in oncologic surgery e.g. for reconstruction of soft tissue defects or vessels. Tumor tissue may interfere with prosthetic devices after implantation. Two cases of malignant soft tissue tumors

are presented exhibiting different reaction patterns to implanted material. Methods: Two cases of malignant soft tissue tumors (aggressive fibromatosis, leiomyosarcoma) were analysed regarding reaction patterns to implanted devices. Macroscopic and microscopic findings were evaluated concerning the implant material and tumor biology. Results: One case of aggressive fibromatosis (female, 39 years-old) with location within the right thoracic wall was resected as a relapse tumor after prior implantation of a goretex net. Tumor cells of desmoidfibromatosis were in close contact to the implant without true degradation or permeation of the net. One case of retroperitoneal leiomyosarcoma (female, 47 years-old) was characterized by an extensive tumor infiltration of a prosthetic vessel device with tumor permeation and gradual degradation of the vessel wall. Conclusions: The two presented cases delineate different reaction patterns of malignant mesenchymal tumors to implanted devices. These reaction patterns seem to be dependent on the material used and the tumor biology. Sarcomas may infiltrate and permeate prosthetic devices more aggressively in contrast to desmoidfibromatosis probably because of exaggerated local invasive potential.

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Atheroembolic renal disease: an underestimated

entity C.A. SEEMAYER, M.J. MIHATSCH Aims: To determine the prevalence of atheroembolic renal disease (AERD) in renal biopsies and to define the typical clinical presentation. Methods: Data files were reviewed for the diagnosis of AERD in renal biopsies (n ¼ 11:703) including clinical data. As controls served 15,274 autopsies. Results: Forty four patients (0.38%) with renal biopsies demonstrated AERD and 123 cases in autopsy (0.81%). For 42 biopsy cases the clinical recordings could be correlated with the histopathological findings. The mean age of the affected patients was 68.3 years (79.2) with a clear male predominance (36:6). The prevalence of AERD in non-glomerulonephritis biopsies increased with age starting with 2.4% of AERD in the age group of 60–69 years, and reaching more than 5% in patients being older than 80 years. As risk factors 30 (71%) patients demonstrated arterial hypertension, 10 (24%) diabetes mellitus, 13 (31%) a peripheral or a coronary arterial occlusive disease, and 16 (38%) patients underwent invasive vascular procedures. eighteen (43%) patients revealed an acute renal failure (ARF) and 24 (57%) a chronic renal failure (CRF). twelve (31%) patients showed a proteinuria of nephrotic range, 19 (49%) patients a proteinuria between 0.5 and 3.5 g per

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day, and 8 (20%) patients only a minor or no proteinuria (n ¼ 39). Nephrotic proteinuria was slightly more common in patients with CRF than in patients with ARF (8:4). Hematuria was present in about 1/4 of the patients (9:33). In histopathology the following unspecific findings were detected: obsolescent glomerula (28.8%), focal segmental glomerulosclerosis (50%), pronounced arteriolosclerosis (43%), intimal fibrotic thickening of the arteries (48%), tubular atrophy (55%) with interstitial fibrosis, and an unspecific interstitial inflammation (17%, n ¼ 42). As associated nephropathies renal diabetic glomerulosclerosis or malignant nephrosclerosis were present in 5 cases each. No differences were found between cases with ARF, CRF and nephrotic syndrome. Of note, 5 patients (n ¼ 39) revealed fibrin within the glomerulus, 2 patient crescent and synechiae respectively, and another patient a mesangiolysis. Conclusions: AERD is not common in kidney biopsies in patients younger than 60 years of age, but the prevalence increases dramatically in patients older than 60 years. AERD results in 30% of the caes in a nephrotic syndrome and in 50% in pronounced proteinuria. Therefore, severe proteinuria specifically of elderly patients should take AERD into account for differential diagnosis, even in the presence of fibrin or cresecents.

holes in the recipient paraffin blocks were drilled using a drill of 0.43 mm in diameter. The PTCBs were punched out of paraffin tissue blocks and manually transferred to the predrilled recipient blocks. According to routine methods, the filled TMAs were cut and the sections stained. Results: The construction of the tissue punches and stylets out of routinely used hypodermic needles was simple and cheap. The tips of these punches of 0.43 mm in inner diameter were strong enough to obtain PTCBs from donor paraffin tissue blocks without breakage. Conclusions: TMAs with PTCBs of 0.43 mm in diameter are technically feasible. This small diameter is sufficient to obtain enough cells for the evaluation of gene expression and gene copy number.

128 Tissue microarrays with paraffin tissue core biopsies of 0.43 mm in diameter are technically feasible

Aims: Neoadjuvant therapy is increasingly being used for the treatment of patients with locally advanced nonsmall cell lung cancer (NSCLC). In patients with extensive therapy-induced tumor regression (less than 10% vital tumor tissue) significantly longer survival times have been established. Up to now, it is not known whether complete therapy-induced tumor regression (regression grade III, no vital tumor tissue) results in an additional survival benefit in comparison with subtotal tumor regression (regression grade IIb). Patients and methods: To answer this question, 212 resection specimens from the German Lung Cancer Cooperative Group (GLCCG) Phase III-study were morphologically analyzed. The established grades of therapy-induced tumor regression (RG) were then correlated with the corresponding survival times. Results: The results were as follows: RG I (no therapyinduced tumor regression): n ¼ 35; median survival time (MST) 18 months, five-year survival rate (5-YSR) 16.5%; RG IIa (morphological evidence of therapyinduced tumor regression, more than 10% vital tumor tissue): n ¼ 102; MST 27 months, 5-YSR 27%; RG IIb (morphological evidence of therapy-induced tumor regression, less than 10% vital tumor tissue): n ¼ 45; MST 50 months, 5-YSR 43%; RG III (complete therapy-induced tumor regression): n ¼ 30; MST 25 months, 5-YSR 36.3%.

U.F. VOGEL, B.D. BU¨LTMANN Institut fu¨r Pathologie, Universita¨t Tu¨bingen Aims: Tissue microarrays (TMAs), as introduced by Kononen et al. in 1998, are especially suited for screening large amounts of tumor samples for those genes which are expressed by most of the tumor cells within a certain sample. The TMAs are intended to harbour as much paraffin tissue core biopsies (PTCBs) as possible to maximize the benefits of the TMA technique. Till now, the minimal diameter of the PTCBs is 0.6 mm.Smaller diameters were said to be impossible due to the breakage of the tissue punch. To increase the number of PTCBs per TMA, we tried to construct TMAs with PTCBs of 0.43 mm in diameter. Methods: Routinely used hypodermic needles (22G, inner diameter: 0.43 mm) were shortened to a length of about two centimeters and resharpened by a drill grinder with a cutting disc. For pushing the punched PTCBs out of the needles stylets were constructed out of hypodermic needles whose outer diameter corresponded to the inner diameter of the needlemade punches (27G; outer diameter: 0.40 mm). The

129 Complete therapy-induced tumor regression in locally advanced non-small cell lung cancer – morphology and prognosis K. JUNKER1, M. THOMAS2, A. LINDER3, H.N. MACHA3, K.M. MU¨LLER1 1

Institut fu¨r Pathologie, Ruhr-Universita¨t Bochum Medizinische Universita¨tsklinik A, Mu¨nster 3 Lungenklinik Hemer 2

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Conclusions: With these results, the prognostic relevance of the applied regression grading system could be confirmed (RGI/IIa vs. RG IIb/III, MST 23 months vs. 45 months; Log-Rank-Test, p ¼ 0:0023). In contrast to this, significantly different survival times could not be established when comparing complete with subtotal therapy-induced tumor regression (RG IIb vs. RG III, Log-Rank-Test, p ¼ 0:168). Thus, complete morphologically established tumor regression does not result in an additional survival benefit.

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Immunohistochemical quantification of lymph vessels, vegf-C and vegf-R 3 in human Sarcomas N. FRIEDRICHS, J. HAHNE, M.S. PEPPER1, U. ROMMERSCHEIDT-FUSS, R. BUETTNER, F. STELZNER2, N. WERNERT Institut fu¨r Pathologie, Universita¨t Bonn 1 School of Anatomical Sciences, University of the Witwatersrand, RSA 2 Abteilung fu¨r Chirurgie, Universita¨t Bonn

Aims: It is long known that human sarcomas only exceptionally metastasize to lymph nodes. We therefore evaluated lymph and blood vessel networks as well as VEGF-C and VEGF-R 3 expression in human sarcomas. Methods: In 32 sarcomas the intratumoral lymphatic (LVD) and for comparison the blood (BVD) microvessel density were determined immunohistochemically by Podoplanin and CD-34stainings and lymphangiosis sarcomatosa evaluated. In addition expression of Vascular Endothelial Growth Factor (VEGF) C and VEGF receptor three were analyzed. five carcinomas were examined for comparison. Results: In 16 of 32 sarcomas (50%) intratumoral lymphatic vessels were detectable ranging from small lymphatic capillaries to large vessels. VEGF-C immunopositivity was detected in 18 of 32 sarcomas. VEGFR 3 was found in 50% of sarcomas containing lymphatic vessels. In only one synovial sarcoma a lymphangiosis sarcomatosa was noted. Four of five carcinomas (breast, lung and colon) expressed VEGF-C and three contained podoplanin-positive lymph vessels. In only one of these cases VEGF-R 3 expression could be demonstrated. In nearly 100% (31 of 32 sarcomas) and in all carcinomas intratumoral CD 34-positive blood vessels were found. Conclusions: An important percentage of human sarcomas contain lymphatic vessels and express VEGF-C but only 50% of tumors which possess a lymphatic network also express the receptor for VEGF-C (VEGF-R 3). The tendency of sarcomas to use this lymphatic network for metastatic spread seems to be extremely low, reflected by

only one case of lymphangiosis sarcomatosa found in a synovial sarcoma.

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M2A expression in lung carcinomas and pleural mesotheliomas A.M. MU¨LLER1, F. BALKAU1, F.E. FRANKE2, K.-M. MU¨LLER1

Institut fu¨r Pathologie 1 Ruhr-Universita¨t Bochum 2 Justus-Liebig-Universita¨t Giessen Aims: The M2A-antigen, known to be expressed in germ cell tumours and lymphatic endothelial cells, has also been found in mesothelial cells and pneumocytes type 1. Up to now there are no larger studies concerning its expression in pulmonary carcinomas and mesotheliomas although the differential diagnosis between epitheloid mesothelioma and pulmonary carcinomas can be difficult. Methods: Seventy six pulmonary carcinomas (30 squamous cell and 30 adenocarcinomas, 11 neuroendocrine and 5 bronchioloalveolar carcinomas) and 36 mesotheliomas (18 with mainly epitheloid differentiation, 18 with mainly sarcomatoid differentiation) were immunostained with the monoclonal antibody D2-40, raised to the M2A-antigen. Results: The 18 epitheloid mesotheliomas showed a strong membranous M2A-expression, in contrast to an indifferent reactivity in sarcomatoid mesotheliomas. In pulmonary carcinomas, 48 showed no reactivity in the tumour cells (in contrast to strongly stained lymph vessels) and 28 carcinomas revealed a weak to moderate plasmatic immunostaining. Conclusions: The strong membranous expression of the M2A-antigen (D2-40) in epitheloid mesothelioma cells can be used as a valid marker for the diagnostic differentiation between epitheloid mesothelioma and pulmonary carcinoma, especially adenocarcinoma. Considering the strong expression of the M2A-antigen in pneumocytes type 1, the weak plasmatic expression in some pulmonary carcinoma cells does no hint at a histogenetical relation between these cells.

132 Fibroblasts as sentinel cells in tumor angiogenesis S. SCHMID, A. GAUMANN, J. SCHRO¨DER, D. WHEATLEY1, F. HOFSTA¨DTER, L.A. KUNZ-SCHUGHART Institut fu¨r Pathologie, Universita¨t Regensburg 1 BioMedES, Hilton College, UK

ARTICLE IN PRESS Symposium: Breast Carcinoma II / Pathology – Research and Practice 201 (2005) 153–300

Aims: The heterologous tumor stroma consists of endothelial cells (EC), immune cells and fibroblasts (F). The latter are the quantitatively most abundant stromal cell type in desmoplastic ductal breast tumors. In order to better understand the impact of stromal fibroblasts in tumor angiogenesis, we have (a) established a new three-dimensional culture system to investigate the interaction of tumor cells, F and EC in a 3-D culture format and (b) applied a two-dimensional co-culture system of F and EC to demonstrate the impact of defined paracrine factors on the formation of vessel-like structure in a fibroblastic environment. Methods: Cell types: normal skin fibroblasts, human umbilical vein EC (HUVEC), different breast tumor cell lines; culture systems: monolayer and spheroid cocultures using EC specific media; analytical tools: immunohistochemistry, digital imaging, semi-automated morphometric image analysis. Results and conclusions: Both, TGF-ß1 and PDGF-BB which induce myofibroblast differentiation in vitro, significantly reduce the formation of vessel-like structures in a two-dimensional fibroblast-EC co-culture system. According to the literature, these co-cultures also respond to VEGF withdrawal whereas the establishment of an EC network with cell junctions, lumen formation and pinocytotic activity in a new spheroid-based 3-D co-culture system does not require endogenous VEGF. Here, EC migration and network formation are critically affected by the status of the fibroblasts, and the addition of various breast tumor cells results in EC-network destruction as a function of time. (study supported by the State of Bavaria)

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Primary and secondary diagnoses in the autopsy reports of the Institute of Pathology at the University Hospital Dresden and their relevance to diagnosis related groups (DRGs) M. EBERLEIN-GONSKA1, A. SCHUMANN2, U. BUCHER3, D. AUST4, G.B. BARETTON4 1

Zentralbereich Qualita¨tsmanagement Universita¨tsklinikum TU Dresden 2 Medizincontrolling Klinikum Hoyerswerda 3 Medizincontrolling, Universita¨tsklinikum TU Dresden 4 Institut fu¨r Pathologie, Universita¨tsklinikum TU Dresden Aims: Clinical autopsies are known to play an important role in the quality assurance of clinical diagnostic and therapeutic measures. Nevertheless, autopsy rates decreased over the last decades with alarming speed. This study was designed to determine the role of clinical autopsies in the quality assurance and to determine their

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potential economic impact with respect to DRG- cost calculation. Methods: All autopsy reports from patients over 18 years between June 2001 and 2004 are being reevaluated with regard to the agreement between clinical primary and secondary diagnoses and the autopsy diagnoses and their relevance not only for quality assurance but also for the patient co-morbidity and complication level (PCCL) when using DRG-calculation. Results: The analysis of the first 50 autopsy reports showed complete agreement of clinical and autopsy diagnoses in only 12 cases (24%), an increase in PCCLvalue in 19 cases (38%) and a decrease in PCCL-value in 11 cases (22%). Furthermore, the autopsy diagnosis changed the DRG primary diagnosis in 24 cases (48%). Conclusions: These preliminary results prove the relevance of this study and raise a number of interesting questions. For instance, should autopsies be considered a ‘‘diagnostic measure’’ with new economic impact in a DRG-based cost calculation system in addition to their important role in quality assurance?

Symposium: Breast Carcinoma II 134 Tissue-based prognostic markers in breast cancer H. KREIPE, R. VON WASIELEWSKI, S. MILDE, F. LA¨NGER, U. LEHMANN Institut fu¨r Pathologie, Medizinische Hochschule Hannover Although 70% percent of node negative breast cancer patients are permanently cured by surgical therapy alone more than 90% of patients receive adjuvant therapy because of uncertain prognostication. Novel diagnostic and therapeutic strategies minimize the material available for analysis and tissue-sparing techniques will be more and more required. This development and heterogeneity of tumour tissues convey major importance to histology-based approaches. Bloom and Richardson grading provide an established method to discriminate high risk types of cancer but reproducibility, especially in core biopsies, is questionable. Combination with objective parameters such as Ki-67 staining enhances the reliability of this morphological procedure. New input has been given by gene expression profiling studies challenging the role of classical morphology in grading. However, there is not a scarcity but a confusing plethora of markers and the deficit in prognostication is rather caused by lack of standardization of well established

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markers such as grading or immunohisto-chemical assessment of proliferation or steroid receptor expression than by a shortage of novel indicators of prognosis. There are two ways discernible by which gene profiling results will be adaptable to histological analysis. One depends on the availability of immunohistochemical reagents and reliable procedures to assess staining results in a quantitative manner. The second approach is provided by quantitative real time PCR in conjunction with tissue dissection. This technique allows quantitative measurements in formalin fixed and paraffin embedded specimens with the necessary certainty and reproducibility required for diagnostic procedures. Because almost all prognostic markers known so far have been yielded by retrospective studies there is a strong demand for prospective studies on promisng tissue-based markers of prognosis.

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Current aspects of the concepts of sentinel lymph

nodes W. JONAT Klinik fu¨r Gyna¨kologie und Geburtshilfe, Universita¨tsklinikum Kiel

136 Genomic profiling and prognosis in breast cancer M.J.v.d. VIJVER Netherlands Cancer Institute, Amsterdam, Netherlands Using gene expression profiling by microaarray analysis we have previously defined a gene expression profile of 70 genes that is predictive for a short interval to distant metastases (o5 yrs) in lymph node negative (LN0) patients. We have subsequently analysed the expression profile for these 70 genes (‘‘poor prognosis signature’’) in a cohort of 261 breast cancer patients younger than 53 years of age; both node negative (LN0) and lymph node positive (LN+)patients were included. In the LN0 patients the prognostic value of the ‘‘poor prognosis signature’’ was confirmed. In the 144 LN+ patients, the metastasis-free percentage at 5 and 10 years are 66% (SE 5%) and 55% (SE 6%) for the ‘‘poor prognosis signature’’ group against 96% (SE 3%) and 83% (SE 8%) for the ‘‘good prognosis signature’’ group (logrank test: P ¼ 0:0001). The hazard ratio for poor/good over the whole follow-up period is estimated to be 4.7 (95% CI: 2.0-11.2). In addition to using these supervised approaches for classification of breast cancer, we are also exploring gene expression signatures that have emerged from other gene

expression profiling experiments. An example is a wound-like gene expression signature, based on the stereotypic transcriptional response of normal fibroblasts to serum, has previously linked wound healing and cancer progression in a variety of common epithelial tumors. We have shown in the same consecutive series of 295 early breast cancer patient that tumors showing an activated wound signature (N ¼ 126) had worse distant metastasis-free probability and overall survival compared to those with a quiescent signature (10 year DMFP ¼ 51% vs. 75% and OS ¼ 51% vs. 84%, P value ¼ 106 and 1010, respectively). We identify and evaluate gene expression centroids of the wound signature that allows the signature to be applied to individual samples prospectively and quantitatively, enabling the signature to be scaled to suit different clinical purposes. The wound signature improves risk stratification independently of known clinical and pathologic risk factors and previously established prognostic signatures based on unsupervised hierarchical clustering (‘‘intrinsic gene signatures’’) or supervised predictors of metastasis (‘‘70 gene prognosis signature’’). More recently, we have performed microarray analysis on a consecutive series of 155 primary breast carcinomas from patients with stage I or II, lymph node negative breast cancer between age 55 and 70. We classified these tumors based on our known ‘70 gene—prognosis profile’ and searched whether a more optimal ‘olderage prognosis profile’ existed by employing supervised classification on whole genome microarrays. We found that the ‘70-gene prognosis profile’ is able to predict outcome of disease in this patient group with an odds ratio for metastasis within 5 years of about 3. Supervised classification on this same patient group, however, showed that a more optimal ‘older-age prognosis signature’ could be derived partially consisting of a different gene set. These results show that gene expression profile may be used in assessing prognosis in breast cancer; and that prognostic profiles may differ in specific subgroups of breast cancer patients.

137 Recent therapeutic options and expectations to the pathologist R. KREIENBERG, A. LEBEAU1 Universita¨tsfrauenklinik, Ulm 1 Pathologisches Institut der LMU Mu¨nchen The treatment of patients with breast cancer has progressively become multidisciplinary. Considering that the establishment of standards of care for medical treatment is a process of building consensus by using the best available scientific evidence, multidisciplinary

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guidelines have been developed in Germany to promote better and more consistent management of breast cancer patients (http://www.krebsgesellschaft.de). These guidelines provide a framework for clinical decision-making and pathological assessment that gives clinically useful and prognostic significant information. The improvement of standards of care is subject to the definition of procedures at the interface between the different involved disciplines. The lecture will focus on special topics at the surgery-pathology interface that are critical for the optimal management of breast cancer, especially with regard to breast conserving therapy: 1. Intra-operative frozen sectioning: (a) the essential criteria that have to be fulfilled to justify rapid frozen sectioning, (b) the diagnostic information that is aimed at with intra-operative examination. 2. Unequivocal marking of the tissue specimens by the surgeon in order to obtain proper orientation. 3. Residual tumour (R) classification (UICC, 2002) and adequate distance to resection margins (for DCIS and invasive carcinomas). 4. Specific requirements on the pathological examination of surgical specimens after primary systemic treatment (neoadjuvant chemotherapy), i. e. the assessment of tumour response. Consensus statements and controversial issues will be presented and discussed.

Symposium: Advances in Molecular Pathology III —Urogenital Pathology

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positive nuclei). FGFR3 mutations were detected by SNaPshot analysis. Results: FGFR3 mutations were found in low-stage/-grade tumors (po0:001), whereas P53 alterations were tied to adverse pathologic parameters (po0:001). FGFR3 mutations and P53 overexpression were exclusive events (po0:001) in pTa and pT1 BC, with a coincidence of only 2% (3/151). Interestingly, pT1 and pTa tumors with adjacent pTis demonstrated significantly less FGFR3 mutations (po0:001). Abnormal CK20 expression was significantly associated with higher tumor grades and stages (po0:001). However, 64% (92/143) of pTa BC revealed an abnormal CK20 pattern. The histologic subgroup of PUNLMP showed dedifferentiation in 52% (11/21) of cases. In the group of pTa and pT1 BC with normal CK20 pattern the FGFR3 gene was mutated in 88% (32/ 36), whereas a mutated FGFR3 gene was found only in 50% (57/58) of cases with abnormal CK20 expression (po0:001). Conclusions: FGFR3 and P53 alterations characterize two alternative genetic pathways in urothelial carcinogenesis. Urothelial dedifferentiation, represented by abnormal CK20 expression, is an early event in the carcinogenesis of papillary non-invasive bladder cancer but seems to occur later than FGFR3 mutations.

139 Gene expression profiling of progressive papillary non-invasive carcinomas of the urinary bladder P.J. WILD1, A. HERR2, C. WISSMANN3, R. STOEHR4, A. ROSENTHAL5, D. ZAAK6, R. SIMON7, G. SAUTER7, R. KNUECHEL8, C. PILARSKY9, A. HARTMANN1 1

138 Characterization of alternative genetic pathways in the pathogenesis of urothelial carcinoma P.J. WILD1, E. ROSSKOPF1, R. STOEHR2, F. HOFSTAEDTER1, J. V. OERS3, E.C. ZWARTHOFF3, A. HARTMANN1 1

Institut fu¨r Pathologie Institut fu¨r Urologie, Universita¨t Regensburg 3 Dept. of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam

Institute of Pathology, Univ. Regensburg Institute of Clinical Genetics, Univ. Dresden 3 Dept. of Internal Medicine, Univ. Berlin 4 Dept. of Urology, Univ. Regensburg 5 Signature Diagnostics AG, Potsdam 6 Dept. of Urology, LMU Munich 7 Institutes of Pathology, Univ. Basel 8 and Aachen 9 Dept. of Surgery, Univ. Dresden 2

2

Aim: To characterize alternative genetic pathways in the pathogenesis of urothelial bladder cancer (BC). Methods: Tissue microarrays were used to analyze CK20, P53 and the Ki-67 labeling index immunohistochemically (n ¼ 263). The expression pattern of CK20 was classified as normal (superficial staining of umbrella cells) or abnormal (negative expression or X10%

Aims: To define gene expression profiles of non-invasive and invasive bladder cancer (BC), and genetic changes relevant for tumor progression of recurrent papillary BC (pTa). Methods: Overall, 67 bladder neoplasias and eight normal bladder specimens were investigated by a combination of laser microdissection and gene expression profiling. Eight of 16 patients with recurrent noninvasive papillary bladder tumors developed carcinoma in situ or invasive BC in the course of time. Results were

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confirmed by quantitative RT-PCR and immunohistochemistry using tissue microarray analysis. Results: Hierarchical cluster analyses revealed no differences between pTaG1 and pTaG2 tumors. However, distinct groups of invasive cancers with different gene expression profiles in papillary and solid tumors were found. Progression associated gene profiles and putative marker genes (e.g. FABP4, CTSE, etc.) could be defined which were already present in the pTa tumors. CTSE expression (p ¼ 0:003) and a high Ki-67 labeling index of at least 5% positive nuclei (p ¼ 0:01) were the only factors that correlated significantly with PFS of pTa BC. Conclusion: Gene expression profiling revealed novel genes with clinical utility to select patients more likely to develop a more aggressive disease.

Conclusions: E2F3 and CDKAL1 are candidate oncogenes in advanced bladder cancer. One or several of these genes may play a crucial role for bladder cancer invasiveness.

141 Molecular analysis of inverted tumors of the urinary bladder M. EIBER1, H. BLASZYK3, J.V. OERS2, E. ZWARTHOFF2, T.V.D. KWAST2, R. STO¨HR1, P. WILD1, M. BURGER1, J. CHEVILLE4, G. SAUTER5, M. AMIN6, S. STO¨RKEL7, R. SCHNEIDER-STOCK8, F. HOFSTA¨DTER1, A. HARTMANN1 1

Institute fu¨r Pathologie u. Klinik fu¨r Urologie der Univ. Regensburg 2 Erasmus-Univ. Rotterdam 3 Univ. of Vermont, Burlington/VT 4 Mayo Clinic, Rochester/MN 5 Univ. Basel 6 Emory Univ., Atlanta/GA 7 Klinikum Wuppertal, Univ. Witten-Herdecke 8 Univ. Magdeburg 1

140 Functional evaluation of 6p22 amplification target genes in urinary bladder cancer is among the most outstanding research communications 2005 M. OEGGERLI, P. SCHRAML, M. BLOCH, M.J. MIHATSCH, G. SAUTER, R. SIMON Institut fu¨r Pathologie, Universita¨t Basel (CH) Aims: About 20% of muscle invasive bladder cancers show high level gene amplifications including the E2F3 locus 6p22.3. However, also other genes may be important in the amplicon. This study was designed to identify possible 6p22.3 target genes and to evaluate their impact on cell growth. Methods: Fluorescence in situ hybridization was applied on a bladder cancer tissue microarray to identify genes inside the amplicon. Expression of candidate genes was analyzed by RT-PCR and Northern blotting. A cell line model and RNA interference was employed to study the effects of target gene knockout on cell proliferation and morphology. Results: FISH mapping identified a genomic region of 1.6Mb presenting the 6p22.3 core amplicon. Two genes, including E2F3 and CDKAL1, showed simultaneous high level amplification and expression. Amplification frequency was highest for E2F3, but CDKAL1 was coamplified in 87.6% of E2F3 amplified cases. Amplification of both genes is strongly associated with high grade and invasive bladder cancers (pTa: 1.25%, pT1: 10.9%, pT2-4: 19.8%; G1: 1.5%, G2: 1.0%, G3: 18.5%). For both genes, expression levels were markedly higher in amplified as compared to non-amplified bladder cancers. RNAi in the 6p22.3 amplified cell line CRL-7930 revealed a significant decrease of cell growth after E2F3 knockout suggesting functional importance.

Aim: Inverted papilloma of the urinary tract (IP) is thought to be benign, but some urothelial carcinomas show a prominent inverted growth pattern (invUC) which may pose a diagnostic dilemma to the pathologist and clinician. Ancillary markers may help to resolve diagnostically challenging cases and may contribute to the ongoing discussion of a possible malignant potential of some IP. Methods: Seventy-two cases diagnosed as IP were reviewed by five uropathologists. Final diagnosis was IP in 63 and invUC in nine cases. Expression of CK20, p53, Mib-1, MSH2, MLH1, and MSH6 was studied by immunohistochemistry. Mutation in FGFR3 was analyzed by SNaPshot. Promotor methylation of p16, DAPK, E-cad, RASSF1 and hMLH1 was investigated by methylationsensitive PCR (MSP). Microsatellite analysis to detect deletions of chromosome 9p/q and 17p (LOH) and microsatellite instability (MSI) using the Bethesda consensus marker panel for HNPCC diagnosis was performed. Results: IP had very infrequently MSI, deletions of chromosome 9p/q and promoter methylation (all o10%). Immunohistochemical analysis of MSH2, MLH1, MSH6, CK20 and p53 revealed no differences between IP and invUC. The Mib-1 proliferation index corre-lated strongly with the histologic impression (po0:0001). FGFR3-mutations were more often seen in invUC compared to IP (p ¼ 0:05). Conclusions: IP is a benign lesion mostly lacking specific genetic alterations of bladder cancer. The presence of FGFR3 mutations and a Mib-1 proliferation index 410% favor a diagnosis of invUC, which could help resolve histologically ambiguous cases.

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Promoter methylation and microsatellite mutation reveals the clonal relationship of multiple urothelial carcinomas with mutator phenotype is among the most outstanding research communications 2005

R. STOEHR1, J.W.F. CATTO2, A. AZZOUZI2, I. REHMANN2, K. FEELEY2, M. MEUTH3, F. HAMDY2, M. BURGER1, A. HARTMANN Institut fu¨r Pathologie 1 Klinik fu¨r Urologie, Univ. Regensburg 2 Royal Hallamshire Hospital, Academic Urology Unit 3 Institute of Cancer Studies, University of Sheffield, UK Aims: The clonality of multiple urothelial carcinomas (UC) is subject to debate and affects treatment. Evidence derived from X-chromosome mosaicism and patterns of molecular alterations supports both a mono- and polyclonal relationship. In contrast to most UC, tumours with the mutator phenotype have frequent mutations in repetitive sequences (MSI) and promoter methylation. The aim of this study was to investigate the clonality of multifocal UC with MSI. Methods: We have screened 400 UC for MSI and found it to occur in 1% of bladder and 15% of upper tract UC. Of these, nine patients, whose tumours had MSI, developed or presented with multiple UC. A total of 32 UC (occurring over 0–6 years, 2–12 TCC per patient), two cases of CIS and nine normal urothelial samples were screened for MSI at 17 loci and aberrant promoter methylation at seven genes. Results: In eight of nine patients, the pattern of microsatellite mutation and promoter methylation suggested that the multiple tumours had a clonal origin. Patterns of aberrant methylation in multiple tumours were more similar than microsatellite mutations, suggesting an earlier carcinogenic timing. MSI and promoter methylation were present in macroscopically normal urothelium from these patients. Conclusions: Aberrant promoter methylation occurs before microsatellite alteration in UC with mutator phenotype. The majority of recurrent UC with MSI are monoclonal in origin and macroscopically normal urothelium harbours multiple molecular abnormalities. Thus, at the time of apparently successful treatment, there is molecular evidence of residual tumour that subsequently develops into recurrent disease.

143 Downregulation of Lysyloxidase L2 gene in prostate

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Gerhard Domagk Institut fu¨r Pathologie, Universita¨t Mu¨nster 1 Institut fu¨r Klinische Chemie und Laboratoriumsmedizin, Universita¨t Mu¨nster 2 Klinik und Poliklinik fu¨r UroIogie, Universita¨t Mu¨nster 3 Pacific Biomedical Research Center, Univ. of Hawaii, Honolulu, USA Aims: In human prostate cancer loss of heterozygosity (LOH) on the short arm of chromosome 8 is frequently observed. Several microsatellite and CGH studies in different tumor tissues identify deletions in this region which lead to the assumption that on chromosome 8p12-22 multiple tumor suppressor genes are located. Therefore the existence of two unknown putative tumor suppressor genes were postulated which plays different roles in prostate cancer progression. Methods: We started a quantitative SNP-screening for deletions and amplifications on chromosome arm 8p21 located between the microsatellite markers D8S258 and NEFL which were investigated too. Comparative immunohistochemical analysis followed. Results: We detected frequent deletions on leucine zipper putative tumor suppressor 1 (LZTS1) and RAN binding protein 16 (RANBP16) nearby D8S258 and an up to now unknown genetic imbalance hot spot on lysyl oxidase-like 2 (LOXL2) which was deleted in 72% of all investigated cases. Comparative immunohistochemical studies demonstrate that LOXL2 shows a high expression in benign prostate tissue and nearly lost in most prostate cancer tissues. Conclusions: A high expression of LOXL2 was first detected in human proliferating tissues like placenta and uterus. It is involved in tumor suppression and cell adhesion. Our results may lead to the implication that LOXL2 is one of the hunted genes on 8p21-22 and loss of this gene is a frequent and early event in prostate cancer progression.

144 A combination of hypermethylated genes as diagnostic and prognostic marker in prostate cancer J. ELLINGER, P.J. BASTIAN1, A. WELLMANN, N. WERNERT, S.C. MU¨LLER1, A. VON RU¨CKER Institut fu¨r Pathologie, Universita¨t Bonn 1 Klinik und Poliklinik fu¨r Urologie, Universita¨t Bonn

cancer E. ELTZE, H. SCHMIDT1, A. SEMJONOW2, K. CSISZAR3, O. BETTENDORF, B. BRANDT1

Aims: Our study was designed to evaluate gene promoter hypermethylation in the diagnosis of prostate cancer.

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Methods: Fifty three prostate cancer and 14 benign prostate tissues were analyzed. We used a quantitative, methylation-specific PCR to identify promoter hypermethylation at GSTP1, APC, PTGS2, MDR1, RASSF1a. Results: At three gene loci (GSTP1, APC and MDR1), hypermethylation was highly prevalent across tumors (483%), whereas hypermethylation at PTGS2 (71.4%) and RASSF1a (67.9%) was less frequent. In noncancerous tissues, we observed no relevant methylation at GSTP1, PTGS2 and APC, but CpG islands at RASSF1a and MDR1 revealed a moderate to high rate of hypermethylation. There was no correlation between the methylation status of a single gene and clinicopathological parameters. In contrast, the methylation of multiple gene loci (i.e. GSTP1+APC+PTGS2, GSTP1+PTGS2 or GSTP1+APC) correlated with locally advanced disease or/and extraprostatic extension (p ¼ 0:04920:035). A high Gleason score correlated with methylation at GSTP1/APC (p ¼ 0:017). Receiver operator curve analysis showed that hypermethylation at GSTP1, APC or PTGS2 or combinations of these genes can distinguish between cancerous and noncancerous prostatic tissue with a sensitivity of 71.1–96.2% and a specificity of 92.9–100%. Conclusions: Our data suggest that evaluation of DNA hypermethylation at GSTP1, APC and PTGS2 is of diagnostic and prognostic value.

acquired and the FISH labeled cell nuclei were interactively segmented. Quantitative 3D-image analysis was done for the radial positioning (HER2, CEP#17, CT#8), volume, surface, roundness and smoothness (CT #8). Results: In cell nuclei of pancreatic cancer significant changes in the roundness (po0:001) and surface (po0:001) were found for CT#8, while the radial positioning and volume revealed no differences (p40:05). Statistically significant differences were found in breast cancer for the radial positioning of HER2 and CEP#17 between neoplastic and non-neoplastic cell nuclei (po0:01). Conclusions: This first report of quantitative analysis of the genome architecture of neoplastic and nonneoplastic cell nuclei within their natural histological context indicates significant changes during malignant transformation of pancreatic and breast cancer. This approach allows the characterization of a higher-order genome organization as a new tool in molecular pathology.

146 Topotecan-resistant and –sensitive renal carcinoma cell lines: A comparative cDNA-array analysis S. HEIKAUS, E. CALISKAN, C. MAHOTKA, C.D. GERHARZ, H.E. GABBERT, U. RAMP Institut fu¨r Pathologie, Universita¨t Du¨sseldorf

145 The 3D genome architecture of neoplastic and nonneoplastic cell nuclei analyzed by quantitative microscopy of archival tissues T. WIECH, S. TIMME, F. RIEDE, M. HAUSMANN, S. STEIN1, C. CREMER1, M. WERNER, A. WALCH Institut fu¨r Pathologie, Universita¨tsklinikum Freiburg 1 Kirchhoff-Institut fu¨r Physik, Universita¨t Heidelberg Aims: Only very little is known about the nuclear architecture of tissue cells within their natural histological context. Here, we show an approach based on quantitative 3D-fluorescence imaging that describes the spatial organization of genes, centromeres and chromosome territories in neoplastic and nonneoplastic cell nuclei in histological sections of archival tissues. Methods: Whole chromosome painting (CT#8), centromeric (CEP#17) and locus-specific (HER2) probes were hybridized to tissue microarrays prepared from formalin-fixed paraffin-embedded blocks of matched pairs (n ¼ 10) from neoplastic and non-neoplastic tissues (pancreatic cancer, breast cancer). 3D-data stacks were

Aims: Renal cell carcinomas (RCC) exhibit a marked heterogenous sensitivity towards the anticancer drug Topotecan-induced apoptosis. Since the underlying mechanisms are not well understood, we performed a comparative cDNA-array analysis in a Topotecanresistant and -sensitive RCC cell line to detect differentially expressed genes which might be responsible for differences in apoptosis-susceptibiliy. Methods and results: (1) In the RCC cell line clearCa-6 exposure to Topotecan (100 mMol ml1) induced apoptosis as seen by light microscopical analysis and reduced cell number to 43.6% of the control. In contrast, clearCa-7 showed no increase in apoptosis and only a weak reduction of cell number to 70.3% of the control after 24 h. (2) In clearCa-6 cDNA-array analysis revealed an up-regulation of multiple E2F1 and p53inducible proapaptotic and cell-cycle regulating target genes (Apo1, Caspase-7, Caspase-8, Caspase-9, Bak1, Bax, APAF1, p53indProtein-RNA, MDM 2, p73, GADD45, p21, CDC25A, Cyclin E). (3) However, transcription of several antiapoptotic NfB dependent target genes were also induced (FLIP, BCL2A1, IAP1, XIAP). (4) In the resistant cell line clearCa-7 only a small number of E2F1 and p53 dependent target genes were differentially regulated (Apo1, Caspase-7, Bax,

ARTICLE IN PRESS Symposium: Breast Carcinoma III / Pathology – Research and Practice 201 (2005) 153–300

p53indProtein-RNA, GADD45, p21). Interestingly, only XIAP as an NfB dependent antiapoptotic genes was induced. Conclusions: Our results suggest that sensitivity of RCC cells to the anticancer drug Topotecan results in transcriptional regulation of a multitude of pro- and antiapoptotic genes. In contrast, in the resistant cell line only a small number of genes was differentially regulated suggesting that Topotecan-resistance is not conducted by de-novo synthesis of antiapoptotic regulators, but by mechanims lying upstream of gene induction.

Symposium: Breast Carcinoma III 147 Minimal residual disease in breast cancer: Diagnosis and biological relevance K. PANTEL Institut fu¨r Tumorbiologie, Universita¨tsklinikum Hamburg-Eppendorf Although many different assays have been developed over the past 10 years to detect disseminated tumor cells (DTC) or minimal residual disease in breast cancer, the two major approaches used involve either immunocytochemical staining or polymerase chain reaction (PCR) analysis. If these assays are sensitive and specific enough, they can detect one DTC in the background of millions of normal cells. For epithelial tumors, cytokeratins have become the best marker for the immunocytochemical detection of DTC. Bone marrow (BM), which can be easily collected from the iliac crest, is the most important site for detecting DTC, which are present in BM samples of 20–40% of patients with breast carcinomas; even in the absence of lymph node metastases (stage N0) or clinical signs of overt distant metastases (stage M0). BM samples can also be monitored for the presence of DTC after primary surgical treatment, to detect tumor recurrence. Several studies have documented the persistence of dormant DTC in BM of cancer patients without clinical signs of overt metastases. Persistence of tumor cells in BM was an independent prognostic factor. These findings indicate that adjuvant systemic treatment may be insufficient to eliminate micrometastatic cells and that at least some of the surviving micrometastatic cells can escape dormancy. It will be now a major challenge to unravel the mechanisms underlying this capacity. Interestingly, the presence of DTC in the BM is not only useful in predicting the development of skeletal metas-

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tases, but also in predicting the development of metastases in other distant organs, such as lung or liver. So BM might be an important reservoir that allows for DTC to adapt and disseminate into other organs. An alternative explanation is that the presence of DTC in the BM might only reflect the general propensity of these cells to disseminate and to survive in organs, rather than just in the BM.

148 Functional role of chemokine receptors in metastasis A. MU¨LLER-HOMEY Klinik und Poliklinik fu¨r Strahlentherapie und Radiologische Onkologie, Heinrich-Heine-Universita¨t Du¨sseldorf The occurrence of metastases within distant organs is the life-threatening event that limits the survival of patients with malignant diseases. To form clinically evident metastasis, tumor cells have to complete a series of highly regulated steps. In its early steps, metastasis mimicks leukocyte trafficking which is critically regulated by chemokines and their receptors. Chemokine receptors interact with small, cytokine-like proteins which have been shown to regulate the firm adhesion of leukocytes to the endothelium and mediate their transendothelial migration. Recently, we have demonstrated that chemokine receptors, in particular CXCR4, influence the organselective pattern of metastasis observed in breast cancer. CXCR4, specifically, is highly expressed in human breast cancer cells. Moreover, CXCL12, the specific ligand for CXCR4, exhibits an organ-selective expression profile with most abundant production at frequent sites of distant metastasis including lung, liver and bone marrow. Signaling through CXCR4 mediates actin-polymerization and pseudopodia formation and, subsequently, induces chemotactic and invasive responses in breast cancer cells. In vivo studies revealed that neutralization of CXCR4 significantly impairs the formation of metastasis to distinct organs implicating a pivotal role for this chemokine receptor in the process of metastasis. Current research focuses on the precise mechanisms of chemokine receptor-mediated tumor cell-host-interactions during early phases of metastasis using intravital microscopy. At this point a variety of therapeutics targeting chemokine receptors, including CXCR4, are under preclinical and clinical evaluation. In the years to come, clinical trials will determine whether interference with chemokine receptor function may control metastasis in cancer patient.

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Plasticity and polarity: How do breast cells remember to make an acini, and why do cancer cells forget

State-of-the-art Lecture

M. J. BISSELL

150

Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA

P. FRIEDL

The microenvironment is emerging as an equal partner to the genome in regulating tissue specificity as well as being a culprit and co-conspirator in cancer induction and progression. I will outline our efforts in devising a physiological context for normal and malignant breast cancer cells to recapitulate the milieu invivo. We show that the integrity of basement membrane (BM) is crucial for breast function, that tissue polarity and BM are determinants of response to chemotherapy and their loss can eventually lead to induction of genetic instability and cancer. Metalloproteinases such as MMP3 and ADAM5 play crucial roles in these processes with the former causing EMT and genomic instability via production of ROS. I will discuss the molecular mechanisms of these processes. Finally, we use the model system for testing of chemotherapeutic agents and also show that restoration of correct balance of different signaling pathways can restore tissue polarity and functional differentiation to all breast cancer cells tested regardless of the degree of mutations and genomic aberrations. Further Reading: [1] JM Bissell, D Radisky. Putting tumors in context. Nat. Rev. Cancer, (2001 Oct); 1(1): 46–54. [2] MJ Bissell, A Rizki, IS Mian. Tissue architecture: the ultimate regulator of breast epithelial function. Curr Opin Cell Biol. Dec; 15(6): 2003 753–62. [3] H Liu, DC Radisky, F Wang, MJ Bissell. Polarity and proliferation are controlled by distinct signaling pathways downstream of PI3-kinase in breast epithelial tumor cells. J Cell Bio 164(4): (2004) 603-12. [4] KL Schmeichel, MJ Bissell. Modeling tissue-specific signaling and organ function in three dimensions. J Cell Sci. Jun 15; 116(Pt 12): (2003) 2377–88. [5] VM Weaver, SA Lelie`vre, JN Lakins, MA Chrenek, JCR Jones, F Giancotti, Z Werb, MJ Bissell MJ.b4 Integrin-dependent formation of polarized 3-D architecture confers resistance to apoptosis in normal and malignant mammary epithelium. Cancer Cell 2: (2002) 205–216. (also see News & Views Nature, 419:790–791; Mini review (Cell) 111:923–925.

Cellular and molecular mechanisms of tumor invasion

Rudolf-Virchow Zentrum fu¨r Experimentelle Biomedizin und Universita¨tshautklinik, Universita¨t Wu¨rzburg Aims: Cancer cell interactions with the extracellular matrix and the migration therein involve the function of adhesion receptors (integrins) and proteolytic mechanisms to overcome tissue barriers. The aim was the timeand space-resolved imaging of these events. Methods: Using time-resolved bright field, confocal, and multiphoton microscopy, molecular live-cell dynamics in tumor and immune cells in 3D tissue culture models and the mouse dermis in vivo were reconstructed. ECM was detected by time-lapse confocal reflection and multiphoton second harmonic generation imaging. Results: Tumor cell migration was characterized by (i) cytoskeletal dynamics of filamentous actin by invading cancer cells, (ii) the subcellular topography of proteolytic cleavage of the extracellular matrix structures, and (iii) integrin-dependent (mesenchymal) as well as integrin –independent (amoeboid) migration modes. Using cocktails of specific protease and integrin inhibitors, we have obtained the following insight into cancer cell migration and cell-cell communication: the difference between individual and collective cancer cell migration; the belt-cleavage concept of pericellular proteolysis in migrating tumor cells; the mesenchymal-amoeboid transition after inhibition of matrix-degrading proteases or b1 integrins; and the collective-amoeboid transition after blocking of b1 integrins. Conclusions: The profile of diversity and adaptation responses suggest an unexpected degree of ‘supramolecular’ plasticity in cancer cell dissemination modes, representing the sectrum from individual to collective invasion, as detected in histopathological specimens.

Symposium: Current Postdoctoral Theses in Pathology 151 Mechanism of allogeneic injury after lung transplantation BITTMANN, IRIS Pathologisches Institut, Universita¨t Mu¨nchen

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Aims: Lung transplantation has evolved from an experimental endeavour to the mainstay of therapy for many end-stage lung diseases. Despite the success of lung transplantation many complications can affect the lung after transplantation, e.g. preservation related injury, mechanical complications of surgery, acute or chronic (obliterative bronchiolitis) rejection, infection, drug toxicity, recurrent disease, de novo disease, lymphoproliferative disorder. Rejection, infection and poor immediate graft function present persistent problems and contribute to a mortality rate of 20–30% at 1 year. The regulation of tissue inflammation and repair mechanisms involving components of the immune systems depends on a number of cell-cell interactions. Today, our understanding of this pathogenetic and functional network is still at the beginning. Methods: Immunohistochemistry, development and establishment of a cDNA MicroArray for analysis of chemokine and cytokine expression. Results: T-cells and their subgroups and the following increase of cytokines and chemokines play an important role during the development of allogeneic lung injury after lung transplantation. T-cell mediated cell injury does not only take place during allograft rejection but seems also to be important for the pathogenesis of reperfusion injury. Furthermore a complex cascade of cytokine interactions develops during graft reaction. Conclusions: The understanding of allograft reaction is still evolving. cDNA MicroArray technique could help to elucidate the pathogenesis of transplant reaction and provide new insights to facilitate the clinical diagnostics of allograft pathology.

152 Evaluation of new diagnostic and prognostic marker genes by microarray analysis in pediatric sarcoma K.-L. SCHAEFER Institut fu¨r Pathologie, Universita¨t Du¨sseldorf Aims: While overall five-year survival rates of childhood malignancies have been raised up to 75% during the last two decades the survival of children suffering from bone and soft tissue sarcoma is still below 50%. In addition the exact diagnosis of this heterogeneous spectrum of tumors may be challenging. Since optimal treatment of patients is highly dependent on reliable diagnosis as well as individual risk assignment the evaluation of genetic diagnostic and prognostic markers is of prime importance. Methods: Primary clinical sarcoma biopsies as well as tumor cell lines were analyzed by comparative genomic hybridization and microarray based expression profiling for characterization of tumor entity or individual risk

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related genetic changes and interesting findings were further investigated to estimate the functional impact of selected genes. Results: Osteosarcoma samples harboring chromosome 17p or 19p gains or 13q14 losses are associated with significant higher risk to relapse and development of metastasis. In Ewing0 s sarcoma loss of chromosome 16q is correlated with adverse prognosis. Clear cell sarcoma of tendons and aponeurosis constitutively overexpress the EGFR related ERBB3 membrane receptor which is activated by phosphorylation in these tumors. This marker gene may therefore become an interesting therapeutical target. Conclusions: Genome wide analysis of genetic and transcriptional changes in bone and soft tissue sarcoma of childhood supplies a more accurate and differentiated characterization of these malignancies and offers an advanced panel of molecular markers to improve diagnosis and risk assignment for optimal patient stratification.

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Investigations on the pathogenesis of reactive, metaplastic and neoplastic lesions of salivary glands S. IHRLER Pathologisches Mu¨nchen

Institut,

Ludwig-Maximilians-Univ.,

Aims: To remedy the lack of understanding of the pathogenesis of regeneration, metaplasia and neoplasia of salivary glands. Methods: Immunohistochemistry (including double staining) and molecular biological techniques were applied to normal glands (n ¼ 8) and reactive (n ¼ 79) and neoplastic (n ¼ 106) lesions for a comparative study of cellular proliferation, differentiation and apoptosis. Results: Low proliferation-rates (0.1%–2.0%) were found in all 5 types of cell of the normal gland (basal, oxyphilic, acinar, myoepi-thelial and intercalated duct cells). There was a 2- to 11-fold in-crease of proliferation in sialadenitis together with increased apop-tosis. Six different forms of metaplasia, including lymphoepithelial metaplasia, were found to develop from basal cells. An intraductal neoplasia was identified in 71.4% of adenocarcinoma, not otherwise specified (ACNOS), and in 77.8% of carcinoma ex pleomorphic adenoma (CEPA). Conclusions: The results demonstrate a unifying morphogenetic concept of cellular proliferation, differentiation and apoptosis in normal glands and in reactive and neoplastic lesions. The regene-rative potential of normal glands is based on low proliferative rates of all differentiated types of cell without the postulated participation of special stem cells. The significant

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increase of proliferation in sialadenitis indicates a clinically important potential for glandular regeneration. The prominent role of basal cells in ductal metaplasia disproves the postulated origin of lymphoepithelial islands from myoepithelial cells (‘‘epimyoepithelial lesion’’) in Sjo¨gren-type sialadenitis. The identification of an intraductal neoplasia in ACNOS and in CEPA demonstrates for the first time preinvasive lesions of salivary carcinomas, indicating analogous mechanisms to those of established models of neoplasia, such as in the mammary gland.

154 Cyclooxygenase-2 (COX-2) is a prognostic marker and a potential new therapeutic target in malignant tumors C. DENKERT Institut fu¨r Pathologie, Charite´ Berlin Aims: Molecular prognostic markers can be determined in tumor tissue and can be used – in addition to conventional clinicopathological parameters – to estimate patient prognosis and to plan the therapy of malignant tumors. In this study, we have focussed on cyclooxygenase-2 and related molecules, which play an important role in tumor biology and inflammation. Methods: Immunohistochemistry, Western blot, RNAi Results: We have investigated the expression of the molecular prognostic factors cyclooxygenase-2 (COX-2) and the human ELAV-like protein HuR, in ovarian carcinoma and breast carcinoma. Increased expression of COX-2 in tumor tissue was associated with poor prognosis in ovarian carcinoma and breast carcinoma. In cell culture models, we have used two different strategies for inhibition of COX-2: pharmacological inhibition and RNA interference. We found that COX-2 inhibitors act on other cellular targets in addition to COX-2 and inhibit proliferation of ovarian carcinoma cells by induction of cell cycle arrest. In further studies we could show that the RNA-stabilizing protein HuR is associated with increased COX-2 expression and is an prognostic factor in ovarian carcinoma, as well. Conclusions: Our results suggest that expression of COX-2 is related to patient outcome and prognosis and provide a basis for further evaluation of COX-inhibitors in tumor therapy. Cyclooxygenase-2 inhibitors are currently investigated in adjuvant tumor therapy in a variety of malignant tumors.

Symposium: Ovarial Carcinoma 155 Molecular carcinogenesis of ovarian carcinoma J. DIEBOLD Pathologisches Institut der Ludwig-MaximiliansUniversita¨t Mu¨nchen Ovarian carcinoma shows a wide range of phenotypic differentiation patterns reflecting different carcinogenetic pathways. On the morphological and molecular level endometrioid carcinoma bears great similarities to type I carcinoma of the uterine endometrium. Mutations of b-catenin (o50%), PTEN (20%), KRAS (25%) and BRAF (25%) are the most frequent alterations. Endometriotic foci are the presumable precursor lesions for some endometrioid carcinomas of the ovary. Apparently mucinous carcinomas of the ovary develop via an adenoma – borderline tumor – carcinoma sequence. On the molecular level KRAS mutations are early events on this pathway. The precursor lesions of conventional high grade serous ovarian carcinomas have not been adequately characterized. Seemingly most cases develop ‘‘de novo’’ from the ovarian surface epithelium as exemplified by rare cases of early stage carcinoma. p53 mutations are found even in these early lesions. Almost all sporadic serous carcinomas show RNA expression patterns that are identical to those of hereditary cases with BRCA1 or BRCA2 inactivation. Chromosomal instability is characteristic for conventional ovarian carcinoma. It leads to complex aberrations, e.g. with frequent gains at 3q26, 8q24 and 20q13 harbouring numerous oncogenes. Serous borderline tumours show lack of p53 mutations, but activation of the KRAS-BRAF signalling cascade in 50–60%, which is rarely found in conventional invasive ovarian carcinomas. Transformation of a serous borderline tumour into invasive serous carcinoma is a rare event (o1%). It is associated with step-wise accumulation of genetic alterations like amplification of oncogenes.

156 Borderline Tumors of the Ovary and Associated Implants M. DIETEL Institut fu¨r Pathologie der Charite´ (CCM), Universita¨tsmedizin, Berlin Aim: The WHO series on Gyn-Pathology (2003) defined to name the group of epithelial ovarian tumors with a dignity between benign and malignant exclusively

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Borderline Tumors of the Ovary (BOT) and to skip the term ‘‘of low malignant potential’’. Although this brings some clarity the group of BOT still cause confusion with regard to a standardized morphological diagnosis, the biological behaviour, the management of the individual case and in particular the importance of the BOT derived peritoneal implants. Method: Sampling is a major topic with min. 1–2 blks. per cm of max. Æ and 1 bl. per 2 cm of largest dimension of an omentum specimen. The report on BOT should include cell type, e.g. serous, mucinous etc., presence or absence of (micro)-invasive areas (quantification), micropapillary growth (significance is still unclear), surface proliferations, capsular rupture, peritoneal implants (invasive/non-invasive), staging UICC/ FIGO, TNM classification and lymph node invasion. Supporting methods for recognizing unfavourable BOT and for distinguishing them from highly differentiated ovarian carcinomas are immunohistochemistry and DNA-cytophotometry. Results: In stage Ia and Ib (confined to the ovaries) almost 100% of BOT show a very favourable prognosis. Microinvasion o5mm has no adverse effect. In more advanced stages (ca. 10–20%) BOT exhibit a worse clinical course depending on micropapillary growth pattern (MPSC), peritoneal implants, the extent of invasion, aneuploid DNA patterns and very important the presence of exophytic growth. Nevertheless only rarely patients die from the tumour within 10 years. If this is the case it is mostly due to local complications like ileus or other gastrointenal complications. A benefit of chemotherapy or radiation have never been shown. Conclusions: The appropriate diagnosis of BOT requires a standardized operating procedure, an excellent cooperation with the clinicians and longstanding morphodiagnostic experience in the field.

157 Prognostic Factors of Ovarian Carcinoma S. HAUPTMANN Institut fu¨r Pathologie, Martin-Luther Universita¨t Halle-Wittenberg A multitude of biological prognostic factors of ovarian carcinoma have been investigated during the last twenty years. These factors include proliferation indices, DNA ploidy, p16, p21, p27, p53, Bax, c-erbB2, EGF-R, VEGF, PKC-isoforms, CD24, COX-2, HuR, and ezrin. Unfortunately, none of these factors has met the criteria necessary to be recommended by the AJCC prognostic factors consensus conference. Grading is an important prognostic factor, however, various grading systems are in use which makes it difficult to obtain homogenous and comparable data. Therefore, AJCC requested to

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establish uniform grading criteria. Meanwhile, the Silverberg system is most widely accepted, and grading seems to be of particular importance in low stage tumors. Although pathogenesis of the various histological types of ovarian carcinomas is different, most studies have failed to show significant differences in survival. The reason for that is probably the lack of reproducible criteria to classify a tumor, particularly in cases with poor differentiation. The inaccurate classification of metastases as ovarian primaries may complicate the situation even more. Currently, stage of disease is the most important prognostic factor. Unfortunately, most patients with ovarian carcinoma present an advanced stage of disease (FIGO stage III/IV). For these patients the residual tumor after primary surgery is the exclusive prognostic factor whereas for patients with organ confined disease other prognostic factors are probably of more importantance and help in the decision whether chemotherapy should be administered. Nevertheless, even in high stage carcinomas, some of the above mentioned factors remain within the cox regression model albeit behind residual tumor. In the future, factors defining the activity of inflammatory pathways or factors mediating the interaction between cancer cells and mesothelium may become more important, both as prognosticators and therapeutical targets. Moreover, predictive factors allowing the individualization of chemotherapy will be of upmost clinical importance.

158 Clinical management of ovarian cancer and the role of the pathologist W. KUHN Ovarian cancer is the fourth most common cancer in women and the leading cause of death in women with gynaecologic malignancies. There are no early symptoms; therefore approximately 70% of patients present with advanced cancer stage FIGO III and IV, (tumour spread out of the pelvis with involvement of middle and upper abdomen and/or retroperitoneal metastasis). Tumour debulking surgery with complete tumour resection often combined with resection of small or large bowl is the prerequisite for good clinical outcome. After operation a platinum-taxane based chemotherapy is standard of care. Complete tumour resection combined with remission to chemotherapy can lead to long term survival. The outcome of patients is essentially dependent on the surgical success therefore treatment of ovarian cancer should only be performed in specialized cancer centers. Recent studies seem to demonstrate that neoadjuvant chemotherapy followed by tumour debulking surgery can improve survival in some subgroups of ovarian cancer patients (e. g. patients with diffuse peritoneal carcinosis, large amount of ascites). Further

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studies are ungoing. The pathologist is essential for treatment decisions in patients with ovarian cancer: The clinician needs a clear differentiation between invasive cancer and ovarian cancer of low malignant potential (borderline-tumour) because treatment modalaties are different. Moreover a clear tumour grading is warranted for treatment decisions in early stage ovarian cancer (chemotherapy in high-grade tumour, no chemotherapy in low-grade tumour). For the future more prognostic and even more important predictive factors would be helpful to tailor individualized treatment in order to reduce the so far high rate of relapse. In order to optimize clinical outcome treatment of ovarian cancer should be performed by a multidisciplinary team of gynaecological oncologist, abdominal surgeon, urologist, internal oncologist, radiologist and pathologist.

Conclusions: This clarifies the discrepancies observed between CGH and LOH for 16q in breast cancer. These different somatic genetic mechanisms may reflect the presence of multiple tumor suppressor genes or oncogenes with that are the target of LOH/ physical loss at chromosome arm 16q. It becomes also obvious that the different, previously described pathogenetic pathways in breast cancer are associated with a specific differential expression of chromosomes 16 and 1 genes.

160 The role of the AP-1 family members c-Fos, FosB, Fra-1 and Fra-2 in the invasion process of breast cancer cells B. ANDRITZKY, H. RO¨DER, T. LO¨NING, A.-M. BAMBERGER, K. MILDE-LANGOSCH

Symposium: Advances in Molecular Pathology IV —Urogenital Pathology 159 Influence of different mechanisms of chromosomal 16q-loss in invasive breast cancer on differential gene expression H. BU¨RGER, E. KORSCHING, A.M. CLETON-JANSEN1, D. KEMMING2, H. SCHMIDT2, N. TIDOW2, B. BRANDT2, W. BO¨CKER Institut fu¨r Pathologie, Universita¨t Mu¨nster 1 Institut fu¨r Pathologie, Universita¨t Leiden, Niederlande 2 Institut fu¨r Klinische Chemie Aims: Comparative genomic hybridization (CGH) demonstrated that well-differentiated breast tumors showed significantly more physical loss of 16q than did poorly differentiated ones. However, polymorphic markers detected no difference in the frequency of 16qLOH between invasive tumors of different histological grade. Methods: We combined data on LOH, fluorescence in situ hybridization (FISH) with chromosome 16 – specific probes, CGH and gene expression analysis using the ABI gene expression platform. Results: We are able to show a preference in welldifferentiated grade I tumors for physical loss of chromosome arm 16q, whereas in poorly differentiated grade III tumors LOH is accompanied by mitotic recombination. Differentially regulated genes in tumors with LOH compared to tumours with physical loss were predominantly seen on chromosomes 16q and 1q.

Institut fu¨r Gyna¨kopathologie, Universita¨tsklinikum Hamburg Aims: Members of the Fos family of transcription factors (c-Fos, FosB, FosB2, Fra-1, Fra-2) bind to AP-1 sites in regulatory regions of target genes. Our own prior analysis has shown correlations of Fos proteins with clinicopathological parameters in mammary carcinomas. As most proteases involved in degradation of the ECM and basal membrane are regulated by AP-1, we analyzed if the expression of Fos proteins might also be important for invasion of breast cancer cells. Methods: In the cell lines MDA-MB231 and MCF7, we performed transient transfections with expression vectors for c-Fos, FosB, FosB2, Fra-1 and Fra-2 followed by Matrigel invasion assays. The influence of Fos proteins on possible target genes (MMP1, MMP9, PAI-1) was studied by double-labelling immuno-cytochemistry and cotransfection experiments with a MMP9-Luciferase construct. Results: Fra-1 transfection resulted in a 2-4fold increase of invasive cells in both cell lines. In a lower degree, the invasive potential was stimulated by c-Fos and Fra-2. By ICC, PAI-1 up-regulation was observed in MCF-7 cells transfected with c-Fos, Fra-1 and Fra-2 expression vectors, whereas MMP1 and MMP9 expression was not affected. Results of cotransfection with a MMP9 promoter construct and AP-1 expression vectors do not indicate a direct up-regulation of MMP9 expression by Fos proteins except a positive effect of c-Fos in MCF7 cells. Conclusions: Our results indicate that changes in the expression of Fos proteins are involved in the regulation of invasion of breast cancer cells. This is in agreement with the results of a prior study on clinical tumor tissue.

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161 C-Kit overexpression in adenoid cystic breast carcinomas is not caused by mutations of the c-kit gene F. OTTERBACH, E.-M. KIND, S.Y. SHEU, K. SCHMITZ, H.A. BABA, K.W. SCHMID Institut fu¨r Pathologie, Universita¨t Essen Aims: C-kit (CD117) is a transmembrane tyrosine kinase which can be overexpressed in non-neoplastic and neoplastic tissues. In gastrointestinal stromal tumors overexpression is caused by activation mutations of the c-kit gene. Ninety percent of the adenoid cystic carcinomas (ACC) of the salivary glands show an overexpression of c-kit but activation mutations could yet not be found. We studied the prevalence of c-kit overexpression and mutations of the c-kit gene in a series of primary ACC’s of the breast. Methods: Eight cases of primary ACC’s of the breast were investigated immunohistochemically using the anti-CD117 antibody (A4502, DAKO). DNA-sequencing of exons 9, 11, 13 and 17 was performed after amplification by PCR. Results: All cases showed an overexpression of c-kit, which was weakly in only one case of well differentiated ACC. However, c-kit was expressed by most of the luminal epithelial cells in adjacent non-neoplastic breast tissue as well. Mutations in exons 9, 11, 13 and 17 could not be detected. Conclusions: Like their counterparts in salivary glands primary ACC’s of the breast are characterized by an overexpression of c-kit which is not caused by activation mutations of the c-kit gene. C-kit overexpression is not specific for adenoid cystic differentiation of breast cancers since it has been detected in 1-13% of all invasive carcinomas, especially of the medullary type. It remains unclear whether primary ACC’s of the breast will respond to an Imatinib (STI571/Glivec) treatment.

162 High expression of focal adhesion kinase (p125FAK) in node-negative breast cancer is related to overexpression of HER-2/neu and activated Akt kinase but does not predict outcome K.J. SCHMITZ, F. GRABELLUS, R. CALLIES2, F. OTTERBACH, J. WOHLSCHLA¨GER, B. LEVKAU1, R. KIMMIG2,3, K.W. SCHMID3, H.A. BABA Institut fu¨r Pathologie 1 Institut fu¨r Pathophysiologie 2 Klinik fu¨r Geburtshilfe und Frauenheilkunde, Universita¨t Essen-Duisburg 3 Westdeutsches Tumorzentrum

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Aims: Focal adhesion kinase (FAK) regulates multiple cellular processes including growth, differentiation, adhesion, motility and apoptosis. In breast carcinoma, FAK overexpression has been linked to cancer progression but the prognostic relevance remains unknown. In particular with regard to lymphnode-negative breast cancer it is important to identify high-risk patients who would benefit from further adjuvant therapy. Methods: We analyzed 162 node-negative breast cancer cases to determine the prognostic relevance of FAK expression and investigated the relationship of FAK with major associated signaling pathways (HER2, Src, Akt and extracellular regulated kinases ERK1/2) by immunohistochemistry and Western blot analysis. Results: Elevated FAK expression did not predict patients’ outcome in contrast to tumor grading (p ¼ 0:005) and Akt activation (p ¼ 0:0383). Significant positive correlations were observed between elevated FAK expression and HER2 overexpression (p ¼ 0:001), as well as phospho-Src Tyr-215 (p ¼ 0:021) and phospho-Akt (po0:001) but not with phospho-ERK1/2 (p ¼ 0:108). Western blot analysis showed a significant correlation of FAK Tyr-861 activation and HER2 overexpression (p ¼ 0:01). Conclusions: Immunohistochemical detection of FAK expression is of no prognostic significance in nodenegative breast cancer but gives evidence that HER2 might be involved in tumor malignancy and metastatic ability of breast cancer through a novel signaling pathway participating FAK and Src.

163 Bone marrow-derived stem cells contribute to breast cancer development in a mouse model R. HUSS, P.J. NELSON1, S. MOOSMANN, U. LO¨HRS Pathologisches Institut, Ludwig-Maximilians-Universita¨t Mu¨nchen 1 Abt. fu¨r klin. Biochemie, Med. Poliklinik, LudwigMaximilians-Universita¨t Mu¨nchen Aims: Stem cells reside in the bone marrow as their primary niche in the adult organism to participate in tissue and organ regeneration mainly by contributing to arteriogenesis and improved micro-circulation at the site of lesion. The same stem cells are also utilized by growing tumours to facilitate tumour neo-angiogenesis. We wanted to investigate the potential role of bone marrow-derived stem cells in the spontaneous breast cancer development in mice. Methods: Female Balb/c mice of different age were stratified into different groups: (a) mice with multiple pregnancies, (b) mice underwent hormonal treatment (17-ß-estradiol) and (c) mice without pregnancy and or

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additional hormonal treatment. Mice from every group received repeatedly a weekly infusion of syngeneic adult stem cells (2  106 cells/mouse), while control mice were left untreated. Results: Older mice after multiple pregnancies and after stem cell infusion spontaneously developed a large tumour of the mammary gland with areas of necrosis, while no spontaneous tumour development was observed in any other group. Transplanted cells were detected primarily in areas with tumour angiogenesis and the surrounding fibrotic tissue. Conclusion: Bone marrow-derived, circulating stem cells contribute to the development of breast tumours in mice by participating in tumour angiogenesis as previously shown in MMTV-transgenic mice and other solid tumours. While stem cells facilitate the proliferation of endothelial precursor cells in tissue regeneration, circulating stem cells integrate into the growing tumour vasculature.

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Quantitative mRNA expression analysis of genes affected by aberrant methylation in breast cancer U. LEHMANN, P. AHRENS, F. LA¨NGER, H. KREIPE Institut fu¨r Pathologie, Medizinische Hochschule Hannover Aims: Gene-specific hypermethylation is a frequent phenomenon in epithelilal malignancies including breast cancer. Under many circumstances it leads to a silencing of transcription which can be assessed by reversetranscriptase PCR or immunohistochemistry. Methods: DNA and RNA were extracted from snapfrozen and formalin-fixed breast cancer specimens. Relative transcript levels were assessed employing quantitative real-time PCR methodology. Several house-keeping genes were examined for their suitability as endogenous control. Immunohistochemical detection of gene products was also used. Selected cases were also laser-microdissected in order to obtain pure tumor cell populations. Results: For several genes shown to be frequently hypermethylated in breast cancer the mRNA levels and, if possible, the protein expression was measured. In the first approach cyclinD2, DAP kinase, GSTp1, and twist were selected. Overall, a good correlation between hypermethylation and silencing of gene expression was found. But for individual cases only a weak or no correlation could be detected. The recently reported down-regulation of the twist gene in ductal breast cancer accompanied by preferential hypermethylation in this histological subtype could not be confirmed.

Conclusions: In breast cancer DNA-hypermethylation is generally associated with transcriptional silencing but exceptions may occur. These can be due to contaminating by-stander cells or selection of the inappropriate region for the assessment of methylation analysis. For expression studies quantitative RT-PCR is superior to immunohistochemistry because of a more precise quantification and independence of available antibodies.

165 uPAR-del4/5, a novel tumour-associated urokinase receptor variant, and the matrix metallo-proteinase inhibitor TIMP-3 in breast cancer M. KOTZSCH (a.G.), P. SPAN3 (a.G.), A. MEYE1 (a.G.), J. FARTHMANN2 (a.G.), F. SWEEP3 (a.G.), V. MAGDOLEN2 (a.G.), T. LUTHER, G. BARETTON Institut fu¨r Pathologie 1 Klinik fu¨r Urologie der TU Dresden 2 Frauenklinik der TU Mu¨nchen 3 Dept. Chemical Endocrinology, University Nijmegen, The Netherlands Aim: Proteases, their receptors and inhibitors are involved in tumour growth, invasion and metastasis. In the present study, we evaluated the association of the expression levels of uPAR mRNA (uPAR-wt) and its splice variants (uPAR-del4/5, uPAR-del5) as well as of TIMP-3 mRNA with clinicopathological parameters and disease progression in breast cancer patients. Methods: Total RNA was isolated from tumour tissues of 205 breast cancer patients. For quantification of uPAR and its variants, and TIMP-3 mRNA levels highly sensitive real-time RT-PCR assays based on LightCycler technology were used. Results: Neither the mRNA levels of uPAR-wt nor of its splice variants were significantly associated with clinicopathological parameters. In estrogen receptor negative tumours, a significantly higher uPAR-del5 expression was found (p ¼ 0:023). However, only high uPAR-del4/5 mRNA expression was significantly associated with shorter disease-free survival of breast cancer patients (p ¼ 0:035). In subgroup analysis we found that high uPAR-del4/5 mRNA levels were significantly correlated with worse prognosis in patients with low TIMP-3 mRNA values (p ¼ 0:0043). The greatest difference in patient survival was found by combining uPAR-del4/5 with TIMP-3 values: patients with high uPAR-del4/5 and low TIMP-3 have a much shorter disease-free survival in comparison to those with low uPAR-del4/5 and high TIMP-3 expression (p ¼ 0:0001). Conclusions: These results suggest that uPAR-del4/5 mRNA may serve as a novel prognostic marker in breast cancer.

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166 BRAF mutations in ovarian epithelial neoplasm: A comprehensive study of invasive carcinomas, borderline tumors (LMP) and extraovarian implants D. MAYR, A. HIRSCHMANN, U. LO¨HRS, J. DIEBOLD Pathologisches Institut der Ludwig-MaximiliansUniversita¨t Mu¨nchen Aims: Mutations of BRAF, a downstream mediator of K-RAS, have been described in serous LMPs of the ovary. Data concerning other types of ovarian tumors are scarce. Therefore we assessed BRAF mutation in a series of 114 different ovarian tumors and additionally searched for K-RAS mutation in all non-invasive neoplasms. Methods: Paraffin embedded material of 114 patients, including 44 serous, 13 mucinous, 13 endometrioid and four clear cell carcinomas, 18 serous and 17 mucinous LMPs, seven implants, two adenomas and three samples of normal tissue was used. DNA analysis was performed by PCR and direct sequencing. Small tumors underwent microdissection prior to PCR. BRAF codon 599 (exon 15) and K-RAS codon 12 (exon 2) were analysed. Results: One hundred cases (87.7%) could be analysed successfully for BRAF mutations. Ninety two cases (92%), including all serous invasive carcinomas (100%), did not show a mutation. Eight cases (8.0%), including five serous LMPs (31.25%) contained a mutation. In all serous LMPs codon 599 was affected. There was no BRAF mutation in mucinous LMPs. Regarding K-RAS 44 of 48 cases (91.6%) could be analysed. Thirty four cases (77.3%) did not show an aberration. The 10 positive LMP cases (22.7%) were of serous (17.6%) and of mucinous type (46.6%). BRAF and K-RAS mutations were mutually exclusive. Six of seven cases (85.8%) with implants could be analysed. No one showed any mutations of BRAF or K-RAS, although one of them had a K-RAS mutation in the ovarian LMP. Conclusions: Mutation of either K-RAS or BRAF is frequent in serous (50%) and mucinous (46.6%) LMPs, but is not found in invasive serous carcinomas and is very rare in other invasive subtypes. These findings suggest different pathological pathways of LMPs and invasive (serous) carcinomas. BRAF mutations could not be found in extraovarian implants.

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Aims: A putative tumor suppressor gene, GABRP has been identified as differentially expressed in breast cancer by analyzing EST databases (see abstract by Dahl et al.: Systematic identifi-cation and molecular characterizationy). GABRP is the pi subunit of the Gamma-aminobutyric acid (GABA) A receptor. The GABRP gene is located on chromosome 5q33-qter, an area frequently lost in BRCA1 tumors (Krop et al.; Cancer Res. 2003 63:2024). Here we present a detailed expression analysis of GABRP. Methods: We performed real time PCR (TaqMan), mRNA in situ hybridization as well as immunohistochemistry with a GABRP specific polyclonal serum. Results: The ‘‘neuronal’’ gene GABRP is expressed in several non-neuronal tissues including the lung, stomach, uterus, ovaries and in the epithelial cells of mammary gland. The 3.3 kb GABRP mRNA transcript is specifically down-regulated in breast tumors. Analysis of dot blot arrays (Clontech) exhibited a more than two fold down-regulation of GABRP mRNA in 76% of breast cancers. This result was confirmed by real-time RT-PCR analysis of matched normal/tumor pairs from invasive mammary carcinoma. In situ hybridization revealed a strong GABRP expression in epithelial breast cells and in benign papilloma cells. No signal was detectable in invasive breast cancer. Currently we are analyzing the GABRP protein expression pattern in normal and malignant breast diseases. The anti-proliferative potential of GABRP will be evaluated after forced expression in established human tumor cell lines. Conclusions: The biological function of this ‘‘neuronal protein’’ in normal breast epitehelial cells is still unclear. Its consistent loss in different types of breast cancer argues for an important function in normal breast tissue homeostasis.

Symposium: Advances in Molecular Pathology V —New Developments 168 Molecular targets for diagnosis and therapy – New challenges for pathologists R. BUETTNER, S. MERKELBACH-BRUSE Institut fu¨r Pathologie der Universita¨t Bonn

167 GABRP, a putative tumor suppressor gene in breast cancer A. BAUER, M. CHOROVICER, R. KNU¨CHEL-CLARKE, E. DAHL Institut fu¨r Pathologie, Universita¨t Aachen

The enormous increase in information on cell growth and differentiation and their genetic basis will make a major impact on classification and diagnostics of human diseases. Although molecular genetic technologies are only beginning to influence traditional morphologybased tumour diagnostics, it is clear that we will

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experience significant individualization of entities and better prediction of tumour behaviour and therapy responses. The impact of genetic information on tumour classification has already significantly changed diagnostic concepts of sarcomas. Many entities, such as Gastrointestinal Stromal Tumors, Synovial Sarcomas and PNETs, are defined by specific activation of signal transduction pathways or characteristic chromosomal translocations, despite a wide spectrum of morphological or immunohistochemical differentiation. Moreover, a number of molecules mediating critical growthpermissive or apoptosis-repressive signals provide interesting new therapeutic targets. In particular signal transduction-related kinases provide attractive targets for individualized therapies. As an example, activation of HER-1 signalling by specific mutations has made it possible to define a subgroup of lung adenocarcinomas responsive to small molecule inhibitors of tyrosine kinases. Therefore, pathologists currently encounter new diagnostic challenges and needs and will critically influence future concepts of individualized therapies.

169 Biochip Analysis in Pathology: Status Quo C. POREMBA, K.-L. SCHAEFER, Y. BRAUN, H.E. GABBERT Institut fu¨r Pathologie, Universita¨t Du¨sseldorf Microarrays allow the analysis of global gene expression in human cells and tissues. Global gene expression analysis helped to identify important genes and signalling pathways in human malignant tumors, that are key players in – – – – – –

loss of repair mechanisms for DNA damage inactivation of tumor suppressor genes activation of oncogenes cell proliferation and immortality tumor neoangiogenesis invasion and metastasis

In addition to basic science research for understanding the initiation and progression of malignant tumors, there is hope that microarrays will make the step from ‘‘the (laboratory) bench to the bedside (of the patient)’’. Recent studies have indeed revealed that analysis of differential gene expression by microarrays may help to identify subtypes of malignant tumors, that allow a risk stratification of the patients. Furthermore, some studies have shown that differential gene expression may help to predict therapy response for particular tumor types. However, there are several issues that need

to be adressed before microarrays may become a tool for routine diagnostics, such as problems with bioinformatic analysis, construction of disease or tissue specific microarrays with only limited numbers of genes of interest, standard operation procedures for tissue preparation to prevent RNA degradation, etc. In this presentation we will address the potential and pitfalls of using microarrays in molecular pathology research and diagnostics, based on our own studies as well as published studies from the literature.

170 CUP syndrome: are there advances? R. MOLL Institut fu¨r Pathologie, Philipps-Universita¨t Marburg Metastatic cancer of unknown primary site (CUP syndrome) comprises 2–5% of all malignant tumors and thus is among the 10 most frequent human cancers. We should distinguish between cancers with initially unknown but later detected primary tumor and cancers with truly unknown primary tumor for life despite complete diagnostic work up. Although being heterogeneous, true CUP share some unique biological features such as early invasion and metastasis. The most important task of histopathology is to help to identify therapy-responsive subgroups (e.g., neck lymph node CUP, axillary lymph node CUP of females, serous papillary carcinoma of peritoneum, neuroendocrine CUP, and germ cell tumor CUP of males). For other CUP, such as those (histologically mainly adenocarcinomas) with primarily liver, lung/pleural, bone or brain metastases and nonpapillary peritoneal carcinomatosis, prognosis is very poor. Autopsy may reveal the primary site in some but not all CUP, the most frequent sites being pancreas and lung. The most important auxiliary method is immunohistochemistry which should be applied in a two-step algorithmic fashion. Next to markers with relative specificity for tumor groups (e.g., CK5, CK7, CK20, p63, ER, GCDFP-15, CA125), an increasing number of markers with high organ specificity (e.g., PSA, PLAP, HMB45, HepPar1, TTF-1, uroplakin III) has become available. Thus many CUPs may be assigned to a distinct immunophenotypic category. PCR detection of EBV or HPV16/18 DNA may identify nasopharyngeal and cervical origin, respectively. Recent studies suggest a diagnostic power of gene expression profiling but its practical value is still limited. Next to new clinical methods (e.g., PET), modern pathology may solve an increasing proportion of CUPs and thus contribute to the development of more specific therapeutic schemes.

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Gene signatures identifying nodal positive breast cancer E. DAHL, E. G. KRISTIANSEN1, K. HERMANN2, I. LOSEN, I. KLAMAN2, G. SAUTER3, R. SIMON3, P.J. WILD4, A. HARTMANN4

Institut fu¨r Pathologie, Universita¨t Aachen 1 Institut fu¨r Pathologie, Charite´, HU Berlin 2 metaGen Pharmaceuticals i.L. Berlin 3 Institut fu¨r Pathologie, Kantonsspital Basel, Schweiz 4 Institut fu¨r Pathologie, Universita¨t Regensburg Aims: The correct determination of a breast cancers lymph node status is critically important for choosing the right therapy. However, axillar lymph node dissection is a major factor of patient morbidity. It would be helpful if the nodal status could be determined directly from the primary tumour, e.g. to support sentinel node diagnostics. We aimed to determine whether gene signatures from primary breast cancer can distinguish nodal positive and nodal negative tumours. Methods: Gene expression profiles of 47 microdissected primary breast cancers were determined using Affymetrix gene chip technology. Using a training data set of 24 tumours (12 pN0 and 12 pN+) pN+ specific gene signatures were identified using K-Nearest Neighbour (KNN-) analysis. Gene signatures were confirmed on an independent data set (test set) of 14 pN0 and 9 pN+ tumours. Results: We identified more than 20 gene signatures with five to 20 genes that can predict lymph node metastasis with an accuracy of more than 85% both in the training and test set. Several gene signatures contained the syndecan-1 gene. Up-regulation of syndecan-1 in pN+ tumours was confirmed on the protein level analyzing a tissue microarray with 2221 breast cancers. Conclusions: Gene expression analysis of primary breast cancers revealed small gene signatures that can be used to distinguish nodal-positive and nodal-negative breast cancer. We will investigate whether a molecular lymph node diagnostics (MLND) can be based on combinations of quantitative RT-PCR experiments for those genes present in small gene signatures.

172 Microarray analyses of microdissected tissues to determine progression-specific molecular markers in breast cancer K. SOTLAR, C. SCHU¨TZ1, H. NEUBAUER1, M. BONIN2, S. CLARE3, O. RIEß2, D. WALLWIENER1, B. BU¨LTMANN, R. KUREK1 Institut fu¨r Pathologie 1 Universita¨ts-Frauenklinik

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2

Institut fu¨r Humangenetik, Universita¨t Tu¨bingen Department of Surgery, Indiana University School of Medicine, Indianapolis, USA 3

Aims: In breast cancer a progression model is postulated, including atypical ductal hyperplasia, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). The transcriptomes of matched DCIS and IDC tumor tissues should be compared in order to identify progression-specific molecular markers. Methods: Because of the heterogeneity of tumor tissues, microarray analyses were combined with laser capture microdissection (LCM) to obtain homogenous cell populations of matched DCIS and IDC from five patients. Total RNA was extracted from about 20,000 cells which were microdissected (Arcturus, PixCell IIe) from hematoxilin/eosin-stained frozen sections. mRNA was linearly amplified (50-100ng starting material) and several quality controls were performed during the experiments by capillary electrophoresis (Agilent, Bioanalyzer). Results: Analysis of data retrieved from full-density microarray hybridization (Affymetrix, human genome U133A) was performed with the Affymetrix data mining tool and the software Genesis (A. Sturn, TU Graz, Austria), thereby identifying 130 genes differentially expressed between DCIS and IDC. Gen expression was in part verified by quantitative real-time PCR (LightCycler, ROCHE). In situ hybridization using tissue microarrays is currently performed for selected candidate genes. Conclusions: Investigating lasermicrodissected tissues molecular markers were identified, potentially characterizing the transition from DCIS to IDC.

173 Systematic identification and molecular

characterization of genes differentially expressed in breast and ovarian cancer

E. DAHL1,3, A. SADR-NABAVI2, E. KLOPOCKI3, B. BETZ4, S. GRUBE5, R. KREUTZFELD6, M. CHOROVICER1,3, G. KRISTIANSEN7, R. GRU¨TZMANN8, J. NA¨HRIG9, M. DIETEL7, R. SCHA¨FER7, A. ROSENTHAL3, R. SCHMUTZLER6, M. DU¨RST5, A. MEINDL2, D. NIEDERACHER4 1

Institut fu¨r Pathologie, Universita¨t Aachen Med. Genetik, TU Mu¨nchen 3 MetaGen, Berlin 4 Frauenklinik, Universita¨t Du¨sseldorf 5 Frauenklinik, Universita¨t Jena 6 Frauenklinik, Universita¨t zu Ko¨ln 7 Institut fu¨r Pathologie, Charite´ (CCM) Berlin 8 Gefa¨ß- und Thoraxchirugie, TU Dresden 9 Institut fu¨r Pathologie, TU Mu¨nchen 2

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Aims: To identify novel disease associated genes in gynecological cancers. Methods: A bioinformatic procedure (electronic Northern) was applied to screen ‘‘Expressed sequence tag’’ (EST)-cDNA libraries for genes differentially expressed in breast and ovarian tumors. Results: We describe the validation of a set of 40 candidate genes in breast and ovarian cancer that are localized in chromosomal regions supposed to be involved in the development of these tumor entities. Dot blot array analysis of matched tumor/normal cDNAs and quantitative RT-PCR detected differential expression in 28 genes. Of these genes, 21 have not been described in the context of breast and ovarian cancer and may constitute novel diagnostic markers. Mutation analysis of ten genes in a set of 20 primary breast tumors selected for gene specific LOH revealed inactivating mutations in only one gene. Conclusions: Our findings indicate that most of the downregulated genes in these gynecological tumors are getting inactivated either by epigenetic mechanisms or by disregulated transacting factors. Our approach detected several genes known to be silenced by gene methylation suggesting that the strategy applied is suitable to quickly identify novel tumor-associated genes in breast and ovarian cancer.

174 Differential analysis of breast cancer proteomes by silver-stained 2D-PAGE H. NEUBAUER, S. CLARE1, K. SOTLAR2, U. VOGEL1, M.A. CAHILL3, M. BERTH4, A. NORDHEIM5, D. WALLWIENER, R. KUREK Universita¨ts-Frauenklinik Tu¨bingen 1 Department of Surgery, Indiana University School of Medicine, Indianapolis, USA 2 Institut fu¨r Pathologie, Universita¨t Tu¨bingen 3 ProteoSys AG, Mainz 4 Decodon GmbH, Greifswald 5 Abteilung fu¨r Molekularbiologie, Institut fu¨r Zellbiologie, Universita¨t Tu¨bingen Aims: The effects of estrogen receptor (ER)-regulated protein expression on tumor cell progression in breast cancer is largely unknown. Therefore, it was the aim of this study to determine and to compare the proteomes of breast cancers with different immunohistochemical (IH) ER expression patterns. Methods: Whole sections obtained from frozen tumor tissues of 26 patients with invasive ductal carcinomas were dissolved and proteins were separated on 2DPAGE. Per patient 5 gels were prepared. After silverstaining the gel images were warped to obtain superimposed master images (Delta2D software, DECO-

DON). Differentially expressed proteins were picked from stored gels and analysed by MALDI-TOF mass spectrometry. Results: Hierarchical cluster analysis allowed the discrimination of ER-positive and ER-negative breast cancers (T-Test: po0:01). Selected differentially expressed proteins are currently validated by IH investigation of breast cancer tissue microarrays. Conclusions: Analyses of differentially expressed proteins in ER-positive and ER-negative invasive ductal breast cancers may identify additional markers of tumor progression, and thus, potential therapeutic targets.

175 Comparative protein profiling of estrogen positive and negative ductal invasive carcinoma F. TRAUB, H. TAMMEN1, A. PICH, P. SCHULZ-KNAPPE1, H.H. KREIPE Institut fu¨r Pathologie, MH-Hannover BioVisioN AG, Hannover

1

Aims: Ovarian steroids, acting through nuclear receptors are crucial players in normal breast development and cancer. Approximately 70% of all breast cancers depend on estrogen stimulation and on a functional estrogen receptor (ER). Estrogen negative tumours often overexpress genes encoding for non steroid growth factors and receptors. Complex interactions with signalling pathways and cell cycle control might explain resistance to endocrine treatment and might lead to novel therapeutic targets. Methods: Peptide extracts from 36 ER+ and 36 ER ductal invasive breast carcinoma tissue samples were investigated. Peptides were analysed using novel Peptidomics technology by employing reversed-phase chromatography fractionation, MALDI-MS and extensive bioinformatics analysis. Results: Despite the heterogeneous nature of cancer tissue, significant differences in protein and peptide expression were determined. Discrimination between ER+ and ER tumors on the basis of profile pattern was achieved with high specificity. Sequence analysis revealed that several peptides are strongly associated with the estrogen receptor status. Especially one peptide was detected in490% of ER cases in elevated, diagnostic concentrations. Conclusions: For the first time we report successful peptidomics analysis in breast cancer tissue specimen for tumormarker-discovery. Novel tumor marker candidates have been found. Peptide and protein profiling of breast cancer thus leads to further insights into the pathogenesis of this still enigmatic tumour entity.

ARTICLE IN PRESS Symposium: Advances in Molecular Pathology VI / Pathology – Research and Practice 201 (2005) 153–300

176 Multiconditional gene expression analysis in microarray experiments supports the characterization of tumor progression pathways on a molecular level E. KORSCHING1, H. BUERGER1, B. BRANDT3, W. BO¨CKER1, R. VOSS2,3 1

Gerhard-Domagk-Institut fu¨r Pathologie, Universita¨t Mu¨nster 2 Institut fu¨r Arterioskleroseforschung an der Universita¨t Mu¨nster 3 Institut fu¨r Klinische Chemie und Laboratoriumsmedizin, Universita¨t Mu¨nster Aims: The exploratory analysis of microarray experiments with replicates in various biological conditions leads to an enormous multiplicity problem. Control of an experiment-wide significance level using stepwise and permutation based procedures to correct for the familywise error (FWER) or the false discovery rate (FDR) is often too conservative and would discard many of the group related biological changes for the genes of interest. Methods: We present a practical approach which combines the Type I errors for n independent genes in two groups with the Type I error from the within-gene analysis to an overall false positive rate. Taking into consideration all possible pairwise tests for each gene, a set of Boolean rules derived from the biological conditions can be used to select subsets of genes with significant changes in some predefined comparisons. Results: We used resampled artificial and real cancer data and a simple model of conditional probabilities to explore the overall Type I error in observed and in permutation based null distributions. It is shown that the expected false positive rate can be reduced markedly simply by increasing the complexity of the design without any need of additional FWER and FDR adjustments. Conclusion: This algorithm opens a principal and reliable way to enhance precision by enhancing complexity.

Symposium: Advances in Molecular Pathology VI —Lymphoma and Leukemia

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Pathologisches Institut, Universita¨tsklinikum Freiburg Aim: Whereas the Biomed II approach for clonality analysis of hematopathological malignancies can be readily applied to fresh tissue samples, its efficiency in fixed, embedded tissues is limited. Here, we examined whether modifications of the Biomed II protocol enable its application to formalin/glutardialdehyde fixed, decalcified bone marrow biopsies with a potential benefit for IgH clonality analysis. Method: IgH clonality analysis of crude DNA extracts was performed by standard semi-nested PCR (FRIII) for 43 fixed, decalcified bone marrow biopsies with histological proven B-NHL. The same DNA extracts were then analyzed by the original Biomed II protocol. Finally, serial sections of all 43 bone marrow samples were subjected to a modified DNA extraction/Biomed II protocol. Results: Of the same crude DNA extracts, standard IgH analysis detected 22/38 (57.9%) of B-NHL, whereas only 17/38 (44.7%) were detected by the original Biomed II protocol. In contrast, the modified DNA extraction/Biomed II protocol improved the detection rate to 32/39 (82.1%). Moreover, in the 16 cases not detected by standard IgH analysis, the modified Biomed II approach was positive in 14/16 (88%) cases. Finally, using the modified Biomed II protocol both FRIII (tube C) and FRII (tube B) regions could be analyzed, with detection rates of 20/38 (52.6%) as compared to 4/38 (10.5%) for the original Biomed protocol. Conclusion: We describe a modified Biomed II protocol for the use in fixed, decalcified bone marrow biopsies. For IgH-FRIII region this method clearly improves the detection rate of B-NHL as compared to the standard procedure.

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NPM-ALK regulated activation of the AP-1 transcription factor in anaplastic large cell lymphomas (ALCL) P. VESELY1, P. STABER2, L. KENNER1, G. MOSER1, S. SCHAUER1, W. DIRKS3, G. HOEFLER1 1

Institut fu¨r Pathologie, Medizinische Universita¨tsklinik Graz 2 Medizinische Universita¨tsklinik Graz, Klinische Abteilung fu¨r Ha¨matologie 3 DSMZ, Braunschweig

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Application of the Biomed II protocol for IgH clonality analysis in fixed, decalcified bone marrow biopsies – technical improvements S. LASSMANN, U. GERLACH, K. TECHNAU-IHLING, M. WERNER, P. FISCH

Aims: More than half of nodal anaplastic large cell lymphomas (ALCL) express the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion, which is a product of the t(2;5)(p23;q35) chromosomal translocation. To identify key genes regulated by this activated

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kinase, we studied consequences of NPM-ALK expression using suppression subtractive hybridization (SSH) and cDNA microarray analysis. Methods: SSH libraries were constructed using mRNA from pools of NPM-ALK positive and negative ALCL cell lines as well as 293 cells transfected with active and kinase-dead NPM-ALK. mRNA expression patterns were analyzed in individual cell lines on cDNA arrays using cDNA clones resulting from the SSH libraries as well as from genes relevant for cancer development. Quantitative RT-PCR of 20 selected genes validated the mRNA expression data. Results: In 293 cells, the active NPM-ALK construct confers significant changes in the expression of 38 genes. Expression of 102 genes distinguishes NPM-ALKnegative (FE-PD, MAC-2A) from NPM-ALK-positive ALCL cell lines (SU-DHL-1, JB-6, SUP-M2, SR-786, DEL and Karpas 299). The majority of these genes are involved in regulation of cell cycle and apoptosis. Interestingly, target genes of AP-1 transcription factor are increased in NPM-ALK expressing cell lines. Electromobility shift assay (EMSA) verified NPMALK dependent AP-1 DNA binding activity. Conclusion: This study identifies AP-1 activation as a target of NPM-ALK signalling which might constitute an essential step in malignant transformation.

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Biallelic mutation of SOCS-1 impairs JAK2 degradation and sustains phospho-JAK2 action in MedB-1 mediastinal lymphoma line is among the most outstanding research communications 2005 T.F.E. BARTH, I. MELZNER, A.J. BUCUR, S. BRU¨DERLEIN, K. DORSCH, C. HASEL, F. LEITHA¨USER, P. MO¨LLER Institut fu¨r Pathologie und Rechtsmedizin, Universita¨t Ulm Primary mediastinal B-cell lymphoma (PMBL) is a welldefined subtype of diffuse large B-cell lymphoma. Molecular cytogenetics revealed frequent gains of 9p24. JAK2, mapping in this region, is presently regarded as a candidate oncogene since expression profiling showed high JAK2 transcript levels and JAK2 was found to be constitutively phosphorylated in mediastinal B-cell lymphomas. We confirm that in the MedB-1 mediastinal B-cell line, harbouring a trisomy 9, JAK2 transcription is elevated and the product is highly phosphorylated. However, JAK2 is not over-expressed at the protein level. On top, JAK2 protein turnover is even delayed. This unexpected finding coincides with a biallelic mutation of the SOCS-1 gene in this cell, which

abrogates SOCS box function of the protein. Ectopic expression of wt-SOCS-1 in MedB-1 leads to growth arrest, dramatic reduction of phospho-JAK2 and its downstream partner phospho-STAT5. Ultimately, the target gene cyclin D1 is repressed in transfectants while RB1, which is silenced in MedB-1, is induced. We conclude that, in MedB-1, action of phospho-JAK2 is sustained due to defective SOCS-1. Hence, SOCS-1 qualifies as a novel tumor suppressor. Of note, SOCS-1 mutations are also present in the parental tumor of MedB-1 and were detected in 9 of 20 PMBL.

180 The grey zone between burkitt and diffuse large B cell lymphomas: Impact of chromosomal breakponts at myc and other loci E. HARALAMBIEVA1,2, E.-J. BOERMA2, G.W. VON IMHOFF3, S. ROSATI2, E. SCHUURING2, H.K. MU¨LLER-HERMELINK1, P.M. KLUIN2, G. OTT1 1

Institut fu¨r Pathologie, Universita¨t Wu¨rzburg Department of Pathology, University Hospital, Groningen 3 Department of Haematology, University Hospital Groningen 2

A prompt distinction of Burkitt lymphoma (BL) versus diffuse large B cell lymphoma (DLBCL) has important clinical implications. Here we report on the analysis of 74 adult lymphoma that shared morphological similarities between BL and DLBCL. Ten pediatric BL were included as a reference group. All samples were stained for molecules of relevance to the diagnosis of BL (Ki-67, CD10, bcl2 and bcl6). The presence of a translocation breakpoint in MYC, BCL2 and BCL6 was studied by interphase Fluorescence in situ Hybridization (FISH). Four hematopathologists reached a final (consensus) diagnosis independently in 80% of the reference BL, 24% of the test BL and 69% of the DLBCL. A MYC breakpoint was detected in all reference BL, 95% of the test BL and of note, in 35% of the DLBCL. BCL2 and BCL6 breakpoints were infrequent in BL (5 and 6%) and but also DLBCL (13% and 16%). Three BL and 5 DLBCL contained double breakpoints. A phenotypic shift to BL in the DLBCL group was indicated by the expression of CD10 and bcl6 in 67% and 91% of the cases, respectively. One third of all lymphomas showed a classical genotype and expression pattern of BL (MYC breakpoint+, Ki-67X90%, CD10+, bcl6+, bcl2-) but only 63% of these cases were classified as BL. Our data indicate that a subgroup of DLBCL shares markers with BL, which is probably due to an overlapping histogenesis of both tumors.

ARTICLE IN PRESS Symposium: Advances in Molecular Pathology VI / Pathology – Research and Practice 201 (2005) 153–300

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T-bet and IRTA1 expression patterns reveal uniqueness of various B-cell subsets homing within and adjacent to marginal zones and epithelia K. JO¨HRENS1, I. ANAGNOSTOPOULOS1, E. TIACCI2, B. FALINI2, H. STEIN1 1

Institut fu¨r Pathologie, Charite´, Campus Benjamin Franklin, Medizinische Universita¨t Berlin, Deutschland 2 Institute of Hematology, University of Perugia, Perugia, Italy Aims and methods: It is controversially discussed whether the marginal zone (MZ) B cells of the spleen and lymph node, the monocytoid B (MC)- and the epithelial associated B (EAB) cells represent separate Bcell subsets or just variants of one B-cell population. To clarify this issue we studied using immunohistology the expression of CD21, CD27, BCL-2, CD45RA, CD75 and of two new molecules T-box expressed in T-cells (Tbet) and immuno -globulin superfamily receptor translocation associated 1 (IRTA1) on MZB cells in spleens and mesenteric lymph nodes, on MCB cells occurring either in toxoplasmic lymphadenopathy or in mesenteric lymph nodes adjacent to MZB-cells, and on tonsillar EAB cells. Results: We found that (i) splenic MZB cells (T-bet, IRTA1, CD21+, CD27+, BCL2+, CD45RA, CD75) have a largely identical immunophenotype as the nodal MZB cells which usually lacked CD21, (ii) the MCB cells have a distinctly different antigen expression profile (T-bet+, IRTA1+, CD21, CD27, BCL2, CD45RA+, CD75+) than the splenic and nodal MZB cells and (iii) the immunophenotype of the EAB cells (Tbet/+, IRTA1+,CD21, CD27, BCL2+, CD45RA+, CD75/+) shares more similarities to that of the MCB cells than that of the MZB cells. Conclusion: These results unequivocally reveal that MCB cells are characterized by the constant T-bet and IRTA1 expression and represent a distinct B-cell subset which differs in terms of differentiation and function from MZB cells but shares features with EAB cells. This data are expected to aid identification of lymphomas originating from these cells which are often subsumed under the heading of marginal zone lymphoma.

182

Expression of cyclin D1 transcript variants in multiple myeloma J. SLOTTA-HUSPNINA1, I. KOCH1, M. RICHTER2, M. KREMER1, K. SPECHT1, J. KRUGMANN3, L. QUINTANILLA-MARTINEZ2, F. FEND1

Institute fu¨r Pathologie 1 Technische Universita¨t Mu¨nchen

2 3

225

GSF Forschungszentrum Neuherberg Universita¨t Innsbruck

Aims: Multiple myeloma (MM) shows upregulation of cyclin D1 (CyD1) in about 50% of cases, due either to a t(11;14) or in associ-ation with trisomy 11. Recently, expression of CyD1 mRNA variants, namely the loss of the 30 UTR region, has been found as poor prognostic sign in mantle cell lymphoma. The significance of the loss of the 30 UTR, which stabilizes the mRNA, as well as of the CyD1 transcript variants A and B, whose levels are influenced by an AG polymorphism at the exon 4/5 boundary, are unknown in MM. Methods: RNA and DNA were isolated from microdissected paraffin embedded bone marrow biopsies or osteolytic lesions (N ¼ 65). Expression of CyD1 mRNAs was quantified by variant-specific TaqMan assays. Proliferation was assessed by MIB1 staining. Results: All MM with high CyD1 expression (D1/TBP ratio 4100; N ¼ 13) showed low proliferation (mean 17%), whereas in MM with low (o10; N ¼ 33) to intermediate (10-100, N ¼ 19) CyD1 expression, proliferation rates varied significantly (mean 25%, range 195%). Loss of the 30 UTR (less than 50% UTR) was associated with very high mRNA levels in the group with D1/TBP4100. Elevated expression of transcript B was found only in samples with AA genotype (N ¼ 12=37), but was not correlated with proliferation rate. Conclusions: Loss of the 30 UTR in CyD1+ MM is associated with very high levels of CyD1 mRNA. In contrast to MCL, however, high CyD1 levels in the presence of a t(11;14) are generally associated with low proliferation rates, in accordance with the more favorable outcome of MM with t(11;14). Increased levels of transcript B were not correlated with total CyD1 levels or proliferation rate. These findings do not support a simplistic view of tumor cell proliferation as direct result of high CyD1 mRNA levels in CyD1+ MM.

183 Frequency and type of c-kit codon 816 mutations in systemic mastocytosis K. SOTLAR, H.-P. HORNY1, P. VALENT2, B. BU¨LTMANN Institut fu¨r Pathologie, Universita¨t Tu¨bingen 1 Institut fu¨r Pathologie, Universita¨t Schleswig-Holstein, Campus Lu¨beck 2 Innere Medizin I, Abteilung fu¨r Ha¨matologie und Ha¨mostaseologie, Universita¨t Wien (A) Aims: Detection of activating c-kit mutation D816 V is one of five criteria for the diagnosis of systemic mastocytosis (SM). The aims of this study was to

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determine the frequency and types of activating codon 816 mutations of the proto-oncogene KIT in patients with SM. Methods: Formalin-fixed and paraffin-embedded bone marrow (bm) biopsies of 55 patients morphologically suspicious for SM and 239 controls were investigated by PNA-mediated PCR-clamping. In 40 of the 55 SM cases, nested PCR amplified DNA of pooled microdissected single mast cells (MC) was investigated by melting point analysis. Results: The prevalence of c-kit mutations in SM accounted for 91% (50/55). The following mutations were detected: D816V (n ¼ 47), D816Y (n ¼ 2) and D816H (n ¼ 1). None of the controls harboured c-kit codon 816 mutations. Of 15 cases with associated clonal hematologic non-MC lineage disorders (SM-AHNMD), in six cases (40%) the mutations were also detected in microdissected cells of the AHNMD, arguing for a monoclonal disease. Conclusions: The c-kit mutation D816V is a typical finding in patients with SM. The reduced effect of tyrosin kinase inhibitor STI571 in the presence of these mutation in some aggressive cases is therapeutic problem.

184

Advanced idiopathic myelofibrosis displays exaggerated osteoprotegerin (OPG) expression O. BOCK, G. LOCH, U. SCHADE, J. SCHLUE´, R.V. WASIELEWSKI, H. KREIPE Institut fu¨r Pathologie, Medizinische Hochschule Hannover

Aims: In advanced chronic idiopathic myelofibrosis (IMF) with osteosclerosis increase and thickening of bone trabeculae is typically contrasted by the absence or sparse presence of osteoblasts as well as osteoclasts. OPG, also named osteoclastogenesis inhibitory factor (OCIF), is the key factor in the control of osteoclast differentiation. Deregulation of OPG expression in IMF could pave the way for inappropriate bone remodelling. Methods: To assess OPG mRNA levels in patients with IMF in the cellular phase (n ¼ 33) and in the advanced stage (n ¼ 31) compared to non-neoplastic haematopoiesis (n ¼ 38), total bone marrow cells were quantitatively analysed by real-time RT-PCR. By applying immunohistochemistry, gene expression of OPG could be correlated with cellular expression pattern of the corresponding protein. Results: IMF with severe fibrosis and osteosclerosis expressed significantly higher (up to 71-fold) OPG mRNA levels (Median 4.0, range 0.42–71.4) as compared to non-neoplastic control cases (Median 0.9, range 0.14–6.8) and the prefibrotic cellular phase of

IMF which not substantially differed from the control group. Stromal cells such as fibroblasts, endothelial cells, adipocytes, and to varying degrees megakaryocytes could be delineated as the source for OPG as demonstrated by immunohistochemistry. Conclusions: We conclude that mainly stromal cells in IMF display up-regulated OPG expression. Osteosclerosis in IMF that is typically accompanied by scarcity of osteoblasts and osteoclasts does not appear to be primarily caused by enhanced bone formation but reduced turnover due to OPG-induced impairment of osteoclast function.

185

Gene expression profiling in rheumatoid arthritis: Molecular evaluation of histologically graded synovitis T. BO¨HME, R. GU¨NTHER, T. HA¨UPL, B. STUHLMU¨LLER, L. MORAWIETZ, G.R. BURMESTER, A. RADBRUCH1, V. KRENN

Institut fu¨r Pathologie, Campus Charite Mitte, Berlin 1 Deutsches Rheumaforschungszentrum (DRFZ), Berlin Aim: Gene expression microarray analyses reflect the molecular complexity of rheumatoid synovitis. The differential cellular composition coincides with disease dependent gene regulation. Therefore the identification of relevant genes is disconcerting. To decipher this complexity, cell type specific expression profiles were applied as baseline information for comparison. Methods: Synovial tissues from patients with rheumatoid arthritis (RA, n ¼ 10), osteoarthritis (OA, n ¼ 10) and normal donors (ND, n ¼ 10) were scored according to ‘‘synovitis score’’ and analyzed by microarrays. Subsequently, they were compared to sorted blood monocytes, granulocytes, CD4+ and CD8+ T-cells from up to 10 ND. Results: We found genes that were differentially regulated into the same direction in all comparisons between two groups (n ¼ 143). They allowed us to correctly classify all patients and normals and to establish diagnostically predictive sets of genes. To determine the percentage of cell type specific infiltration into the synovial tissues the signal levels of markers found by comparing expression profiles between different cell populations served as a measure. Through a new developed algorithm, the complex tissue profiles were rebuilt from profile components of the contributing cell types. Differences between the virtual profiles and the real profiles revealed a measure for truly regulated genes. Discussion: This study substantially improved the quality of array interpretation by identification of tissue associated gene regulation. It would help to identify

ARTICLE IN PRESS Posters: Gastrointestinal Pathology I / Pathology – Research and Practice 201 (2005) 153–300

markers common between blood and tissue. It may provide candidates for disease classification and activity scoring. The principle of this algorithm is applicable to many other complex samples with variable cellular composition including tumors and blood.

Posters: Gastrointestinal Pathology I 186

The histopathology of esophageal (Barrett’s) adenocarcinoma M. SARBIA Institut fu¨r Pathologie, Technische Universita¨t Mu¨nchen Aims: To determine whether the histopathological appearance of Barrett’s cancer differs significantly from that of cardiac and gastric adenocarcinoma (AC). Methods: HE-stained slides of 215 primarily resected esophageal AC as well as 108 cardiac and 184 gastric AC were classified according to Lauren’s classification, WHO classification, Carneiro’s classification and a variety of other clinico-pathologic parameters. Results: According to Lauren’s classification, esophageal AC (1.4%) less frequently belonged to the diffuse type than cardiac (2.8%) and gastric AC (23.9%; po0:0001). Tubular and papillary AC, as defined by the WHO classification, were more frequent among esophageal (94.4%) than among cardiac (87.0%) and gastric AC (59.2%; po0:0001). Solid carcinomas according to Carneiro’s classification were less frequent among esophageal (2.8%) than among cardiac (10.2%) and gastric AC (9.2%; po0:0001). Esophageal AC were graded more frequently G1/G2 (53.9%) than cardiac (30.6%) and gastric AC (27.7%; po0:0001). Patients with esophageal AC were male more frequently than patients with cardiac or gastric AC (90.2% vs. 82.4% vs. 55.9%; po0:0001). The above described differences in the histological appearance remained significant when esophageal (n ¼ 50) and gastric (n ¼ 20) AC confined to the mucosa were compared. Among esophageal AC, carcinomas graded G1 or G2 were more frequent among mucosal cancers (90.0%) than among more advanced cancers (30%; po0:0001). Conclusions: Although esophageal (Barrett’s) AC display the same histological spectrum than cardiac and gastric AC, the relative proportion of differentiated, gland-forming carcinomas is much more frequent in the esophagus than in the cardia and in the stomach.

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187 Development of overall and site-dependent incidence of gastrointestinal stromal tumors compared to other mesenchymal tumors of the gastrointestinal tract M.M. GOLAS, B. GUNAWAN, S. LUKAS, B. SANDER, L. FU¨ZESI Zentrum Pathologie, Universita¨t Go¨ttingen Aims: Gastrointestinal stromal tumor (GIST) appears to be the most frequent mesenchymal neoplasm of the gastrointestinal tract. Little is known about the incidence of GIST in the pre-c-kit era. The aim of this study was to analyze the incidence of GIST between 1991 and 2003 compared to the incidences of other mesenchymal tumors of the gastrointestinal tract. Methods: Gastrointestinal mesenchymal tumors occurring in a population of 210,000 inhabitants of southeastern Lower Saxony diagnosed at the University of Go¨ttingen between 1991 and 2003 were re-evaluated using histomorphological and immunohistochemical markers. In cases of uncertain diagnosis comparative genomic hybridization and mutational analysis of KIT and PDGFRA genes were additionally performed. Results: In total, 173 mesenchymal tumors (except lipomas) were reclassified (115 GIST, 35 leiomyomas, 9 leiomyosarcomas). There was a tenfold increase in incidence of GIST from 1991 to 2003. Overproportional increase in incidence was observed in low-risk gastric GISTs, whereas the incidence of high-risk gastric GISTs and intestinal GISTs (duodenum, small and large bowel) increased only slightly. However, non-GIST mesenchymal tumors showed a stable incidence in the same observation period. Conclusions: The overall incidence of GIST in contrast to other mesenchymal tumors of the gastrointestinal tract increased between 1991 and 2003. This increase in GIST incidence is mainly due to an overproportional increase in frequency of small gastric GIST of the lowrisk category.

188

Reclassification of gastrointestinal mesenchymal tumors: Underdiagnosis or overdiagnosis of GIST? U. PAUSER, S. HINZ1, G. KLO¨PPEL

Institut fu¨r Allgemeine Pathologie, Universita¨t Kiel 1 Klinik fu¨r Allgemeine Chirurgie und Thoraxchirurgie, Universita¨t Kiel Aims: Gastrointestinal mesenchymal tumors include leiomyomas, leiomyosarcomas, schwannomas, MPNSTs and GISTs. The diagnosis of GIST increased rapidly after the introduction of c-kit immunostaining. Because they can be treated with the tyrosinekinase

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inhibitor Glivec, they must be distinguished from leiomyosarcomas and MPNSTs. On the other hand, ckit positive tumors other than GIST should not be misdiagnosed as GIST. Methods: One hundred and forty two mesenchymal tumors of the gastrointestinal tract diagnosed between 1983 and 2004 were investigated by immunohistochemistry and reclassified on the basis of their histological and immunohistochemical differentiation. Results: Thirty three GIST were diagnosed before 2000. Twenty five were confirmed as GIST by their c-kit expression. One GIST was reclassified as leiomyoma. Seven tumors were c-kit negative and did not show any neural or muscular differentiation. Seven of 8 leiomyosarcomas and 3 neurogenic sarcomas were reclassified as GIST. Fifty five mesenchymal stromal tumors were diagnosed as GIST after 2000. We confirmed the diagnosis in 48 cases. One tumor was reclassified as leiomyosarcoma, one as schwannoma and 5 tumors were negative for c-kit and neural or muscular differentiation. One of four leiomyosarcomas was reclassified as GIST. Conclusions: On the basis of c-kit immunostaining malignant gastrointestinal stromal tumors can be recognized and the patients can be given adjuvant therapy with Glivec. A small group of c-kit negative tumors may represent a subset of GIST with pathogenetic alteration in PDGFRa gene.

189

Comparative analysis of risk categorizations of gastrointestinal stromal tumors B. SANDER, B. GUNAWAN, M. M. GOLAS, D. KOVAC1, S. GASPAROV2, R. BOLLMANN3, L. FU¨ZESI Zentrum Pathologie, Universita¨t Go¨ttingen Department of Pathology, University Rijeka, Croatia 2 Department of Pathology, University Zagreb, Croatia 3 Institut fu¨r Pathologie, Bonn

1

Aims: The malignant potential of gastrointestinal stromal tumors (GIST) is unclear. Various classification schemes have been proposed to predict the biological behavior of these tumors, in particular Franquemont et al. (Am J Clin Pathol 1995,103), Fletcher et al. (Hum Pathol 2002,33) and Miettinen et al. (Hum Pathol 2002, 33). The aim of this study was to analyze which classification most accurately predicted clinical behavior. Methods: One Hundred and fifty five GISTs reevaluated at the University of Goettingen were classified according to Franquemont, Miettinen, and Fletcher. Events monitored for survival analysis included relapse and death caused by tumor disease.

Results: The disease-free survival rate was about 55% and overall survival rate about 70%. In the postoperative observation period, no relapse (100%) was observed in the category of low/very low risk (Fletcher) and benign/low-malignant (Miettinen) tumors. In contrast, the relapse rate for low risk (Franquemont) tumors was about 80%. Tumor relapse was predicted most accurately by Miettinen compared to Franquemont and Fletcher. Conclusions: The classification schemes proposed by Miettinen and Fletcher correctly identified the subgroup of tumors with benign clinical behaviour. Overall, the advantage of the Miettinen scheme lies in the consideration of tumor site. The precise risk evaluation may facilitate a proper selection of patients who will benefit from adjuvant therapy.

190 Characterization of macrophage subpopulations and vessels in cancers of the gastrointestinal tract D. SICKERT (a.G.)1, A.E. DANIELA2, (a.G.) P. DIETER1, G.B. BARETTON2 1

Institut fu¨r Physiologische Chemie, TU Dresden Institut fu¨r Pathologie, Klinikum Carl-Gustav-Carus, TU Dresden 2

Aims: The functional relationship between immune and cancer cells appears to be a critical factor in the development, progression and dissemination of cancer. The aim of our study was to determine the number of tumor associated macrophages (TAM) of different subtypes in cancers of the gastrointestinal tract to elucidate the complex relationship between TAMs and tumor cells and to analyse its contribution to angiogenesis. Methods: Cancers of the gastrointestinal tract were arrayed into tissue microarrays (TMA). TAMs and vessels were characterized by immunohistochemistry using the antibodies PG-M1, KP-1, MRP8, MRP14, MRP8/14 and CD31, CD34, respectively. Results: The number of macrophages was significantly higher in tumor tissues than in tumorfree tissues. There was a positive correlation between the five macrophage subtypes as well as the number of macrophages and number of microvessels in all tumors. The number of KP-1+ and PG-M1+ TAMs as well as the number of microvessels were significantly higher in adenocarcinomas than in squamous cell carcinomas. Low numbers of MRP8+-, MRP14+- and MRP8/14+-macrophages and microvessels tended to occur with more advanced cancers. Conclusions: Our results suggest that different cancers of the gastrointestinal tract show very similar characteristics with regard to recruitment and differentiation of

ARTICLE IN PRESS Posters: Gastrointestinal Pathology I / Pathology – Research and Practice 201 (2005) 153–300

macrophages. The increased number of macrophages in cancers compared to tumorfree tissue indicates that macrophages are attracted to the tumor site. The distribution of TAMs and microvessels differs in adenocarcinomas Versus squamous cell carcinomas. Further functional studies are needed to elucidate the complex interaction between tumor cells and macrophages.

191

Reactive lymphoid hyperplasia or primary hepatic MALT-type lymphoma: A differential diagnosis in a patient presenting with three liver tumours K. WILLENBROCK, S. OESCHGER, S. KRIENER, M.-L. HANSMANN

Senckenbergisches Institut fu¨r Pathologie, Universita¨t Frankfurt am Main Three different tumours of the liver were detected in a single patient. In addition to two common lesions (focal nodular hyperplasia and haemangioma) a nodular lymphoid mass was found. The lymphoid lesion was composed of small lymphocytes forming follicles, plasma cells, few immunoblasts and centroblasts, few macrophages, epitheloid cells and giant cells. The lymphoid tissue displaced the adjacent hepatic parenchyma. Few lymphoepithelial lesions were detected. By immunohistochemistry and length fragment analysis of immunoglobulin- and T-cell receptorgene rearrangements the lymphoid infiltrate was found to be polyclonal. The diagnosis of reactive lymphoid hyperplasia was made. Reactive lymphoid hyperplasia is a rare but unique entity and has also been termed nodular lymphoid lesion of the liver. The discrimination of lesions like this from primary hepatic malignant nonHodgkin lymphoma of MALT-type is important, may be difficult and may require the use of molecular techniques.

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developed HSV-hepatitis in his graft. Despite immediate intravenous high-dose ayclovir therapy he developed acute liver failure, was retransplanted and developed recurrent and fatal HSV-hepatitis in his second graft despite continuous antiviral therapy. Results: Liver histology obtained from the first graft showed focal necrotizing hepatitis, Cowdry type A nuclear inclusions and immuno-histochemistry for HSV antigen was positive in the necrotic areas and the adjacent vital hepatocytes. In the explanted liver, no nuclear inclusions were detectable and immunoreactivity for HSV-antigen was limited to necrotic areas indicating therapy response. Liver biopsy obtained one month after retransplantation revealed again necrotizing hepatitis, Cowdry type A nuclear inclusions and immunohistology for HSVantigen was again strongly positive in necroses and adjacent vital hepatocytes. Conclusions: HSV-hepatitis may reoccur after liver transplantation despite continuation of high-dose antiviral therapy. Restriction of HSV-immunoreactivity to necrotic areas in explanted liver indicates initial therapy response. The reason for recurrence may be explained by selection of acyclovir resistant strains. This case demonstrates the necessity to develop strategies to monitor and treat HSV-infections in transplant setting.

193 Receptor for advanced glycation end products in chronic hepatitis C D. NEUREITER*, C. LOHWASSER1,*, Y. POPOV1, F. STICKEL1, M. PISCHETSRIEDER2, D. SCHUPPAN1,3 *contributed equally Pathologisches Institut, Universita¨t Erlangen-Nu¨rnberg 1 Medizinische Klinik 1, Universita¨t Erlangen-Nu¨rnberg 2 Institut fu¨r Pharmazie und Lebensmittelchemie, Universita¨t Erlangen-Nu¨rnberg 3 Department of Medicine, Harvard Medical School, Boston, USA

192 Fatal recurrent herpes simplex virus hepatitis after liver retransplantation T. LONGERICH1, C. EISENBACH2, T. KREMER3, C. FLECHTENMACHER1, B. HELMKE1, J. ENCKE2, T. KRAUS3, P. SCHIRMACHER1 1

Pathologisches Institut, Universita¨t Heidelberg Medizinische Klinik IV, Universita¨t Heidelberg 3 Chirurgische Klinik, Universita¨t Heidelberg 2

Introduction: A 61 year old man received orthotopic liver transplantation for alcoholic liver cirrhosis and

Aims: Advanced glycation end products (AGEs) were shown to induce localized inflammation and profibrinogenic reactions via its receptor RAGE. In the present studies we aimed to investigate whether RAGE plays a role in AGE-induced effects in the liver of patients with chronic hepatitis C. Methods: Human liver specimens of control patients and of patients with chronic hepatitis C infection were immunohistochemically analyzed with monoclonal antibodies against RAGE, lymphocytes, macrophages and stellate cells/myofibroblasts. Additionally, mRNA levels of RAGE and markers of inflammation (TNFa;

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Interleukin (IL)-12, interferong; IL-10) and fibrosis (CTGF, TGF-b1, procollagen-a1(I), matrix-metalloproteinase (MMP)-2, MMP-3, MMP-9, tissue inhibitor of MMP (TIMP)-1) were correlated. Results: RAGE was overexpressed in patients with progressive disease states of chronic hepatitis C. Colocalization studies revealed that RAGE was more associated with lymphoid cells than with myofibroblasts. Overall, we observed disease dependent significant correlations of RAGE mRNA expression with the different applied markers of inflammation (e.g. IL-12, IL-10) and fibrosis (e.g. procollagen-a1(I)). Conclusions: AGE-RAGE interactions may influence the process of inflammation and fibrosis in chronic hepatitis C patients through modulation of inflammatory and profibrogenic gene expression.

38% and 79% vs. 55%). The heterozygous genotypes in the -597 and -6208 Loci are also more frequent than in the control (each 92% vs. 40% and 78% vs. 48%). Conclusions: SNPs of the IL-10 gene might be useful in selecting patients at high risk for developing HCV related cirrhosis and HCC.

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Hepatocellular carcinomas after intraportal transplantation of ovarian fragments in ovarectomized rats – a new model of hepatocarcinogenesis

F. DOMBROWSKI1, C. FLASCHKA1, B. VON NETZER1, L. KLOTZ2, M. EVERT1 1

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Single-nucleotide polymorphisms (SNPs) of the interleukin-10 gene as potential risk factors for the development of liver cirrhosis due to chronic hepatitis C virus (HCV) infection N. BEKTAS1, A. CEOLIN SCHMITT, D. KUBE3, H.P. DIENES1, A. ZUR HAUSEN2 1

Institut fu¨r Pathologie, Universita¨t zu Ko¨ln Institut fu¨r Pathologie, Universita¨t Freiburg 3 Zentrum fu¨r Innere Medizin, Universita¨t Go¨ttingen 2

Aims: Only a subset of patients (20–30%) with chronic HCV infection develop cirrhosis of which approx. 75% develop hepatocellular carcinoma (HCC). One reason for the different susceptibility to develop cirrhosis and HCC may be genetic variability (polymorphism) in immunomodulatory cytokine genes. Interleukin-10 (IL-10) is reported to downregulate immune response by suppressing Th1-type cell development, which contributes to viral persistence because Th1 cells are important in eliminating HCV. Therefore SNPs of the IL-10 gene could help to identify HCV patients at higher risk for the development of HCV-related cirrhosis and HCC. These patients might benefit from an early monitoring, diagnosis and therapy. Methods: 56 paraffin embedded specimens of Caucasians with HCV cirrhosis were DNA-extracted and analyzed for SNPs in the following loci of the IL-10 gene by fluorescence based multiplex polymerase chain reaction: -6752A/T, -824C/T, -1087A/G, -597A/C, -3538AT, -6208C/G, -1354A/G. Regarding the -1087A/ G SNP an additional TaqMan assay was performed. Genotype data of 212 healthy patients were used as control. Results: Homozygous genotypes in the -6752 and -824 loci are significantly more frequent in the cirrhosis group than in the control healthy population (each 65% versus

Institut fu¨r Pathologie, Universita¨t Magdeburg Neurologische Klinik, Universita¨t Bonn

2

Aims: The hepatocarcinogenic potential of natural steroid hormones is not yet clear. We have previously shown that after portal-embolic transplantation of ovarian fragments, the liver acini, draining the blood from the ovarian grafts, show alterations similar to chemically induced amphophilic preneoplastic foci (APF). In this study, we investigated the destiny of these APF in the long term. Methods: Four hundred and forty three male and female Lewis rats were divided into one main group (MG, female) and 11 control groups (CG 1–7 female, 8–11 male). MG animals were ovarectomized and received transplants of ovarian fragments into the right part of the livers. In the CGs, different combinations of ovarectomy or castration, transplantation of ovarian or testicular fragments and administration of the antiestrogen toremifen were used. Animals were killed after 6–30 months. Results: Transplants in the MG showed follicle maturation, were hormonally active and larger than in the CGs. APF developed only in the MG. Hepatocellular adenomas were significantly more numerous in the MG animals (n ¼ 12) than in all CGs together (n ¼ 4). 18 hepatocellular carcinomas (HCC) developed in the MG, so that 78% of MG animals showed at least one hepatocellular carcinoma after 24 and 30 months. However, no single HCC emerged in any of the CGs. Toremifen administration completely abolished hepatocarcinogenesis in this model. Testicular grafts showed no influence on the liver tissue. Conclusions: Preneoplastic liver acini, draining the blood from intraportally transplanted and hormonally stimulated ovarian fragments, may develop into HCA and HCC, most likely caused by a receptor-mediated carcinogenic influence of natural estrogens, derived from the ovarian grafts.

ARTICLE IN PRESS Posters: Gastrointestinal Pathology I / Pathology – Research and Practice 201 (2005) 153–300

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Absence of PRDX1 gene mutations in human hepatocellular carcinomas J. GISIN, A. PERREN, M. BAWOHL, W. JOCHUM Institut fu¨r Klinische Pathologie, Universita¨t Zu¨rich

Aims: Oxidative stress due to free radicals has been implicated in the pathogenesis of many cancers. Various chronic liver diseases display formation of reactive oxygen species (ROS) and are associated with a high risk of hepatocellular carcinoma (HCC) suggesting that defects in ROS scavenging systems may contribute to the initiation and/or promotion of HCC. Peroxiredoxin (PRDX) 1, located on chromosome 1p34, is an intracellular anti-oxidant protein with thioredoxindependent peroxidase activity which protects cells against oxidative stress by inactivating H2O2. PRDX1 hemizygous mutant mice develop HCC with high frequency (Nature 424:561-5, 2003). Since chromosome 1p deletions are frequent in human HCC, we have tested the hypothesis that mutations or polymorphisms of the PRDX1 gene contribute to hepatocelluar tumorigenesis. Methods: A series of 27 HCC and two hepatocellular adenomas (HA) was analysed for PRDX1 allelic deletions and sequence alterations by loss of heterozygosity (LOH) analysis, PCR/denaturing gradient gel electrophoresis (DGGE) and DNA sequencing. Results: LOH of at least one of four microsatellite markers of the 1p34.1 region located within 1 Mb of the PRDX1 gene was observed in two out of 27 informative HCCs (7.4%), but was absent in the two HA. No mutations or polymorphisms in the translated exons 2-6 of the PRDX1 gene were found in the HCC and HA analysed. Conclusions: We conclude that mutations of the PRDX1 gene are rare events in hepatocellular tumors indicating that other genes on chromosome 1p contribute to liver tumorigenesis.

197 Expression of the interleukin-12 p40 homologue EBI3 in Hepatitis C and B virus related hepatocellular carcinogenesis A.C. SCHMITT, N. BEKTAS1, M. MU¨LLER1, G. NIEDOBITEK2, H.P. DIENES1, A. ZUR HAUSEN Institut fu¨r Pathologie, Universita¨t Freiburg 1 Institut fu¨r Pathologie, Universita¨t Ko¨ln 2 Institut fu¨r Pathologie, Universita¨t Erlangen Aims: Epstein-Barr virus induced gene 3 (EBI3) is a homologue of the interleukin (IL)-12 p40 subunit which by forming heterodimers with the p35 subunit of IL-12 is able to inhibit the development of a Th1 immune

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response. The clearance of some viral infections, e.g. HCV, is characterized by a predominant and sustained Th1 response. Weak or absent Th1 response and the presence of Th2 cytokines has been linked to the development of chronic HCV infection. We tested the expression of EBI3 in virus related, i.e. HBV and HCV, hepatocellular carcinogenesis. Methods: Paraffin embedded tissues of liver biopsies and resections specimens (n ¼ 138) were investigated for the expression of EBI3 by immunohistochemistry using a monoclonal EBI3 antibody. Patients included 52 cases with chronic viral hepatitis (30 HCV+, 20 HBV+ and 2 HBV/HDV+) and 41 with unspecific reactive changes as control, 26 cirrhosis (17 HCV+, 6 HBV+, 1 HCV and HBV+ and 2 without viral etiology), 19 patients with HCC (13 HCV+, 3 without viral etiology and 3 with unknown viral status). Results: EBI3 expression was restricted to portal dendritic cells (DC) and sinudoidal liver macrophages. In contrast to HBV-positive specimens and controls, EBI3 was highly expressed in all stages of HCV related hepatocellular carcinogenesis. Conclusions: The marked increase of EBI3 expression in HCV-related hepatocellular carcinogenesis points to an important role of EBI3 in the context of an inappropriate response for the viral clearance of HCV, thus contributing to the establishment of chronic HCV infection.

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Liver Metastases. Incidence and Distribution Regarding the Primary Tumors in the Liver Register of the University of Cologne H.U. KASPER, V. DRIES, U. DREBBER, H.P. DIENES Institut fu¨r Pathologie der Universita¨t zu Ko¨ln Aims: Metastases are the most common malignant tumors of the liver. In a greater amount of cases the primary tumor is known. In about 60%, however, they can also be the first symptom of a metastastic tumor disease. Knowledge of the percentage of primary tumors will help in the differential diagnosis. Methods: We evaluated the files of the institute of pathology of the university of cologne for liver metastases and their primary tumors. Results: In the files of the institute of pathology 12.161 liver tissue cases are registered. Of them, 1.357 cases (11.2%) showed tumors or tumor like diseases. Liver metastases of solid tumors were the largest group of the neoplasias with 611 cases (5.0%) followed by hepatocellular carcinoma (380 cases; 3.1%). Other entities were rare and include cholangiolar carcinoma (0.5%), vascular tumors (0.4%;), 72 Lymphomas (0.4%), focal

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nodular hyperplasias (0.36%) and liver cell adenomas (0.23%). Adenocarcinoma the largest group of metastases with 400 cases (65.5%). 48.2% of this group were metastases of colorectal cancer, 13.5% of pancreatic cancer, 13% of breast cancer, 6.2% of gastric cancer, 4.5% of lung cancer and 3.7% of esophagic cancer. Neuroendocrine Carcinoma were the second largest group with 16% of liver filiae. Other entities were rarely found. Metastases in cirrhotic livers were seldom. Conclusions: Liver metastases dominate the malignant liver disease and are seen twice as often as primary liver carcinoma. Most liver metastases derive from colorectal carcinoma. Surprisingly, neuroendocrine carcinoma are the second largest group. This knowledge will help in the differential diagnosis of liver tumors.

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Apoptosis investigation in liver metastases with precision cut tissue slices (PCTS) H.U. KASPER, E. KONZE, D. STIPPEL1, U. DEBBER, H.P. DIENES Institut fu¨r Pathologie Klinik fu¨r Visceral- und Gefa¨ßchirurgie, Universita¨t Ko¨ln 1

Aims: Apoptosis induction in tumor cells, e.g. by tumor infiltrating lymphocytes, is one of the main host mechanisms for cancer defense. Liver metastases are a frequent event in patients with colon cancer. The host defense in the liver is not yet fully understood. We used PCTS of liver metastases of colon carcinoma to look into the reagibility of tumor tissue to apoptotic stimuli. Methods: PCTS were prepared from 3 human liver metastases of colorectal carcinoma using Brendel Vitron Tissue Slicer and incubated submersed in supplemented RPMI medium. Apoptosis was induced by CD95 activating antibody (0,1 actinomycin D [AD]) and by TNF a (1 ng/ml, 10 ng/ml, 10 ng/ml and 1 mg/ml AD). The slices were formalin fixed and paraffin embedded after 6, 12, and 24 h. Apoptosis was detected immunohistochemically with mg/ml, 1 mg/ml and 1 mg/ml and 1 mg/ml M30CD (caspase-cleaved cytokeratin-18 fragments). Results: Using CD95, a slight increase in apoptosis was seen after 6 h. After 12 h, the apoptotic rate was significantly elevated with all 3 concentrations compared to the spontaneous apoptosis rate. After 24 h, only costimulation with AD revealed a significant apoptotic stimulus. Using TNF alpha 10ng/ml with AD, a significant apoptosis rate was seen after 6 h and with 10ng/ml with or without AD also after 12 h. After 24 h

or with low dose TNF alpha no significant effect was seen. Conclusion: Activating CD95 antibody and TNF alpha can induce apoptosis with a peak at 12 h stimulation time. Thus, tumor tissue of liver metastases of colorectal cancer shows sensitivity to these apoptotic pathways. Stimulation of both pathways via TIL or in artificial setups would allow prevention or defense of metastases. PCTS will allow deeper insight in the pathomechanism of apoptosis in cancer tissue.

200 Cytotoxic equipment of tumor infiltrating lymphocytes (TIL) in liver metastases H.U. KASPER, E. KONZE, D. STIPPEL1, H.P. DIENES Institut fu¨r Pathologie Klinik fu¨r Visceral- und Gefa¨ßchirurgie, Universita¨t Ko¨ln 1

Aims: Metastases are the most common malignant tumors in the liver. For host defense, the adequate function of the immune system is necessary. TIL are the immune cells responsible for destruction of tumor cells. The cytotoxic potential of TIL is variable. Therefore we investigated the cytotoxic ability of TIL in liver metastases. Methods: Frozen tissue of 40 liver metastases of different primary tumors were investigated immunhistochemically for the expression of granzyme B, perforine, TIA, CD95 ligand and CD95. Apoptosis rate was determined by staining with M30CD. The intratumoral and peritumoral TIL were assessed regarding of their equipment of the cytotoxic enzymes separately. Results: Intratumoral lymphocytes were detected with the following equipment: TIA: 0,4TIL/HPF, Perforine 1TIL/HPF, Granzyme 2,2 TIL/HPF, CD95L 3,1TIL/ HPF, CD95 5,8TIL/HPF. Peritumoral lymphocytes were detected with the following equipment: TIA: 0,3TIL/HPF, Perforine 0,9TIL/HPF, Granzyme 2,7 TIL/HPF, CD95L 7 TIL/HPF, CD95 54,2 TIL/HPF. The mean apoptotic rate was 2,4/HPF. In 9 cases tumor cells also expressed CD95 and in 3 cases tumor cells also expressed CD95L. There was no correlation between histological subtype, diameter of metastases, apoptosis rate and cytotoxic equipment of TIL. Conclusion: TIL in the tumor itself as well as in the interface region between tumor and liver are equipped with a very low amount of cytotoxic enzymes. The CD95/CD95L-pathway seems to be the main antitumoral mechanism. An insufficient cytotoxic capacitiy of the immune system in the liver could be one of the main reasons for development of liver metastases.

ARTICLE IN PRESS Posters: Gastrointestinal Pathology II / Pathology – Research and Practice 201 (2005) 153–300

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Intraductal papillary mucinous carcinoma in an explanted liver: A case report. M. KOSMAHL, F. BRAUN1, J. LU¨TTGES, G. KLO¨PPEL

Institut fu¨r Pathologie, Universita¨t Kiel 1 Klinik fu¨r allgemeine Chirurgie und Thoraxchirurgie, Universita¨t Kiel Introduction: Liver transplantation is a live-saving procedure, that includes an extensive diagnostic work up before transplantation. Case report: A 55-year-old male patient presented with pain in the upper right abdomen, weight loss and progressive liver dysfunction. Liver histology showed progressive purulent cholangitis and cholestasis with focal necrosis. The patient underwent liver transplantation under the suspicion of liver cirrhosis and druginduced cholangitis. Macroscopically, the explanted liver showed multiple nodules in the liver capsule and fibrosis around the bile ducts. No cirrhosis was present. Histologically, the bile ducts were dilated and showed a proliferation of columnar epithelial cells with papillary projections. An invasive mucinous component with signet-ring cells was seen. Both intraductal and invasive tumor components were positive for MUC1 and negative for MUC2. The diagnosis was intraductal papillary mucinous carcinoma of the intrahepatic biliary tract. Conclusion: Intraductal papillary mucinous neoplasms of the biliary tract are rare. The nomenclature for such lesions is not yet standardized. They have been designated as biliary papillomatosis, papillary cholangiocarcinoma or mucin-hypersecreting bile duct tumors. They reveal clinical, radiological and histological similarities to pancreatic intraductal papillary mucinous neoplasms.

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gastrointestinal tract, via the processing of signalling peptides, such as angiotensin II. Angiotensin II mediates the decrease in gastric mucosal blood flow observed in gastric ulcers, and ACE inhibitors improve ulcer healing in animal models. Methods: The expression of ACE in human gastric tissues was investigated immunohistochemically, using ACE-specific monoclonal antibodies on paraffinembedded sections obtained from biopsies of the gastric antrum and corpus (14 patients with H. pyloriassociated gastritis and 17 control patients with no or mild gastritis and without any evidence of H. pylori infection) and resection specimens of gastric ulcers (13 patients). Results: The non-lesional luminal and foveolar epithelium did not express ACE, while fundic chief cells showed weak to moderate cytoplasmic staining, and the mucin-secreting cells of the antral and pyloric region exhibited membranous expression at the apical cell surface. The expression of ACE in the antral glands was related to inflammation, with ACE being more commonly expressed in inflamed mucosa than in noninflamed mucosa. ACE was also found in endothelial cells of the granulation tissue in each specimen from gastric ulcerations. Conclusions: The localisation of ACE in the microvasculature of gastric ulcers may account for the ulcer healing effect of ACE inhibitors observed in animal models. Inhibition of angiotensin II production by treatment with well-established ACE inhibitors may also be a therapeutic option to assist the healing process for gastritis and gastric ulceration in humans.

203 Quantitative analysis of cellular components of carditis in patients with reflux and helicobacter pyloriinfection H.-U. SCHILDHAUS, C. KROGEL, A. ROESSNER

Posters: Gastrointestinal Pathology II 202 The distribution of angiotensin converting enzyme in gastritis and stomach ulcer may have implications for ulcer healing S.CARL-MC GRATH1, U. LENDECKEL2, M. EBERT3, A. ROESSNER1, C. RO¨CKEN1 1

Institute fu¨r Pathologie Experimentelle Innere Medizin 3 Klinik fu¨r Gastroenterologie, Universita¨t Magdeburg 2

Aims: Angiotensin converting enzyme (ACE) is involved in the regulation of growth and differentiation in the

Institut fu¨r Pathologie, Universita¨t Magdeburg Aims: Inflammation of the gastric cardia is controversial. Its pathogenetic relationship to reflux and H.p.infection still remains unclear. We aimed at characterizing the type, grade and cellular components of inflammation in the gastric cardia. Methods: A total of 80 biopsy specimens (sets consisting of antrum and cardia biopsies) from 20 patients affected by antrum dominant H.p.-gastritis and 20 patients with clinical symptoms of GERD (lacking H.p.-infection) were analyzed. The degree of inflammation in cardia and antrum was graded according to the Sydney system. Granulocytes, lymphocytes, plasma cells, eosinophils, and CD3, CD4, CD8, CD56, CD57, CD68 and granzymeB immunopositive cells were counted in the

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lamina propria (cells/0.2 mm2) and in the epithelium (cells/300 epithelial cells). Results: The grade of acute carditis was higher in cases of antrum dominant H.p.-gastritis (po0:001; H.p.: 25.876.7, reflux: 6.072.2 granulocytes/0.2 mm2; p ¼ 0:01), whereas chronicity of carditis did not differ significantly among both groups. Numbers of CD57positive (NK and NK-like) cells per 0.2 mm2 lamina propria were higher in H.p. infection compared with reflux (cardia: 8.870.9 vs. 3.970.77, po0:001; antrum: 8.171.2 vs. 2.970.6; po0:001). Among the intraepithelial inflammatory cells in the cardiac mucosa, only eosinophils occurred more frequently in H.p. cases (p ¼ 0:015). Cytotoxic (granzymeB-positive) cell counts in the antrum were significantly higher in H.p. than in reflux patients (42.776.4 vs. 20.571.8 cells/0.2 mm2; p ¼ 0:003), but did not differ in the cardia, as was seen for the CD4/CD8-ratio (antrum: p ¼ 0:03; cardia: n.s.). Conclusions: Carditis occurred in both reflux and H.p. patients, but differs in terms of their cellular components. CD57-positive cells appear to be associated with H.p. infection rather than reflux.

204 Hepatoid type of gastric cancer: Diagnostic and prognostic relevance A. DELLMANN1, H. HANNIG2, N. HAGEN1, L. FUEZESI3, K. DONHUIJSEN1 1

Institut fu¨r Pathologie, Klinikum Braunschweig Zentrale Einrichtung fu¨r molekulare Diagnostik, Klinikum Braunschweig 3 Zentrum Pathologie, Universita¨t Go¨ttingen 2

Aims: Despite their histological inhomogeneity gastric cancer is classified with respect to cohesiveness of tumor cells (Lauren). However, the hepatoid type is associated with worse prognosis although the pattern is cohesive. Methods: We observed a 67-year-old man with gastric cancer of hepatoid type combined with features of muzin producing adenocarcinoma in small areas. After microdissection the different parts of the tumor were analysed for immunohistochemistry (CK7, 20, MUC1, MUC2, CDX2, CEA, AFP, HepPar1) and by PCR for microsatellite instability in 5 loci, loss of heterocygosity (LOH), K-ras and p53 mutations and by comperative genomic hybridisation (CGH). Results: The hepatoid differentiated carcinoma exhibited an excessive angioinvasion in venoles of T. submucosa although the carcinoma was growing widely limited to T. mucosa. Immunohistochemically the hepatoid part reacted positive for HepPar1 antibody but not for MUC1, CEA and CK20 as the mucinous

part did. The molecular-pathological results did not reflect such differences and some of the chromosomal imbalances were identical. Conclusions: The histopathologist should pay attention to the very rare hepatoid type of gastric cancer, which is showing as well strong cohesiveness as an excessive angioinvasion.

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Cocarcinogenic effects of islet hormones and NNitrosomor-pholin in hepatocarcinogenesis after portal embolic transplan-tation of pancreatic islets in streptozotocin-diabetic rats M. EVERT, C. JOST, S. MANEKELLER, F. DOMBROWSKI Institut fu¨r Pathologie, Universita¨t Magdeburg

Aims: A morphologically very similar process of hepatocarcino genesis in rats can be induced either by administration of N-Nitrosomorpholin (NNM) or by low-number transplantation of islets of Langerhans into the livers of diabetic rats. In both models, hepatocellular adenomas (HCA) and carcinomas (HCC) develop stepwise from preneoplastic lesions, beginning with the formation of clear-cell foci of altered hepatocytes (CCF). In this study, we investigated both models for their cocarcinogenic effects. Methods: Three different protocols of NNM-administration were used, i.e., a single NNM-dose of 200 mg/kg (experiment A), a daily medium NNM-dose of 16 mg/kg for 6 weeks (experiment B) or a low NNM-dose of 5 mg/ kg for the whole experiment (experiment C). These were each combined with different transplantation procedures, i.e., low-number (n ¼ 350; main group [MG]) and high-number (n ¼ 1000; control group [CG] 1) islet transplantation or low-number transplantation of latex particles (CG 2) in streptozotocin-diabetic animals as well as low-number islet (CG 3) or sham (CG 4) transplantation in non-diabetic rats. 305 animals were sacrificed between 4 and 52 weeks after transplantation. Results: In the low-dose experiment A, cocarcinogenic effects were not observed until week 52, visible as an increase in the number of CCF in the MG. However, corresponding to the higher NNM-doses, cocarcinogenic effects were much stronger in B and in C, leading to an increase in number and to a considerably earlier appearance of HCA and HCC in the MG animals. Conclusion: The local influence of islet hormones, i.e., insulin, on the islet-graft-adjacent hepatocytes accelerates NNM-induced hepatocarcinogenesis in diabetic rats.

ARTICLE IN PRESS Posters: Gastrointestinal Pathology II / Pathology – Research and Practice 201 (2005) 153–300

206 Cytogenetic characterisation of primary and metastatic pancreatic acinar cell carcinomas F. BERGMANN, S. AULMANN, I. ESPOSITO, R. PENZEL, H. FRIESS1, P. SCHIRMACHER Pathologisches Institut 1 Abteilung Allgemein-, Viszeral- und Unfallchirurgie, Universita¨t Heidelberg Aims: Pancreatic acinar cell carcinomas (PAC) are associated with a poor prognosis. At the time of diagnosis, approximately half of the patients display metastatic spread, usually affecting regional lymph nodes and the liver. To date, cytogenetic and molecular analyses were limited to few cases of primary PAC, however, no data have been obtained for their metastases. Therefore, the present study was designed to detect possible differences between primary and metastatic PAC. Methods: Three primary PAC, one corresponding regional lymph node metastasis and two corresponding hepatic metastases were characterised by means of comparative genomic hybridisation, fluorescence in situ hybridisation, and immunohistochemistry. Results: Chromosomal imbalances were detected in all tumors, most consistently affecting gains of 1q, 8q, 12, 17, 20q and 22q, as well as losses of 1p, 4q, 11q and 18q. In general, similar chromosomal imbalances were seen in primary and metastatic PAC. However, some changes, including gains of 3p, 8q, and 17q, were more frequent or limited to metastatic PAC. Low level amplifications of c-MYC (8q24) were detected in the primary tumor and in one hepatic metastasis of one patient. Neither primary nor metastatic PAC revealed expression of Her2/Neu (17q21). Conclusions: PAC display a consistent chromosomal profile, which shows a large overlap between primary and metastatic PAC. Chromosomal imbalances, more frequently or exclusively occurring in metastatic PAC, indicate chromosomal loci possibly harboring candidate genes that are involved in tumor progression and spread.

207 Trefoil factor family protein 1 (TFF1, pS2) –

the missing link between gallstones and gallbladder carcinoma? C. LANGNER, M. LEMMERER1, P. REHAK2, P. KORNPRAT1

Institut fu¨r Pathologie, Medizinische Universita¨t Graz, Austria 1 Chirurgische Klinik, Medizinische Universita¨t Graz, Austria

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2

Abteilung fu¨r Biomedizinische Technik und Datenverarbeitung, Medizinische Universita¨t Graz, Austria Aims: Trefoil factor family protein 1 (TFF1) interacts with mucins to protect gastrointestinal epithelium against injury and contributes to mucosal repair promoting epithelial cell migration and restitution. Moreover, TFF1 has antiproliferative and antiapoptotic effects and promotes cell scattering and invasion. Our study aimed to investigate TFF1 expression in gallbladder cancer in comparison to non-neoplastic gallbladder mucosa. Methods: TFF1 immunoreactivity was investigated in healthy and inflamed non-neoplastic gallbladder mucosa as well as gallbladder carcinomas (n ¼ 57) and corresponding metastases (n ¼ 18) using a tissue microarray technique. Results: TFF1 was absent in healthy mucosa, commonly present in epithelium with inflammatory alterations, and seen in 35% of primary and 24% of metastatic cancer tissues. Immunoreactivity decreased with increasing tumour category (p ¼ 0:009) and increasing tumour grade (p ¼ 0:001). Patients with TFF1 positive tumours showed a more favorable outcome compared to patients with TFF1 negative tumours in univariate analysis (p ¼ 0:006). However, multivariate analysis proved resection status and tumour grade as the only independent prognostic factors. Conclusions: TFF1 is expressed in inflamed nonneoplastic gallbladder epithelium and in low stage and low grade gallbladder carcinomas. Thus, TFF1 may be the missing link between gallstones, chronic cholecystitis, and gallbladder cancer. Further studies are needed to evaluate whether TFF1 immunostaining can be used as a diagnostic tool to identify patients with a more favourable outcome.

208 Nodal stage dependent mRNA expression of matrix metalloproteinases and their inhibitors in T3 stage colorectal carcinomas and corresponding normal mucosa F. HALLER, H.-J. SCHULTEN, S. SCHWAGER, B. GUNAWAN, L. FU¨ZESI Zentrum Pathologie, Universita¨t Go¨ttingen Aims: The family of matrix metalloproteinases (MMPs) is characterized by the ability to degrade extra-cellular matrix. Physiologically, their members are involved in wound healing or tissue structure, but, due to their proteolytic activity, they may also play an important role in tumor invasion, especially in lymphogenic metastasis. The aim of this study was to assess the role of MMPs and corresponding tissue inhibitors of MMPs (TIMPs) in colorectal carcinomas and specifically,

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to evaluate their role as predictive markers for lymphogenic metastasis. Methods: By using quantitative RT-PCR, we analyzed the expression of 7 MMPs (MMP-1, -2, -9, -14, -15, -16, -24) and 3 TIMPs, (TIMP1, -2, -3) in 17 node-negative and 11 node-positive T3 stage colorectal carcinomas and in five normal mucosa samples. Results: Comparing node-negative with node-positive tumors no significant association could be demonstrated for differential expression of any of the MMPs or TIMPs analyzed. Comparing tumor tissue with normal mucosa, MMP-1, -14, -15, and TIMP-1 were strongly overexpressed (2–120 fold) in the carcinoma tissue. Conclusions: These results indicate a role for MMP1, 14, -15 and TIMP-1 in tumor development and progression rather than in lymphogenic metastasis, and may contribute to recent observations that overexpression of at least some MMPs in tumor is limited to the invasive margin.

209 Expression and prognostic significance of the deathinducing CD95/CD95L-system in curatively resected colon carcinomas J. STRA¨TER, U. HINZ1, C. HASEL, U. BHANOT, G. MECHTERSHEIMER2, T. LEHNERT1, P. MO¨LLER Abteilung Pathologie, Universita¨t Ulm 1 Chirurgische Universita¨tsklinik, Universita¨t Heidelberg 2 Institut fu¨r Pathologie, Universita¨t Heidelberg Aims: Loss of CD95 expression in colorectal carcinoma cells may be associated with disease progression while neo-expression of CD95L in tumour cells may contribute to immune evasion. To further explore the role of the CD95-system in colon carcinomas Methods: CD95 and CD95L expression was examined by immunohistochemistry in 128 R0-resected UICC stage II/III colon carcinomas and correlated with disease-free survival. Results: CD95 expression was retained in all tumour cells in 30 carcinomas (23.4%), whereas it was reduced or completely lost in the others. Loss of CD95 in tumour cells was related to adverse prognosis in uni- and multivariate analysis (p ¼ 0:046 and p ¼ 0:036; respectively). Tumour-infiltrating lymphocytes (TIL) were the major source of CD95L in colon carcinomas. CD95L+ TIL were present in 83% of cases, whereas CD95L was found in tumour cells in only 12% of cases. Moreover, a high rate of CD95L+ TIL correlated with prolonged disease-free survival of patients in UICC stage II (p ¼ 0:05), but not in stage III.

Conclusions: Loss of CD95 in tumour cells may be an independent prognostic factor in colon carcinomas. The CD95L counterattack is not a relevant feature in colon carcinoma, but CD95L+TIL may contribute to tumour control in the early stages of the disease.

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Expression of pS2 in colorectal carcinomas: correlations with histopathological classifications and prognosis S.E. BALDUS, S.P. MO¨NIG1, B. HACKENBROICH, S. LANDSBERG, P.M. SCHNEIDER1, A.H. HO¨LSCHER1, H.P. DIENES

Institut fu¨r Pathologie, Universita¨t Ko¨ln 1 Klinik fu¨r Viszeral- und Gefa¨ßchirurgie, Universita¨t Ko¨ln Aims: pS2/ trefoil factor 1 (TFF1) represents a member of the trefoil peptide family. TFF proteins are secretedtype proteins expressed in the mucous layer of the gastrointestinal tract. They may associate with mucins as well as other unidentified membrane receptors on cell surfaces. TFF1 plays a key role in mucous-barrier formation and in mucosal repair through promotion of repair after injury. In the present study, its role in colorectal tumor biology was investigated. Methods: A series of 239 patients with colorectal cancer was characterized immunohistochemically applying a pS2 specific monoclonal antibody and a labelled streptavidin biotin procedure. After a semiquantitative evaluation of the staining results, statistical correlations with clinicopathological variables as well as overall survival were performed. Results: In a series of 239 colorectal carcinomas, 149 (62.3%) did not show a pS2 expression in more than 5% of the tumor cells. Only 13.8% exhibited a pS2 strong positivity (in more than 35% of the tumor cells). At a cut-off point of 35% positive tumor cells, statistically significant correlations with localization, staging as well as grading were not observed. However, carcinomas exhibiting an elevated mucinous component were characterized by a stronger pS2 immunoreactivity. In uni- as well as multivariate survival analysis, pS2 expression in more than 35% of the tumor cells revealed as an (independent) factor indicating a lower survival probability. Conclusions: According to our results, a strong expression of pS2 (TFF1) in colorectal carcinomas is associated with an elevated mucinous tumor component as well as a worse prognosis.

ARTICLE IN PRESS Posters: Gastrointestinal Pathology II / Pathology – Research and Practice 201 (2005) 153–300

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Problems in the definition and application of the diagnostic terms hamartoma’’ and adenoma’’ for rare ’’ ’’ gastrointestinal polyposis syndromes (i.e. Peutz Jeghers syndrome) W. BACK

Pathol. Institut, Univ.-klinikum Mannheim, Universita¨t Heidelberg Aims: The Peutz-Jeghers syndrome (PJS) is a rare genetic syndrome characterized by multiple intestinal polyps, labial pigmentations and a spectrum of benign and malignant tumours. Microscopically the polyps of PJS are regarded to be diagnostic due to their branching streaks of smooth muscle cells and dysplasia-free epithelium. Since in few studies the accuracy of the histopathological typing in hamartomatous polyposis syndromes has been questioned, the polyps of a German cohort of PJS patients were studied. Methods: Based on a combined clinical, morphological and genetic approach 24 patients from 21 different families were diagnosed as PJS patients. Clinical histories, molecular genetic findings and follow up data (mean 12 years) of all these patients were analyzed. From these patients 30 gastric, 120 intestinal and 80 colorectal polyps could be reviewed as paraffin tissue. Results: Characteristic histopathological findings of PJStype polyps were present in virtually all intestinal polyps, but only in 2/3 of the colorectal polyps and in about 1/2 of the gastric polyps. Moreover 10 polyps of the small intestines and the colon showed low grade dysplastic features qualifying for an adenoma‘‘. Two intestinal ’’ adenocarcinomas developped in PJS polyps. Conclusions: The PJS polyps in the stomach often show unspecific findings. Congeners of PJS polyps in the colorectum are especially serrated type adenomas‘‘ (if ’’ low grad dysplasia is present) and polypoid lesions in the mucosa prolaps syndrome‘‘. PJS polyps should be ’’ considered potentially neoplastic. The actual definitions of the colorectal adenoma‘‘ and hamartoma‘‘ should ’’ ’’ be changed.

212 Wnt-signaling and apoptosis in rectal cancer after short-term neoadjuvant radiotherapy N. GASSLER, I. HERR1, M. KEITH, J. KARTENBECK1, P. SCHIRMACHER, F. AUTSCHBACH Pathologisches Institut, Universita¨t Heidelberg 1 Deutsches Krebsforschungszentrum, Heidelberg Aims: Recent concepts in the treatment for primary rectal cancer include the combination of surgery and

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short-term neoadjuvant radiotherapy (STNR). STNR is usually given in a dose of 25 Gy over 5 days in order to reduce local recurrence rates, but clinical studies have shown that local recurrence is found in some patients despite STNR. Methods: Molecular patterns of the Wnt- and apoptosis pathways as well as expression of junction-associated molecules were examined by immunohistochemistry and molecular techniques such as LightCycler RT-PCR and Western blot analysis in 25 sporadic rectal adenocacrinoma specimens derived from STNR-patients or nonpretreated donors, respectively. Results: The molecular pattern in response to STNR found was heterogeneous. Responders from STNR did show activation of apoptosis and cellular remodeling, whereas the group of non-responders from STNR did not show such reactions and was very similar to untreated controls. In detail, enhanced expression of beta-catenin was generally mediated by STNR, but exclusively in the responder group impaired expression of c-Myc and junction-associated molecules as well as cleavage of poly-ADP-ribose polymerase and of the caspase substrate cytokeratin 19 were found. Conclusions: The molecular profiles suggest that STNR interferes with Wnt-signaling and c-Myc expression. STNR in its present form is not suitable to fully complete the sequence of apoptosis in all rectal adenocarcinomas.

213 Prognostic value of histopathological tumor regression in locally advanced rectal cancer after neoadjuvant 5-FU based chemoradiotherapy C. JAKOB1, T. LIERSCH2, D.E. AUST1, W. MEYER1, G.B. BARETTON1 1

Institut fu¨r Pathologie, Universita¨tsklinikum der TU Dresden 2 Klinik fu¨r Viszeralchirurgie, Universita¨tsklinikum Go¨ttingen Aims: In locally advanced rectal cancer neoadjuvant 5fluorouracil (5-FU) based chemoradiotherapy (CRT) leads to marked tumor reduction and improves local control. However, little is known about the influence of neoadjuvant CRT on disease-free survival. The local effect of CRT is usually assessed by histopathological tumor regression. The aim of this study was to correlate histopathological tumor regression in surgical resection specimens after neoadjuvant CRT with disease-free survival. Methods: Surgical resection specimens (n ¼ 40) from patients with rectal cancers stage II or III (UICC 2003) receiving neoadjuvant CRT were assessed for tumor

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regression according to Dworak et al. Follow-up ranged from 29 to 77 months. Results: After neoadjuvant CRT 32 patients showed locally marked (grade 2–4) and 8 patients no or little (grade 0–1) tumor regression. In 15 patients (37.5%) downstaging was achieved with a switch from N+ to N0. During follow-up period 10 patients (25%) developed a tumor recurrence (median time to progression 11.5 months). Interestingly, all these patients showed locally marked tumor regression (grade 2–4), but, remained lymph node positive (ypUICC stage III). Conclusions: These data suggest that post-treatment lymph node status is the crucial factor for diseasefree survival independently of the local tumor regression.

214 Immunohistochemical analysis of thymidylate synthase, thymidine phosphorylase, and dehydropyrimidine dehydro-genase expression in rectal cancer: correlation with histopathological tumor regression after neoadjuvant chemoradiotherapy C. JAKOB1, D.E. AUST1, T. LIERSCH2, W. MEYER1, G.B. BARETTON1 1

Institut fu¨r Pathologie, Universita¨tsklinikum der TU Dresden 2 Klinik fu¨r Viszeralchirurgie, Universita¨tsklinikum Go¨ttingen Aims: In locally advanced rectal cancer neoadjuvant 5fluorouracil (5-FU) based chemoradiotherapy (CRT) leads to marked tumor reduction and improves local control. Thymidylate synthase (TS), thymidine phosphorylase (TP), and dihydropyrimidine dehydro-genase (DPD) are important markers to predict tumor response to 5-FU therapy. This study determines the correlation between TS, TP, and DPD protein expression and the response to neoadjuvant treatment assessed by histopathological tumor regression. Methods: Pre-operative biopsies (n ¼ 22) and surgical resection specimens (n ¼ 40) from patients with rectal cancers stage II or III (UICC 2003) receiving neoadjuvant radiochemotherapy were studied for TS, TP, and DPD protein expression by immunohistochemistry using three different scoring systems. Results were compared with histopathological assessment of tumor regression. Results: A significant correlation between protein expression and tumor response could only be seen when both, staining intensity and staining pattern, were considered. TS protein expression was above the median in pre-treatment biopsies and resection specimens in all non-responders (p ¼ 0:04). TP protein expression was below the median in all non-responders (p ¼ 0:02).

Conclusions: These data indicate that immunohistochemistry is a suitable method to determine the correlation between TS-, TP-protein expression and histopathological tumour regression. However, precise results can only be achieved if staining intensity as well as staining pattern within the tumor is considered.

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Endometriosis of the vermiform appendix – A systematic analysis of 60 cases C. LANGNER, M. THALHAMMER1

Institut fu¨r Pathologie, Medizinische Universita¨t Graz, Austria 1 Chirurgische Klinik, Medizinische Universita¨t Graz, Austria Aims: The vermiform appendix represents one of the commonest sites of occurrence of intestinal endometriosis. Our study aimed to analyze the epidemiology, macroscopic and histopathologic changes of appendiceal endometriosis. Methods: A survey of the archives of our institute was performed (1/1984–12/2003), and the original slides of cases of appendiceal endometriosis were reviewed. Results: Sixty cases of appendiceal endometriosis were identified among 31.596 females who underwent appendectomy (0.2%) and 3847 patients with endometriosis (1.6%). Mean and median age was 37.2 and 36.5 years (range 20–90), respectively. In 18 (30%) cases the macroscopic appearance was suggestive of ‘‘tumour’’; however, in only one of these cases diagnosis of endometriosis was suspected. In 41 (68%) cases the endometriosis was localized in the distal end, while in the other cases the localization remained unclear. The muscularis propria was involved in 54 (90%), the subserosa in 39 (65%), the mesenteriolum in 8 (13%) and the submucosal layer in 6 (10%) cases, respectively. Only 13 (22%) cases showed acute appendicitis, whereas 29 (48%) specimens showed fibrosis (including 6 cases of neurogenic appendicopathy) without signs of acute inflammation. Eighteen appendices were entirely normal apart from endometriosis. In 21 (35%) patients synchronous foci of endometriosis were identified during the operation. Conclusions: Endometriosis of the vermiform appendix is a rare disease that is mainly discovered as a result of incidental appendectomy. It prevails in the distal end, and the muscularis propria is mainly involved. Histologic signs of acute inflammation are seen only in a minority of cases, whereas fibrotic changes (including neurogenic appendicopathy) are common.

ARTICLE IN PRESS Posters: Molecular Pathology I / Pathology – Research and Practice 201 (2005) 153–300

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Mutations of the KIT gene and KIT (CD117) protein expression in anorectal melanoma B.M. HELMKE, G. MECHTERSHEIMER, P. SCHIRMACHER, R. PENZEL Institut fu¨r Pathologie, Universita¨t Heidelberg

Aims: Anorectal melanoma (AM) is a rare and highly malignant tumor, that is histologically and immunhistochemically similar to cutaneous melanoma. In addition, amelanotic AM can be misinterpreted as sarcoma or gastrointestinal stromal tumor. In this study, the frequency of mutations in the KIT gene (exons 9,11 and 13) and the protein expression of KIT (CD117) were investigated in a series of 23 AM. Methods: KIT exons 9, 11 and 13 were screened for mutations using PCR amplification of genomic DNA and subsequent SSCP analysis, followed by direct sequencing. Immunohistochemistry was performed using a polyclonal anti-KIT (CD117) antibody (Dako; A4502). Results: KIT mutations detected in 3 out of 23 AM were exclusively found in exon 11 and consisted of two activating point mutations (CTT4CCT resulting in a L576P transition), and of one deletion (C1755 4589X). 18 out of 23 AM showed KIT (CD117) protein expression in variable amounts while five cases were KIT-negative throughout. Conclusions: Expression of KIT (CD117) is a frequent event in AM. Since a subset of AM also exhibit axtivating KIT mutations, these tumors might be candidates for a therapy with the tyrosine kinase inhibitor imatinib (Gleevecs).

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protocol for the detection of HCV in FFPE tissue for routine diagnostic purposes. Methods: Thirty FFPE specimens of cirrhotic liver tissue (12 HCV-positive, 18 HCV-negative) were submitted to a 1 day RISH protocol using a 16 bp-FITClabelled Peptic Nucleic Acid (PNA) probe directed to the UTR of HCV. Detection was performed using a commercial PNA ISH Detection Kit. Specificity of RISH was assessed by RNAse digestion and HCV-/ßactin RT-PCR. Results: All tissues revealed sufficient RNA-quality to test for the presence of HCV. In 11 of 12 cirrhotic HCV-positive liver tissues specific cytoplasmic HCVtranscripts were detected in hepatocytes at the periphery of cirrhotic nodules. Specificity was proved by RNAse digestion prior to RISH. Nine HCV-positive cases were detected by HCV RT-PCR. Interestingly, HCV-RISH detected specific transcripts in three additional specimens. Conclusions: We introduce a rapid and reliable protocol for the detection of HCV in FFPE tissues for diagnostic purposes. Since the RNA in FFPE tissues is strongly fragmented and HCV is present in low copy number the positive HCV-RISH results are very likely due to the bias of the short 16 bp HCV-probe used in HCV-RISH which renders HCV-RISH superior to RT-PCR in this diagnostic setting.

218 Western blot analysis from paraffin-embedded tissues by application of the HOPE*-technique T. GOLDMANN1, U. UHLIG2, S. UHLIG3, D. BRANSCHEID3, J. GALLE1, E. VOLLMER1 1

Klin. & Exp. Pathologie, Forschungszentrum Borstel Lungenpharmakologie, Forschungszentrum Borstel 3 Thoraxchirurgie, Krankenhaus Großhansdorf 2

Posters: Molecular Pathology I 217 Fast and reliable RNA in situ hybridisation protocol for the detection of Hepatitis C virus in formalin-fixed paraffin embedded tissues M. ODENTHAL1, A. C. SCHMITT2, M. MU¨LLER1, H.P. DIENES1, A.Z. HAUSEN2 1

Institut fu¨r Pathologie, Universita¨t Ko¨ln Institut fu¨r Pathologie, Universita¨t Freiburg

2

Aims: The diagnostic use of Hepatitis C virus (HCV) RNA in situ hybridisation (RISH) in formalin fixed and paraffin embedded (FFPE) tissue is hampered by time consuming protocols. We established a fast and reliable

Aims: Fixation of specimens with formalin, the most commonly used fixative, usually prevents further molecular analysis, since it leads to degradation of nucleic acids and denaturation of the antigenic determinants of proteins. To overcome these problems, the HOPEfixation technique has been developed, which preserves nucleic acids and antigenic determinants of proteins, thus expanding the applicability of immunohistochemical methods. It was the aim of the present study to investigate whether HOPE-fixed tissue can be analyzed by Western blotting and to compare this with conventionally fixed and frozen material. Methods: The specimens used were tumor-free materials from lobectomies due to lung cancer. Results: All four antibodies tested, i.e. antibodies specific for focal adhesion kinase, surfactant protein A, PI-3kinase, and IKKa, worked well if used for immunoblotting

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of HOPE-fixed and frozen tissue. In contrast, these antibodies showed no or only very weak specific binding if formalin-fixed specimens were analyzed. Conclusions: Our findings show that HOPE-fixation maintains the antigenicity of proteins better than formalin fixation. The possibility to perform Western blotting with archived paraffin-embedded specimens extends the options for diagnostic and scientific analyses of fixed tissues. *Hepes-glutanic-acid-buffer-mediated-Organic-solventProtection-Effect

219 Quantification of gene expression in paraffinembedded tissues – relevance of variation in mRNA stability I. KOCH, J. SLOTTA-HUSPENINA, F. FEND Institut fu¨r Pathologie, Technische Universita¨t Mu¨nchen Aims: RNA isolated from formalin-fixed paraffinembedded (FFPE) tissue samples is suitable for quantitative RT-PCR assays, and results are usually normalized to housekeeping genes. However, it is unknown whether different mRNA species are more susceptible to fixation-induced degradation, which could significantly influence interpretation of qRT-PCR from FFPE. Methods: Using a standardized approach with paired samples of fresh and FFPE pellets of 5 cell lines, the influence of formalin fixation on 15 different TaqMan assays (2 for 18S RNA, 1 for TBP, 4 for different isoforms of CyclinD1 and 8 for other genes) were performed and shifts in Ct values were monitored. Results: As expected, Ct values were significantly higher for RNA obtained from FFPE than from fresh cells, with a mean shift of 6.5 delta Ct. Shifts for each assay were constant and reproducible in repeat experiments and for different cell lines, but varied considerably from assay to assay (range from 4.1 to 10.7 delta Ct). Importantly, shifts also varied for the 4 different CyclinD1 assays: D1 total (exon1/2) 6,2; D1a (exon4/ 5) 7.5; D1b (exon4/intron4) 4.1 and D1 30 UTR 6.7. Conclusions: qRT-PCR results obtained from FFPE samples normalized to a housekeeping gene do not always reflect the expression ratios in vivo, indicating that formalin fixation does not equally affect all mRNA species. Ideally, the Ct shift of the housekeeping gene and of the target gene should be in the same range to obtain reliable results. Otherwise, over- or underestimation of gene expression by a factor of up to 2d Ct (normalizer to gene) can occur. These results need to be taken into account for qRT-PCR from archival tissues, especially when expression levels of different genes or isoforms of the same mRNA are compared.

220 Fluorescence in situ hybridization of already stained paraffin sections mounted on uncoated slides – what to do? U.F. Vogel, B.D. Bu¨ltmann Institut fu¨r Pathologie, Universita¨t Tu¨bingen Aims: Especially concerning small biopsies, the whole paraffinized material may be consumed for step sectioning and routine stainings. Therefore, no material is left for additional stainings like fluores-cence in situ hybridization (FISH). Under these circumstances, FISH may be performed on the already routinely stained sections. However, because of the use of uncoated slides in routine laboratories, these sections will float off the slide during the pretreatment procedures for FISH. Therefore, we looked for techniques to overcome this problem. Methods: Already routinely H&E stained and coverslipped sections were placed in xylene to remove the mounting medium and the glass coverslip. Then three different methods were used: (1) The stained sections were scraped off the slide with a trimming knife, reembedded in paraffin, cut and mounted on coated slides. (2) The stained sections were covered with a thin layer of liquid hot paraffin, cooled down to about 25 1C, lifted off the slide by using the trimming knife with a sharp microtome blade, transferred to a warm water bath (42 1C) and then mounted on a coated slide. (3) A sticky, so-called tape window was put on the stained sections. Then the sections were lifted off the slide by using the trimming knife with a sharp microtome blade and transferred to a specially coated slide (Paraffin Tape Transfer System, Instrumedics, Hackensack, NJ, USA). By using these techniques, finally, the sections were mounted on coated slides and further processed using routine FISH pretreatment protocols and DNA-directed probes. Results: With all three techniques, FISH signals could be detected. Conclusion: With the described techniques, it is possible to use routinely mounted and stained paraffin sections for additional examinations like FISH.

221 Quantum dots for multicolor and photostable immunofluorescent labeling K. BINK1, P. HUTZLER1, U. BUCHHOLZ1, L. QUINTANILLA-MARTINEZ1, H. HO¨FLER1,2, F. FEND2 1

Institut fu¨r Pathologie, gsf Forschungszentrum, Neuherberg

ARTICLE IN PRESS Posters: Molecular Pathology I / Pathology – Research and Practice 201 (2005) 153–300 2

Institut fu¨r Pathologie, Technische Universita¨t Mu¨nchen Aims: Immunofluorescence microscopy is a standard technique for the simultaneous detection of multiple antigens in tissue sections. Currently available organic fluorochromes, such as FITC, cyanine dyes, and AlexaFluor dyes, are limited by photobleaching. Semiconductor nanocrystals, also known as quantum dots (QDs) are newly available fluorophores of high brightness, photostability and narrow emission spectra. These particles can be used for multicolor fluorescent labeling without spectral overlap. We tested QDots-conjugated secondary antibodies as alternative reagents for paraffin-section immunofluorescence. Methods: Serial sections of human tonsil were used for multicolor labeling with Qdots655, Qdots565, Qdots605 or organic fluorochromes FITC, Cy3, and Texas Red. Primary antibodies MIB1, p27, Cyclin E, Cyclin D1, CD3, CD20, CD68 and Vs38c were used to label targets in different cell compartments. Stains were evaluated with an LSM 510 NLO meta microscope. Results: We sucessfully performed single, dual and triple color labeling with both types of fluorochromes. Compared to organic fluorochromes, an intensified antigen retrieval was necessary for optimal results with Qdots conjugates. Furthermore, Qdotss could be employed for the simultaneous combination of FISH and immunofluorescence on tissue sections (FICTION). Conclusions: For multicolor labeling (more than 3 colors) and FICTION, Qdots-labeled reagents show several advantages over organic dyes. However, optimization of tissue pretreatment and careful selection of primary antibodies are necessary to obtain optimal results.

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drying effects are presumed. To prove this assertion we tried to provoke degradation of immunoreactivity by chemical oxidation, photooxidation and artificial drying. Methods: A paraffin block of an estrogen receptor (ER) positive invasive lobular breast carcinoma (IRS 12) was freshly cut. Prior to ER-immunhistochemistry (IHC) tissue sections were (a) pretreated in a hydrogen peroxide (H2O2) bath of different concentrations (b) stored under dry heat at 561C temperature and (c) exposed to ultraviolet irradiation (360 nm). The durations for each series of experiments was adjusted for appropriate time periods varying from single minutes up to several weeks. Staining results were analysed in comparison to untreated control slides. Results: Specimens were partly destroyed by the peroxide treatment but even higher concentrations and long duration did not have any effect on the ER staining intensity. Similarly, extended drying at least for several days did not show any detectable diminution. In particular ultraviolet irradiation indicated a decrease of intensity of the immunohistochemistry although no entire loss of antigenicity was observed. Conclusion: From our experiments, photooxidation seems to be the key parameter for the deterioration of antigenicity in immunohistochemistry. Additional studies will be performed to validate these findings.

223 Icon-TMA* for internal control of immunohistochemistry I. PETERSEN, A. KOEPENIK1, M. DIETEL, V. KRENN Institut fu¨r Pathologie, Charite´ Campus Mitte, Berlin 1 Oligene GmbH, Charite´ Campus Mitte, Berlin

222 Investigation of parameters with potential influence on antigenicity loss in immunohistochemistry C. BLIND, A. KOEPENIK1, M. PACYNA-GENGELBACH, N. DEUTSCHMANN, T. KNOESEL, M. DIETEL, V. KRENN, I. PETERSEN Institut fu¨r Pathologie, Charite´ Campus Mitte, Berlin 1 Oligene GmbH, Charite´ Campus Mitte, Berlin Aims: Formalin-fixed tissues embedded in paraffin blocks keep antigenicity for a long time under normal storage conditions, whereas tissue sections may show a diminution in immuno-histochemical reactivity over time. However, little is known about the key processes that are responsible for the loss of antigenicity and how tissue sections should be appropriately conserved for extended storage. In the literature oxidation and

Aims: Immunohistochemistry (IHC) has become an essential adjunct in daily histopathological diagnosis. However, quality control is difficult and becoming critically important considering the need for standardization and quantification of immunoreactivity. Methods: To help solving these problems we developed small size tissue microarrays (TMAs) with 2–4 punch biopsies from well characterized specimens of sizes between 0.6 and 2 mm diameter. In its simple variant these internal control (Icon) TMAs consist of two tissue cylinders, one being clearly positive and the other clearly negative for a specific immunohistochemical marker, e.g. cytokeratins or hormone receptors. In its more elaborate format, the Icon-TMAs carry additional tissue spots providing a scale for quantitatively assessable markers, e.g. breast cancer biopsies representing the Her2/NEU scores 0, 1+, 2+, 3+. The specimen with the yet

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unknown immunoreactivity is then applied to the IconTMA section and both are simultaneously analyzed. Results: A successful IHC analysis could almost be judged by the macroscopical inspection of the two +/ spots representing the positive and negative sample of the Icon TMA. While the spot with positive control provided an internal standard for strong immunoreactivity, the negative one provided an estimate to differentiate weak positivity from unspecific background staining. The technique proved to be particularly useful for markers that were not necessarily present in the uncharacterized specimen, e.g. BCL2 in lymphoma. Conclusion: Icon-TMAs provided a convenient measure for internal quality control and may become an indispensable tool for standardization and quantification of gene expression by immunohistochemistry or related techniques like mRNA in-situ hybridization. *Icon-TMA was filed for a registered mark.

224 Comparison of ductal and lobular breast cancer by DNA microarray analysis D. STANGE1, B. RADLWIMMER1, P. LICHTER1, F. TRAUB2, LA¨NGER2, U. LEHMANN2, H. KREIPE2 1

Abt. Molekular Genetik, Deutsches Krebsforschungszentrum 2 Institut fu¨r Pathologie, Medizinische Hochschule Hannover Aims: Ductal (IDC) and lobular (ILC) breast cancer provide the major histological subtypes of mammary cancer. IDC and ILC are characterized by particular morphological and clinical features, the genetic basis of these differences are only partly understood. To address the question we analyzed chromosomal aberrations in 21 ILC and 19 IDC. Methods: For genome-wide profiling of copy number imbalances, we constructed a DNA microarray consisting of about 6000 large-insert genomic clones covering the whole genome with an average resolution of 0.5 Mb. On the microarray differentially labelled genomic tumor DNA was hybridized against normal reference DNA and the signal ratio was measured to assess copy number changes. Results: We detected 207 recurrent aberrations, including some smaller than 1 Mb. Although the overall concordance of the aberration patterns was high between the two subtypes, we were able to identify four distinct regions on 1q and 16p where imbalances occurred at significantly higher frequency in ILC than IDC. Unsupervised hierarchical clustering resulted in the formation of three groups, one consisting primarily of IDC and two mainly of ILC.

Conclusions: Invasive lobular and ductal breast cancers can be distinguished based on the frequencies of occurrence of some chromosomal aberrations. Unsupervised hierarchical cluster analysis furthermore suggests that ILC can be subdivided in two groups.

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Improved detection of mycobacteria in HOPE*fixed tissues T. GOLDMANN1, D. HILLEMANN2, T. KUBICA3, G. ZISSEL3, J. MU¨LLER-QUERNHEIM3, R. SEN GUPTA1, J. GALLE1, E. VOLLMER1 1

Klin. & Exp. Pathologie, Forschungszentrum Borstel NRZ f. Mykobakterien, Forschungszentrum Borstel 3 Abt. Pneumologie, Universita¨tsklinik Freiburg 2

Aims: Standard PCR-based detection of mycobacterial DNA in paraffin-embedded specimens may lack sufficient sensitivity due to degradation of nucleic acids by routinely used formalin fixation. We therefore set up an approach aimed at improving the results by application of the novel HOPE-fixative in PCR-detection of mycobacteria in paraffin-embedded tissues. Methods: PCR, Real-time PCR, Spoligotyping Results: Comparison of PCR-results using DNA extracted either from HOPE- or formalin-fixed specimens in BCG-infected SCID-mice revealed a more than 100 fold enhanced sensitivity for the HOPE-fixed material. Due to the preservation of DNA from degradation in HOPE-fixed tissues even differentiation within the M. tuberculosis complex was possible by spoligotyping. Conclusions: We therefore conclude that the HOPE-fixative is a useful tool for molecular pathology, which enhances the sensitivity of PCR-based methods for detection of pathogens in paraffin embedded tissues compared to formalinfixation. Due to the better preserved DNA improved differentiation of mycobacteria from archived materials is possible. These results promise new and substantially enlarged possibilities in the field of molecular pathology. *Hepes-glutanic-acid-buffer-mediated-Organic-solventProtection-Effect

226 Inter-laboratory validation of PCR-based detection of Mycobacterium tuberculosis in paraffin-embedded tissue C. SCHEWE1, T. GOLDMANN2, M. GROSSER3, A. ZINK4, S. PAHL1, A. NERLICH4, G.B. BARETTON3, M. DIETEL1, E. VOLLMER2, I. PETERSEN1 1

Institut fu¨r Pathologie, Universita¨tsmedizin Berlin Klin.& Exp. Pathologie, Forschungszentrum Borstel

2

ARTICLE IN PRESS Posters: Molecular Pathology I / Pathology – Research and Practice 201 (2005) 153–300 3

Institut fu¨r Pathologie, Technische Universita¨t Dresden 4 Institut fu¨r Pathologie, KH Mu¨nchen-Bogenhausen Aims: The present study is based on an initiative for quality assurance of the German Society of Pathology and the Professional Association of German Pathologists. Four panel laboratories with experience and expertise in PCR detection of Mycobacterium tuberculosis were selected to establish the prerequisites for continuous external laboratory trials, in particular by providing pre-tested specimens and evaluation criteria for participating institutes. Methods: In a first step, the four panel laboratories performed an internal trial to test their own reliability and reproducibility. Results: Paraffin sections and DNA preparation from 32 clinical specimens and two experimentally generated tissues totalling to 66 samples were evaluated by each panel institute according to their own protocols using different methodologies. Despite these differences, a high degree of inter-laboratory reliability was achieved. The results will be published providing recommendations for applying PCR-based methodology for the detection of mycobacterial DNA in surgical specimens of pathology. Conclusions: Pre-tested specimens are now available for the external trail and can be ordered from the steering institute1 via Oligene (www.oligene.com). All molecular pathology laboratories are invited to participate in this quality assurance initiative.

227 Comparison of histology, volume CT, and compliance measurements in bleomycin-induced lung fibrosis M. KOENIGSHOFF, A. WILHELM, S. GRESCHUS1, G. DAHLEM, A. JAHN, K. PETRI, R.M. BOHLE2, A. GUENTHER, W. SEEGER, L. FINK2, F. ROSE Medizinische Klinik II, Universita¨t Giessen 1 Zentrum fu¨r Radiologie, Universita¨t Giessen 2 Institut fu¨r Pathologie, Universita¨t Giessen Aims: Determination of parameters suited for a guided selection of bleomycin-treated mice for subsequent experiments. Methods: Lung fibrosis was induced by inhalative application of 5U/kgbw bleomycin. The development of fibrosis was characterized by CT scans, compliance measurements, and histological analysis after 7, 14, and 21 days after exposition to bleomycin. Isolated FB were used for proliferation assays in coculture with AEC from healthy lungs and for real-time PCR-based

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quantification of transcripts of genes relevant in the development of lung fibrosis. Results. Fibrotic lungs consistently showed slight or pronounced groundglass opacities and/or reticular patterns after 14 days with varying regional/ lobal predominance. Histological status matched well with CT findings. Compliance measurement ranged between 0.04–0.13 ml/kPa (healthy: 40.15) correlating to CT findings with decreasing values. Proliferation of FB was inhibited by AEC. Decreased AEC-dependent inhibition of proliferation was observed when FB were isolated from fibrotic lungs with a compliance of o0.08. Conclusion: CT scan, compliance measurement and histology are feasible tools for accurate detection of the heterogenous distribution of bleomycin-induced lung fibrosis. Consideration of these techniques, in particular the compliance measurement allows a selection of bleomycin-treated mice lungs with cells showing typical fibrotic properties.

228 Down-regulation of heat shock protein hsp70 and hsp84 mRNA in the chronic irradiation response of the lung M.G. HAASE, A. KLAWITTER, G.B. BARETTON Institut fu¨r Pathologie, Technische Universita¨t Dresden Aims: We have previously shown that the transcription factors AP-1 and Sp1 are inactivated at 4.5 to 5.5 weeks after irradiation, and NF-is activated in the chronic radiation response of the lung. Fibrosing alveolitis starts at 8 weeks after irradiation. The aim of the study was to identify potential targets of those transcription factors that might play a role in chronic radiation response. Methods: The right lungs of Fischer rats were irradiated with a single dose of ionizing irradiation. Fresh lung tissue was obtained at 4 to 12 weeks after irradiation. A subtractive DNA library was produced from lung tissue obtained at 5.5 weeks after irradiation (+cDNA) and 4 weeks after irradiation (-cDNA) to find genes that are down-regulated at 5.5 weeks after irradiation. The resulting clones were sequenced. Quantitative RT-PCR reactions were done at various time points from selected genes. Results: Two of the genes that are down-regulated at 5.5 weeks after irradiation are hsp(heat shock protein) 70 and hsp84. Hsp70 is down-regulated after at 5.5 weeks to 6 weeks after irradiation. In contrast, hsp 80 is down-regulated after 5.5 weeks after irradiation and does not return during the time of observation of 12 weeks. Conclusions: The results suggest that both hsp70 and hsp84 are under control of transcription factors that are

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inactivated after irradiation (like AP-1 and Sp1). In addition, p70 seems to be under control of NF-kB, either directly or indirectly, because the mRNA reappears at later time points after irradiation. Hsp70, a molecular chaperone and hsp84, a stabilizer of mutated proteins of the hsp90 family, might play an important role in the de-stabilization of cellular homeostasis in the long-term response to irradiation.

230 Microsatellite analysis as useful tool in diagnostics of pleural and bronchial fluids from patients with nonsmall cell lung cancers M. WOENCKHAUS1, U. GREPMEIER1, P. J. WILD1, B. WERNER2, M. PFEIFER2,3, C. SCHULZ3, F. ROCKMANN4, U. WOENCKHAUS4, G. RO¨CKELEIN5, F. HOFSTA¨DTER1, W. DIETMAIER1 1

229 Chromosomal alterations in preneoplastic and early stages of malignant mesothelioma M. KRISMANN, F. SIMON, K.-M. MU¨LLER Institut fu¨r Pathologie der Ruhr-Universita¨t Bochum an den BG-Kliniken Bergmannsheil, und Deutsches Mesotheliomregister Aims: To evaluate the molecular pathological progression of pre-neoplastic and early neoplastic stages of serosal lesions, we first classified lesions as (1) clear benign reactive mesothelial hyperplasia (RMH), (2) atypical mesothelial proliferation (AMP) with slight cytolo-gical atypia, (3) mesothelioma in situ (MIS) with compact multilayer-ed atypical mesothelia without infiltrative growth, or a superficial pa-pillary growth pattern and (4) early mesothelioma (EM) with infiltration into the adjacent fat tissue. Methods: Twelve cases (2 RMH, 4 AMP, 2 MIS, 4 EM) were analysed after laser-assisted microdissection (approx. 200–500 cells) and DOP-PCR amplification by classical comparative genomic hybridisation (CGH) and the results were compared with our findings in more advanced stages of epithelioid mesotheliomas. Results: No distinct chromosomal imbalances were detectable in RMH. In AMP, up to 8 losses and 1 gain could be demonstrated (4.3 defects/ case). MIS were characterised by defect patterns (8.5 def./ case) comparable to more advanced mesothelioma stages, as well as EM (5.3 def./ case). Losses were more common than gains. Conclusions: By means of classical CGH analysis, chromosomal imbalances can be observed even in mesothelial lesions that show no morphologically unambiguous signs of malignancy. The defects in early and preneoplastic mesothelial lesions are heterogenous altogether, but in detail they reflect changes known from more advanced stage malignant mesotheliomas. According to our findings, there is no single pattern of defects specific for early mesothelial neoplasia. In problematic cases, the method may be used in future as additional differential diagnostic aid.

Institut fu¨r Pathologie, Universita¨t Regensburg Institut fu¨r Innere Medizin, Lungenfachklinik Donaustauf 3,4 Institut fu¨r Innere Medizin II,I, Universita¨t Regensburg 5 Pathologisch-Anatomische Institut, Regensburg 2

Aims: Allelic losses at chromosome 3p and 12p are early genetic events in carcinogenesis of non-small cell lung cancer (NSCLC). Alterations at chromosome 2p, 5p, 12q and 17q can be observed in advanced stages of tumor disease. Our aim was to determine whether microsatellite analysis of pleural and bronchial fluids is able to detect early molecular alterations and to improve diagnostic sensitivity. Methods: We applied 15 microsatellite markers localized at the chromosomal regions (2p, 3p, 5p, 12p, 12q, 17q) that have been shown to be altered in preneoplastic lesions and early NSCLC. We investigated microsatellite alterations (LOH and MSI) in pleural (n ¼ 25) and bronchial fluids (n ¼ 22) of patients with and without NSCLC. Both supernatants and cell pellets were examined and the results were compared with cytological and histological findings. Results: LOHs in supernatants of pleural fluid from tumor patients with positive and negative cytological findings were detected in 71% and 57%, respectively. The supernatants from bronchial fluids showed LOH in 60% of patients with malignant disease. In contrast no LOHs were found in the according cell pellets. Conclusions: Microsatellite analysis could provide a useful tool in diagnostics of pleural and bronchial fluids. Detection of microsatellite alterations is significantly higher in supernatants of fluid material than in cell pellets.

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Methylation assay for molecular cytological diagnosis of lung cancer on bronchial aspirates. Results from a cohort study J. GROTE H., V. SCHMIEMANN, M. KAZIMIREK, H.E. GABBERT1, R. KAPPES2, A. BO¨CKING

Institut fu¨r Cytopathologie Institut fu¨r Pathologie, Universita¨t Du¨sseldorf 2 Florence-Nightingale-Krankenhaus, Du¨sseldorf 1

ARTICLE IN PRESS Posters: Molecular Pathology I / Pathology – Research and Practice 201 (2005) 153–300

Aims: Recently we established a molecular assay for the diagnosis of lung cancer applying a quantitative methylation specific PCR (QMSP) on bronchial aspirates. In case-control-studies promoter hypermethylation of APC, p16, RASSF1A, and RARB2 proved to be a useful biomarker. We now wanted to verify our QMSP-assay in a retrospective cohort study design. Methods: The cohort comprised 240 bronchial aspirates from patients who underwent bronchoscopy for the diagnosis of lung cancer or its exclusion. The methylation status of marker genes (APC, p16, RARB2, RASSF1A) was studied using our QMSP-assay. The results were compared with QMSP on corresponding microdissected tumor tissue and with sequencing of cloned promoter regions in three cases. Results: Considering 12 months of follow-up, 107 patients presented with lung cancer, 94 had benign lung disease, and 39 showed other diagnoses. Sensitivity of cytology, histology and QMSP assay (APC, p16, RASSF1A) was 43%, 64% and 54%, respectively. The combination of all three methods yielded a sensitivity of 88% for the diagnosis of lung cancer with one single bronchoscopy. Specificity was 499%. Adding RARB2 to the marker panel raised the negative predictive value of the QMSP-assay to 70%. Sequencing of cloned promoter regions confirmed specificity of QMSP. Conclusions: Our methylation assay is a promising complementary method for lung cancer diagnosis and may be used as a reflex test if morphology is negative or doubtful.

232 Differential gene expression in mouse lings after short exposure to hypoxia J. WILHELM, J. BEST1, G. KWAPISZEWSKA1, W. SEEGER, N. WEISSMANN, L. FINK1, R.M. BOHLE1 Medizinische Klinik II, Universita¨t Giessen 1 Institut fu¨r Pathologie, Universita¨t Giessen Aims: Genes involved in the response to early hypoxia should be identified by expression profiling with DNA microarrays. Methods: Groups of 18 mice were exposed to normoxic and hypoxic (3 h and 24 h FiO2 ¼ 0.1) conditions. Total RNA was isolated from lung homogenate of these mice and labeled cDNA was generated by incorporation of Cy3- and Cy5-dCTP during RT. The labelled cDNA was hybridized on 44 K 60mer oligonucleotide microarrays. The data were analyzed using the limma package in R. Potentially regulated genes were filtered by moderated Bayes statistics.

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Results: From the top-50 genes considered most likely to be differentially expressed under hypoxia, 45 (45) genes were upregulated and 5 (5) genes were downregulated after 3 h (24 h) hypoxia. Most genes were regulated by a factor of 2–4; only few genes showed higher factors. Fourteen genes (mainly endothelin-related, metallothioneins) were upregulated after 3 h as well as after 24 h. After 3 h, a set of genes for stress response was upregulated, whereas after 24 h VEGF, TGFb, TIMPs and collagens were found to be upregulated. Conclusions: Three hours after exposition to hypoxic conditions, the cells show a stress response, mainly as a reaction to radical oxygen species. Endothelins seem to be the key players for pulmonary vasoconstriction. Already after 24 h of hypoxia, inducers of lung remodelling can be identified in the expression profiles.

233 Differential protein expression in mouse lungs after short exposure to hypoxia G. KWAPISZEWSKA, S. SCHMITT1, M. LINDER1, W. SEEGER, R.M. BOHLE2, N. WEISSMANN, L. FINK2 Medizinische Klinik II, Universita¨t Giessen 1 Institut fu¨r Biochemie, Universita¨t Giessen 2 Institut fu¨r Pathologie, Universita¨t Giessen Aims: Hypoxia-induced changes in protein expression are analyzed by 2D-gel electrophoresis for a better insight into the early response of lung cells to low oxygen tension. Methods: Mice were exposed to 3 hours or 24 hours hypoxia (FiO2 ¼ 0.1) or normoxia. Protein extract from lung homogenate was analyzed by 2D-gel electrophoresis. Coomassie Blue stained proteins were detected in the range of 10-220 kDa. Differences in protein abundance were determined with Proteomweaver Software 2.2.2. Western blots were performed to confirm the protein expression levels. Cellular localisation of 3 selected proteins was determined by immunohistochemistry. Real-time PCR was used for relative quantification of transcript levels. Results: Out of 9379 detected spots, 92(129) proteins were found to be significantly up (down) regulated in hypoxia. 35 proteins were identified from these spots by MALDI-TOF analysis. Most regulated proteins were responsible for electron transport and ROS balance. The differential regulation of Eef1a1 (x10), cat1 (x2.2) and HMGB1 (x0.27) was confirmed by Western blot analysis but could not be shown on transcript level. Conclusion: 2D-gel analysis can be used to identify proteins involved in hypoxia response. Even short exposure to hypoxia (FiO2 ¼ 0.1) induces many changes in protein levels. Interestingly, many of these

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changes were not seen on the transcriptional level. Future studies might show that protein stabilization and modifications are major steps in early response to hypoxia.

C. VON WEYHERN1, R. HAUBNER2, M. SCHWAIGER2, H. HO¨FLER1, M. SARBIA1 1

Institut fu¨r Pathologie Nuklearmedizinische Klinik, Technische Universita¨t Mu¨nchen 2

234 Expression of cyclooxygenases (COX-1, COX-2) and vascular endothelial growth factors (VEGF-A, VEGF-C) is correlated In Barrett’s cancer B.H.A. VON RAHDEN1, H.J. STEIN1, F. PU¨HRINGER, R. LANGER, J.R. SIEWERT1, J. THEISEN1, M. SARBIA Institut fu¨r Pathologie 1 Chirurgische Klinik und Poliklinik, Technische Universita¨t Mu¨nchen Aims: To investigate the role of both cyclooxygenaseisoforms (COX-1 and 2) and their link with proangiogenetic factors (VEGF-A and C) in esophageal adenocarcinomas (Barrett’s cancer). Methods: Expression of COX-1 and 2 mRNA as well as VEGFA- and C was determined in 97 primary resected esophageal (Barrett’s) adenocarcinomas , using RTPCR. COX protein expression was analyzed using immunhistochemistry. Two esophageal cancer cell lines were exposed to selective inhibitors of COX-1 (SC-560), COX-2 (rofecoxib) or non-selective COX-inhibitors (diclofenac) in order to substantiate a potential regulatory relationship between COX and angiogenesis. Results: All carcinomas were positive for COX-1 and COX-2 protein expression. mRNA expression levels varied between tumor tissues of different patients. The expression levels of both COX-isoforms were strongly correlated with each other (R ¼ 0:758; po0:001) as well as with VEGF-A (r ¼ 0:777; po0:001; r ¼ 0:788; po0:001) and VEGF-C (r ¼ 0:811; po0:001; r ¼ 0:668; po0:001). Exposition of esophageal cancer cell lines OE-33 and OSC-1 with COX-inhibiting drugs lead to a significantly reduced expression of VEGF-A and VEGF-C. Conclusions: The variable expression of both COXisoforms and the strong significant correlation with the proangiogenetic factors VEGF-A and C suggests a pathogenetic role in Barrett’s carcinogenesis, via an effect on angiogenesis and lymphangiogenesis. The functional relationship was substantiated by cell cultural inhibition experiments. Thus, chemoprevention of esophageal cancer by COX-inhibition may be – at least in part – antiangiogenetic in nature.

235 Expression of integrin a5b1 correlates with loss of tumor cell cohesion in adenocarcinomas of the upper gastrointestinal tract

Aims: Integrins are a class of proteins which play an important role in cell/matrix interaction including cell adhesion during tumor angiogenesis and invasion. Here we determine the expression of the integrin subtype a5b1 in adenocarcinomas of the upper gastrointestinal (GI) tract. Methods: Using a tissue microarray, 250 primarily resected adenocarcinomas of the upper GI tract (54 Barrett’s carcinomas, 102 cardiac carcinomas and 94 gastric carcinomas) were investigated for expression of integrin a5b1 by using immunohistochemistry. Results: Expression of integrin a5b1 was present more frequently in gastric (60.6%) than in cardiac (37.3%) and in Barrett’s carcinomas (33.3%; p ¼ 0:0026). Expression of a5b1 was present more frequently in signet-ring cell carcinomas (56.0%) than among other adenocarcinoma subtypes (42.5%) according to the WHO classification (p ¼ 0:0092). According to Lauren’s classification, a5b1 expression was present more frequently among diffuse carcinomas (69.6%) than among mixed carcinomas (50.0%) and intestinal carcinomas (40.5%; p ¼ 0:0042). In contrast, a5b1 expression did not correlate with pT-category, nodal status, tumor stage, grading, and tumor size. Conclusions: Our data indicate that expression of integrin a5b1 is correlated with loss of cohesiveness in upper GI tract adenocarcinomas and may therefore be involved in invasiveness of these tumors.

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Identification of insulin-like growth factor (IGF)-II as a therapeutic target in human liver cancer

T. NUSSBAUM, S. VREDEN1, K. BREUHAHN, P. SCHIRMACHER Institut fu¨r Pathologie, Universita¨t Heidelberg 1 Institut fu¨r Pathologie, Universita¨t Ko¨ln Aims: High level overexpression of insulin-like growth factor (IGF)-II, a foetal and mainly liver-derived cytokine is well established in hepatocellular carcinomas (HCCs) as well as in other malignant tumour entities. Here we want to elucidate the role of IGF-II overexpression in hepatocarcinogenesis which may have substantial mechanistic and therapeutic impact. Methods: Human HCCs (n ¼ 43) and HCC cell lines (HUH7, HepG2, Hep3B) were analysed by cDNAmicroarray profiling. Specific in vitro reduction of

ARTICLE IN PRESS Posters: Molecular Pathology II / Pathology – Research and Practice 201 (2005) 153–300

IGF-II expression by IFN treatment and RNA interference was confirmed using real-time-PCR. Functional aspects were analysed by MTT-vitality and BrdUELISA assays. Results: IGF-II overexpression defines a subgroup of HCCs (16.3%), which displays less tumor cell apoptosis, lower number of tumor infiltrating lymphocytes and suppression of IFN-regulated genes. All analyzed HCCcell lines with intrinsic IGF-II overexpression coclustered with IGF-II overexpressing HCCs. HUH7 cells show a significant down regulation of IGF-II after IFNg-treatment. A specific reduction of IGF-II mRNA (85%) was obtained after transient and stable transfection of siRNA. Diminished IGF-II bioavailability was associated with a significant decrease of tumour cell vitality (60%) and proliferation (20%). Conclusions: IGF-II overexpression exemplifies a relevant molecular subgroup of HCCs. Since inhibition of IGF-II signalling is associated with reduced tumour cell vitality and proliferation, it may offer prospects for therapeutic intervention, e.g. by IFN-treatment.

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different from hARLP-1 by one amino acid (D313N), indicating two allelic forms of the human aldose reductase-like gene. A novel antibody directed against hARLP revealed hARLP positivity in human HCC by immunohistochemistry. Conclusions: Of all investigated human HCC samples 95% were positive for hARLPs as proven by 2-DE analysis and/or by the use of the antibody directed against hARLP. Thus, hARLP is a strong candidate as immunohistochemical diagnostic marker of human HCC.

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Helicobacter pylori infection influences expression of matrix-metalloproteinase 1 (MMP-1) in gastric cancer cells T. HUNDERTMARK1, T. KALINSKI1, U. PEITZ2, A. ROESSNER1, S. KRU¨GER1 1

Institut fu¨r Pathologie, Universita¨t Magdeburg Institut fu¨r Gastroenterologie/Hepatologie,Universita¨t Magdeburg 2

Posters: Molecular Pathology II 237 Identification of new tumor markers in human hepatocellular carcinomas E. ZEINDL-EBERHART, S. HARAIDA, S. LIEBMANN, P.R. JUNGBLUT1, H.M. RABES Pathologisches Institut, Ludwig-Maximilians-Universita¨t Mu¨nchen 1 Max-Planck-Institut fu¨r Infektionsbiologie, Berlin Aims: The proteomic approach provides an excellent tool to detect and to identify proteins which are associated with cancer. We focused on human hepatocellular carcinomas (HCC) resulting from Caucasian male and female patients for the detection and identification of new tumor-associated protein variants. Methods: Comparative proteome analysis was performed with normal liver tissue and human HCC. Several new tumor-associated protein variants were detected by two-dimensional electrophoresis (2-DE) and identified by MALDI-TOF-MS. Results: These new tumor-associated protein variants represent members of the aldo keto reductase superfamily. The human aldose reductase-like protein-1 (hARLP-1) was the most prominent tumor-associated variant. The enzyme was found in various distinct protein species located at different Mr and pI in the majority of human HCC. A so far unknown human aldose reductase-like protein (hARLP-5) was identified,

Aims: Gastric colonization by Helicobacter pylori provokes host inflammatory responses and may be of prognostic relevance in gastric carcinomas. Therefore, the aim of this study was to clarify whether H. pylori infection of gastric epithelial cells influences the expression of genes known to be involved in extracellular matrix remodelling, tumor progression and metastasis, and which molecular pathways are required for these events. Methods: AGS gastric cancer cells co-cultured with or without H. pylori were examined for their differential gene expression using cDNA microarray analysis. Results were confirmed by quantitative real time PCR. MMP-1 expression was further analyzed in cell cultures and biopsies using western blots and immunohistochemistry. Functional significance of MMP-1 in cellular migration was tested in boyden chambers using antisense-ODNs. Cytokines and inhibitors of signal transduction pathways related to H. pylori infections were tested for their ability to possibly regulate MMP-1 expression Results: Expression of MMP-1 was upregulated more than 10-fold in co-cultures of AGS cells with H. pylori. Immunohistochemistry revealed strong MMP-1 expression at sites of bacterial attachment. MMP-1 upregulation could be induced by IL-8 and TGF-ß, inflammatory cytokines upregulated by H. pylori infection. Inhibition of the ERK1/2 pathway, also activated by H. pylori, decreased MMP-1 production. AGS cells treated with asMMP-1 showed significantly reduced migration. Conclusions: The increased expression of MMP-1 in H. pylori-infected gastric cancer cells suggests a potential

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role of MMP-1 in progession and metastasis of gastric cancer.

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Infection of primary human gastric epithelial cell preparations and cultured gastric cells with H. pylori – Modulations of gene expression levels and migratory capabilities

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Immunoprofiles of 11 biomarkers using tissue microarrays identifies prognostic subgroups in colorectal cancer T. KNO¨SEL1, A. EMDE1, K. SCHLU¨NS1, Y. CHEN1, K. JU¨RCHOTT1, M. KRAUSE2, M. DIETEL1, I. PETERSEN1

1

Institut fu¨r Pathologie, Charite´—Campus Mitte, Berlin Abteilung fu¨r Chirurgie und Onkologie, RRC, Charite´, Berlin 2

S. KRUEGER1, T. HUNDERTMARK1, T. KALINSKI1, U. PEITZ2, A. ROESSNER1 1

Institut fu¨r Pathologie, Universita¨t Magdeburg 2 Institut fu¨r Gastroenterologie/Hepatologie, Universita¨t Magdeburg Aims: Most studies investigating H. pylori induced gastritis in vitro have used the gastric cancer cell line AGS. However, this model differs markedly from the situation in vivo since AGS cells lack the ability to form a polarized epithelium. Using primary gastric epithelial cell cultures and 3D-organotypic coculture systems consisting of a collagen I matrix with integrated fibroblasts and macrophages and gastric epithelial cells, effects of H. pylori infection on the integrity and migration of the gastric epithelium were studied under in vivo-like conditions. Methods: Gastric epithelial cells were isolated from 2–4 biopsies in a collagenase/dispase solution and maintained in Quantum 286 culture medium on collagen I-coated culture dishes for 1 week. The single cell cultures and the 3D-cultures were examined for CK20 and E-cadherin expression. The expression of genes encoding adhesion molecules and proteases was examined by quantitative RT-PCR. Cellular integrity with and without H. pylori infection was analyzed using electron microscopy and fluoresceinconjugated phalloidin staining. Motogenic response to H. pylori was monitored by phase-contrast microscopy. Results: The tightly packed colonies of primary epithelial cells stained positively for CK20 and E-cadherin, which was negative in AGS cells. H. pylori increased protease mRNA’s in primary and AGS cells, whereas adhesion molecules were unaffected on mRNA level in AGS cells. In both, primary and AGS cells, the organisation of the actin cytoskeleton and the cell-cell contacts were disturbed as shown by fluorescence and electron microscopy with distinct morphological differences. Motogenic response was much higher in AGS than in primary cells, possibly due to the lack of adherence and tight junctions. Conclusions: Use of primary gastric epithelial cells would give new insights in studies of the pathogenesis of H. pylori.

Aims: Genome-wide expression profiling has identified a number of genes expressed at higher levels in colorectal carcinoma (CRC) than in normal tissue. Our objectives in this study were (1) to test whether genes found to be differentially expressed in CRCs by cDNA expression profiling were also distinct on the protein level, (2) to evaluate these potential biomarkers in a series of well–characterized CRCs, (3) to apply hierarchical cluster analysis to the immunohistochemical data. Methods: TMAs comprising of 351 CRC specimens from 270 patients were constructed to evaluate the genes Adam 10, Cyclin D1, Annexin II, NFKB, Casein kinase 2 b; YB-1, P-32, Rad 51, c-fos, IGFBP4 and Connexin 26 (Cx26). In total, 3797 samples were analyzed by immunohistochemistry. Results: The data confirmed that all proteins were differentially expressed between normal and cancerous tumors. Furthermore, unsupervised hierarchical clustering discovered subgroups of CRC that differed by tumor stage and survival. The validation of specific biomarkers by Kaplan–Meier analysis showed that reduced Cx26 expression was significantly associated with a shorter patients survival and higher tumor grade (G1/G2 vs. G3, p ¼ 0:02). Furthermore Adam 10 expression was significantly associated with a higher tumor stage (pT1/ T2 vs. pT3/4, p ¼ 0:04). Conclusions: Our study highlights the potential of TMAs for a higher dimensional analysis of multiple candidate genes in CRC by evaluating serial sections of the same tissue core (3D TMA analysis). In addition, it endorse the use of immunohistochemistry supplemented by hierarchical clustering for the identification of tumor subgroups with diagnostic and prognostic signatures.

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Detection of microsatellite instability in colon carcinoma by microfluidics based chip technology N. BARTA, R. SALOWSKY1, H.P. DIENES, S. LAMMERS, S.E. BALDUS, M. ODENTHAL Institut fu¨r Pathologie, Universita¨t Ko¨ln 1 Agilent Technologies, Waldbronn

ARTICLE IN PRESS Posters: Molecular Pathology II / Pathology – Research and Practice 201 (2005) 153–300

Aims: Microsatellite instability (MSI) is caused by a failure of the DNA mismatch repair system and occurs frequently in various types of cancer. Since sporadic MSI, associated with ca. 10% to 15% of colorectal, gastric or endometrial carcinoma, impact clinical prognosis, MSI analysis is an important tool of molecular pathology. This study aimed to develop a simple and efficient procedure of MSI detection. Methods: Forty cases with no (27), low (l) or high (h) MSI (13), preidentified by conventional fluorochromeassociated PAGE technology, were selected out of a panel of 150 patients with colon carcinoma. Microdissected non-tumor (N) and tumor (T) tissue areas of one or two 4 mm-sections were deparaffinized and DNA were extracted by Qiagen kit methodology. Primer sets recognizing the five microsatellite loci, BAT25, BAT26, D5S346, D17S250, D2S123, which are recommended at the 1997 National Cancer Institute-sponsored conference on MSI for the identification, were applied in labelfree duplex or single PCR assays for DNA amplification. one ml of the amplicons were analysed by microfluidics based on-chip electrophoresis of the Agilent 2100 bioanalyzer. Results: In all 40 cases, chip linked microcapillary electrophoresis of the amplicons, arisen from tumor and non-tumor DNA, resulted in highly resolved, distinct patterns of each of the five microsatellite loci. Label-free microfluidic detection of MSI could be demonstrated by microsatellite loci-associated, well defined deviations in the electropherogram profiles of tumor and non-tumor material, and confirmed the prediagnosis of the MSI cases by conventional technology. Conclusion: The microfluidic chip technology is a simple and robust technology for MSI detection, which allows label-free microsatellite analyses of amplicons within 30 minutes.

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Smac/for tumour progression in renal cell carcinomas (RCCs). Methods and results: XIAP and SmacmRNA and protein expression was analysed in primary tumour tissue from 66 RCCs of all major histological types by quantitative real-time PCR, Western blot and ELISA. (1) XIAP and SmacmRNA expression was found in all RCCs. (2) The relative XIAP mRNA expression levels significantly increased from early (pT1) to advanced (pT3) tumour stages (p ¼ 0:0002) and also with tumour dedifferentiation (p ¼ 0:04). (3) Western blot analysis confirmed the tumour stage-dependent increase of XIAP expression on the protein level. (4) In contrast, mRNA and protein expression levels of Smacdid not significantly change between early and advanced tumour stages or between low and high tumour grades. (5) The mRNA expression ratio between XIAP and Smacmarkedly increased during progression from early (pT1) to advanced (pT3) tumour stages. Conclusions: Our investigation demonstrates that the balance between antiapoptotic XIAP and proapoptotic Smac/expression is gradually disturbed during progression of RCCs, resulting in a relative increase of XIAP over Smac/thereby probably contributing to the marked apoptosis resistance of RCC.

243 Detection of chromosomal aberrations—a sensitive method for the diagnosis of bladder cancer (preliminary study report) S. FRIGERIO, R.T. STREBEL1, B.C. PADBERG, D. LENGGENHAGER, H. MOCH, H.A. JOHN1, D.R. ZIMMERMANN Department of Pathology, University Zu¨rich, Switzerland 1 Department of Urology, University Zu¨rich, Switzerland

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XIAP and Smac/DIABLO expression during tumour progression in renal cell carcinomas U. RAMP, Y. YAN, C. MAHOTKA, S. HEIKAUS, T. SHIBATA, N. WETHKAMP, J. LIEBMANN, Y. GUO, C.D. GERHARZ, H.E. GABBERT Institut fu¨r Pathologie, Universita¨t Du¨sseldorf Aims: Dysregulation of apoptosis plays an important role in tumour progression. The X-linked inhibitor of apoptosis (XIAP) is considered to be one of the most potent caspase inhibitor of all known inhibitor of apoptosis (IAP)-family members. Only recently, an antagonist of XIAP has been identified, termed Smac/ The aim of the present study was to explore the relevance of antiapoptotic XIAP and proapoptotic

Aims: Successful treatment of bladder cancer depends largely on early detection of both primary and recurrent disease. Our study aims at validating and comparing two molecular biological methods (LOH and FISH analysis), which will provide a specific and sensitive tool to aid routine cytology. Methods: Matched biopsies and urines from patients scheduled for tumor resection were collected prospectively and examined by routine histology and cytology. LOH analysis was performed using a set of 10 microsatellite markers which were amplified by PCR using fluorescence-labelled primers and analyzed by capillary electrophoresis. Results: Preliminary analysis detected LOH events in 25 out of 33 urine samples from carcinoma patients (75%). Most genetic aberrations were associated with LOH on

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chromosomes 9p, 9q and 17p. Remarkably, the sensitivity of LOH for low grade tumors was superior to cytology (75% vs. 12% for G1 tumors; 53% vs. 6% for G2 tumors). In addition, LOH analysis detected aberrations in 2 out of 27 TUR-B considered to be tumor-free by standard morphological criteria. This might reveal a greater sensitivity of LOH in the detection of minimal residual disease, a notion that will be verified by the clinical follow-up of these patients. In addition, we would like to extend the analysis of voided urines by comparing LOH with FISH analysis (UroVysion), a method that is currently being established in our laboratory. Conclusions: Preliminary data show that for its high sensitivity, LOH analysis represents an excellent tool to aid cytological diagnosis of bladder cancer.

by a change in PPIX-production, which cannot be explained in simple parameters of hem metabolism. Protein identification based on 2D differential expression will reveal the final crucial factors.

245 RNA-expression profiling of normal and tumor cells following photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) induced protoporphyrin IX (PPIX) in vitro P.J. WILD1, R. KRIEG2, J. SEIDL1, R. STOEHR3, C. HOFMANN4, J. LOUHELAINEN5, A. ROSENTHAL6, A. HARTMANN1, C. PILARSKY7, A. BOSSERHOFF1, R. KNUECHEL2 1

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New insights into photodynamic therapy (PDT): Generation and metabolic characterization of a PDTresistant cell line R.C. KRIEG, M. GROTENRATH, K. SCHWAMBORN, K. REHER1, R. KNUECHEL

Institut fu¨r Pathologie, Universita¨t Aachen Klinik fu¨r Urologie, Universita¨t Regensburg

1

Aims: Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) induced protoporphyrin IX (PPIX) as the photosensitizer is a promising tumor treatment modality. This method is now in focus of several clinical trials. However fundamental mechanisms still need to be addressed in detail. Therefore we generated and characterized a cell line resistant to PDT-treatments. Methods: Human HT29 [G2] were treated with ALA induced PDT using a prior defined LD90 dose. Surviving cells were cultured for 8 d and treated again. This was repeated over 60 cycles, giving HT29R. Sham treated cells were cultured accordingly giving HT29K. Cellular PPIX content, porphobilinogendeaminase (PBGD)- , ferrochelatase (FC)- and mitochondrial activity were quantified as well as CD71 expression using various extraction methods and flow cytometry. Differential protein expression was analyzed using high resolution 2D-SDS-PAGE. Results: HT29R are resistant to PDT. Their intracellular PPIX-content is 50% of HT29K. No differences were found in PBGD and mitochondrial activity nor in CD71 expression. Interestingly HT29R showed only 20% of FC activity in comparison to HT29K. 2D-SDS-PAGE revealed a significant differential protein expression pattern. Conclusions: It is possible to generate a cell line resistant to the per se lethal PDT. Resistance seems to be caused

Institutes of Pathology, Univ. Regensburg and Aachen 3 Depts. of Urology 4 and Internal Medicine 5 Univ. Regensburg; Cancer Research UK Clinical Centre, Univ. Leeds 6 Signature Diagnostics AG, Potsdam 7 Dept. of Surgery, Univ. Dresden 2

Aim: To understand the mechanism of photodynamic therapy (PDT) using 5-aminolevulinic-acid (ALA). Methods: A normal cell line (UROtsa, urothelial) and two tumor cell lines (RT4, urothelial; HT29, colonic) were treated with LD50 doses of light after exposure to ALA, and harvested for RNA extraction 0, 10 and 30 min after irradiation. The RNA was hybridized to a custome-made Affymetrix GeneChip containing 2,800 genes. Results: Comparing the gene expression profiles between the different samples, 40 genes (e.g. SOD2) were significantly expressed. We selected CASP8 and DUSP1 for further validation of the array findings, and compared their expression with the expression of the immediate early gene FOS by quantitative RT-PCR. RNA expression of CASP8 stayed unchanged whereas DUSP1 RNA was upregulated in normal and tumor cells starting 30 min after irradiation. In contrast, FOS RNA was found continuously upregulated over time in all three cell lines. Induction of DUSP1 protein expression was shown after one hour using western blot analysis. No changes of CASP8 and CASP3 protein expression but activation of catalytic activity was detected only in UROtsa cells one hour after PDT, but no changes were seen in both tumor cell lines. Conclusion: Combined data analysis suggests that PDT in vitro leads to apoptosis in UROtsa but to necrosis in the tumor cell lines.

ARTICLE IN PRESS Posters: Molecular Pathology II / Pathology – Research and Practice 201 (2005) 153–300

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Functional Analysis of Endothelial Signaling Systems in Experimental Tumors G. BREIER, A. NICOLAUS, M. LABELLE, S. KIRSNEROVA, R. HEIDENREICH1

Institut fu¨r Pathologie, Technische Universita¨t Dresden 1 Universita¨ts-Hautklinik, Universita¨t Tu¨bingen Aims: Endothelial receptor tyrosines (RTK), in particular the receptors for vascular endothelial growth factor (VEGFR), and transcriptional regulators that control their expression are thought to be key mediators of angiogenesis in human tumors. We have investigated the function of these signalling molecules in different tumor models. Methods: Expression of RTK mRNA and protein was investigated in Ras-transformed mouse mammary tumor (MMT) cells grown in culture or in syngeneic mice. Functional studies were performed by retrovirusmediated gene transfer of dominant-negative (dn) VEGFR mutants or of a dn Ets-1 transcription factor mutant in experimental BFS-1 fibrosarcoma or B16 melanoma. Results: Cultured MMT cells expressed VEGFR-1 following epithelial to mesenchymal transition. In vivo, MMT showed endothelial expression of VEGFR-1 and VEGFR-2. Gene transfer of dn VEGFR inhibited growth and vascularization of BFS-1 fibrosarcoma but not of B16 melanoma. Gene transfer of dn Ets-1 reduced (BFS-1) or abolished (B16) VEGFR-2 expression but not tumor growth. Conclusions: Angiogenesis in different tumors depends to a variable degree on VEGFR signalling. Endothelial RTK may trigger not only tumor angiogenesis but also tumor cell growth, survival, and/or invasion. Although Ets-1 is expressed in angiogenic tumor endothelium and regulates the expression of angiogenic receptors, its function is not essential for tumor angiogenesis.

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Activation of NF-jB pathway is essential during Taxols- induced apoptosis in human epithelioid sarcoma cells M. HASSAN, P. REINECKE, H.E. GABBERT, C. MAHOTKA Institut fu¨r Pathologie, Universita¨t Du¨sseldorf

Aims: Paclitaxel (Taxols), a naturally antimitotic agent, has shown significant cell-killing activity in a variety of tumor cells through induction of apoptosis. Although the human epithelioid sarcoma (ES) is resitant to many anticancer drugs, the ES cell line GRU-1 and its clonal subpopulations (GRU-1A, GRU-1B, GRU-1C) have

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been shown to be sensitive to paclitaxel treatment. So far, the mechanism by which paclitaxel induces cell death is not entirely clear. In this study, we examined the effect of paclitaxel on the NF-signaling pathway that thought to be involved in the modulation of paclitaxelinduced cell death in ES cells. Methods: MTT assay, H&E staining, Western blot, Kinase assay, and EMSA. Results: Paclitaxel was found to induce apoptosis in all ES cell lines, the effect being most pronounced in GRU1 and GRU-1C. Although Paclitaxel did not appear to affect the expression of the IkappaB kinase alpha (IKKa), the treatment with Paclitaxel increased markedly the basal activity of IKKa as well as the activity of the nuclear transcription factor NF-kB in ES cells. Interestingly, the activation of IKKa NF-kB was closely related to the frequency of Paclitaxel-induced apoptosis in ES cells. Pretreatment of ES cells with NF-kB inhibitor, Bay11-7082, abolished paclitaxel-induced activation of NF-kB, and inhibited the apoptosis induced. Conclusions: Our data provide evidence that activation of the NF-kB/IkB-a signaling pathway is an essential part of the mechanism by which the anticancer drug, Paclitaxel induces apoptosis in human epithelioid sarcoma cells.

248 Chromosomal alterations in melanoma cell lines lacking presentation of HLA class I N. ARENS, A. SUCKER1, A. PASCHEN1, D. SCHADENDORF1, R. HILDENBRAND Institut fu¨r Pathologie, Universita¨tsklinikum Mannheim 1 Klinische Kooperationseinheit fu¨r Dermato-Onkologie des Deutschen Krebsforschungszentrums am Universita¨tsklinikum Mannheim Aims: Numerous clinical studies aim to create immunotherapeutic strategies to inhibit tumor growth. This is achieved by stimulation of cytotoxic T cells targeting the tumor cells. It is shown that tumor cells are able to develop mechanisms to escape recognition of cytotoxic T-cells by exhibiting a phenotype that is characterized by a lack of HLA class I presentation on the surface. We questioned whether this HLA-negative phenotype is associated with chromosomal aberrations especially of chromosome 15 where the coding gene for ß2-microglobulin is located. Methods: From three melanoma patients a primary tumor cell line was established. Also peripheral blood mononuclear cells (PBMCs) were isolated from each patient and grown in cell culture. From each sample pair DNA was isolated and LOH analysis was performed using the microsatellite markers

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D15S642, D15S818 and D15S1015, all located on chromosome 15. Metaphase spreads were prepared from the primary tumor cell lines and characterized cytogenetically. A DNA sample specific for the a-satellite region and the subtelomeric region of chromosome 15 was hybridized to the metaphase spreads. Results/Conclusions: Molecular cytogenetic and LOH analyses revealed numerous chromosomal rearrangements including complete and partial deletions of chromosome 15. These results show that lack of HLA class I presentation correlates with genomic alterations.

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Molecular analysis of a medulloblastoma mouse

model O. SHAKHOVA, C. LEUNG, S. MARINO Institute of Clinical Pathology, University of Zu¨rich, Zu¨rich, Switzerland Aims: Medulloblastoma are common tumors of childhood which typically arise from granule cell precursors in the cerebellum. We have developed a mouse model of medulloblastoma by Cre-LoxP-mediated inactivation of Rb and p53 tumor suppressor genes in cerebellar granule cell precursors. Here we set out to dissect the crucial role of p53 pathogenesis in our mouse model. Methods: GFAP-Cre; RbLoxP/LoxP mice were bred with ATM+/ to obtain GFAP-Cre; RbLoxP/LoxP ;ATM+/ which were intercrossed to generate GFAP-Cre; RbLoxP/LoxP ;ATM/ . A similar breeding sheme was used to generate mice simultaneously mutant for Rb and p19 ARF/ , p21/ (GFAP-Cre; RbLoxP/LoxP; p19 ARF/ or GFAP-Cre; RbLoxP/LoxP; p21/ ). Results: No medulloblastomas have been detected in mice simultaneously mutant for Rb and p19 ARF/ or p21 or ATM, therefore pointing at a prime role of lack of p53 in the pathogenesis of medulloblastoma in our model. In addition, we show that at postnatal day 15 a significant percentage of mutant granule cell progenitors lacking Rb and p53 are still actively proliferating, are located in external granule layer (EGL) and showed delayed terminal differentiation. These results point towards a deregulation of the crucial balance between proliferation and differentiation in granule cell precursors lacking Rb and p53 well before the development of the neoplasms. Conclusions: The lack of p19 or p21 is dispensable for neoplastic transformation of Rb deficient EGL precursor cells. Deregulation of the proliferation/differentiation switch of EGL precursor cells lacking Rb and p53 precedes neoplastic transformation.

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Ets-1 expression enhances the transformed phenotype of epithelial HeLa cells by inducing migration, invasion and anchorage-independent growth J.C. HAHNE1, A.F. OKUDUCU1,2, A. KAMINSKI1, A. FLORIN1, F. SONCIN3, N. WERNERT1 1

Institut fu¨r Pathologie, Universita¨t Bonn Institut fu¨r Neuropatho-logie, Charite´ Berlin 3 Institut de Biologie de Lille, Lille (F) 2

Aims: Ets-1 is the prototype of the ETS family of transcription factors. In human tumours Ets-1 is expressed in endothelial cells and fibroblasts of the tumour stroma and is suggested to play a role for both tumour vascularization and invasion probably by upregulating several matrix-degrading proteases. In human carcinomas Ets-1 can also be expressed by the neoplastic cells but little is known about the functional implications of this finding. We therefore adressed the roles of Ets-1 in the epithelial HeLa tumour cell line which shows low levels of Ets-1 expression. Methods: HeLa cells were transfected with plasmids encoding human ets-1 m-RNA in sense and anti-sense (RNAi) orientation, respectively. From these transfections stably Ets-1 over- and under-expressing HeLa cell lines were selected by neomycin addition. Ets-1 based effects were evaluated using soft- agar, wound and Boyden Chamber assays. In addition expression of different integrins were analysed. Results: We found that Ets-1 overexpression increases the transformed phenotype of HeLa cells by promoting anchorage independent growth in the soft-agar assay as well as cell migration and invasion in wound and Boyden Chamber assays. In contrast Ets-1 down-regulation reduced cell attachment. In line with this finding we found that Ets-1 up-regulation increases integrin-b2 expression. Expression of integrins- b4, -a6, -a3b1 and -b3 were not affected. Conclusions: The present results suggest that Ets-1 can favor tumour development and progression by increasing neoplastic properities of the tumour cells in addition to its well establised roles in the tumour stroma for tumour vascularization and invasion.

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Parvovirus B19-associated inflammatory cardiomyopathy: Distinct viral proteins are involved in apoptosis and ion channel activation T. BOCK1, A. LOUPESCU2, P. LANG1,2, F. LANG2, R. KANDOLF1

1

Abteilung fu¨r Molekulare Pathologie, Tu¨bingen Institut fu¨r Physiologie, Tu¨bingen

2

ARTICLE IN PRESS Posters: Molecular Pathology II / Pathology – Research and Practice 201 (2005) 153–300

Aims: Parvovirus B19 (PVB19) has been described to be associated with inflammatory cardiomyopathy. As target cells of PVB19 endothelial cells of small vessels have been demonstrated. The aim of our study was to investigate the interaction of distinct PVB19 proteins with inflammatory and apoptotic processes with respect to endothelial dysfunction. Methods: We generated inducible stable cell lines which express the PVB19 proteins NS1, VP1 and VP2. To determine apoptotic processes annexine-binding, cyto16 and TUNEL assays were performed. To investigate ion channel activation (ICRAC) Fura-2 immunofluorescence was established. Results: After induction of NS1 and VP2 significant annexine binding and DNA fragmentation was demonstrated. It has been described recently that phospholipase A2 (PLA) has an impact on the regulation of the Ca2+ channel ICRAC and therefore on the activation of endothelial cells. A viral VP1-PLA activity has been recently shown. Using Fura-2 immunofluorescence we can show that after totally depletion of intracellular CA2+ the Ca2+ influx under the control of VP1 was significantly enhanced in comparison to VP1-PLA mutant (negative) cell lines. Conclusion: Our findings demonstrate that the PVB19NS1 and VP2 proteins are involved in apoptotic processes. Additionally the VP1 protein can activate ICRAC and therefore modulate the Ca2+ homeostasis by its PLA activity. We therefore conclude that the major PVB19 proteins VP1, VP2 and NS1 demonstrate functional characteristics in respect to apoptosis and Ca2+ dependent signalling which are important for the development of PVB19-associated endothelial dysfunction.

252 Cell contact mediates VEGF expression via b-integrin, FAK and IRS molecules M. NEID, A. TANNAPFEL, K. DATTA1, D. MUKHOPADHYAY1 C. WITTEKIND Institut fu¨r Pathologie, Universita¨tsklinikum Leipzig Mayo Clinic Cancer Center and Department of Biochemistry and Molecular Biology, Gugg 1401A, Mayo Clinic, 200 First Street SW, Rochester MN 55905, USA 1

Aims: Investigation on the impact of cell-cell contact vs. extra cellular matrix on VEGF-expression and the influence of b1-integrin, its kinase FAK and the IGF1R effector molecules IRS-1 and IRS-2. Methods: AsPC-1 pancreatic cancer cells were cultured in low and high confluency and on plastic or fibrinectin/ ECL coated plates. 0.35kb (SP1) vs 2.6kb (HIF-1) VEGF promoter activity was measured by luciferase assays. Expression of phospho-FAK and b1-integrin was

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detected via Western blot and RT-Real time PCR. dN FAK was transfected into the cells and VEGF transcription as well as IRS-1 and IRS-2 expression was measured by luciferase assay or RT-Real time PCR. b1-integrin was blocked by antibody and IGF-1R, IRS-1, IRS-2 and VEGF expression was deteced by RT-Real time PCR. Results: Fibronectin or ECL did not change VEGF transcription but inreased cell-cell contact did. In more confluent cells, there was more b1-integrin and more active FAK. Blocking b1-integrin resulted in increased expression of VEGF, IGF-1R and IRS-2, but not IRS-1. After blocking FAK, increased 0.35kb VEGF transcription was detected but 2.6kb promoter activity decreased. At the same time, IRS-2 was up- and IRS-1 was down regulated. Conclusions: In low confluent AsPC-1 cells, Sp-1 but not HIF-1 dependent VEGF transcription is activated via b1-integrin and its kinase FAK through differential expression of IRS proteins. Fibronectin and ECL components had no impact on VEGF expression.

253 Lipid raft protein composition distinguishes stem cells from tumour cells A. OSTERHUES, E. ZEINDL-EBERHART, L. GRAEVE1, R. HUSS Pathologisches Institut, Ludwig-Maximilians-Universita¨t Mu¨nchen 1 Institut fu¨r Biologische Chemie, Universita¨t Hohenheim Aims: A stem cell line was established from the canine bone marrow, which could differentiate into different tissue lineages upon growth factor signalling or cell-cell interactions. The transfection of the stem cell line with a gene of a heterodimeric MHC protein led to malignant transformation and tumour growth in NOD/SCID mice suggesting functional and compositional changes in cell signalling associated lipid rafts. Methods: Comparative proteome analysis was performed with lipid raft samples isolated from stem cell—and tumour cell lines as detergent insoluble membrane fractions by sucrose density ultracentrifugation. Lipid raft samples were analyzed by two-dimensional electrophoresis (2-DE). Protein spots different in their intensities were identified by MALDI-TOF-MS. Results: Caveolin-1 and flotillin-1 were identified and represent characteristic raft-associated marker proteins. Galectin-3, involved in cell migration, cell adhesion and cell-cell interaction, was found in increasing amounts in tumour cells as compared with the original stem cell line. Vimentin, a member of the intermediate filament family, functioning like caveolin-1 as a scaffolding protein, was found to be strongly reduced in tumour cells. Structural

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similarities between vimentin and several transcription factors like c-Fos, Fra1, CREB and c-Jun suggest an involvement of vimentin in regulatory cell functions. Conclusions: Stem cells and associated tumour cells can be distinguished by differences in lipid raft protein composition.

254 Reversible methylation of SOCS-3 in head and neck squamous cell carcinoma and its precursor lesions A. WEBER1, U. HENGGE3, I. TISCHOFF2, F. SOMMERER2, A. MARKWARTH, A. DIETZ1, CH. WITTEKIND2, A. TANNAPFEL2 1

Klinik fu¨r Hals-Nasen-Ohrenheilkunde, plastische Operationen 2 Institut fu¨r Pathologie, Universita¨t Leipzig 3 Klinik fu¨r Dermatologie, Universita¨t Du¨sseldorf Aims: This study was performed to elucidate the role of Suppressor of Cytokine Signaling (SOCS)-3 in the tumorigenesis of squamous cell carcinoma of the head and neck (HNSCC) and its precursor lesions. Methods: After microdissection, promoter methylation of SOCS-3 was studied by using methylation-specific PCR in 94 HNSCC, corresponding normal mucosa, lymph node metastases and 16 high and 21 low grade dysplasias. The presence of SOCS-3 mRNA transcripts was confirmed by semiquantitative real-time PCR, and the SOCS-3 protein was analyzed immunohistochemically. Results: SOCS-3 hypermethylation was found in 85/94 HNSCC (90%) and in 10/16 high and 9/21 low grade dysplasias. Lymph node metastases exhibited an identical methylation status as the primary tumors. In the cell lines tested, SOCS-3 transcripts increased upon treatment with the demethylation compound 5-aza2-deoxycytidine. Overexpression of wild type SOCS-3 in carcinoma cells with methylated SOCS-3 resulted in the induction of apoptosis and growth suppression as well as downregulation of STAT3, bcl-2 as well as bcl-x. Conclusions: Our data suggest, that promoter methylation of SOCS-3 is involved in the carcinogenesis of HNSCC. Due to its involvement in tumor growth, restoration of SOCS-3 may hold treatment potential for HNSCC.

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Versicans are potent inhibitors of cell and axon migration during embryonic development and in the adult central nervous system M.T. DOURS-ZIMMERMANN, S. DUTT, E.T. STO¨CKLI1, L. SOMMER2, R. FA¨SSLER3, D.R. ZIMMERMANN

Institute of Clinical Pathology 1 Institute of Zoology, University of Zurich (CH) 2 Institute of Cell Biology, ETH Zurich (CH) 3 Department of Molecular Medicine, MPI Martinsried Aims: We have previously shown that the extracellular matrix proteoglycan versican is present in tissues that are non-permissive for axonal growth. During the development of the peripheral nervous system, the large splice-variants V0 and V1 are expressed in tissue barriers that block migratory neural crest cells and direct extending axons. Furthermore, versican V2 is exclusively expressed in the mature CNS in association with the axonal growth inhibiting myelin sheaths. We are currently exploring the potentially inhibitory properties of versicans in cell and axonal migration and regeneration processes in vitro and in vivo. Methods: We have isolated versican V0, V1 and V2 and have tested their inhibitory capacity in stripe choice assays in vitro and by ectopical injections into axon growth pathways in chick embryos in ovo. In addition, we prepared a versican V0/V2 specific KO-mice. Results: All proteoglycan isoforms of versican are potent inhibitors of axonal growth and neural crest cell migration in vitro and in ovo. Versican V2 knockout mice are viable, fertile and display no overt phenotype. The myelinated fiber tracts appear unchanged, however, the lack of versican V2 affects the deposition of other extracellular matrix components at the nodes of Ranvier. Conclusions: Versicans act as potent inhibitors of neural crest cell migration and axonal growth by negatively modulating substrate-adhesion. These findings suggest a functional involvement of versican in axonal guidance, in impeding CNS regeneration and in controlling cell migration processes.

256 Frequent mutations of the exon 3 of the b-catenin gene differentiate between superficial and deep fibromatoses. B. BODE, B. ODERMATT, D. ZIMMERMANN Institut fu¨r Klinische Pathologie, Universita¨tsspital Zu¨rich, Schweiz Aims: Determination and comparison of the protein expression and the frequencies of beta-catenin gene mutations in deep and superficial fibromatoses. Methods: The distribution of the b-catenin expression was evaluated using imunohistochemistry. Mutations in exon 3 of the b-catenin gene were detected by PCR amplification and subsequent sequencing of the DNA

ARTICLE IN PRESS Posters: Orthopedic Pathology / Pathology – Research and Practice 201 (2005) 153–300

extracted from formalin fixed, paraffin embedded routine archival tissues. Results: Twelve cases of superficial (7 palmar and 5 plantar; 4 female and 8 male patients; mean age 47y) and 15 cases of deep fibromatosis (7 intraabdominal and 8 extraabdominal tumors; 3 patients with known familial adenomatous polyposis (FAP); 8 female and 7 male patients; mean age 45y) were investigated. Moderate to strong nuclear and cytoplasmatic expression of bcatenin was found in the majority of cells in all examined cases of superficial and deep fibromatoses. Two types of non-synonymous point mutations (codon 41 and 45) in exon 3 of the beta-catenin gene were identified in 9/15 (60%) of deep fibromatoses. In tumors of the 3 patients with FAP no b-catenin gene mutations could be detected. Furthermore, no mutations were found in any of the plantar and palmar fibromatoses (0/12). Conclusions: These data suggest that superficial and deep fibromatoses are two different kinds of myofibroblastic proliferations. The cause of the nuclear and cytoplasmatic overexpression of b-catenin in the plantar and palmar fibromatoses remains to be elucidated.

Posters: Orthopedic Pathology 257 Apoptosis resistance in pigmented villonodular synovitis I. BERGER, S. AULMANN, V. EHEMANN, H. WECKAUF, M.L. GROSS, B. HELMCHEN Institut fu¨r Pathologie, Universita¨t Heidelberg Objective: Pigmented villonodular synovitis (PVNS) is a proliferative lesion originating from synovial tissue with a locally aggressive behaviour. We analysed the pathogenetic role of apoptosis resistance for sustained cell proliferation in PVNS. Methods: The expression of bcl-2, p53 and Ki-67 was examined in 80 cases of PVNS using immunohistochemistry. In 43 of these cases, DNA content and distribution of cell-cycle phases were investigated by flow cytometry. Additionally, 10 cases of PVNS were analysed by multiparametric flow cytometry for expression of p53, caspase3, and bcl-2 and by TUNEL to detect DNA fragmentation. Results: No apoptotic cell fractions were detected in any investigated cases. Expression of bcl-2 was found in 84% of cases (up to 6.5% of cells) and was significantly associated with DNA-fragmentation observed by TUNEL (p ¼ 0:037). Orthologous p53 expression was observed in 37% of cases. The level of p53 expression correlated with the proliferative activity and the expres-

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sion of both caspase3 (p ¼ 0:017) and bcl-2 (p ¼ 0:0013). (No statistically significant correlations between expression of bcl-2, p53, caspase3, DNA fragmentation or proliferative index and age, sex of patients, disease recurrence, growth pattern or size of lesion were found.) Conclusion: Apoptosis resistance is a critical event in the progression of PVNS and may contribute to the survival of the proliferating synovial cells in PVNS and to the permanent slow progression of these lesions.

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Expression of cyclooxygenases and prostaglandin receptors in growth plate and articular cartilage of the rat: An in situ study CH. BROCHHAUSEN, P. NEULAND1, R. NU¨SING1, G. KLAUS1, C.J. KIRKPATRICK Institut fu¨r Pathologie, Universita¨t Mainz 1 Zentrum fu¨r Kinderheilkunde, Universita¨t Marburg Aims: The role of Prostaglandin E2 (PGE2) for proliferation of growth plate chondrocytes was demonstrated by in vitro studies. The effects of PGE2 are mediated by the receptors EP-1 to EP-4. However, expression of EP receptors and COX-1 were not analysed in growth plate and articular cartilage in situ to confirm their physiological and pathophysiological role in vivo. Methods: Frozen, paraformaldehde fixed sections from the rat proximal tibia were fixed and stained by the alkaline-phosphatase-anti-alkaline-phosphatase (APAAP) method. Polyclonal rabbit antibodies against COX-1, COX-2, EP-1 and EP-2 were used. Staining for collagen II and collagen X functioned as positive controls. Results: A homogenous expression of COX-1, EP-1 and EP-2 receptor in all differentiation zones of the growth plate could be demonstrated. For COX-2 a lack of staining in the reserve zone chondrocytes was found. Chondrocytes of articular cartilage showed the expression of EP-1, EP-2, COX-1 and COX-2 in a markedly different pattern with less expression in apical cell layers. Conclusions: The present study demonstrated the in situ expression of EP-1, EP-2 and COX-1 and COX-2 in growth plate and articular chondrocytes and revealed their physiological role. These findings warrant further studies on the relevance of the prostaglandin system in developmental disorders and its possible role in tumorgenesis of chondrogenic tumors.

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Analysis of the dedifferentiating effect of IL-1 on human chondrocytes T. SCHUBERT1, A.K. BOSSERHOFF1, S. GRA¨SSEL3, W.FALK2, J. RADONS2

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1

Institut fu¨r Pathologie, Universita¨t Regensburg Klinik fu¨r Innere Medizin I, Universita¨t Regensburg 3 Klinik fu¨r Orthopa¨die, Universita¨t Regensburg 2

Aims: Interleukin-1 (IL-1) activates numerous signalling pathways that may be involved in cartilage destruction and dedifferentiation of chondrocytes. Thus, identification of the cartilage-degrading mechanisms remains an exciting challenge. Methods: Experiments were carried out in the chondrosarcoma cell line SW1353 and cartilage explants. After determining the expression of certain members of the IL-1 signalling cascade, we analysed the effect of IL-1 on the different systems. To block specifically the IL-1-induced signalling pathways mediating chondrocytic dedifferentiation, we incubated SW1353 cells and explants with IL-1 in the presence or absence of inhibitors of p38MAPK (SB203580), p44/p42MAPK (PD98059), PI3 K (Wortmannin, LY294002), and JNK (CEP11004, SP600125). We analysed expression of Aggrecan at the RNA level as well as secretion of IL-6 and pro-MMP1. Results: After IL-1 exposition we found a reduction in the expression of Aggrecan in SW1353 cells and cartilage explants at the RNA level. SB203580 attenuates this effect in SW1353 cells. PD98059 partially reverses this effect in cartilage explants. Furthermore, inhibitors of p38MAPK and/or PI3 K reduced the IL-1induced IL-6 secretion and pro-MMP1 release in SW1353 cells. Conclusion: The dedifferentiating effects of IL-1 on chondrocytes are mediated by differing pathways in chondrosarcoma cells and cartilage explants. The pathways regulating IL-1 induced IL-6 and proMMP1 secretion by SW1353 cells partially overlap with the pathways regulating IL-1 induced dedifferentiation. This knowledge may help us to develop new modulation strategies in autologous chondrocyte transplantation.

260 Translocation detection in extraskeletal myxoid chondrosarcoma using formalin-fixed paraffin-embedded tissues G. WEIRICH1, N. KOLEGANOVA1, K. BINK2, H. HO¨FLER1, F. FEND1 1

Institut fu¨r Pathologie, Technische Universita¨t Mu¨nchen 2 GSF Forschungszentrum Neuherberg Aims: Extraskeletal myxoid chondrosarcoma (EMC) is a rare low-grade soft tissue sarcoma with a high propensity to recur and metastasize. Cytogenetically, 75% of EMC harbour chromosomal translocations involving

the EWS and TEC genes. The differential diagnosis of EMC may be challenging and includes skeletal myxoid chondrosarcoma, myxoid liposarcoma, and Ewing/PNET sarcomas. This study aimed to establish an RNA-based method to detect the EWS/TEC fusion transcript in formalin-fixed paraffin-embedded tissues. Methods: RNA was extracted from formalin-fixed paraffin-embedded tissues including 5 EMC and 3 negative controls (1 skeletal myxoid chondrosarcoma, 1 myxoid liposarcoma, 1 ES). To detect the 3 major types of the tanslocation t(9;22)(q22-31;q12), namely EWS12/TEC (type 1), EWS7/TEC (type 2), EWS12nt1262/TEC (type 3), three novel sets of primers were used to amplify short cDNA fragments (o300 base pairs). PCR products were directly sequenced. Results: The type 1 transcript was detected in 2/5 EMC, type 2 in 1/5 EMC. A type 3 transcript was not found. In 2/5 EMC and in the 3 negative controls neither one of the transcripts was detected. Conclusions: This RNA-based method will be a useful diagnostic adjunct for EMC and expands the range of molecular tests for the differential diagnosis of sarcoas using routinely processed material.

261 TNF activates the mitogen activated protein kinases p38 and ERK in the synovial membrane in vivo B. GO¨RTZ1, S. HAYER2, B. TUERK2, K. REDLICH2, J. ZWERINA2, J.S. SMOLEN2, G. SCHETT2 1

Institut fu¨r Pathologie, Universita¨t Gießen Abteilung fu¨r Rheumatologie, Klinik fu¨r Innere Medizin III, Allgemeines Krankenhaus Wien, A 2

Aims: Tumor-necrosis-factor (TNF) is one of the major factors of synovial inflammation in rheumatoid arthritis. TNF effects are mediated through activation of mitogen-activated protein kinases (MAPKs), such as ERK, p38MAPK and JNK. Methods: In the present study we examined human TNF transgenic (hTNFtg) mice, representing an in vivo model of TNF-induced arthritis for activation of these MAPKs. Paraffin sections of the paws were examined immunohistochemically for the expression of the phosphorylated forms of ERK, JNK and p38MAPK in untreated hTNFtg mice and after systemic blockade of TNF, IL-1 and RANKL as well as in wild-type mice. Results: Increased activation of p38MAPK and ERK was found in the synovial membrane of hTNFtg mice. Activated p38MAPK was predominantly found in synovial macrophages, whereas ERK activation was present in both synovial macrophages and fibroblasts.

ARTICLE IN PRESS Posters: Orthopedic Pathology / Pathology – Research and Practice 201 (2005) 153–300

Systemic blockade of TNF reduced activation of p38MAPK and ERK, whereas inhibition of IL-1 only affected p38MAPK. Blockade of RANKL did not show any decrease of MAPK activation in the synovial membrane. Expression of activated JNK was generally low in all cell types and not modified by the various treatments. Conclusion: TNF mainly activates p38MAPK and ERK in the synovial membrane exposed to TNF. Blockade of TNF reduces the activation of these two MAPKs. A direct pharmacological inhibition of p38MAPK and ERK may have beneficial effects on synovitis in TNFmediated arthritis by blocking the TNF effects in the joints.

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infiltrate with CD3 (r ¼ 0:754) and CD138 (r ¼ 0:744) and for the stroma activation with CD14 (r ¼ 0:744). Conclusions: The proposed synovitis-score is based on well definable histopathologic criteria and contributes to the diagnosis of rheumatic and non-rheumatic joint diseases.

263 CD52 expression on osteogenic tumors R. GU¨NTHER, K.-D. SCHASER1, I. MELCHER1, V. KRENN Department of Pathology, Charite Campus Mitte, Berlin 1 Center for Musculoskeletal Surgery, University Hospital Charite, Berlin

262

Standardized histopathological grading of chronic synovialitis

L. MORAWIETZ, T. BO¨HME, T. HA¨UPL1, V. KRENN Institut fu¨r Pathologie, Charite´ Berlin 1 Klinik fu¨r Rheumatologie, Charite´ Berlin Aims: To establish a feasible, standardized histopathological graduation system for chronic synovialitis irrespective of the etiology. The system evaluates (1) the enlargement of the lining cell layer, (2) the lymphocytic infiltrate and (3) the stroma activation semiquantitatively from absent (0 points) to strong (3 points). Methods: Four hundred and eighty three synovial specimens (resections n ¼ 462; biopsies n ¼ 21) were graded by two independent observers. Clinical diagnoses were osteoarthrosis (OA; n ¼ 153), posttraumatic arthritis (PoA; n ¼ 31), rheumatoid arthritis (RA; n ¼ 246), psoriatic arthritis (PsA; n ¼ 32), reactive arthritis (ReA; n ¼ 7), and controls (Co, n ¼ 21) from necropsies of patients without joint damage. Additionally, the gene expression profile of n ¼ 30 synovial biopsies graded, according to the score, were analyzed with Affymetrix HG-U133A microarrays. Results: The correlation between two observers was high (po0:001). Median synovialitis scores when correlated to clinical diagnoses were: Co 0.5, OA 2, PoA 2, PsA 3 ReA 4, RA 5. The differences in scores between Co and all other groups were highly significant (po0:001). Also, the differences between degenerative and inflammatory joint diseases were significant (po ¼ 0:005). A synovialitis-score of 5 points and more is strongly associated with primary inflammatory, rheumatic joint diseases. Validation of the synovialitis-score by gene expression data showed good correlations for the lining cell enlargement with MMP1 (r ¼ 0:685), for the leukocytic

Aim: In a previous microarray analysis we found CD52 on giant cell tumors of the bone (GCT). Surprisingly, our group detect that certain solid tumours and bonerelated tumours in particular chondrosarcomas (CS) and osteosarcomas (OS) express the cellular antigen CD52 in vivo and in vitro. CD52 is a GPI-anchored antigen, also named CAMPATH-1 antigen, and it is abundantly expressed on lymphocytes, monocytes, macrophages and eosinophils as well as within the male genital tract. The sperm CD52 antigen differs from CD52 lymphocyte antigen in its carbohydrate structure. Methods: RNA was prepared from tissue samples and cell lines each of GCTs, OSs and CSs for analysis by RT-PCR, real time PCR and Western blot. Paraffinembedded tissue of GCTs (primary tumour n ¼ 16; relapse tumour n ¼ 6), OSs (tumor n ¼ 4; metastasis n ¼ 5) and CSs (n ¼ 4) were used for immunohistochemical analysis. For each case HE, CD3, CD20, CD34, CD52, CD68 and Ki67 staining were performed. All tumours were semi-quantitatively scored. Results: In tissue equally in cell lines of GCTs, OSs and CS both RNA and protein could be detected. Immunohistochemical analyzation show, that CD52 was expressed on giant cells, macrophage- and stromal-like cells in GCTs. For OSs a CD52 expression could be detected in osteoblast- and chondroblast-like tumor cells as well as in osteclasts. A mean value of 7 by the immunoreactivity score were detected for CSs. Discussion: Antibody directed to CD52 is capable of complement activation and it can be clinically useful to treat lympho-proliferative disorders. Therapeutically, osteogenic tumours can pose particular problems in that they can be localised in areas in which they can not be removed. Therefore we suggest, that the use of CAMPATH-1s can be an important tool for bone tumors that are currently difficult be treated at all.

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264 Validation of microarray data differentially expressed genes in giant cell tumors of bone R. GU¨NTHER, C. SERS, L. MORAWIETZ, I. MELCHER1, K.D SCHASER1, H.U.KASPER2, R.-J. KUBAN3, U. UNGETHU¨M3, V. KRENN Institut fu¨r Pathologie, Charite Campus Mitte, Berlin 1 Center fu¨r Unfallchirurgie, Charite Virchow Klinikum, Berlin 2 Institut fu¨r Pathologie, Universita¨t Ko¨ln 3 Labor fu¨r funktionelle Genomforschung, Charite Campus Mitte, Berlin Aims: Giant cell tumors of the bone (GCT) are local osteolytic neoplasms with variable degrees of aggressiveness. The objective of the current study was the molecular characterization of primary and relapsed GCTs. We established gene profiles and discovered a small number of genes that may be causatively involved in the pathogenesis of the GCT recurrence process. Methods: RNA was prepared from seven frozen GCT samples (primary tumor n ¼ 5; relapses n ¼ 2) and used for the microarray analysis, whereas Paraffin-embedded tissue (primary tumor n ¼ 16; relapse tumor n ¼ 6) were used for immunohistochemical analysis of CD10, CD52, Ephrin A1, AMFR, FGFR3 and Claudin 7. Results: A total of 66 genes were scored as significantly differentially expressed in microarray analysis. Many of these genes recognized by our analyses concerned an important biological process, such as receptor tyrosine kinases, suggesting that many of these genes be part of the neoplastic and recurrence progression. We found immunohistochemically a significant down-regulation of CD52, Claudin 7, Ephrin A1 and AMFR when compared primary with relapse GCT tissue Conclusion: In summary we have identified a set of genes that shed light on the molecular basis underlying the pathogenesis and progression of GCT. More studies are needed to identify which mechanisms are involved in GCT. Further characterization of these genes mayrefine our understanding of GCTs and enhance our ability to diagnose and manage these patients appropriately.

265 Correlation of quantitative histopathological morphology and quantitative radiological analysis during aseptic loosening of hip endoprothesis S. BERTZ, J. KRIEGSMANN1, A. ECKARDT2, M. OTTO1 Institut fu¨r Pathologie, Universita¨t Regensburg 1 Gemeinschaftspraxis fu¨r Pathologie Trier 2 Orthopa¨dische Klinik, Universita¨t Mainz

Aims: Aseptic hip prostheses loosening, caused by polyethylene wear, are the most important longterm complication in total hip arthroplasty. The single-shot radiological analysis (EBRA) is a computerized method for early radiological prediction of aseptic loosening. In this study EBRA parameters were correlated with histomorphological parameters of the periprosthetic membrane. Methods: Periprosthetic membranes obtained from 19 patients of loosened ABG I-type total hip prostheses were analysed histologically and morphometrically. The EBRA parameters thickness of PE debris layer and dimension of inclination and anteversion were compared with the density of macrophages and giant cells. Additionally, the semiquantitatively determined density of lymphocytes, plasma cells, giant cells and size of necrotic areas was correlated with the EBRA results. Results: All periprosthetic membranes were classified as debris-induced type membranes. We found a positive correlation between number of giant cells and thickness of PE layer. There was no significant correlation between number of macrophages or all semiquantitative parameters and EBRA parameters. The number of giant cells decreases with implant duration. Conclusions: The number of foreign body giant cells reflects most suitable the results of the EBRA, but only in morphometrical analysis. The density of macrophages, lymphocytes, plasma cells and size of necrotic areas does not correlate with the EBRA indicating that there is no correlation with aseptic loosening.

266 Osteoarthritic chondrocytes are catabolically more active, but less responsive to catabolic stimulation with IL-1ß T. AIGNER, Z. FAN, S. SOEDER, TH. KIRCHNER Institut fu¨r Pathologie, Universita¨t Erlangen-Nu¨rnberg Aims: IL-1 is thought to be one of the most important cytokines relevant for osteoarthritic joint disease. The aim of this study was to investigate whether there are different effects of low and high concentrations of IL-1ß on the expression level of anabolic and catabolic genes as well as cytokines and whether there is any difference in reactivity of normal and osteoarthritic chondrocytes. Methods: Gene expression levels (collagen type II, aggrecan, MMP-1, MMP-2, MMP-3, MMP-13, ADAMTS-4, IL-1ß, IL-6, LIF) were detected by realtime PCR in primary human articular chondrocytes (normal (n ¼ 6); osteoarthritic (n ¼ 7)) after stimulation with 0.01 ng, 0.1 ng, 1 ng and 10 ng/ml IL-1ß. Results: In normal adult articular chondrocytes the expression of aggrecan and collagen type II genes were

ARTICLE IN PRESS Posters: Urogenital Pathology / Pathology – Research and Practice 201 (2005) 153–300

both significantly down-regulated by Il-1ß. In contrast, matrix degrading proteases – except MMP-2 – as well as the investigated cytokines IL-1ß, IL-6, and LIF were induced by IL-1ß dose-dependently. The strongest regulation was found for IL-6 and LIF. Osteoarthritic chondrocytes, showed strongly increased basal levels of catabolic enzymes and mediators investigated, but appeared to be less responsive to further stimulation with IL-1ß. Conclusions: IL-1ß activity is critically dependent on the applied concentration and the reactivity of the cells stimulated and appears to be significantly reduced in osteoarthritic chondrocytes. From our study, it remains open whether this indicates that osteoarthritic chondrocytes are hardly influenced by IL-1 or are already so strongly stimulated during the disease process that they are refractory to further stimulation with IL-1ß.

Posters: Urogenital Pathology 267 Frequency of positive surgical margins in different methods of radical prostatectomy J. ZUMBE, E. SHEREMET1, H.-J. TERPE1 Klinik fu¨r Urologie 1 Institut fu¨r Pathologie, Klinikum Leverkusen Aims: Nowadays the radical prostatectomy is the gold standard in the treatment of locally confined prostate cancer. Patients with positive surgical margins have a high risk of recurrences, and are often considered for adjuvant therapy. The purpose of this retrospective study was to determine the correlation between the positive margins and the different methods of radical prostatectomy. Methods: About a period of 24 months radical prostatectomies (203 laparoscopic, 48 retropubic and 67 perineal cases) were implemented from 318 patients. The distribution of grading and staging was similar in all cases. All surgical specimens were completely examined for histology. A positive surgical margin was defined as the extension of tumour to the inked surface of the resected specimen. For the documentation we used a standardization protocol. Results: A total of (1) 25% (52 cases) of the 203 laparoscopic cases had positive margins, whereas in the last 50 cases only 14% were positive. (2) (46%) (31 cases) of the 67 perineal cases had positive margins. (3) (50%) of the 48 retrobucic cases had positive margins. (4) In all cases the positive margins were correlated with higher grade and stage. (5) In laparoscopic and retropubic cases we found positive margins predominantly in

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the apex region. (6) In the perineal cases we determined more frequently positive margins in the basis region. Conclusions: Our results confirm that compared to the other methods the cases with laparoscopic prostatectomy had a lower rate of positive surgical margins.

268 Prospective multicentric radiation therapy study of locally advanced prostate cancer after radical prostatectomy (RP): Comparison of morphological and morphometric data of the local and central pathology and correlation with PSA follow up R. GOLZ, G. MAKHLOUF, T. WIEGEL1, U. STEINER2, A. SIEGMANN1, W. HINKELBEIN1, K. MILLER2, S. STO¨RKEL Institut fu¨r Pathologie, Universita¨t Witten/Herdecke 1 Klinik fu¨r Strahlentherapie u 2 Urologische Klinik, UKBF, Berlin Aims: A total of 15–60% of patients with pT3a/b or pT4 prostate cancer have signs of a progress within an intervall of 3–5 years after RP. The benefit of radiation therapy for these patients is being evaluated in a multicenter trial. Morphological results between local pathologist (LP) and central pathologist (CP) are compared. Correlation of morphological and morphometric data of RP specimen with PSA course is done. Methods: Slides of 257 RP (stage: XpT3a) from 20 centers were evaluated. Gleason Grading and TNM – classification from 1992 was used for more detailed stratification (pT3a,b,c). Morphometric analysis included measurement of extraprostatic extension (EPE) and length of positive surgical margins (SM+). Results: Comparison of Gleason-Grading in 167 cases exhibited discordances in 47%. Discrepancies in pT, pN and status of SM were found in 25%, 4% and 1 2%, when comparing LP and CP. On average 2.7 localisations of EPE were found/case, base and left neurovascular bundle being most often affected. Apex and base showed most frequent SM+ with an average of 1.5 positive SM in 171 cases with R1 resection. The length of the SM+ increased with stage of disease, but did not correlate with postop. PSA. Cases with postoperative persistently raised PSA (26% of cases) vs. normal PSA revealed a greater length of SM+ (+ 10.14 vs. 6.35 mm) and EPE (+ 3.29 vs. 2.25 mm). Conclusions: (1) Gleason Grading is affected by a high interobserver variability. (2) A central pathology is justified by high discrepancies in staging and grading. (3) The length of SM+ and EPE may be risk indicators for PSA failure.

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Gleason score, DNA ploidy and other prognostic parameters in carcinomas of the prostate M. REBEL, A. SPIETHOFF, M. KOSER1, M.H. BOHRER Institut fu¨r Pathologie, Klinikum Ludwigshafen 1 Urologische Klinik, Klinikum Ludwigshafen

Aims: In carcinomas of the prostate the relationship of the Gleason grading system to DNA ploidy was investigated. Both parameters and other factors were related to local recurrence and metastasis. Methods: Hundred and twenty one untreated patients with radical prostatectomies were included. Mean observation time was 37.5 months. DNA analysis was performed by image analysis. Prognostic parameters were the histologic grading systems by Gleason and Helpap, stage, surgical margins and lymphnode status. Results: During the observation time in 35 patients signs of tumour relapse and metastasis (verified histologically or serologically by an increase of PSA level) were detected. These risks significantly correlated to both histologic grading systems, to DNA ploidy, to stage, to surgical margins, and to lymphnode status. In a multivariate analysis however, the pathologic stage of the tumours was the only significant prognostic factor (po0:05). There was a significant relationship between DNA ploidy and Gleason score (po0:05). In Gleason sum score 7 tumours (n ¼ 76) DNA ploidy was markedly heterogeneous. In these tumours no significant relation-ship of DNA ploidy to the endpoints of our study could be verified. Besides, the predominant pattern (comparison of score 3+4 with score 4+3) had no significant influence on risk. Conclusions: The results of our study reveal that in carcinomas of the prostate tumour staging has superior importance for risk assess-ment. DNA ploidy was significantly correlated to the Gleason grading system. In the Gleason score 7 group DNA ploidy did not yield additional information for risk.

270 CSE1L protein expression in prostate carcinoma: Correlation with tumor stage, Gleason score, cell cycle proteins and 20q13.2 gain M. MUDERS, M. TOMA, I. HAUPT, G.B. BARETTON Institut fu¨r Pathologie, TU Dresden Aims: The CSE1L gene located on 20q13 is involved in cell cycle control and apoptosis. CSE1L protein is overexpressed in a variety of tumors. In this study, we investigated the cytoplasmic expression of CSE1L in

prostate cancers and correlated it with TNM stage, Gleason score, 20q13.2 gain, proliferation and expression of p53, cyclin D1 and cyclin A. Methods: Samples for immunhistochemical analysis were obtained from 42 patients with prostate cancer. CSE1L protein expression was detected by staining paraffin embedded tissue with a monoclonal antibody against CSE1L protein (Novocastra) and scored semiquantitatively. Cutoff values for abnormal cytoplasmatic staining were defined by the mean percentage of stained cells in the normal controls72 standard deviations (X20%). The immunhistochemical expression of Ki67, p53, cyclin D1 and cyclin A was detected by tissue microarray technique. 20q13gains were detected by FISH (Vysis label;ZNF217) using a nuclear suspension. Results: Most tumors showed cytoplasmic overexpression of CSE1L protein (74%; mean expression level 54%). Only two cases of normal tissue showed CSE1L expression levels higher than 30%. In DNA-diploid cases CSE1L expression correlated significantly with nuclear expression of p53 and cyclin A. Proliferation (Ki67) tends to be correlated with CSE1L protein expression. Tumor stage, Gleason score and 20q13.2 gain did not correlate with CSE1L expression. Conclusions: CSE1L is overexpressed in most prostate cancers. It seems to play a role in carcinogenesis as demonstrated by the correlation with p53 and cyclin A expression.

271 Cytogenetic changes and (LOH) in atypical adenomatous carcinoma of the prostate and in the prostate using comparative (CGH) and multiplex-PCR.

loss of heterozygosity hyperplasia (AAH), in nonneoplastic tissue of genomic hybridization

O. BETTENDORF, H. SCHMIDT, E. ELTZE, I. GOCKEL1, A. SEMJONOW2, H. BU¨RGER, W. BO¨CKER, B. BRANDT1 Institut fu¨r Pathologie, Universita¨t Mu¨nster 1 Institut fu¨r Klinische Chemie und Labormedizin, Universita¨t Mu¨nster 2 Klinik fu¨r Urologie, Universita¨t Mu¨nster Aims: High grade prostatic intraepithelial neoplasia (PIN) and atypical adenomatous hyperplasia (AAH) are discussed to be precursors of prostate cancer (PC). Unlike high grade PIN the relation between AAH and PC is however unclear. Methods: In the present study we analyzed AAH, corresponding prostate carcinomas and carcinomas of the transitional zone after microdissection using comparative genomic hybridization (CGH) and multipelxPCR with 10 microsatellite polymorphic markers.

ARTICLE IN PRESS Posters: Urogenital Pathology / Pathology – Research and Practice 201 (2005) 153–300

Nonneoplastic prostatic tissue was investigated for the same allelic imbalances. Results: Two AAH showed allelic imbalances in multiplex-PCR. These imbalances did not correlate with the corresponding tumours and furthermore were different to the LOH found in the investigated prostate tumours of the transitional zone. One AAH showed loss on chromosome 22q. We found allelic imbalances in over 50% of nonneoplastic tissue adjacent to prostatic carcinoma. Conclusions: Our findings support the idea that AAH does not seem to be linked closely to PC and should not be considered as an obligate premalignant lesion.

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Aneusomy of chromosomal region 20q13.2 in prostate cancer - correlation with DNA ploidy M. TOMA, M. MUDERS, K. FRIEDRICH, W. MEYER, G. BARETTON Institut fu¨r Pathologie, Universita¨tsklinikum CarlGustav-Carus der TU Dresden Aims: DNA ploidy plays a significant prognostic role in prostate cancer. Overexpression of STK15/BTAK/Aurora-A kinase, a centrosome-associated serine-threonine kinase located on 20q13, is associated with aneuploidy and centrosome amplification in a variety of solid tumors, however, little is known about the role of 20q13.2 gains in prostate cancer. The aim of this study was to determine the frequency of relative or absolute 20q13.2 gains and its correlation with DNA-ploidy. Methods: DNA image cytometry was performed on Feulgen stained disintegrated 50mm paraffin sections by an OPTIMAS based image cytometry workstation. FISH was performed on suspended nuclei of 30 cases prostate cancers (15 diploid, 15 non-diploid). We used the 20q13.2 probe from Vysis Inc., labelled with Spectrum Orange, co-hybridized with centromeric probes specific for chromosome 20, labelled with Spectrum Green. For the statistical analyses, the Student’s T-test (significance level po0,05) was used. Results: We analysed the loss or gain of chromosome 20 and the isolated gain on 20q13 in relation to DNAploidy. We considered aneusomy when more than 20% of the tumor cells showed loss or gain of chromosome 20 or isolated gain of 20q13 (relative or absolute gain of 20q13). From 15 diploid cases only 4 cases showed a gain of 20q13, the other 11 did not. From 15 nondiploid cases 9 cases showed a gain of 20q13. Conclusions: The absolute gain of 20q13 is a relatively rare event in prostate cancers, but appears to be associated with aneuploidy. Further studies are needed in order to elucidate the role of aneuploidy and genes on chromosome 20q13.2 in prostate carcinogenesis.

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273 Usefulness of PTEN and p27 as predictive markers in prostate core needle biopsies R. GROBHOLZ1, T. DREHER1, H. ZENTGRAF2, U. ABEL3, A. KAPPELER1, M.S. MICHEL4, U. BLEYL1 1

Pathologisches Institut, Universita¨tsklinikum Mannheim 2 Abt. ATV, Deutsches Krebsforschungszentrum, Heidelberg 3 Abt. Medizinische Biometrie und Statistik, Universita¨t Heidelberg 4 Urologische Klinik, Universita¨tsklinikum Mannheim Aims: To determine the impact of PTEN and p27 expression in prostatic carcinoma as predictive markers. Methods: PTEN/p27 were studied immunohistochemically in 93 core needle biopsies and corresponding prostatectomies. Immuno-histochemistry was evaluated using an immunoreactive score. Tumors were classified as high grade (HG, Gleason scoreZ7) and low grade (LG, Gleason scoreo7). Results: PTEN expression rate correlated to tumor grade (LG vs. HG: p ¼ 0.0005; Gleason score o7 vs. ¼ 7 vs. 47: po0,0001). Localization of p27 expression correlated to tumor grade (LG vs. HG: p ¼ 0.0008; Gleason score o7 vs. ¼ 7 vs.47: po0,0001). The portion of HG tumors increased while LG tumors decreased throughout the following three groups of tumors with: (1) both tumor suppressors intact; (2) either one tumor suppressor intact, (3) both tumor suppressors inactive (LG versus HG and Gleason score o7 vs. ¼ 7 vs. 47: po0.0001). Statistical analyses of the corresponding prostatectomies revealed a significant correlation for each cal-culation (po0.0001). Analyses of sensitivity (se) and specificity (sp) of the following three parameters revealed: Gleason score: se ¼ 0.688, sp ¼ 0.672; p27: se ¼ 0.970, sp ¼ 0.390; PTEN: se ¼ 0.966, sp ¼ 0.356. Correct prediction of tumor grade in prostatectomies by para-meters in biopsies was seen (1) Gleason score: in 68% (63/93), (2) p27: in 60% (55/92) and (3) PTEN: in 56% (49/88) of all cases. Conclusions: PTEN and p27 expression in core needle biopsies of prostatic carcinoma are of limited value as predictive markers and do not improve the predictive value of the Gleason score.

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CD99 Expression is a Marker of Favourable Outcome in Patients with Prostate Cancer

G. KRISTIANSEN1, M. BURKHARDT, F. FRITZSCHE, T. GANSUKH, K. JUNG, S. LOENING, M. DIETEL1

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1

Institut fu¨r Pathologie, Charite´ (CCM) Universita¨tsmedizin Berlin 2 Urologische Klinik, Charite´ (CCM) Universita¨tsmedizin Berlin Aims: CD99/MIC-2 is a transmembrane cell surface protein characteristically expressed in tumours of the Ewing sarcoma/PNET group. We aimed to evaluate its expression in sporadic adenocarcinoma of the prostate and to correlate with clinico-pathological parameters. Methods: 110 adenocarcinomas of the prostate were immunostained with a monoclonal CD99 antibody using a standard detection system. Staining intensity was graded semiquantitatively (0-3+). Fishers exact test, bivariate correlation and Kaplan Meier analysis were applied for statistical analysis. Results: 27% of cases showed a weak CD99 expression, 18% stained moderately and 3% stained strongly. 52% of cases were negative. No significant correlations to tumor grade or pT stage were demonstrable. CD99 negative cases showed a trend to suffer earlier PSA relapses than CD99 positive cases (p ¼ 0.06). On stratified analysis, this finding became significant in the group of larger (4pT2) or high-grade(4Gleason 6) tumors. Conclusions: CD99 expression is not only a marker of tumours of the ES/PNET group but is observed in 48% of prostate cancer cases and might be a marker of a better prognosis, which appears to be especially valid in more aggressive or more progressed tumours.

275 Increased Tiam1 expression is a new and independent prognostic factor in human prostate cancer R. ENGERS1, A. WALTER1, D. NGUYEN1, M. MU¨LLER2, J.G. COLLARD3, H.E. GABBERT1 1

Institut fu¨r Pathologie u. Urologische Klinik, Universita¨t Du¨sseldorf 3 The Netherlands Cancer Institute, Amsterdam, The Netherlands 2

Aims: Tiam1 specifically activates the Rho-like GTPase Rac and plays an important role in tumor invasion and metastasis as well as in oncogenicity. However, all of these studies have been performed in vitro or in animal models, respectively. Therefore, the aim of the present study was to test the potential diagnostic and prognostic value of Tiam1 and Rac in human tumors, using human prostate cancer (PCa) as the first tumor model. Methods: Semiquantitative immunohistochemistry, cell culture, immunoprecipitation. Results: We investigated the expression of Tiam1 and Rac in benign prostate, high-grade prostatic intraepithelium neoplasia (HG-PIN) and PCas of 60 R0resected radical prostatectomy specimens. By semiquan-

titative immunohistochemistry Tiam1 and Rac proved significantly overexpressed in both HG-PIN (po0:001) and PCas (po0:001) when compared with their expression levels in benign secretory epithelium. In line with this two different human prostate cancer cells lines exhibited markedly stronger Tiam1 expression levels than a benign prostate epithelium cell line. By KaplanMeier analysis, in addition to pre-operative PSA-levels (p ¼ 0:0457), tumor stage (p ¼ 0:0016), and Gleason grading (p ¼ 0:008) overexpression of Tiam1 in human PCas also had a significant impact on relapse-free survival (p ¼ 0:0108) and this effect proved to be independent by multivariate Cox regression analysis (p ¼ 0:037). Conclusions: Tiam1 is a new and independent prognostic factor in human prostate cancer, which is already upregulated in HG-PIN lesions.

276 Trends in gleason grading in Germany: Evaluation using prostate cancer tissue microarrays – final report R. ENGERS, M. BURCHARDT, M. MUELLER, T. BURCHARDT, R. ACKERMANN, H. E. GABBERT, J.I. EPSTEIN1, M.A. RUBIN2 Institut fu¨r Pathologie, Universita¨t Du¨sseldorf 1 Johns Hopkins School of Medicine, Baltimore, MD 2 Harvard Medical School, Boston, MA Aims: Due to PSA screening and increased awareness, prostate cancer (PCa) is identified earlier resulting in smaller diagnostic samples on prostate needle biopsy. Because Gleason grading is vital to treatment planning, the ability of German pathologists to grade small PCas was evaluated using a series of tissue microarray (TMA) images. Methods: We have previously demonstrated excellent agreement in Gleason grading using TMAs amongst expert genitourinary pathologists. In the current study, we identified 331 TMA images (95% PCa and 5% benign) to be evaluated by an expert PCa pathologist and then reviewed by practicing pathologists throughout Germany. The evaluations were kept anonymous. Results: Twenty nine German pathologists completely reviewed all 331 images. There was excellent consenus on which TMA samples contained PCa (mean ¼ 93.4%) with the expert. The median percentage of TMA images assigned the exact Gleason scores (GS) as the expert was 45% (range 17–70%). GS differed by no more than one point (+/1) in 82.5% of the TMA samples evaluated. GS were correctly assigned into clinically relevant categories (i.e. o7, 7, or 47) in 68% of cases (range 56-81%). The most significant trend was that 22 of 29

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( ¼ 76%) respondents under graded the TMA images significantly in contrast to only 3 ( ¼ 10%) significantly over graded cases. Conclusion: The vast majority of practicing pathologist under graded these small tumors. Clinically relevant GS categories were correctly assigned in only 68% of cases. This raises a potentially significant problem for pathologists, who have not had as much experience evaluating small PCas.

277 BRAF mutations are independent from RAS but not from microsatellite instability in germ cell tumours of the testis F. SOMMERER1, A. MARKWARTH1, S. VOMSCHLOß1, J.-U. STOLZENBURG2, C. WITTEKIND1, A. TANNAPFEL1 1

Institut fu¨r Pathologie, Universita¨t Leipzig Klinik fu¨r Urologie, Universita¨t Leipzig

2

Aims: The BRAF gene, one of the human isoforms of RAF, is activated by oncogenic Ras. Recently, somatic missense mutations in the BRAF gene have been detected in a variety of human tumors. We have studied male germ cell tumours (GCT) for probable mutations of the BRAF and Ras oncogene. Methods: Microsatellite instability (MSI) was analyzed using mono- or dinucleotide marker. Mutational analysis of 62 GCT (30 seminomas, 32 non-seminomas) was performed after microdissection of the different tumour components. The expression of ERK1/2 was assessed immunohistochemically. Results: Activating BRAF missense mutations were identified in three out of 32 cases of non-seminomas (9%), but not in seminomas. The mutations were 1796T4A mutations and were found in the embryonic carcinoma component of these tumors. Two out of 30 seminomas (7%) and three out of 32 non-seminomas (9%) exhibited KRAS gene mutations. MSI was observed in four out 62 tumours (7%) (one seminoma and three non-seminomas [embryonal carcinoma]). Microsatellite instable embryonal carcinomas had a mutated BRAF gene. All five GCT with RAS mutations had an intact BRAF gene. We identified activated ERK in almost all tumours tested. Conclusions: BRAF gene mutations are rare events in GCT and are independent of KRAS mutations. In embryonal carcinomas, BRAF mutations may be linked to the proficiency of these tumours in repairing mismatched bases in DNA. The finding of activated ERK suggests a causative role for MAPK activation in GCT independent of activating BRAF or RAS mutations.

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278 Putative preneoplastic

clear cell tubular lesions of human kidneys show immunohistochemical similarities to the distal tubular system C. SEILER, M. EVERT, F. DOMBROWSKI Institut fu¨r Pathologie, Otto-von-Guericke-Universita¨t, Magdeburg

Aims: The histomorphogenesis of clear cell renal cell carcinoma (RCC) has not yet been fully clarified. It is generally assumed, that human clear cell RCC arises from the epithelium of the proximal tubule system. Investigations on clear cell RCC experimentally induced in the rat, however, indicate that it may originate from the distal tubule and the collecting duct system. Clear cell tubules have occasionally been reported as putative precursor lesions of clear cell carcinoma in both species. Methods: Eighty six kidneys, removed for clear cell RCC, were morphologically investigated for the incidence of clear cell tubular lesions, and subsequently immunophenotyped, using antibodies, directed against the proximal nephrogenic renal antigen (PNRA), epidermal growth factor-receptor (EGF-R), CD10, CK7, CK8, CK18, CK 19 and Ki-67. Results: One or multiple clear cell tubules were identified in the renal cortex in 12 out of the 86 kidneys. Morphologically, these tubules were strikingly similar to clear cell RCC. The immunohistochemical marker profile corresponded predominantly with the distal tubule system of the adjacent renal parenchyma: no expression of PNRA and CD10, weak expression of EGF-R, and moderate to strong expression of cytokeratins. Conclusions: Clear cell tubular lesions of putative preneoplastic potential displayed a striking histomorphological similarity with clear cell RCC and a close immunophenotypical relationship with the distal tubule system.

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Local complement C3 expression contributes to humoral and cellular rejection of renal allografts

M. MENGEL1, E. SERINSO¨Z1, O. BOCK1, W. GWINNER2, A. SCHWARZ2, H. HALLER2, H. KREIPE1 1

Institut fu¨r Pathologie, Medizinische Hochschule Hannover 2 Abteilung Nephrologie, Medizinische Hochschule Hannover Evidence on the role of complement system in transplantation pathology has recently been accelerated by the discovery of C4d as an in situ marker of

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antibody-mediated rejection. However, a local or systemic source of complement expression during acute rejection is still under discussion. Thus, we quantitatively analysed the local RNA expression of complement component C3 as a pivotal molecule in active humoral as well cellular rejection of renal allografts. After laser microdissection, real-time RT-PCR was performed for C3 using RNA extracted from tubuli and glomeruli of 68 paraffin-embedded renal allograft biopsies. Protocol and indication biopsies with signs of humoral and/or cellular rejection were investigated. We observed a significant increase in the median expression level of C3 RNA in glomeruli and tubuli of C4d-positive indication biopsies, and in tubuli from indication biopsies with signs of T-cell-mediated cellular rejection. Highest expression levels were found in indication biopsies that were both C4d-positive and had signs of cellular rejection. Analysing sequential protocol biopsies, no predictive value of C3 expression was found concerning upcoming or resolving rejection episodes. We conclude that locally synthesised complement component C3 contributes to both humoral and cellular rejection, with tubular epithelial cells being a major source.

280 Phenotyping of infiltrates in protocol biopsies from renal allografts M. MENGEL1, R. BAJESKI2, W. GWINNER2, I. FRANZ2, A. SCHWARZ2, H. HALLER2, H. KREIPE1 1

Institut fuer Pathologie, Medizinische Hochschule Hannover 2 Abteilung Nephrologie, Medizinische Hochschule Hannover A high percentage of protocol biopsies from stable renal allografts show to various extent interstitial infiltrates. Frequently, these infiltrates do not reach cut off levels for cellular rejection applying the Banff classification. Whether the cellular composition and localization of such subclinical infiltrates can provide relevant diagnostic or predictive information needs further proof. We re-evaluated 1278 renal allograft biopsies (Bx) (917 protocol Bx, 361 indication Bx) from 366 patients for the morphological pattern and localization of cortical infiltrates. 322 protocol Bx were immunophenotyped for Granzyme B, CD20, CD68, and activated lymphocytes (ZAP70). Results were correlated to grade of rejection and allograft function. More than 80% of stable renal allografts have cellular infiltrates in protocol biopsies. The frequency of infiltrates is comparable between protocol and indica-

tion Bx from allografts with dysfunction. Currently in the Banff classification not considered nodular infiltrates of CD20-positive cells are more frequent in protocol Bx and are related to an impaired allograft function 1 year after transplantation. The amount of infiltrating cells of each investigated immunophenotype correlated significantly with the degree of acute cellular rejection following the Banff-classification. We conclude that cellular infiltration in protocol Bx from stable allografts is a common finding. Cortical nodular infiltrates of CD20-positive cells are more frequent in allografts with deteriorating function. Increasing numbers of T-cells, as well as B lymphocytes and histiocytes indicate higher degrees of subclinical immunological activity in surveillance Bx.

281 Different methods for protein recovery from urine: feasibility study for high resolution 2D-gelelectrophoresis K. SCHWAMBORN, R.C. KRIEG, J. GROSSE1, R. KNUECHEL Institut fu¨r Pathologie, Universita¨t Aachen Urologische Klinik, Universita¨t Aachen

1

Aims: The gold standard in urologic tumor diagnosis is a cystoscopically driven biopsy. Besides tissue based approaches a high sensitive and specific method for protein detection in more easily accessible body fluids e.g. urine is necessary to decrease the number of invasive and costly cystoscopies. Normal urine contains less than 150 mg protein/liter. Therefore an efficient protein enrichment technique has to be performed, prior to the proteomic analysis itself. Methods: Healthy midstream urine was subjected to the following protein enrichment techniques: centrifugation, precipitation (TCA, acetone, methanol), 2D-Clean up Kit, Amicon centrifugal filter device, 2D-lysis buffer. Following protein enrichment a high resolution 2D-SDS-PAGE was performed. Gel images were analyzed by software regarding for detection and quantification. Results: Average spotcounts ranged from 55 to 350 per gel, depending on the protein recovery method used. Best results according to spot count and total spot intensity were found with acetone and methanol precipitation at -201C, followed by the commercial 2D cleanup kit, the Amicon filter devices, TCA-precipitation, regular centrifugation and simple 2D-buffer. Conclusions: High sensitive proteomic analysis out of normal urine with 2D-SDS-PAGE is possible following proper protein enrichment. The successful establishment of protein detection is promising for future studies, especially for pinpointing potential tumor related marker proteins.

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BMP-7 expression and signalling in adult human kidney P. WETZEL, J. HAAG, V. CAMPEAN, K. AMANN, TH. KIRCHNER, T. AIGNER Institut fu¨r Pathologie, Universita¨t Erlangen-Nu¨rnberg

Aims: BMP-7 plays an important role during fetal kidney development and BMP-7 knockout mice die as result of severe kidney dysplasia. Recent experimental evidence suggests that BMP-7 is able to protect tubular epithelial cells from ischemic damage and prevents or reverses interstitial fibrosis. The aim of this study was to further characterize the expression pattern as well as potential target cells of BMP-7 in the adult kidney. Methods: Immunostainings on normal kidney sections were performed for BMP-7, Phospho-Smads 1,5,8 as well as for calbindin (distal nephron) and uromucoid (thick ascending limb) using standard immunofluorescence technologies. Results: BMP-7 expression was localized specifically to the epithelia of the distale tubulus as well as of the collecting ducts as identified by co-immunostaining with calbindin. No expression was detected in the proximal tubulus and the glomeruli. Phospho-Smad1,5,8 expression colocalized with BMP-7 and was also restricted to the distale tubulus and the collecting ducts. Conclusions: Our data indicate that BMP-7 expression in the adult kidney is much more restricted than in the fetal situation and selectively found in the distale nephron, in which also cellular reactivity to BMPstimulation was detectable. Our findings suggest that BMP-7 plays a physiological role mostly in this part of the kidney. Still, as reported previously, proximale tubular cells might be responsive to BMP-7, but not in an autocrine or paracrine mode, but may be only if externally given.

283 Comparative genomic hybridization in clear cell renal cell carcinomas and their metastases: Gain at 5q is associated with longer overall survival B. GUNAWAN, A.V. HEYDEBRECK1, J. EBSCHNER, H.-J. SCHULTEN, R.-H. RINGERT2, L. FU¨ZESI Zentrum Pathologie, Universita¨t Go¨ttingen 1 Abteilung fu¨r Bio- and Chemoinformatik, Merck KGaA, Darmstadt 2 Abteilung Urologie, Universita¨t Go¨ttingen Aims: To evaluate the cytogenetic events underlying metastatic progression of renal cell carcinomas (RCC), we performed comparative genomic hybridization in 20

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primary clear cell RCCs and their corresponding adrenal metastases. Methods: The associations between clinico-pathological variables and cytogenetic abnormalities were evaluated using the two-sample Wilcoxon test and Fisher’s exact test for contingency tables, Survival rates were plotted using the Kaplan-Meier method. Results: Overall, the frequencies of individual imbalances did not differ significantly between primary RCCs and their metastases. The most common changes were losses at 3p (88%), 14q (53%), 6q (33%), 9p (33%), 13q (33%), 4q (30%), 18q (28%), 8p (23%), and gains at 5q (30%), 16p (15%), and 8q (13%). Interestingly, patients with gains at 5q in their tumors were revealed to have a three-fold longer overall survival (mean, 28 months) than patients without 5q in their tumors (mean, 9 months). Conclusions: The present data indicate that gain at 5q as an early cytogenetic event identifies a cytogenetic variant of clear cell RCC with more favourable outcome even in the setting of metastatic disease. This finding supports previous observations in a larger series of primary clear cell RCCs suggesting a better overall survival for tumors with gains of 5q as identified by conventional cytogenetic analysis (Gunawan et al., Cancer Res 2001;61:7731-8).

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Concommittant deregulation of HIF1a and cell cycle proteins in VHL mutated renal cell carcinomas D.J. ATKINS1, C. GINGERT1, C. JUSTENHOVEN2, G.E. SCHMAHL1, M.S. BONATO3, H. BRAUCH2, S. STO¨RKE1 1

Institut fu¨r Pathologie, Universita¨t Witten/ Herdecke, Helios-Kliniken Wuppertal 2 Dr. Margarete Fischer-Bosch-Institut fu¨r kinische Pharmakologie, Stuttgart 3 Fach-Hochschule, Mu¨nster, Germany Aims: Renal cell carcinomas (RCC) of the clear cell type are associated with alteration of the von Hippel-Lindau (VHL) tumour suppressor gene, subsequent stabilization and over-expression of hypoxia inducible factor (HIF), which in turn causes up-regulation of cyclin D1. Based on their ability to interact with cyclin D1 we investigated a number of cell cycle proteins to further shed light on the downstream effects of HIF dysregulation. Methods: Expression of HIF1a, cyclin D1, cyclin dependent kinase 4, cyclin dependent kinase inhibitors p16, p21 and p27 was studied using immunohistochemistry. Since NFkB1/RelA have been shown to bind to the cyclin D1 promotor mRNA expression of these trans-cription factors was further analyzed by quantitative PCR.

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Results: In VHL negative tumours over-expression of HIF1a was paralleled by up-regulation of cyclin D1, CDK4 and down-regulation of p21 and p27. Moreover, p27 expression was inverse correlated with tumour cell differentiation. Comparison of non-tumorous autologous kidney tissues revealed a significant downregulation of NFkB1 mRNA expression in patients harbouring RCC with VHL mutations. Conclusions: Our data support the notion of a link between VHL deficiency/HIF dysfunction and disturbances of cell cycle control in the tumorigenesis of VHL negative RCC.

retrospective reports that FGFR3 mutations and expression of CK20 and p53 can be useful in determination of recurrence rate and survival in bladder cancer patients.

Symposium: Inflammatory Liver Diseases 286 Alcoholic and non-alcoholic steatohepatitis H. DENK

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Frequency and clinical significance of EMAST in bladder cancer

Institut fu¨r Pathologie, Medizinische Universita¨t Graz, AT

J. BURGER, M. BURGER1, P.J. WILD, R. STO¨HR1, W.F. WIELAND1, F. HOFSTA¨DTER, A. HARTMANN

Major morphologic criteria of steatohepatitis are steatosis, ballooning of hepatocytes, often but not constantly associated with Mallory bodies, pericellular fibrosis and inflammation (neutrophilic and mononuclear). These alterations vary in extent and proportion. Liver cirrhosis follows in about 20–50%. With respect to etiology an alcoholic and non-alcoholic type can be distinguished, the latter being the characteristic hepatic lesion associated with the metabolic syndrome (type II diabetes, insulin resistance, obesity, dyslipidemia). Ballooning of hepatocytes as well as Mallory body formation are associated with a disturbance of the keratin intermediate filament cytoskeleton. Mallory bodies are protein aggregates consisting of keratin (particularly keratin 8), p62, a stress-induced adapter protein involved in signal transduction pathways, heat shock proteins, and ubiquitin. Mallory bodies thus resemble the product of a disturbed unfolded stress protein response in which oxidative stress is involved. Major sources of oxidative stress in alcoholic and nonalcoholic steatohepatitis are the microsomal biotransformation system (cytochrome P-450), the mitochondria as well as impaired antioxidant defense systems in the hepatocytes. Oxidative stress leads to misfolding/ unfolding and abnormal phosphorylation of proteins (particularly keratins) and thus interferes with intermediate filament formation. Moreover, impairment of cellular defense systems against abnormal proteins, i.e. chaperone action and proteasomal degradation, leads to the accumulation of abnormal aggregation prone keratins which after ubiquitination finally associate with the stress-induced ubiquitin binding protein p62 to form Mallory bodies. Thus, Mallory body formation closely resembles an "off-folding" protein response of the amyloid type. These pathogenetic principles of the human disease are supported by immunohistochemical and gene expression studies in experimental animals and by transfection experiments in tissue culture cells.

Institut fu¨r Pathologie 1 Klinik fu¨r Urologie, Universita¨t Regensburg Aims: MSI at selected tetranucleotide markers (EMAST) was recently reported to be common in urothelial carcinoma (UC). The clinical role of EMAST in UC however is unclear. The aim of the present study was to evaluate the clinical significance of EMAST in UC and its relation to common molecular alterations. Methods: A prospective series of 114 consecutive unselected UC patients was investigated. After microdissection and DNA isolation, microsatellite analysis to detect microsatellite instability (MSI) and chromosomal deletions (LOH) was performed using 12 tetranucleotide microsatellite markers reported to show MSI in tumors with EMAST and 5 additional markers (NCI consensus panel for HNPCC detection). FGFR3 mutations, proliferation index (MIB), and expres-sion of CK20, p53 and MMR proteins were investigated. Results: EMAST was found in 10/114 (8,8%) cases, whereas MSI in the HNPCC marker panel and loss of MMR protein expression was not detected. LOH in EMAST markers was detected in 6/114 (5,3%) cases. EMAST was not correlated to histopathological stage, grade, clinical outcome or other parameters. FGFR3 mutations were related to low stage and grade (p ¼ 0:0001), to overall survival (OS- p ¼ 0:001), but not to recurrence free survival (RFS). Normal umbrella cell staining of CK20 showed a trend to prolonged RFS (p ¼ 0:06), but not to OS. In contrast, p53 positivity was correlated with OS (p ¼ 0:02), but not with RFS. Conclusions: EMAST is rare in UC and is not correlated to other histopathological or molecular parameters. Nevertheless it can be distinguished as a distinct form of MSI. The prospective study con-firmed

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287 Chronic viral hepatitis P. SCHIRMACHER Institut fu¨r Pathologie, Universita¨t Heidelberg Chronic viral hepatitis is one of the most frequent severe diseases worldwide and affects about 1.5% of the german population. It is caused by Hepatitis B Virus (HBV) (potentially with syn- or metachronous infection with Hepatitis d Virus (HDV)) or Hepatitis C Virus (HCV) infection. It is one of the major causes of liver cirrhosis and its sequelae as well as hepatocellular carcinoma (HCC). Chronicity of HBV- (5–10%) and HCV-infection (70–80%) is determined by viral mechanisms and the state of the host immune response. Liver pathology plays a major role in typing of chronic hepatitis (including comorbidity) and assessing the inflammatory activity (grading) and especially state of fibrosis and architectural disturbance (staging). Diagnostic semiquantitative and qualitative scores have been developed and contribute significantly to prognostic evaluation, therapy planning, and clinical studies and are part of clinico-pathological consensus on diagnosis and therapy of viral hepatitis. HBV-antigens (immunohistology) as well as HCV genomic RNA (RT-PCR) can be reliably detected in formalin fixed routine biopsies supporting diagnosis in problematic cases. Future morphology based studies will aim at predictors of therapeutic response including upcoming therapeutic agents, improving prognostic algorhythms, and further evaluation of fibrogenic mechanisms.

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diagnosis should be confirmed by a scoring system including histopathology. Variants of autoimmune hepatitis cover seronegative cases, acute onset autoimmune hepatitis and autoimmune hepatitis with centrilobular necrosis. Differential diagnosis of autoimmune hepatitis includes drug induced chronic hepatitis that may mimick autoimmune hepatitis by clinical course and serology. Histopathology may give helpful hints for the correct diagnosis. Autoimmune lesions of the biliary tract are PBC in the first line. The target antigen of the autoimmune response has been identified, natural history of the disease is well known and histopathology is pathognomonic in about a third of the cases. In clinical practice liver biopsy is taken to exclude other etiologies when AMA is present in the serum, staging the disease at first diagnosis and to establish diagnosis in cases of AMA negativity. The autoimmune nature of PSC has been discussed in the literature ever since the first description and the answer is not settled yet. Histopathology is relevant for the diagnosis in excluding other etiologies and confirming the diagnosis of small duct PSC. The term autoimmune cholangitis has been used to designate AMA-negative PBC, however, based on research experience and the clinical data it should be reserved to the overlap syndrome of AIH and PSC in children that seem to make up a disease entitiy of its own.

289 Tumour development in chronic inflammatory liver disease

288 Autoimmune Hepatitis and Autoimmune Cholangitis

CH. WITTEKIND Institut fu¨r Pathologie, Universita¨t Leipzig

H.P. DIENES Institut fu¨r Pathologie, Universita¨t Ko¨ln Autoimmune liver diseases encompass autoimmune hepatitis, primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) as lesions of the biliary tract. The term autoimmune cholangitis has not been generally accepted, so it remains an entitiy waiting for precise definition. AIH is a chronic progressive necroinflammatory liver disease mostly occuring in female individuals and leading to ultimate autodestruction of the liver if not treated. Histopathology of the liver reflects the general understanding of the underlying immune especially self reactive CD4+ T-helper cells mediated mechanisms in destruction of liver cells displaying a typical but by no means pathognomonic histopathological pattern. Since there are no specific and generally valid tests the

Aims: Several tumour entities have been described to develop in chronic inflammatory disease, e.g., bile duct carcinoma in chronic inflammations caused by parasites, extrahepatic bile duct carcinoma in primary sclerosing cholangitis, and hepatocellular carcinoma in chronic hepatitis. This review will focus only on the latter disease entities, particularly on chronic hepatitis caused by viral disease (HBV and HCV infection). Methods: Clinical and molecular analyses using a variety of techniques yielded a considerable amount of information about liver carcinogenesis. Recent results were obtained by high-output gene analysis using cDNA-microarrays. Results: The multistep process of HCC may be divided into several steps: chronic liver injury, chronic inflammation, cell death, regeneration and cirrhosis, DNA damage, dysplasia and finally HCC. Until now, true specific genetic (or epigenetic) changes have not been

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identified for these stages of carcinogenesis. The genetic changes identified so far include gene amplification (leading to activation of proto-oncogenes), gene deletion or mutation (leading to inactivation of tumor-suppressor genes) and reactivation of telomerase activity. Finally, at late stage of carcinogenesis, genetic changes expressed as chromosomal aberrations, are observed. To date, more than a dozen genes have been shown to be altered in HCC. Conclusions: Several genes among which are those of the complex growth regulatory TGF-b /IGF pathway could be altered during hepatocellular carcinogenesis. Activation of TGF-b and IGF signalling is observed even in liver cirrhosis.

290 Indication of the biopsy and expections of the pathologist

In both alcoholic and non alcoholic fatty liver disease, liver biopsy does not play an important role in the diagnosis and provides only prognostic information since histology will not influence treatment recommendations. Finally, a biopsy of focal lesions in chronic liver disease is necessary if non-invasive methods of diagnosing hepatocellular carcinoma including alpha fetoprotein and imaging procedures are not sufficient in establishing the diagnosis. Although it should be mentioned, that biopsy of carcinoma carries the risk of needle-track seeding. In summary, liver biopsy can be helpful in diagnosis, but remains a more important tool for grading and staging of chronic liver disease as well as for the indication of treatment and prediction of the disease.

Lecture

T.O. LANKISCH, MICHAEL P. MANNS Abteilung fu¨r Gastroenterologie, Hepatologie und Endokrinologie, Medizinische Hochschule Hannover

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Health risks and side effects

M. LU¨TZ Liver biopsy in general is a useful tool for the diagnosis of liver disease in particular to determine aetiology but even more for grading and staging of the disease. In combination with non-invasive diagnostic tests such as viral serological tests and hepatic biochemical tests, history, clinical examination and imaging procedures liver histology can be helpful to establish the diagnosis, severity of disease, prognosis and treatment decisions. However, in some conditions liver biopsy is not necessary. In hepatitis C, diagnosis is made by noninvasive serologic tests. Liver biopsy plays a major role in staging and grading the extent of disease and predicting disease progression and whether a treatment may be helpful. The serum alanine aminotransferase (ALT) shows only poor correlation with liver histology. Liver biopsy is especially more useful for genotype 1 patients to determine indication for treatment, since therapy is longer and less successful. In hepatitis B, liver biopsy is only a minor component and gives some prognostic insights as viral serological tests remain the gold standard for diagnosis and treatment of hepatitis B. The diagnosis of autoimmune hepatitis (AIH) is based on several diagnostic criteria and liver biopsy may be helpful to distinguish AIH from other disease entities or to diagnose overlap syndromes. Here, biopsy may be helpful if the diagnostic criteria of the International Autoimmune Hepatitis Group reveal a borderline score. On the other hand, liver biopsy is important for the indication of treatment and follow-up of patients. This is especially relevant for patients who are not treated due to mild disease. Here biopsy every three to five years is necessary.

Allgemeinpsychiatrie, Alexander Krankenhaus Ko¨ln

Study Group: Dermatopathology 292 Cutaneous metastases and specific tumor markers I. MOLL, R. MOLL1 Klinik und Poliklinik fu¨r Dermatologie und Venerologie, Universita¨ts-Klinikum Hamburg-Eppendorf 1 Pathologie der Philipps-Universita¨t Marburg Two percent to 5% of all internal neoplasms give rise to cutaneous metastases. Among them breast cancer is most frequent, followed by cancers of lung, colon, ovary, kidney and melanomas. Cutaneous metastases usually represent a late stage in tumor disease; early cutaneous metastases are rare and mostly derived from lung, kidney or ovary. Cutaneous metastases frequently are localized to the thorax, periumbilical area and scalp. Most of them are solid tumor nodules, grow rapidly, and are restricted to the dermis. Ulcerations are rare and late events. Differential diagnosis includes primary cutaneous tumors, e.g. of adnexal origin which may be hard to distinguish from skin metastases. In some cases of cutaneous metastases, the primary tumor is unknown. To establish the diagnosis, routine histopathology and

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immunohistochemistry are both important. Cutaneous metastases of breast cancer often show in H & E staining the same pattern as the primary tumor. As immunohistochemicals, gross cystic disease fluid protein–15 and estrogen receptor are important. However these two markers do not differentiate such metastases from eccrine and apocrine gland carcinomas. The expression of the EGF-R which is usually absent from breast cancer may be helpful. Mucinous carcinomas of colon and rectum are characterized by CK 20 which is negative in mucinous breast cancer. Cancers of lung are often the origin of cutaneous metastases. A specific marker of adenocarcinomas of lung is TTF-1 while CK 20 mostly is absent. Cutaneous metastases of various other internal carcinomas are rare and mostly retain their typical morphological and immunohistochemical characteristics. Thus a typical pattern of tumor markers may help in clarifing the origin of cutaneous metastases.

293 Pitfalls in the diagnosis of cutaneous lymphomas P. V. D. DRIESCH Bereich fu¨r Dermatopathologie, Zentrum fu¨r Hautkrankheiten, Klinikum Stuttgart Aims: Cutaneous B- and T-cell lymphomas are difficult to be diagnosed and should be worked out by a certain clinical and pathological algorithm using the new EORTC/WHO classification. Methods: Certain characteristics of cases are demonstrated by means of their clinical and (immuno)pathological features. Results: Infiltration pattern analysis and certain pathological and immunopathological features allow the pathological diagnosis of cutaneous lymphomas according to the new classification. The right diagnosis has certain impact on the prognosis and thus on the individual therapy in the patients. Demonstration of clonality by molecularbiological methods should be the last and convincing step to ascertain the right classification of each case. Conclusions: The diagnosis of cutaneous lymphomas needs a multistep diagnostic procedure working with clinical, histological and immunohistological as well as molecularbiological methods. Each step is needed to ensure the diagnosis.

294 Characterization of novel melanocyte differentiation marker PNL2 and comparison with HMB45, A103, T311, and D5 A.A. JUNGBLUTH1, D. KUCUGO¨L2, E. SATO1, D. FROSINA1, K.J. BUSAM2

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1

Ludwig Institute for Cancer Research, New York Dept. of Pathology, Memorial Sloan-Kettering Cancer Center, New York, USA 2

Aims: PNL2 is a mAb to a yet unknown antigen present in melanocytic cells and tumors. However, no extensive analysis of its reactivity pattern has been performed yet. In the present study, we analyzed the PNL2 immunoreactivity in various normal tissues, melanocytic tissues and tumors as well as related lesions. In malignant melanomas (mM), we compared its reactivity pattern to conventional melanocyte differentiation markers. Methods: IHC was done on standard archival tissues using an ABC detection system. Conventional sections and tissue micro-arrays were employed using the following mAbs: PNL2, D5 (MITF), A103 (Melan-A), T311 (tryosinase), and HMB45 (gp100). Results: PNL2 stained normal melanocytes, and melanocytic lesions such as compound, and dysplastic nevi in a consistent fashion. Staining was also seen in 10/10 angiomyolipomas while 30 non-melanocytic carcinomas and sarcomas were all negative. In mM, the mAbs stained as follows: Metast mM (38): PNL2 87%, T311 92%, HMB45 74%, A103 82%, D5 84%. Desmoplastic mM (13): PNL2 1/13, T311 0/13, HMB45 1/13, A103 1/ 13, D5 1/13. Non-desmoplastic spindle cell mM (5): PNL2 3/5, T311 1/5, HMB45 3/5, A103 2/5, D5 4/5. In 13 MPNSTs, immunoreactivity was as follows: PNL2 1/ 13, T311 0/13, HMB45 0/13, A103 1/13, D5 3/13. Conclusions: PNL2 appears to be a valuable new reagent to a not yet isolated antigen which is present in melanocytic cells and tumors as well as in related lesions. PNL2 is a highly sensitive marker for the diagnosis of metastatic melanoma while it appears to offer no superior immunoreactivity to the conventional markers in the desmoplastic and spindle cell variants. PNL2 should prove useful as a new reagent for the diagnosis of melanocytic lesions.

295 Absence of BRAF gene mutations differentiates Spitz nevi from malignant melanoma D. MIHIC- PROBST, A. PERREN, S. SCHMID, P. SAREMASLANI, P. KOMMINOTH1, P.H.U. HEITZ Institut fu¨r Pathologie, Universita¨t Zu¨rich 1 Institut fu¨r Pathologie, Kantonsspital Baden Aims: Distinction of Spitz nevus from malignant melanoma is sometimes difficult on the basis of conventional histology. As a high rate of BRAF gene mutations in malignant melanomas (66%) and nevi (82%) has recently been reported, we investigated whether BRAF mutations occur in Spitz nevus and

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whether they could be used as a marker for differentiating Spitz nevus from melanoma. Methods: We screened a series of 20 Spitz nevi for BRAF mutations in exons 11 and 15 by denaturing gradient gel electrophoresis (DGGE). Five metastases of malignant melanomas were used as positive controls. Results: BRAF exon 15 DGGE analysis of 20 Spitz nevi exhibited no additional bands. On the other hand, three of the five melanoma metastases showed two distinct additional bands representing heteroduplexes. Cycle sequencing of the three melanomas revealed a nucleic acid exchange T4A in codon 599 leading to a mutation in the BRAF activation segment (V599E). The analysis of exon 11 showed an identical single band pattern in Spitz nevi as well as in all controls. Conclusions: (1) BRAF mutations rarely if ever occur in Spitz nevi, (2) activating BRAF mutations do not play an important role in the pathogenesis of Spitz nevi and (3) the exclusion of BRAF mutations may be helpful to differentiate Spitz nevi from malignant melanoma.

296 Comparison of promoter methylation status of skin of young and old adults M. KORABIOWSKA, U. BRINCK, C. CORDON-CARDO1, P. BORTKIEWICZ, H. SCHULZ, K. HOMBURG2, G. FISCHER Institut fu¨r Pathologie, Reinhard-Nieter Krankenhaus, Wilhelmshaven 1 Division of Molecular Pathology, Memorial Sloan Kettering Cancer Center, New York, USA 2 Klinik fu¨r Dermatologie, Santa Ponsa, Mallorca Aims: Methylation of promoter region occurs not only in malignant tumours but also in aging tissues. Methylated promoter regions are blocked and results in nonsufficient transcription and protein synthesis .The main aim of this study was to compare the methylation status of promoter regions of selected DNA repair genes (MLH1, APC) and cell cycle regulators (PTEN, p16) in skin from young and old adults. Methods: DNA was isolated from paraffin embedded tissue and after GC modification, PCR assay was performed. Results: In skin from young adults (20–35 years old) promoter methylation was not present. In skin from old adults several methylations of promoter region were observed including: MLH1 in 8/10 cases, APC 6/10, PTEN 4/10, p16 3/10. Conclusions: Presence of methylated promoter region in skin from old adults correlates with increased risk of occurrence of skin cancer and hypothethically with poor answer to antiaging therapy.

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Relation of promoter methylation status to the tumour progression of cutaneous carcinomas M. KORABIOWSKA, U. BRINCK, C. CORDON-CARDO1, P. BORTKIEWICZ, H. SCHULZ, K. HOMBURG2, G. FISCHER

Institut fu¨r Pathologie, Reinhard-Nieter Krankenhaus, Wilhelmshaven 1 Division of Molecular Pathology, Memorial Sloan Kettering Cancer Center, New York, USA 2 Klinik fu¨r Dermatologie, Santa Ponsa, Mallorca Aims: Down regulation of MLH1, APC, p16 and PTEN reported by our research group previously for cutaneous carcinomas stimulated us to perform promoter methylation analysis of these tumours. Methods: DNA was isolated from paraffin embedded tissue and after GC modification, PCR assay was performed. Material investigated included 15 solar keratoses, 10 carcinomas in situ and 12 squamous cell carcinomas. Results: In solar keratoses methylations of promoter region were observed in 8/15 cases-MLH1, 9/15-APC, 2/ 15-PTEN, 7/15-p16. In carcinomas in situ methylation was present in 9/10 cases-MLH1, 7/10-APC, 1/10-PTEN and 4710-p16. Similar results were obtained in squamous cell carcinomas of the skin. Conclusions: Methylation of promoter region is an early finding in the progression of cutaneous carcinomas and may contribute to carcinogenesis.

Study Group: Cytopathology 298 Application of Liquid Based Preparation in NonGynaecological Cytology- Shared Routine Experience and Critique H.H. NEUMANN Institut fu¨r Pathologie, Nordhorn Aims: to report on our experience in adapting a liquid based cell preparation for various applications in nonGYN cytology. Methods: After introducing one of the approved liquid based cytology (LBC) system (TriPath0 s SurePath system TM) into our laboratory for cervical cytology we consecutively applied this technique for urines, effusions, brush biopsies, bronchial washings and BAL, CSFs and, finally fine needle aspiration biopsies (FNA). Specific fixation and preparation routines were

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developed in order to optimise cell enrichment while minimizing loss of background information. Results: LBC-slides present standardized features. In general, cellularity is higher, preservation of nuclear and cytoplasmic detail superior to our conventional techniques, frequently even to tissue sections. Important background clues remain available. 3-D microbiopsies are frequently obsereved, especially in brushings and FNAs. These large structures are the LBC equivalent to street-like formations in smears.LBC in many organs needs a systematic approach to understand quantitative and sometimes qualitative changes in background information. Hemolytic fixatives in FNAs of the thyroid result in slides with numerous neutrophils and lymphocytes without remaining erythrocytes. Remaining cell pellets have proven valuable for ancillary immunocytological or- employing cell blocks- immunohistochemical tests. These are especially useful in effusions. Normally Giemsa stains cannot be performed on these slides. Conclusions: LBC can in our experience greatly enhance diagnostic accuracy in non-GYN cytology. Time for examination of one LBC slide is far shorter than for a series of smears. At least as a spin-off when employing these techniques in cervical cytology non-GYN appliances are worth the effort to readjust one0 s diagnostic clues.

299 ZyPaKu: A web-based course in cytopathology at http://alf3.urz.unibas.ch/zypaku K. GLATZ, D. GLATZ1, P. DALQUEN, G. FEICHTER, L. BUBENDORF Institut fu¨r Pathologie, Universita¨t Basel (CH) 1 Rechenzentrum, Universita¨t Basel (CH) Aims: To construct an online cytopathology training course (ZyPaKu) for residents and cytotechnicians covering all important topics in the field of cytopathology. Methods and results: Over several years, a large collection of cytologic preparations of all types representing a wide spectrum of diagnoses has been built up. All in all 726 images were taken of the most common cytologic diagnoses. The course covers the following topics: – Gynecologic cytology – Extragenital exfoliative cytology – Extragenital fine needle aspiration cytology – Immunocytochemistry – Pitfalls Each diagnosis in the web course comprises a short introductory text (in German) as well as images with a description and annotations illustrating the cytological

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findings. Additional images of corresponding normal cytological findings and of the histology are useful for comparison. The game MatchingPair serves as a selfevaluating tool. ZyPaKu is freely accessible in the world wide web at http://alf3.urz.unibas.ch/zypaku. Furthermore, all images are available in our online image database PathoPic at http://alf3.urz.unibas.ch/pathopic/ (German and English). Discussion: Our high quality web based learning tools in the field of (cyto-)pathology are intensively used and highly accepted by the target groups. Web-based courses represent a good opportunity for self-guided learning independent of time and location.

300 Ambiguous cellular atypia in lung cytology – expert knowledge versus FISH in an online quiz K. GLATZ, S. SAVIC, P. SPIELER1, P. DALQUEN, G. FEICHTER, L. BUBENDORF Institut fu¨r Pathologie, Universita¨t Basel (CH) 1 Institut fu¨r Pathologie, Kantonsspital St. Gallen (CH) Aims: Ambiguous cellular atypia in lung cytology can be a diagnostic challenge. In such cases FISH may be used for the analysis of chromosomal aberrations to allow a reliable distinction of benign and malignant cells. Methods: An online picture gallery of 30 lung cytologies comprising 23 "atypical" specimens as well as 5 positive and 2 negative controls had been prepared and is accessible at http://www.unibas.ch/patho/lungenzyto/ loesung/. The diagnoses had been confirmed by clinical follow up and/or biopsy. Each of the shown cell groups had been analyzed by multi-target FISH (LA Vysions, Vysis/Abbott) after PAP image capturing and automatic relocalization. Positive FISH results were restricted to carcinoma patients. We compared the performance of cytologists/cytotechnicians and lay persons (after video instruction) in solving the online quiz. Results: The online questionnaire had been completed by 137 experts from all continents and 40 lay persons. At least 15% of the experts made a false positive diagnosis in 5/8 benign atypias and a false negative diagnosis in 2/ 15 malignant atypias. In two patients with benign conditions the rate of false-positive answers was remarkably high (31.4% and 62.8%). Of the ‘‘atypical’’ cases 11 were solved better by the lay persons and 19 by the experts. As expected, control cases were correctly answered more often. Conclusions: Ambiguous cellular atypia in lung cytology may preclude a firm diagnosis by analyzing cytomorphology alone. The selective application of FISH in such cases can markedly improve the diagnostic performance.

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301 Immunocytochemical identification of carcinomas of unknown primary (CUP) in serous effusions N. POMJANSKI, H. J. GROTE, P. DOGANAY, V. SCHMIEMANN, B. BUCKSTEGGE, A. BO¨CKING Institut fu¨r Cytopathologie, Universita¨t Du¨sseldorf Aims: Metastases from carcinomas of unknown primary site (CUP) in serous effusion are a common clinical problem. Immuncytochemistry was applied as an adjunct to the cytological diagnosis of metastatic carcinomas in serous effusions. Methods: Subject of this study were 118 pleural, 53 peritoneal, and nine pericardial effusions originating from a cohort of 180 patients routinely investigated between November 2001 and November 2003 in the Institute of Cytopathology. Specimens were primarily stained according to Papanicolaou.The avidin-biotincomplex method was secondarily applied for the visualisation of immunologic reactions. Results: Using a panel of six monoclonal antibodies (CK 5/6, CK 7, CK 20, CA 125, TTF-1 and cdx 2) we determined marker prevalences of metastatic carcinoma cells in serous effusions and defined an algorithm of immunocytochemical marker constellations. In 86/101 (85.1%) patients with a CUP syndrom we were able to establish the correct diagnosis of primary tumour site prospectively. The best result was achieved regarding the correct identification of metastatic carcinomas of the ovaries (94.7%) and the lungs (88.1%). Conclusions: We identified an algorithm comprising six immunocytochemical markers and achieved a correct diagnosis of primary tumour sites in 85.1%. The panel studied could be useful in diagnostic routine for the identification of primary tumours of unknown origin metastatic to the serous membranes.

302 Application of DNA-chip technology for detection of malignancy associated patterns of gene expression in cytological smears from metastatic lesions M. KORABIOWSKA, U. BRINCK, C. CORDON-CARDO1, H. SCHULZ, P. BORTKIEWICZ, G. FISCHER Institut fu¨r Pathologie, Reinhard-Nieter Krankenhaus, Wilhelmshaven 1 Division of Molecular Pathology, Memorial Sloan Kettering Cancer Center, New York, USA Aims: The disadvantage of cytology is limitation of material for immunocytochemical investigations. The

main aim of this study was to apply the DNA-chip with full panels of genes coding intermediate filaments and tumour associated genes in cytological diagnosis of metastatic lesions. Methods: RNA was isolated from cytological smears and hybridized with Cy3 and Cy5 on a chip. Signals were evaluated applying Gene-Pix software. Results: DNA chip analysis demonstrated typical expression profiles for malignant melanomas (4/16 cases), hepatocellular carcinomas (3/16), prostate cancer (6/16) and soft tissue sarcomas (3 cases). DNA chip analysis correlated highly significant with immunohistochemical results. Conclusions: Application of DNA chip technology to cytological specimens is a valuable diagnostic tool for detection of malignancy-associated patterns of gene expression and may be used as an adjunct to diagnosis of malignancy in cytology

303 Crazy chromosomes: caveats and pitfalls of chromosomal analysis in urinary cytology S. SAVIC, B. GRILLI, A. BARASCUD, M. HERZOG, P. SPIELER1, G. FEICHTER, K. GLATZ, L. BUBENDORF Institut fu¨r Pathologie, Universita¨tsspital Basel 1 Institut fu¨r Pathologie, Kantonsspital St. Gallen Aims: Fluorescence in situ hybridization (FISH) has been shown to improve the sensitivity of urinary cytology for detection of urothelial tumors at a high specificity. Nevertheless, false-positive results have been reported. Here, we evaluated the prevalence and importance of chromosomal aberrations in selected non-neoplastic conditions of the urogenital tract. Methods: Papanicolaou (PAP)-stained cytological specimens from 70 patients with non-neoplastic urogenital tract conditions were evaluated by a multitarget FISH assay for enumeration of chromosomes 3, 7, 17, and the locus 9p21 (UroVysion, Abbott/Vysis). Results: Polysomies were detected in 57% of cytologies from irritative bladders (n ¼ 42), mostly revealing a tetraploic pattern of umbrella cells. Chromosomal abnormalities were also found in 11 of 12 patients with radiation cystitis and in seminal vesicle cells. A transient aneuploidy confirmed by FISH and DNA cytometry was found in a 42 years old patient with urolithiasis but no evidence or history of malignancy. Urinary decoy cells from seven renal transplant recipients were highly aneuploid by DNA-cytometry due to accumulation of viral DNA, but negative by FISH. Conclusions: Chromosomal aberrations in urothelial cells are not restricted to neoplasia but can occurr in non-neoplastic conditions. Interpretation of FISH

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results should always include clinical findings and cytomorphology.

R. SCHOENEGG3, M. MIRLACHER, N. NOVOTNY, C. CATHOMAS1, M.J. MIHATSCH, G. SAUTER, L. BUBENDORF

304 Relevance of DNA image cytometry in the diagnosis of carci-noma of the bile duct, the pancreatic duct and Vater’s papilla

Institut fu¨r Pathologie, Universita¨t Basel 1 Kantonales Institut fu¨r Pathologie, Liestal 2 Viollier, Abteilung Histopathologie, Basel 3 Institut fu¨r Pathologie, St. Gallen

S. BIESTERFELD, M. MAYER Institut fu¨r Pathologie, Universita¨t Mainz Aims: Increasingly cytological specimens from diagnostic ERCP procedures have to be investigated by means of conventional cytology. Especially brush smears from the bile duct are diagnostically difficult as frequently degenerative and inflammatory changes have to be considered. In this study, the DNA distribution pattern of benign and malignant specimens of the bile duct (48 benign; 19 malignant), the pancreatic duct (42; 22) and Vater’s papilla (28; 11) was interpreted diagnostically. Methods: Specimens were either cytological smears or monolayer smears, prepared from three 50 mm thick paraffin sections by a cell separation technique. After Feulgen staining using a standard protocol, the DNA content of 250 epithelial cells, chosen per random, was determined using a TV-image analysis system CM-1 (Hund, Wetzlar, Germany). The DNA content of 30 lymphocytes served as an internal standard for the normal diploid value in every individual case. Different ESACP-accepted DNA cytometric variables were calculated. Results: Analyzing the 128 benign specimens, slightly increased values for the 2cDI, the mean ploidy and the entropy were obtained in correlation to inflammatory changes indicating inflammation-induced cell proliferation. Euploid polyploidization up to 8c was present in some specimens; the highest rate (8.3%) was found in the bile duct group. Requesting a specificity of490%, the sensitivity rates for the diagnosis of malignancy were calculated as 65% (entropy), 76% (stemline ploidy), 78% (5cEE), 82% (2cDI) and 95% (mean ploidy), respectively. Conclusion: Our results indicate that DNA single cell cytometry represents a relevant and clinically useful tool in the differential diagnosis of benign and malignant lesions of the bile duct, the pancreatic duct and Vater’s papilla.

305 HER-2 status by FISH in breast cancer: A comparison between primary tumors and distant metastases C. TAPIA, B. GRILLI, M. HERZOG, A. BARASCUD, S. SAVIC1, U. WAGNER2,

Aims: The Her-2 status is a predictive and prognostic marker in breast cancer. Only breast cancers with an HER-2 amplification respond to the therapy with trastuzumab (Herceptins). The HER-2 status is commonly determined in the primary tumor even in patients with metastatic disease. Here, we explored the HER-2 status in cytologic specimens of distant metastases. Methods: The HER-2 status was determined by FISH (PathoVysion, Vysis). The result from cytology of distant metastases (pleura, peritoneum, liquor, skin and lung) were compared to the results from the corresponding primary tumors (n ¼ 48 patients). The median time period between first surgery and occurence of distant metastases was 5.3 years (range 0-18 years). Results: HER-2 Amplification was found in 20.8% (10 patients) of the 48 patients. There was a 100% concordance of the HER-2 status between primary tumors and their metastasis pairs that have been tested so far. Conclusions: The HER-2 gene status remains highly conserved as breast cancers metastasize. Cytology of distant metastases is well suited for HER-2 FISH analysis.

306 Aberrant promoter methylation as a tumor marker in problematic cases of pulmonary cytology V. SCHMIEMANN, A. BO¨CKING, M. KAZIMIREK, H.E. GABBERT1, R. KAPPES2, H.J. GROTE Institut fu¨r Cytopathologie 1 Institut fu¨r Pathologie, Universita¨t Du¨sseldorf 2 Florence-Nightingale-Krankenhaus, Du¨sseldorf Aims: Aberrant promotor methylation plays an important role in the development of cancer. This study investigates whether a combination of methylation specific markers may improve accuracy of conventional cytology for the diagnosis of lung cancer. Methods: The case-control study comprised 162 bronchial aspirates assigned to the following categories: rare tumor cells (n ¼ 54), highly suspicious atypical cells (n ¼ 26), doubtful (n ¼ 25), false negative (n ¼ 27) and true negative (n ¼ 30). The aspirates were screened for

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aberrant promoter methylation of the genes APC, p16, RARB2, RASSF1A using a quantitative methylation specific real time PCR. Results: The analysis was performed using a three marker panel with a specificity of 100% (APC, p16, RASSF1A) or a 4 marker panel with a specificity of 77% (APC, p16, RARB2, RASSF1A). Hypermethylation in bronchial aspirates with rare tumor cells could be detected in 65% (35/54) and 87% (47/54) using the 3 and 4 marker panel, respectively. Specimens with highly suspicious atypical cells showed methylation in 69% (18/ 26) and 92% (24/26), respectively. False negative and doubtful cases were diagnosed for lung cancer in 40% with 3 markers and in 67% with four markers. Conclusions: The case-control study demonstrates that methylation specific biomarkers enable a highly specific diagnosis of lung cancer and may reduce the number of false negative cytologic diagnoses. Therefore, methylation markers are complementary to conventional pulmonary cytology.

310 Gene expression analysis of DLBCL M. HUMMEL Institut fu¨r Pathologie der Charite´ (CBF), Universita¨tsmedizin Berlin

311 Gene expression analysis of Burkitt’s and Burkitt’slike-lymphoma H. STEIN Institut fu¨r Pathologie der Charite´ (CBF), Universita¨tsmedizin Berlin

312 Gene expression analysis with lymphochips G. OTT, A. ROSENWALD Institut fu¨r Pathologie, Universita¨t Wu¨rzburg

Study Group: Hematopathology 307 Morphology and immunophenotype of high-grade

313 High-grade B-cell-lymphoma in children W. KLAPPER, L. HARDER, R. SIEBERT, R. PARWARESCH

B-cell lymphomas Institut fu¨r Ha¨matopathologie, UK-SH, Campus Kiel H.W. BERND, A. FELLER Institut fu¨r Pathologie, UK-SH, Campus Lu¨beck

314 Morphology, genetics and gene expression of mediastan lymphoma T. BARTH, P. MO¨LLER

308 Cytogenetik and molecular genetic results in highly malignant B-cell lymphomas

Institut fu¨r Pathologie und Rechtsmedizin, Universita¨t Ulm

R. SIEGBERT, T. BARTH1 Institut fu¨r Pathologie, UK-SH, Campus Lu¨beck 1 Institut fu¨r Pathologie und Rechtsmedizin, Universita¨t Ulm

Study Group: Pediatric Pathology 315 Parvovirus B19 infection as a triggering factor for

309

placentitis, hydrops fetalis and intrauterine fetal death (IUFD): A PCR Analysis

M. FRANK

K. SCHARDT, M. ODENTHAL, M. MUELLER, H.P. DIENES, J.W.U. FRIES

Institut fu¨r Pathologie, Universita¨t Frankfurt/Main

Institut fu¨r Pathologie, Universita¨t Ko¨ln

Basic methods and comparison of different gene expressions

Aims: Infection with Parvovirus B19 is an often misdiagnosed disease which, apart from placental

ARTICLE IN PRESS Study Group: Pediatric Pathology / Pathology – Research and Practice 201 (2005) 153–300

inflammation and others, can cause fetal hydros and thus lead to IUFD. Up to now diagnostic is mainly based upon characteristic core alterations which are mostely infrequent or hard to find. Numerous of such cases with discreate placental findings in form of minimal villitis and rare characteristic core alterations on one hand and ambiguous fetal anaemia with hydrops and IUFD on the other hand leaded us to do this study. Methods: We established a qualitative semi nested PCR analysis specific for Parvovirus B19 using formalin fixed and paraffin embedded placenta specimen. DNA from 25 cases selected out of our tissue bank (2001-2004) was extracted and purified. Subsequently PCR was performed and data from gel electrophoresis of PCR amplicons were compared with our results of corresponding histology. Results: Parvovirus B19 DNA could be detected in several tissue samples by qualitative PCR analysis whereas the prediagnosis of Parvovirus B19 infection due to morphological/clinical findings was only sporadically confirmed. Currently we examine more cases of ambiguous fetal hydrops/IUFD from our tissue bank to investigate the frequency of Parvovirus B19 infections. Conclusions: A PCR analysis for Parvovirus B19 infection should necessarily be done for clarification of every placentitis, fetal hydrops or IUFD of unknown genesis

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Survey: Med. Univ. Innsbruck (Prof. Ch. Marth, Prof. Ch. Brezinka, Prof. G. Simbruner), Hall in Tyrol (Prim. Dr. B. Abendstein), Zams (Prof. Dr. Schro¨cksnadel), Schwaz (Prim. Dr. H. Maneschg), St. Johann in Tyrol (Prim. Dr. Trockenbacher), Reutte (Prim. Dr. Pinzger), Kufstein (Prim. MR Dr. G. Schuchter) and other associated clinics (e.g. Kitzbu¨hel). Results: Sixty three perinatal autopsies have been performed in this 6-month period. Morphological findings at autopsy were correlated to clinical histories and laboratory findings during pregnancy to determine causes of intrauterine growth or post-neonatal death. Conclusions: The assessment of the quality of health care is sometimes fraught with difficulties. Clinical audit is seen as a vital and essential component for improving the quality of care. Criteria for quality audit are a regular series of clinical meetings, audit cycle completed for at least four audit topics, a regular audit of case notes, discharge letters, and summaries has occurred, conduction of a patient satisfaction survey, a regular perinatal mortality meeting at a frequency appropriate to the size of the hospital, multidisciplinary audit involving professions other than midwives, paediatricians, and pathologists has occurred.

318 Bilateral brachial amela, symmetrical cheilognathopalatoschisis holoprosencephaly and disrupted aortic arch

316 Intrauterine enterovirus-induced myocarditis as a rare cause of fetal death

A. ZIMPSER, P. MINY1, U. DOMBROWSKI2, E. BRUDER

A. QUAAS, H. SCHA¨FER Institut fu¨r Pathologie, Universita¨t Basel Medizinische Genetik, Universita¨ts-Kinderspital Basel 2 Frauenklinik Rheinfelden

Institut fu¨r Pathologie, Universita¨tsklinikum HamburgEppendorf

1

317 Tyrolean model of perinatal mortality survey

319

Relevance of b1-integrin in mouse limb bud for outgrowth and patterning

C. SERGI, G. MIKUZ1 Pa¨dopathologie, Institut fu¨r Pathologie, Univ. Innsbruck, O¨sterreich 1 Institute of Pathology, Universita¨t Innsbruck, O¨sterreich Aims: To review perinatal mortality in Tyrol using Audit Clinical Meetings on a regular basis. Methods: Clinical data and anatomic-pathological results following a complete perinatal autopsy from March 2004 to August 2004 were reviewed. Audit resources and meetings were evaluated. Audit Leading was monitored on a collegial base. The following hospital departments of Obs. & Gyn. and Neonatology take part to the Tyrolean Model of Perinatal Mortality

B. HERMANNS-SACHWEH1,2, A. ASZODI2,3, M. MORGELIN4, R. FASSLER2,3 1

Department of Pathology, RWTH Aachen, Germany Department of Experimental Pathology, Lund University, Sweden 3 Max-Planck-Institut of Biochemistry, Martinsried, Germany 4 BMC, Lund University, Sweden 2

Aims: We have been focused on ECM molecules in limb diseases and development. Therefore, it is of crucial interest to find out which role integrins, especially b1Integrin plays for limb outgrowth in interaction with ECM molecules and neighboring cells.

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Methods: To investigate the role of b1-Integrin and its interaction to cell membrane and extracellular matrix proteins in early limb development we generate conditional knockout mice under use of the loxP system to produce b1-Integrin floxed mice under the control of the Msx-2 cre promotor. Msx-2 is a homeobox related gene highly expressed in the apical ectodermal ridge (AER) of the embryonic limb bud. The AER is the responsible signaling center for limb outgrowth and, in interaction with the zone of polarization activity (ZPA) and the dorsal mesoderm, for patterning. Results: A selective b1-Integrin knockout on the apical ectodermal ridge results in a flattening of the AER and a diverging of the ectodermal cells. Furthermore the integrity of the basement membrane is disturbed and the expression of fgf-4, fgf-8 and bmp-4 are reduced. Conclusions: b1-Integrin is necessary for a normal outgrowth of the extremities and an adequate digit separation in early embryonic development. The integrin causes the regulation of the ectodermal cell shape, intercellular contact, and function of the apical ectodermal ridge (AER). Expression pattern of fgf-4, fgf-8, and bmp-4 are impair, syndactylia and synostosis with fusion of the flexor tendons are resulting.

320 Expression of laminin, tenascin and collagen IV in the developing ductal plate of normal foetuses and foetuses affected with trisomies 13, 18 and 21 and foetuses with meckel-gruber syndrome C. BAKER, L. KRAUS1, P. KAHL2, S. ADAM2, C. SERGI1,2 Paediatric Pathology, University of Bristol, United Kingdom 1 Institut fu¨r Pathologie, Universita¨t Innsbruck, O¨sterreich 2 Institut fu¨r Pathologie, Universita¨t Heidelberg, Deutschland Aims: Intrahepatic bile ducts arise from primitive, immature and undifferentiated hepatocytes. The development of the bile ducts has been studied and can be classified into three distinct stages of development that occur in a specific order: ductal plate, remodelling ductal plate and remodelled bile ducts. Methods: Liver sections from different cases were studied: congenitally normal (n ¼ 9), trisomy 13 (n ¼ 8), trisomy 18 (n ¼ 9), trisomy 21 (n ¼ 10) and Meckel-Gruber Syndrome (n ¼ 9). Avidin-Biotin-Complex method of immunohistochemistry was used. Results: Collagen IV was observed in each of the five genetic groups although the intensity of the expression

varied. Collagen IV was also observed throughout the ductal plate development. The strongest levels of expression were observed within the normal group and the weakest levels of expression were observed within trisomy 13. Laminin was observed within each of the genetic groups studied and at each stage of bile duct development. The strongest intensity of expression was observed within the normal and trisomy 18 groups and the weakest intensity of expression was observed within trisomy 13. Tenascin was observed to be weak in most of the cases where expression occurred. Conclusions: A fascinating interplay of mesenchymal markers occurs during the development of ductal plate in foetuses with chromosomal imbalances.

321

Tumor-like marginal zone hyperplasia of MALT with light chain restriction in the terminal ileum, coecum and appendix vermiformis C. TAEGE, H.-J. HOLZHAUSEN, G. OTT1, G. KLOHS2, S. HAUPTMANN

Institut fu¨r Pathologie, Universita¨t Halle 1 Institut fu¨r Pathologie, Universita¨t Wu¨rzburg 2 Klinik fu¨r Kinderchirurgie, Universita¨t Halle Aims: We report on a tumor-like lesion of the ileocoecal region in a 4-year-old boy, which caused the clinical symptoms of invagination. By conventional histology, the remarkable finding was a massive submucosal lymphatic proliferation with reactive and progressively transformated follicles and an excessive broadening of the marginal zone of the mucosa-associated lymphoid tissue (MALT). Methods: Immunohistochemical and molecular analyses were performed to differentiate between an unusual lymphatic hyperplasia and a marginal zone B-cell lymphoma. Results: Immunohistochemistry revealed a slight predominance of lambda light chain expression, although the overall expression was that of an organoid marginal zone hyperplasia. Molecular analyses yielded polyclonality for both the IgH and the T-cell receptor g chain. Conclusions: The diagnosis of an unusual and massive marginal zone hyperplasia of the mucosa associated lymphoid tissue was rendered in spite of an immunohistochemically perceived light chain restriction, because the overall aspect of the lesion was that of an organoid lymphatic proliferation and because the molecular data were in favour of a reactive process. There are few reports with similar features in the literature. Although a favourable prognosis was credited to the patients even without chemotherapeutic intervention. A long-term follow-up is recommended.

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322 Loss of chromosome 22q in an infantile rhabdoid tumour of the liver C. RAINER, I. VERDORFER, A. ZIMMERMANN1, B. MEISTER2, A. KLEIN-FRANKE2, J. HAGER3, G. MIKUZ, C. SERGI Inst. fu¨r Pathologie, Univ. Innsbruck, O¨sterreich Institut fu¨r Pathologie, Univ. Bern, Switzerland 2 Pa¨d. Onkologie, Kinderklinik, Univ. Innsbruck, O¨sterreich 3 Kinderchirurgie, Chirurgische Klinik, Univ. Innsbruck, O¨sterreich 1

Aims: Rhabdoid tumours are highly malignant tumours with rhabdoid cells and facultative neoplastic areas. Rhabdoid tumours can be located either at renal or at extrarenal sites. Treatment includes chemotherapy and frequently surgery. We report on a 1-year old child with a rhabdoid tumour of the liver. The child was treated by chemotherapy and partial hepatectomy following no response to the medical treatment. Methods: Immunohistochemistry was performed on formalin-fixed and paraffin-embedded tissue. Comparative genomic hybridization (CGH) was performed on frozen tissue. Results: We found a high malignant rhabdoid tumour of the liver with pronounced infiltration of the liver parenchyma without formation of a pseudocapsule. Immunohistochemistry revealed a positivity of the tumour cells for pancytokeratin, cytokeratin 8, (focal) vimentin, epithelial membrane antigen, CD34 and CD99. The tumour was negative for cytokeratin 7 and 19 and b-catenin as well as for a-fetoprotein, c-kit, S-100 protein, chromogranin A, synaptophysin and CD56. Ki67 showed at least 50% of tumour cells in a proliferative phase. CGH showed loss of 22q chromosome. Conclusions: Loss of 22q and 19 have been described in rhabdoid tumours of the brain. To the best of our knowledge this is the first youngest case of rhabdoid tumour of the liver showing loss of 22q chromosome.

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Aims: To characterize the cytogenetic effects related to chemotherapy in Wilms tumors we performed CGH on Wilms tumors of blastemal type which were treated with preoperative chemotherapy (preop-CT-group) or not (non-preop-CT group). Methods: Genomic tumor DNA was isolated from 19 Wilms tumors of the preop-CT-group and 22 of the nonpreop-CT group. CGH was performed according to established protocols. Results: Overall, imbalances could be detected in 32 tumors, with +1q, +7q, +7p, and -7p as the most common changes. Tumors in the non-preop-CT group had more changes (mean, 3.8) than those in the preopCT-group (mean, 2.7). Regarding individual chromosomal imbalances, +1q was very common in both the non-preop-CT group (7/22) and the preop-CT group (10/19). However, while in the non-preop-CT group, +7q (9/22) and imbalances at 7p (10/22) were the most frequent imbalances and appeared to be correlated with high-stage tumors (p ¼ 0:077 and p ¼ 0:047; respectively), +7q and imbalances at 7p were significantly less frequent in tumors after preoperative CT (9/22 vs. 1/19, p ¼ 0:011; and 10/22 vs. 2/19, p ¼ 0:019; respectively). Conclusions: The results suggest that CT in Wilms tumors apparently does not affect tumor populations with +1q, while cytogenetically more complex clones with +7q and/or imbalances at 7p are more sensitive to CT and are more likely to be eliminated by chemotherapeutic treatment.

324

Molecular biological manipulation of defensive inflammatory reaction in an experimental rhabdomyosarcoma model - immunohistochemical investigations

325 EGF receptor, PDGF receptor and c-Kit expression in neuroblastoma. Are these receptors targets for therapy? K. ERNESTUS1,3, I. LEUSCHNER2, B. HERO3, H. P. DIENES1, F. BERTHOLD3 1

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Effects of chemotherapy on the cytogenetic constitution of wilms tumor H.-J. SCHULTEN1, T. SCHLOMM2, B. GUNAWAN1, B. SANDER1, N. GRAF3, I. LEUSCHNER4, L. FU¨ZESI1 1

Zentrum Pathologie, Universita¨t Go¨ttingen Abteilung Urologie, Universita¨t Hamburg-Eppendorf 3 Abteilung Pa¨diatrische Onkologie, Universita¨t Homburg/Saar 4 Institut fu¨r Pathologie, Universita¨t Kiel 2

Institut fu¨r Pathologie, Universita¨t Ko¨ln Institut fu¨r Pathologie, Universita¨t Kiel 3 Zentrum fu¨r Kinderonkologie u. -ha¨matologie, Universita¨t Ko¨ln 2

Aims: Advanced neuroblastoma demonstrates poor outcome despite intensive chemotherapy. Tyrosine kinase inhibitors have been shown to be potent alternatives for treatment. Neuroblastic tumors were analyzed for expression of the tyrosine kinases epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR) and c-Kit as potential therapeutic targets and prognostic factors.

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Methods: A multitissue array from 160 paraffin embedded neuroblastomas treated in the German neuroblastoma trials NB90/95 and NB 97 was prepared. Immunohistochemistry was performed for EGFR, PDGFR and c-Kit. The slides were evaluated semiquantitatively. Results: EGFR was not expressed in the neuroblastic tumors (n ¼ 150). C-kit was expressed in 46/145 neuroblastomas (32%). PDGFRß was positive in 113 from 145 cases (78%) demonstrating a homogenous staining in most tumor cells. PDGFRß expression was distributed nearly equally between the different stages. There was a positive correlation between PDGFRß and c-kit expression. Expression of PDGFRß and c-Kit showed no statistically significant correlation with event free survival or overall survival or histology group according to the Shimada classification. Conclusions: EGFR seems not to play a role in neuroblastoma. C-kit and PDGFRare expressed in neuroblastomas and exspecially PDGFR was expressed in a high percentage of neuroblastomas suggesting the potential usefulness of tyrosine kinase inhibitors in the treatment of advanced or progressing neuroblastoma.

326 Technical status and perspective of virtuell pathology

Methods: Evaluation of the strengths and weaknesses of conventional on-line telepathology by 13 pathologists in a series of 104 sessions using the the ZEISS Axiopath system. Development of a new digital microscope system for off-line telepathology. Results: In the Axiopath sessions the correct diagnosis or differential diagnosis was reached in 94 of 104 cases (90.4%). The quality of the images was judged as satisfactory. Most serious limitations were slowness of transmission and delay in image construction after changing magnifications. This led to loss of confidence of the analysing pathologists who were not sure that the complete visual information of the slides had been appreciated. Therefore a DMS was developed generating virtual slides that can be evaluated in off-line mode. Main features are LED illumination, automatic generation of images with Z-stacks enabling a focus function at high magnification for cellular details and continuous magnification (zoom function) without loss of speed leading to almost simultaneous appreciation of image information from low and high magnifications. Conclusions: Evaluation of the shortcomings of on-line telepathology led to the development of a new digital microscope system which promises to overcome these limitations by generating high quality virtual slides that can be analysed in off-line mode. This DMS may also serve as a tool for histopathology teaching and consultation services and pave the way towards a fully digitized diagnostic pathology.

K. KAYSER1, P. HUFNAGL2, W. SCHLAKE3, G. KAYSER4

328 The german fieldstudy of telepathology – prelimin-

Study Group: Informatics

ary results and perspectives 1

UICC-TPCC, Institut fu¨r Pathologie, Universita¨tsmedizin Berlin 2 Institut fu¨r Pathologie der Charite´ (CCM), Universita¨tsmedizin Berlin 3 Institut fu¨r Pathologie, Gelsenkirchen 4 Institut fu¨r Pathologie, Universita¨t Freiburg

327 New digital microscope system (DMS) for telepathology V. MORDSTEIN1, A. NOLTE2, E. STELZER3, N. SALMON4, U. LO¨HRS1, J. DIEBOLD1, 1

Pathologisches Institut der Universita¨t Mu¨nchen ZEISS Go¨ttingen, 3 EMBL Heidelberg, 4 SLS Heidelberg 2

Aims: The present study had two aims: (1) to define the shortcomings of conventional telepathology, (2) to design a tool that is able to overcome these limitations.

S. VON GERLACH, A. SCHULZ, A. BATTMANN Institut fu¨r Pathologie, Universita¨t Giessen Aims: Telediagnostic of frozen sections is a relatively new possibility of diagnostics. Intention of the study is to get information about the reproducibility and reliability of telepathologically obtained diagnoses on frozen sections. The questions concern the quality of diagnosis based on digitised images, the reliability and the interobserver variability. Methods: Participants of the study placed the digitised images captured during the diagnostic procedure on a consultation server. Main questions concerned either the dignity or resection margins free of tumour. Diagnoses were compared to the final diagnoses obtained on the paraffin section. Furthermore they were asked to evaluate the other cases. Results: Cryosections of 10 different organ systems have been examined. Only in a few cases the interpretability was limited. We found a change of diagnosis (paraffin

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vs. cryosection) in 4.94% of all cases, esp. false negative 3.8% and false positive 1.06%. Interobserver variability could not be examined due to an insufficient response by the study participants. Conclusions: In the majority of cases telepathology/ telediagnostic of frozen sections led to the correct diagnosis, compared to the final examination. However, interobserver variability, a core element of the study, could not be examined. A new attempt to gather sufficient data is made by using a cd-rom. This should now permit fast offline diagnoses in a limited number of randomly selected cases by the study participants.

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Conclusions: The results show the potential of the developed software package for quantitative characterization of cell nuclei by appropriate image parameters for 3D genome organization.

330 Automated measurements of images obtained from immunohistochemically stained slides (EAMUSTD) K. KAYSER1, E. VOLLMER2, M. MAEURER3, R. SOMMER1, P. BZDYL4, D. RADZISZOWSKI4, G. KAYSER5 1

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A software package for 3D fluorescence image analysis of the spatial genome organization in FISH labelled cell nuclei M. HAUSMANN, S. STEIN1, S. TIMME, F. RIEDE, T. WIECH, M. SCHURIKE1, J. FINSTERLE1, C. CREMER1, M. WERNER, A. WALCH Pathologisches Institut, Universita¨tsklinikum Freiburg 1 Kirchhoff-Institut fu¨r Physik, Universita¨t Heidelberg

Aims: The higher-order, spatial genome organization (genome architecture) in 3D conserved cell nuclei is of functional meaning. The study of the genome architecture and potential variations during tumor genesis requires quantitative measurements of specific fluorescence labels in 3D microscopic images. Therefore, a software package was developed, which allows 3Dimage analysis of more than 10 different parameters, e.g., the radial position of the labelled sites, and its variations in FISH labelled cell nuclei. Methods: 3D-fluorescence images of FISH labelled cell nuclei were recorded using a high resolution epifluorescence microscope equipped with an Apotome and a b/w CCD camera for optical sectioning. After automated or interactive object segmentation, the nuclei were subjected to the newly developed versatile software package for image analysis and 3D-reconstruction. Several threshold dependent and independent image parameters were calculated and compared between normal and aberrant cell nuclei. Results: The software was successfully applied to cell nuclei after whole chromosome painting or FISH labelling of specific domains. For haematological blood samples as well as for tissue microarrays prepared from formalin-fixed and paraffin-embedded blocks of various tissues, it was possible to get quantitative information of the genome organization. According to several different image parameters signicant differences were found between morphologically normal and altered cells.

UICC-TPCC, Institut fu¨r Pathologie, Universita¨t Berlin 2 Institut fu¨r Pathologie, Forschungszentrum Borstel 3 Institut fu¨r Klinische Mikrobiologie, Universita¨t Mainz 4 AGH University of Science and Technology, Krakow 5 Institut fu¨r Pathologie, Universita¨t Freiburg Aims: To introduce an internet-based, automated image measurement system for immunohistochemically stained slides including fluorescence images in an on-line and off-line mode. Methods: An image analysing system has been developed that automatically measures images obtained from immunohistochemically stained slides. It is divided into a common server platform and a specific image quantification system based upon DIAS (DIAS, University Jena). After registration, the user fills in an input data form and attaches images to be measured. The server periodically transfers the data to the measurement system. The measurement works on fluorescence and conventional visualized images, and includes stereological algorithms, object quantification, syntactic structure analysis, and quality assurance. Results: The system has been tested for DAB, AP, and fluorescence images (FITC, etc.). The reproducibility and stability of the system accounts to498%. The series of successfully measured images comprises41000 images in total in the on-line and off-line mode. Conclusions: An internet-based automated image measurement system has been developed that offers worldwide access to the major requests of quantification of immunohistochemically stained slides, i.e., tissue array analysis, nuclear stains (MIB, hormones), membrane stains (Herceptin), vascularization, and FISH.

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Sensitivity and accuracy of automated histomorphologic diagnosis of primary bronchial carcinomas, a feasibility study for virtual slide analysis

G. KAYSER1, A. MAYER1, M. WERNER1, K. KAYSER2

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1

Institut fu¨r Pathologie, Universita¨t Freiburg UICC-TPCC, Institut fu¨r Pathology, Universita¨t Berlin 2

Aims: To analyze the accuracy of a developed texture analysis algorithm and the influence of its specific parameters on automated cell type classification of common bronchial carcinomas. Methods: The image texture is analyzed by a recursive algorithm, and the basic information content is extracted, and was compared with the data of artificial images generated by random. The analyzed parameters include: (1) analyzed area fraction of original image, (2) number of repeated random steps, (3) image magnification. According to Voronoi’s formula, randomly distributed, non-overlapping squares were calculated. The image information of these squares was compared with that of the artificial images. The diagnoses include tumor-free lung tissue and the four major carcinoma cell types. Multivariate discriminate analysis served for statistical analysis Results: A 20 objective magnification revealed the most significant results to distinguish between the different cell types. The discrimination between the different tumor cell types was 495% in both the teaching and analyzing set. The accuracy of the algorithm remained nearly constant to area fractions 10%. It was found to be independent from the number of sequential random procedures. Conclusions: Histomorphological automated ‘‘crude’’ diagnosis extraction in lung tissue can be performed on less than 10% of acquired image area without significant losses of diagnosis – relevant information. The data indicate, that analysis algorithms of virtual slides can be undertaken on only 10% of original image area, and at moderate magnifications only.

332 Pathology in developing countries Need, solution, limits – example Phnom Penh-Projekt T.H. KUAKPAETOON1, G. STAUCH2, C.H. VATHANA3, K.-D. KUNZE4, C. HAENER3 1

Department of Pathology, Rajavithi Hospital, Bangkok 2 Institut fu¨r Pathologie, Aurich 3 Sihanouk Hospital Center of HOPE, Phnom Penh 4 Technische Universita¨t, Dresden Aims: Only four Laboratories with five Pathologists are offering a service to about 12 million inhabitans in Cambodia, diagnosing 3000 morphological cases per year. The technical equipment of laboratories is low, the experience of technicians and pathologists is not equivalent to international standards. The financial

support by the government is very poor. SHCH is a NGO bases hospital, offering Cambodia people a charge free treatment. From 2002 for the diagnostic work by telepathology via internet. Methods: Analysing the co-operation of 2 years between SHCH and an international expertround we evaluated the need and the limit for morphologic diagnostic in developing countries and discribe the solutions we found in our project in order to enlight the situation of morphology under poor economic conditions. Results and conclusion: (1) There is a need for morphology even in low medical conditions for therapeutic decisions, even in some instances as triage diagnosis, rational understanding of diseases, for establishing tumour registers. (2) There is also a need for establishing international networks with personal contacts to break through the political induced isolation of intellectual persons. (3) Pathologic diagnostic should be adopted to actual medical treatment possibilities to prevent vesting financial resources. (4) Training on pathologists should be focused on needed specialised fields. The training should be done in locally by international experts to consider local situation. (5) The aim of foreign aid should be establish an independent medical system in those countries.

333 The prototype of a semantic based image retrieval system T. SCHRADER, T. LEUTHOLD, S. NIEPAGE, K. SAEGER, P. HUFNAGL Institut fu¨r Pathologie, Charite´, Universita¨tsmedizin Berlin Aims: Images are an important component of both clinical care and medical education. The use of the digital virtual microscope (DVM) with stores and processes digitalised histological glass slides offers new advantage in image retrieval. In the Prototype of the project ‘‘Semantic Web in Pathology’’ a textanalyser split the text into words and sentence and map these to semantic concepts stored in an ontology. Using the diagnostic path and the semantic concepts an image content retrieval system was developed. Methods: The DVM stores and processes digitalised images in a relational database (SQL-Server, Microsoft). The pathology reports are stored in a Database too. After a linguistic analysis of 100 selected reports we extracted a subset from the UMLS Metathesaurus of lung anatomy and lung pathology related concepts. These concept where transferred to OWL to prepare the semantic network for image retrieval system. The

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prototype was tested with 50 random selected reports and 75 slides of the DVM including the diagnostic path. Results: For the test set of pathology report specific parameters (precision and recall) of retrieval properties were calculated. The result set consists of reports containing the search term or synonym and presents related reports to the search term. Conclusion: The application of techniques of natural language processing and the implementation of ontology is very useful for the focus on image retrieval on very large images like the digital slides. The main problem is the high variability of the text in pathology whether this is a highly specific subset of natural speech.

be improved by training of technicians and improving the standard of technical equipment (2) Image quality by inefficient field selections and dataquality of electronic images (3) Interpretation quality due to complexity of question. Heterogenity of terminology and lack of clinical data. Conclusions: Most important factor for diagnostic accuracy in Telepathology is (1) Slide quality (2) Clinical information and 3. Technical equipment.

334 Diagnostic accuracy in teleconsultation via internet between developing countries and experts: The PNH an VTN Project

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Study Group: Oral Pathology Prognostic significance of HPV-DNA detection, expression of P16 and EGFR in oropharynx carcinoma N. REIMERS, J.P. KLUSSMANN1, H.U. KASPER

1

2

J. SETTAKORN , G. STAUCH , T.H. KUAKPAETOON3, K.-D. KUNZE4, C. HAENER5, C.H. VATHANA5, S. BOUNTHOME6 1

Department of Pathology, Chiang Mai University, Chiang Mai 2 Institut fu¨r Pathologie Aurich 3 Department of Pathology, Rajavithi Hospital, Bangkok 4 Institut fu¨r Pathologie, Technische Hochschule, Dresden 5 Sihanouk Hospital Center of HOPE, Phnom Penh 6 Department of Pathology, National University, Laos Aims: Developing countries are suffering from lack of laboratory facilities and experienced pathologists to provide an adequate service. The turn around time of second opinion of experts is unacceptably prolonged by long mailing time. Consultation via internet by Telepathology seems to be an acceptable solution to some extend. Methods: There are two projects in Phnom Penh (PNH) Cambodia and Vientiane (VTN) Laos, using Telepathology via internet for second opinion. Aim of the study is to evaluate the diagnostic accuracy and the limit in special questions of diagnostic pathology. Totally 240 requests, 220 from PNH and 20 from VTN related mostly to surgical pathology were analysed between 01.04 and 06.04. Results: The diagnostic security of experts differ from 95% in breast lesions to 30% in bone marrow cases and diagnostic concordance was found between 83% in non complex histological cases. 90% in cytological noncomplex questions and 57% in complex histology respectively 47% in complex cytology. The turn around time was within 24 h for 50% of all cases. The reasons for diagnostic problems are (1) Slide quality which can

Institut fu¨r Pathologie, Universita¨t Ko¨ln 1 Institut fu¨r HNO, Universita¨t Ko¨ln Aims: The aim was to analyze the prognostic significance of HPV-DNA Detection, EGFR and P16 expression in squamous cell carcinoma of the Oropharynx (OC). Methods: We analyzed 107 newly diagnosed OC treated between 1997 and 2002. After reassessment all were tested for HPV-DNA by multiple PCR assays, expression of p16 and epidermal growth factor receptor (EGFR) by immunohistochemistry. Disease-free and overall survival was calculated in relation to these molecular markers using the Kaplan-Meier method and log-rank tests. Results: Of the tested carcinomas 22% contained oncogenic HPV and 34% were positive for p16. p16 expression was highly correlated with the presence of HPV-DNA (po0.001). EGFR expression and HPV detection were inversely correlated (p ¼ 0:051) and 71% of the HPV positive carcinomas were EGFR negative. Survival analysis revealed significant better outcome for p16 positive tumors for 5-year disease-free (76% vs. 49%, p ¼ 0:01) and overall survival (84% vs. 44%, p ¼ 0:01). There was a trend for better prognosis for EGFR negative tumors for disease-free (73% vs. 48%, n.s) and overall survival (70% vs. 47%, n.s.). Remarkable highly significant was the inverse combination of p16 and EGFR leading to 5-year disease-free survival estimates of 92% for p16 positive/EGFR negative tumors vs. 44% for p16 negative/EGFR positive cases (p ¼ 0:008). Conclusions: These data indicate that p16 is an accurate surrogate marker for HPV positive OC and has strong prognostic impact with p16+/EGRF- carcinoma having a better outcome. HPV positive/p16 positive

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carcinomas showed decreased EGFR expression. Using both immunohistological markers has strong prognostic value applicable for routine pathologic histology.

336 Subcellular localization of ß-catenin and laminin-5 c2 chain (Ln5/c2) deposition in oral squamous cell carcinoma (OSCC) A. BERNDT, K. KATENKAMP, P. RICHTER, F.D. BO¨HMER1, P. HYCKEL2, H. KOSMEHL3 Institut fu¨r Pathologie 1 AG Molekulare Zellbiologie 2 Klinik fu¨r Mund-, Kiefer- und Gesichtschirurgie, Universita¨t Jena 3 Institut fu¨r Pathologie, Helios-Klinikum Erfurt Aims: Cytoplasmic Ln5/g2 is one of the best markers for carcinoma cell invasion. In colorectal carcinomas, the expression of Ln5/g2 is regulated by nuclear ß-catenin (Hlubek et al. 2001). The study is aimed at the definition of the relation between localization of ß-catenin and cytoplasmic deposition of Ln5/g2 in OSCC. Methods: Paraffin embedded material of normal, hyperplastic oral mucosa and 23 OSCC (6xG1, 11xG2, 6xG3). Immunohistochemical detection of ß-catenin using the ß-catenin antiserum C2206 (Sigma) and the ß-catenin antibody 17C2 (Novocastra) and comparison to histological criteria of invasion and cytoplasmic Ln5/ g2 deposition (clone D4B5). Results: Normal/hyperplastic oral mucosa: ß-catenin mainly in the epithelial cell membrane, cytoplasmic ßcatenin in the basal/parabasal region as well as in blood vessels. Using the antiserum, nuclear staining of keratinocytes was seen in the stratum granulosum. Ln5/ g2 exclusively in the BM. OSCC: Loss of membranous staining and cytoplasmic accumulation of ß-catenin mainly in the invasive front. Nuclear ß-catenin only in few less differentiated OSCC in central areas and in inflammatory cells (polyclonal antiserum). In G1 OSCC cytoplasmic Ln5/g2 in basal keratinocytes and invading carcinoma cells. With raising malignancy grade the whole tumour compartiment becomes positive. No spatial correlation with nuclear ß-catenin. Conclusions: (1) Nuclear ß-catenin is neither involved in regulation of tumour cell invasion nor of Ln5/g2 expression in OSCC but seems to reflect terminal differentiation/apoptosis of keratinocytes. (2) Whereas cytoplasmic accumulation of Ln5/g2 is a common feature of invading carcinoma cells, regulation of its expression differs among carcinoma entities.

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EGFR pathways and laminin-5 c2 chain (Ln5/c2) expression in oral squamous cell carcinoma (OSCC)

P. RICHTER, F.D. BO¨HMER1, P. HYCKEL2, L. BORSI3, D. KATENKAMP, H. KOSMEHL4, A. BERNDT Institut fu¨r Pathologie 1 AG Molekulare Zellbiologie‘‘ ’’ 2 Klinik fu¨r Mund-, Kiefer- und Gesichtschirurgie, Universita¨t Jena 3 Istituto Nazionale per la Ricerca sul Cancro, Genoa, It. 4 Institut fu¨r Pathologie, Helios-Klinikum Erfurt Aims: Overexpression of epidermal growth factor receptor (EGFR) was shown for the majority of OSCC and correlates to tumour size, stage and cytoplasmic accumulation of the laminin-5 g2 chain (Ln5/g2), which is known as a marker of invading tumour cells. There is only limited knowledge if and how EGFR signalling pathways are important for invasion associated processes and the regulation of Ln5/g2. Methods: Paraffin embedded material of normal, hyperplastic, dysplastic oral mucosa and 23 OSCC (6xG1, 11xG2, 6xG3). Phosphorylated Erk1/2, p38MAPK and Akt were immunohistochemically detected using activation state specific antibodies (all Cell Signalling Technol.) and compared to histological criteria of invasion and cytoplasmic Ln5/g2 deposition (clone D4B5). Results: With raising histological grade, there is a slight increase in nuclear pErk1/2 stained tumour cells (p ¼ 0:398) and a loss of nuclear (p ¼ 0:593) and increased cytoplasmic staining (p ¼ 0:144) of pAkt mainly in invading OSCC cells. Nuclear pp38MAPK could only be sporadically detected in few cases. In case of pErk1/2 and pAkt only a partial colocalisation to cytoplasmic Ln5/g2 could be revealed in cases with abundant kinases and Ln5/g2 expression. Conclusions: Among the investigated kinases only pAkt shows a relation to histological grade and invasion in OSCC. pErk1/2, pp38MAPK and pAkt do not represent a direct link between EGFR and Ln5/g2 synthesis. Therefore, enhanced Ln-5/g2 may be a secondary phenomenon of EGFR induced tumour cell proliferation and dissemination.‘‘

338 Monophasic synovial sarcoma of the neck after therapy of torticollis spasmodicus with botulinum toxin D. JANSSEN1, C. SCHRADER2, M. KNEBA2, S. GOTTSCHLICH3, U. JA¨NIG4, M. KRAMS4, R. PARWARESCH5, G. KLO¨PPEL1 1

Institut fu¨r Allgemeine Pathologie, Universita¨t Kiel II. Medi-zinische Klinik, Universita¨t Kiel

2

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Klinik fu¨r Hals-Nasen-Ohren-heilkunde, Universita¨t Kiel 4 Institut fu¨r Paidopathologie, Universita¨t Kiel 5 Institut fu¨r Ha¨matopatholgie, Universita¨t Kiel Aims: Monophasic synovial sarcoma is a rare disease with a typical chromosomal translocation t(x;18). The etiology is still unknown. This is a case-report of a 67year old patient with a long history of torticollis spasmodicus treated with botulinum toxin in the right musculus sternocleidomastoideus. In the same area the patient developed a synovial sarcoma 11 years after injection of botulinum toxin. Methods: The tumor biopsies were stained with hematoxylin and eosin and Giemsa. Immunohistochemical analysis was performed with the following antibodies: Vimentin, cytokeratin, EMA, CD34, CD45, CD99, S100, smooth muscle actin, bcl-2 and Ki-S5. RT-PCR was performed to detect SYT/SSX-fusion transcripts. Results: The MRT scanning showed an infiltrating tumor close to the musculus sternocleidomastoideus. Histological analysis revealed a monophasic fibrous synovial sarcoma. Immunohistochemically, the tumor cells were positive for vimentin, CD99, bcl-2, and focally for EMA. The results of the RT-PCR were in line with a t(x;18). Conclusions: This is the first report on a synovial sarcoma developing after a long history of repeated botulinum toxin injections in the site of the tumor. Botulinum toxin may play a role in the pathogenesis of this tumor.

339 Novel aspects of the histogenesis of Warthin’s tumor T. HANSEN, A. SCHAD, A. WEBER, CH. BROCHHAUSEN, C.J. KIRKPATRICK Institut fu¨r Pathologie, Universita¨t Mainz Aims: Warthin’s tumor is regarded as a unique neoplasm among the salivary gland tumors, since it almost exclusively occurs in the parotid gland. The pathogenesis of this disease is still a matter of debate. In brief, one concept is that the tumor is an epithelial neoplasm inciting a lymphocytic response; the other theory is that it develops from heterotopic salivary ducts, mainly striated ducts, within pre-existing lymphoid tissue or intraparotid lymph nodes. In this study, we show new aspects concerning the histogenesis of Warthin’s tumor. Methods: Tissue specimens from 28 patients with Warthin’s tumor (13 female, 15 male; mean age: 64 y) were routinely processed and paraffin-embedded. Immunohistochemistry was performed including antibodies against CD31, CD34, FVIII, and CD117. Results: We found strong expression of CD117 in almost all oncocytic epithelial cells and in a few mast

283

cells embedded in the lymphoid stroma. Interestingly, in normal salivary gland tissue the striated ducts exclusively revealed a distinct expression of this molecule. Furthermore, by using anti-CD31, anti-34, and anti– FVIII numerous sinus-like vessels could be detected in the subcapsular zone and in close vicinity to the epithelial lining. Conclusions: The presented expression of CD117 confirms the view that the epithelial component of Warthin’s tumor is histogenetically related to the striated ducts of the salivary gland. It remains to be investigated whether the finding of sinus-like vessels in subcapsular and subepithelial localization is in line with the idea that Warthin’s tumor originates from heterotopic intralymphoid or even intranodal salivary gland tissue.

340 HIF1-a expression indicates a good prognosis in squamous cell carcinomas of the oral floor H. BU¨RGER, R. WERKMEISTER1, P.J. VAN DIEST2, B. BRANDT3, U. JOOS4, T. FILLIES4 Institut fu¨r Pathologie, Universita¨t Mu¨nster 1 Bundeswehrzentral-krankenhaus Koblenz 2 Institut fu¨r Pathologie, Universita¨t Utrecht, Niederlande 3 Institut fu¨r Klinische Chemie 4 Klinik fu¨r MKG-Chirurgie, Universita¨t Mu¨nster Aims: Hypoxia-inducible factor 1a (HIF-1a) is a transcription factor, which plays a central role in biologic processes under hypoxic conditions, especially concerning tumour angiogenesis. It is mostly coexpressed with CA IX and Glut1, indicating a response to hypoxic stimuli. Methods: Eighty five patients with histologically proven surgically treated T1/2 squamous cell carcinoma (SCC) of the oral floor were eligible for the study. Tumor specimens were investigated by means of tissue micro arrays (TMAs) and immunohistochemistry for the expression of HIF-1a, CA IX and GLUT 1. Correlations between clinical features and the expression of HIF-1a , CA IX and GLUT were evaluated by KaplanMeier curves, Log-Rang—and w2 Tests. Results: The expression of HIF-1a was related with a significantly improved 5-year survival rate (po0:01) and a significantly increased disease free period (p ¼ 0:01). In primary nodal negative T1/T2, SCC of the floor of the mouth, absence of HIF-1a expression specified a subgroup of high- risk patients (po0:05). HIF-1a expressionwas independent of CA IX and GLUT 1. Conclusions: Our data show for the first time that the HIF-1a overexpression is an indicator of favorable

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prognosis in T1 and T2 squamous cell carcinomas of the floor of the mouth and is regulated independently fromm hypoxic stimuli.

341 Undifferentiated salivary gland carcinoma with lowgrade myoepithelial component: EBV infection, viral gene expression and immunophenotype H. HERBST, G. NIEDOBITEK1 Institut fu¨r Pathologie, Universita¨t Mu¨nster 1 Pathologisches Institut, Universita¨t ErlangenNu¨rnberg Aims: Salivary gland lymphoepithelial carcinomas (LEC) are associated with Epstein-Barr virus (EBV) in endemic areas, whereas most sporadic cases are EBVnegative. LEC may be accompanied by benign lymphoepithelial lesions (BLEL), and it has been proposed that BLEL may be LEC precursor lesions. We have studied two EBV-associated LEC from Caucasian patients for EBV gene expression profile and immunophenotype. Methods: EBV infection was detected in paraffin sections using EBER-specific in situ hybridisation. Viral gene expression pattern and cytokeratin expression profile were studied by immunohistochemistry. Results: In case 1 a high-grade component expressing glandular cytokeratins was associated with a low-grade spindle cell lesion with an immunophenotype of myoepithelial cells. Case 2 expressed basal cell and glandular cytokeratins. In case 1, expression of EBNA1, LMP1 and LMP2A was seen in the high-grade tumour. By contrast, the spindle cell lesion of case 1 and the high grade tumour in case 2 showed only EBNA1 in the absence of LMPs. Conclusions: The detection of EBV in both histologically distinct components of case 1 supports the concept that some LEC may develop through a low grade myoepithelial intermediate. EBV infection may occur more frequently in sporadic salivary gland LEC than previously anticipated. Expression of LMP2A in salivary gland LEC is of interest because it may make such cases amenable to immunotherapy with cytotoxic T-cells targeting LMP2A.

1

Institut fu¨r Pathologie HNO-Klinik, Universita¨t Mu¨nster 3 Institut fu¨r Pathologie, Klinikum Osnabru¨ck 2

Aims: Salivary gland tumors are characterized by a wide spectrum of structural differentiation. Therefore they are especially suitable to study differences as well as a possible common origin in stem cells. Our study focused on an immunohistochemical panel of various cytokeratins, receptor proteins and nuclear antigens to correlate it with different tumor entities. Additionally to true differences and true coexpressions we hoped to find mathematically detectable associations between such immunprofiles that could be of important value in a possible stem cell concept of tumorigenesis. Methods: Semiquantitatively scored immunohistochemical findings detected on a tissue micro array of 57 malignant parotideal tumors was underwent a mathematical cluster analysis. Results: In relation to tumor entities the immunprofiles were distributed on four divergent clusters that were characterized especially by the degree and pattern of expressed cytokeratins 5/6 and 8/18. Using a biomathematical cluster analysis the immunprofiles were found to be in significant relations of each other. Cytokeratin 5/6 and 14 frequently inverse related to CK 8/18 were significantly associated with low levels of androgen receptor, Cyclin D1, a-Catenin and c-erb B2, but with high levels of p53, g-Catenin, bcl-2 and p63. In contrast, CK 8/18 showed an inverse relation to these proteins. Conclusions: The results suggest to assume common stem cells in origin of malignant parotideal tumors. The tumor entities in the result of various differentiations are related to clusters of immunprofiles.

Study Group: Gastrointestinal Pathology

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Mastocytosis in the GI-tract: Eva Wardelmann,

Bonn Serrated polyps/adenomas: D. Aust, Dresden

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Biomathematical cluster analyses of immunohistochemical results on a tissue microarray of 57 malignant salivary gland tumors: The importance of a new method in stem cell concept of tumorigenesis C.H. AUGUST1, J. ALBERTY2, J. PACKEISEN3, H. TAPPE1, H. BU¨RGER1, D. HUNGERMANN1, W. BO¨CKER1, E. KORSCHING1

Gastric atrophy and metaplasia: G. Faller, Erlangen GIST: R. BU¨TTNER, Bonn

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344 Angiogenesis G. BREIER

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E. WARDELMANN, S. MERKELBACH-BRUSE, K. PAULS, E. PAGGEN, C. KUHNEN1, P. HOHENBERGER2, G. SEITZ3, K. HARTMANN4, J. RU¨SCHOFF5, S.M. KRO¨BER6, R. BU¨TTNER

Institut fu¨r Pathologie, Universita¨tsklinikum Dresden

345 Angiotensin converting enzyme (ACE) is regulated by cytokines and may be involved in invasion S. C.-M. GRATH1, S. KRU¨GER1, U. LENDECKEL2, M. EBERT3, A. ROESSNER1, C. RO¨CKEN1

Institut fu¨r Pathologie, Universita¨tsklinikum Bonn 1 Institut fu¨r Pathologie, Ruhr-Universita¨t Bochum 2 Chirurgische Klinik Mannheim, Universita¨t Heidelberg 3 Institut fu¨r Pathologie, Klinikum Bamberg 4 Klinik fu¨r Dermatologie, Universita¨t Ko¨ln 5 Institut fu¨r Pathologie, Klinikum Kassel 6 Institut fu¨r Pathologie, Universita¨t Tu¨bingen

1

Institute fu¨r Pathologie Experimentelle Innere Medizin 3 Klinik fu¨r Gastroenterologie, Universita¨t Magdeburg 2

Aims: Angiotensin converting enzyme (ACE) cleaves angiotensin I to form angiotensin II, which is involved in the regulation of growth and differentiation via the angiotensin II receptors, Type 1 (AT1) and Type 2 (AT2). ACE is up-regulated in gastric cancer, and the increased mRNA expression associated with the ACE polymorphism (Ro¨cken et al.; abstract submit-ted) may contribute to the development of lymph node metastases in gastric cancer. Methods: Using immunohistochemistry, we investigated the expression of ACE in lymph node metastases of gastric cancer. Gastric cancer cell lines were evaluated for the mRNA expression of ACE, AT1, and AT2. After in vitro treat-ment with cytokines, real-time RT-PCR was performed to quantify the resulting changes in ACE mRNA expression. In vitro cell proliferation and invasion assays were carried out after ACE- and AT1 inhibition. Results: ACE was expressed in 26 of 45 (58%) lymph node metastases, and in four of five gastric cancer cell lines. AT1 and AT2 were both strongly expressed in three and weakly expressed in two of the five cell lines. Incubation with cytokines lead to both up- and downregulation of ACE. Inhibition of ACE and AT1 resulted in significant upregulation of proliferation in MKN45 and NCI-N87 cell lines, which was combined with a significant reduction in their invasive ability. The MKN28 cells showed no changes in proliferation, but the extent of invasion was significantly increased. Conclusions: The effect of ACE and AT1 inhibition on cell proliferation and invasive ability seems to depend on the balance of expression of ACE, AT1 and AT2, whose expression may be regulated by cytokines.

346 Pathological and molecular characteristics of multiple synchronous gastrointestinal stromal tumors (GISTs)

Aims: Gastrointestinal stromal tumors (GISTs) most often occur solitarily. However, in a minority of cases, more than one lesion is found. It is still unknown whether these tumors develop indepen-dently or if they represent one primary GIST and its metastases. Methods: We evaluated 16 cases with more than one tumor for their clinical, morphological, immunhistochemical and molecular features. GISTs with primary location outside of the gastrointestinal tract (E-GISTs) were excluded. Results: Two patients with multiple GISTs carried a germline mutation in exon 8 of KIT and suffered of an associated systemic mastocytosis. In two females (one of them with Carney’s triad), multiple nodules in the stomach and associated hyperplasia of cells of Cajal suggested a germline mutation. Two others had a known neurofibromatosis type I. However, wild type sequences were found in all exons of KIT and PDGFRa evaluated up to now. In three patients, different KIT or PDGFRa mutations were identified in the different lesions. All other patients had wild type sequences in all examined exons of the different lesions. Conclusions: Multiple synchronous GISTs may be associated with a germline mutation in KIT, a neurofibromatosis type I or Carney’s triad. Rarely, synchronous tumors carry different KIT or PDGFRa mutations. The predominance of GISTs with wild type sequences in both KIT and PDGFRa as compared to sporadic GISTs strongly suggests an additional alternative molecular pathogenesis.

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Cryopreservation of precision cut tissue slices (PCTS): Investigation of morphology and reagibility H.U. KASPER, E. KONZE, N.K. CANOVA1, U. DEBBER, H.P. DIENES

Institut fu¨r Pathologie der Universita¨t zu Ko¨ln, Deutschland 1 Institute of Pharmacology, 1 Faculty of Medicine, Charles University, Prague, Czech Republic

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Aims: We adapted PCTS for functional morphological research in liver tissue. To overcome the shortage of liver tissue, to optimize standardization and to get independence from the time of operation, a storage system of PCTS would be useful. Therefore, we investigated the possibility of cryopreservation of PCTS. Methods: PCTS were prepared from 4 porcine livers using Brendel Vitron Tissue Slicer. Half of the PCTS were exposed to ice cold RPMI medium with supplements for 15 min with 10% DMSO and the other half with 30% DMSO. Half of each group were shock frozen with medium and the other half without medium. The slices from two livers were thawn after 24 h and cultivated for 48 h. From the other two livers, the slices were thawn after 24 h, cultivated and apoptosis stimulation was done by 25 ng/ml TNF a and 1 mg/ml actinomycin D. Non-frozen PCTS were incubated parallel with and without apoptosis stimulation. Apoptosis and proliferation was assessed immunohistochemically (M30CD; Ki67). Results: After cryopreservation, morphology of PCTS is well preserved. The rate of spontaneous apoptosis and proliferation was very low. Compared to non-frozen PCTS, the rate of spontaneous apoptosis and proliferation is significantly reduced. The reagibility of apoptotic stimuli was also significantly reduced. There were no differences between the four freezing techniques. Conclusion: Cryopreservation of PCTS does not change morphology of the slices. The reagibility, however, is dramatically disturbed. Apoptotic stimuli cannot induce the same amount of cell deaths compared to non-frozen sections. Thus, cryopreservation of PCTS does interfere with pathomechanisms in the slices. Cryopreserved PCTS can not be a source for further investigations. K.

348 In situ analysis of acyl-CoA-synthetase 5 expression in gluten sensitive enteropathy N. GASSLER, M. KEITH, P. SCHIRMACHER, F. AUTSCHBACH Pathologisches Institut, Universita¨t Heidelberg Aims: The most severe injury of small intestinal tissue architecture in gluten sensitive enteropathy is villus atrophy. Molecules reflecting the state of villus architecture are not well characterized at present. Methods: Expression of acyl-CoA-synthetase 5 (ACS5) was studied in gluten sensitive enteropathy as well as in unaffected human intestinal tissues and by several methods including molecular techniques and an in situ approach using a novel established monoclonal antibody directed against human ACS5.

Results: Strong expression, synthesis, and enzymatic activity of ACS5 was found in normal small intestinal mucosa. ACS5 preferentially located to the epithelium covering villi. In gluten sensitive enteropathy, expression of ACS5 in the surface epithelium of villi was apparently independent of the grade of villus maturation. Thus, ACS5 was seen in the villus epithelium of the small intestine with gluten sensitive enteropathy Marsh I, II, IIIa, or IIIb, respectively. In Marsh IIIc lesions of gluten sensitive enteropathy, comprising of total villus atrophy, hyperplasia of crypts, and intraepithelial lymphocytosis, strong expression of ACS5 was neither detectable in the surface epithelium of the mucosal plateau nor in the epithelium lining the hyperplastic crypts. Reconstitution of Marsh IIIc tissue injury within about 10 month under a gluten-free diet into Marsh 0 was associated with a strong re-expression of ACS5 in the villus epithelium. Conclusions: These data suggest that ACS5 is a marker molecule for the detection of villus atrophy in the small intestine.

349 Prognostic value of protein expression in Barrett0 s adenocarcinoma of the esophagus-a tissue microarray study R. LANGER, B.H.A. VON RAHDEN1, R. REITER, M. FEITH1, H.J. STEIN1, J.R. SIEWERT1, H.HO¨FLER, M. SARBIA Institut fu¨r Pathologie, TU Mu¨nchen Chirurgische Klinik und Poliklinik, Klinkum Rechts der Isar, TU Mu¨nchen 1

Aims: To correlate immunohistochemical expression patterns and prognosis in Barrett0 s adenocarcinoma (BCA) of the esophagus. Methods: A tissue microarray was constructed from 98 primarily resected BCA (pT1: 39 cases, pT2: 20 cases, pT3: 39 cases). The expression of C-erbB-2, p53, p16, p27, Cyclin D1 and EGFR was studied by immunohistochemistry. Expression patterns were correlated to tumor stage, grade and lymph node status, as well as to patients0 survival. Results: Eleven tumors (11.3%) showed an overexpression of C-erbB-2 oncoprotein. Overexpression of EGFR was observed in 52 cases (54.1%), accumulation of p53 in 46 cases (46.9%). Accumulation of Cyclin D1 was present in 68 tumors (69.4%). Loss or inactivation of p16 and p27 was observed in 79 cases (80.6%) and 71 cases (72.4%), respectively. Expression of these proteins did not correlate with tumor stage, grade, Lauren0 s or WHO classification or lymph node status. C-erbB-2 overexpression was significantly associated with p53 accumulation (p ¼ 0:012). Loss of p16 was significantly correlated to accumulation of Cyclin D1 (p ¼ 0:05).

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In univariate and multivariate analysis lymph-node involvement (p ¼ 0:008) and C-erbB-2 overexpression (p ¼ 0:007) were independent negative prognostic factors for survival. Conclusions: Overexpression of C-erbB-2 oncoprotein is a valuable prognostic factor in patients with primarily resected BCA. Induvidualized adjuvant therapy-regimes (e.g. Trastuzumab) based on immunohistochemical determination of C-erbB2 expression may improve clinical outcome of patients with BCA.

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350 Aberrant expression rather than mutation of Desmoglein 2 in familial and sporadic gastric cancer

Aim: The aim of the study was to evaluate the DNApolymorphisms in the TS- and MTHFR-genes for an association with response and survival of locally advanced gastric cancer patients treated with a 5-FU based preoperative chemotherapy. Methods: DNA of 135 patients was isolated from blood or from formalin fixed nontumorous tissues. The polymorphic tandem repeats (double or triple repeats) in the promotor region of the TS-gene were analyzed by PCR and separation of the PCR products on an agarose gel. Genotyping of the C677T-polymorphism of the MTHFR-gene was performed by an allelic discrimination assay with the TaqMan system. Genotypes were analyzed for an association with chemotherapy response and patient‘s prognosis. Results: The TS-genotypes were not associated with response, but were statistically significant related to prognosis for all patients (p ¼ 0:002) as well as for the 102 completely resected patients (p ¼ 0:007). Patients with the 2rpt/3rpt-genotype showed a noticeable difference in respect to survival between responding and nonresponding patients (p ¼ 0:0007), in contrast to the patients with the 2rpt/2rpt- (p ¼ 0:11) and 3rpt/3rptgenotype (p ¼ 0:08). No association with response or prognosis was found for the MTHFR C677T-polymorphism. Conclusion: The prognosis of the patients was primarily dependent on the individual TS genotype and was only modulated by neoadjuvant chemotherapy. The TS gene seems to play a major role for tumor progression.

K. BIEDERMANN, H. VOGELSANG1, I. BECKER, S. PLASCHKE, J.R. SIEWERT1, H. HOEFLER, G. KELLER Institut fu¨r Pathologie, Technische Universita¨t Mu¨nchen 1 Chirurgische Klinik, Technische Universita¨t Mu¨nchen Aims: The aim of our study was to investigate the desmosomal cell adhesion molecule Desmoglein 2 (DSG2) for a potential role in gastric carcinogenesis. In particular we were interested, if it might be involved in genetic predisposition for familial diffuse type gastric cancer, similar to the cell adhesion molecule ECadherin. Methods: Seventy five formalin-fixed, paraffin-embedded familial (n ¼ 34) and sporadic (n ¼ 41) gastric cancer samples were analyzed immuno histochemically for DSG2 expression. DNA samples from 31 familial gastric cancer patients were analyzed for germline DSG2 mutations by DHPLC. Somatic mutation analysis was performed from 5 sporadic gastric carcinomas with an abnormal protein expression pattern. Results: An overall aberrant expression pattern was statistically significant more frequently found in nonintestinal than in intestinal type tumors (p ¼ 0:046), with the highest frequency in the familial, non-intestinal group (50%). Germline mutation analysis revealed one missense variant leading to a non-conservative amino acid change in exon 15 (DSG2 c.2810 C4A, T937N). None of the five sporadic gastric tumors showed somatic mutations in DSG2. Conclusions: Alterations in DSG2 expression suggest an implication of DSG2 in the carcinogenesis of a subset of gastric carcinomas, in particular of the non-intestinal type. No clear pathogenic germline or somatic mutations were found, suggesting that DSG2 is not a major gastric cancer predisposition gene and that altered protein expression is not due to somatic mutations. The significance of the identified missense variant in exon 15 has to be considered as unclear.

TS and MTHFR polymorphisms and association with response and survival of neoadjuvant treated gastric cancer patients K. OTT1, K. BECKER, H. VOGELSANG1, F. LORDICK1, M KOBL2, J.R. SIEWERT1, H. HO¨FLER, G. KELLER Institut fu¨r Pathologie, TU-Mu¨nchen 1 Chirurgische Klinik, TU-Mu¨nchen 2 Institut fu¨r Epidemiologie und Statistik, TU-Mu¨nchen

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The number of lymph node metastases in gastric cancer correlates with the angiotensin I-converting enzyme (ACE) gene insertion/deletion polymorphism C. RO¨CKEN1, U. LENDECKEL2, J. DIERKES3, S. WESTPHAL3, S. CARL-MCGRATH1, S. KRU¨GER1, A. ROESSNER1, M. EBERT4 1

Institute fu¨r Pathologie, Exp. Innere Medizin und 3 Klinische Chemie, 4 Klinik fu¨r Gastroenterologie, Universita¨t Magdeburg 2

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Aims: We aimed to substantiate the putative significance of ACE on gastric cancer biology, by investigating the influence of its gene polymorphism on gastric cancer progression. Methods: One-hundred-thirteen gastric cancer patients operated on between 1995 and 2002 were included. Genomic DNA was purified from peripheral blood mononuclear cells or tissue specimens. Amplified ACE gene fragments were separated on agarose gels. D or I alleles were identified by the presence of 190 bp or 490 bp fragments, respectively. Local expression of ACE was investigated by immunohistochemistry. Results: Twenty-four (21%) of the 113 gastric cancer patients had the II-, 57 (51%) the ID-, and 32 (28%) the DD-genotype. The distribution of the ACE genotypes did not differ significantly from the control group of 189 patients without gastric cancer. However, the ACE genotypes correlated with the number of lymph node metastases and the UICC tumor stage. Patients with the II genotype had a highly significantly smaller number of lymph node metastases (po0:001) and a significantly lower UICC tumor stage (p ¼ 0:01) than patients with the DD genotype. No correlation was found between tumor type, tumor location, local tumor growth, distant metastases, and the ACE genotype. ACE was detected immunohistochemically in endothelial cells (100/100) and tumor cells (56/100). Conclusions: Our study shows that ACE is expressed locally in gastric cancer and that the gene polymorphism influences metastatic behavior.

353 Genetics of small intestinal adenocarcinomas H. BLA¨KER, P. SCHIRMACHER Institut fu¨r Pathologie, Universita¨t Heidelberg Aims: To elucidate the molecular mechanisms underlying tumor development in the small intestine. Methods: Sequence analysis of APC, CTNNB1, k-ras/braf, SMAD4, microsatellite analysis, and immunohistochemistry for b-catenin and SMAD4 was performed on 21 small intestinal adenocarcinomas. Results: Mutations of either APC or CTNNB1 were only found in three cases. Approximately 60% of cases harboured k-ras or b-raf mutations, 30% showed SMAD4 mutations. Ten percent displayed microsatellite instability. Nuclear accumulation of b-catenin was observed in 20% of cases but was not linked to APC mutations. Loss of immunohistochemical SMAD4 staining correlated with homozygous gene deletions. In cases with missense mutations of SMAD4 in the cterminal domain staining was preserved.

Conclusions: Small intestinal adenocarcinomas frequently display activating mutations in the ras-rafpathway and inactivating mutations in the TGFbpathway. Activation of wnt/wingless contributes to the pathogenesis of a subset of small intestinal adenocarcinomas but is infrequently associated with mutations in APC or CTNNB1. Adenocarcinoma development in the small intestine follows a different genetic pathway than colorectal carcinoma. The pathway is most similar to ampullary adenocarcinoma. This is possibly explained by the exposure of both, the ampulla and the small intestine to bile acids which are quantitatively removed from the nutritional content before it enters the large intestine.

354

Diagnostic value of MSI screening in colorectal HNPCC adenomas

A. MU¨LLER1,2, C. BECKMANN1, T.H. BRODDEGER2, T.H. HENKEL2, J. RU¨SCHOFF2 1

Abteilung Allgemeinchirurgie, Universita¨tsklinikum Go¨ttingen 2 Institut fu¨r Pathologie und Biomedizinische Forschung, Klinikum Kassel Aim: The prevalence of microsatellite instability in adenomas of HNPCC patients has yet not been studied in detail. It is the aim of this study to demonstrate the frequency and thus the potential diagnostic value of the MSI analysis in HNPCC adenomas. Methods: For this purpose 15 syn- or metachronous adenomas from proven HNPCC-patients have been evaluated in a first step. The MSI status was correlated to the protein expression of MSH2, MLH1, MSH6 and PMS2 measured by IHC. Additionally, in a second cohort collected within a follow up study over three years the frequency of MSS adenomas in HNPCCpatients was determined. Results: In 5/15 cases the precise MSI-result (MSI-H) was only detectable after laser microdissection, allowing selective examination of adenomatous cells, but not after manual microdissection, which is the recommended standard. Three out of fifteen adenomas revealed a MSS-status and a regular expression of the mismatch-repair proteins, so that these adenomas had to be classified as sporadic. In the follow up study we found that adenomas (n ¼ 40), which developed in close vicinity to carcinoma or as so-called carcinoma in adenoma, revealed a MSI-pathway, either showing a MSI-H status or/and loss of protein expression corresponding to the HNPCC-carcinoma. However, 6 out of 31 adenomas, which developed in a distance of more than 5 cm from the primary tumor syn- or

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metachronously, turned out to be MSS following another than the MIN/MSI pathway. Conclusions: To our knowledge this is the first study which shows, that intralesion heterogeneity in adenomas of HNPCC may lead to false negative MSI testing and more importantly that sporadic adenomas occur next to HNPCC-associated lesions.

355

p16ink4a promoter methylation and 9p21 LOH in colorectal carcinomas: Relation with expression and with tumor budding F. PRALL1, C. OSTWALD1, V. WEIRICH2, H. NIZZE1

1

Institut fu¨r Pathologie, Universita¨t Rostock Institut fu¨r Rechtsmedizin, Universita¨t Rostock

2

Aims: In colorectal carcinomas p16INK4a inactivation is known to occur by allelic loss and by promoter methylation, but mutations are rare. p16INK4a is upregulated in tumor buds, and the consequent shut-down of proliferation may be a prerequisite for tumor budding. Methods: p16INK4a promoter methylation and 9p21 allelic loss were investigated in a series of 57 colorectal carcinomas. This was related to p16INK4a expression as determined by a quantitative evaluation of immunostains, and to tumor budding as quantified on pancytokeratin immunostains. CpG island methylator phenotype (CIMP) and microsatellite status were determined in addition. Results: p16INK4a promoter methylation was seen in 17/ 57 cases (14 of these CIMP-positive), and a total loss of expression was seen in six of these. Quantification of immunohistochemical p16INK4a expression for the remaining 11 cases revealed statistically lower frequencies of expression as compared to cases without p16INK4a promoter methylation. 9p21 allelic loss was observed in nine cases but p16INK4a expression in these carcinomas was not reduced. Attempted linear regression of p16INK4a expression in tumor buds on the degree of tumor budding did not show an association of statistical significance. Conclusions: p16INK4a promoter methylation can completely abrogate p16INK4a expression in colorectal carcinomas. In many cases, however, it has an appreciable but only modulatory influence on p16INK4a expression. Possibly, methylations are heterozygous and/or quite mosaic in colorectal carcinomas and/or methylations are not totally stable but can be lost between carcinoma cell replication cycles. Up-regulation of p16INK4a does not seem to be a strict requirement for tumor budding, hence the absence of a correlation.

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Expression and prognostic value of DNA repair proteins ATM, BRCA1, BRCA2, Ku70 and Ku80 in colorectal cancer – an immunohistochemical study on 342 tumours W. MU¨LLER, M. DATTANI1, L. BARKER2, N. MAUGHAN2, H.E. GABBERT3, H. GRABSCH1

Gemeinschaftspraxis Pathologie Starnberg 1 Acad. Unit of Pathol., Univ. of Leeds, UK 2 Dept. of Histopathol., Leeds Teaching Hosp. NHS Trust, UK 3 Institut fu¨r Pathologie, Universita¨t Du¨sseldorf Background: Failure of DNA double strand break [DSB] repair may result in chromosomal instability, a common phenomenon in many cancer. DNA DSB repair is achieved by two pathways: non homo-logous end joining and homologous recombination. The expression of DNA DSB repair proteins involved in both pathways has not been investigated in detail in colorectal cancer [CRC]. Methods: Expression of DNA repair proteins (ATM, BRCA1, BRCA2, Ku70 and Ku80) was investigated by immunohistochemistry in 342 CRC and compared with clinicopathological data and patient survival. Results: Expression of ATM, BRCA1 and BRCA2 was observed in 96%, 88% and 96% of CRC, ranging from 1% to 95% positively stained tumour cells (median: 11%, 58% and 38%). In contrast, all tumours showed expression of Ku70 and Ku80 in nearly all tumour cells. Apart from a lower BRCA1 expression in poorly differentiated tumours, no correlation was found between expression of DNA re-pair proteins and clinicopathological data. Low expression of ATM or BRCA1 prove to be a predictor of poor survival independent from established parameters of the pTNM system (p ¼ 0:023; p ¼ 0:029). Conclusions: Reduced protein expression of ATM, BRCA1, and BRCA2 is an early and frequent event in CRC suggesting an impaired capability of DNA DSB repair by homologous recombination whereas the alternative DSB repair pathway seems to be unaffected. Furthermore, reduced protein expression of ATM and BRCA1 identifies a subgroup of CRC patients with poor prognosis which may benefit from a more tailored chemotherapeutic approach.

357 High prevalence of mycobacterium avium subspecies paratuberculosis IS900 DNA in Crohn’ disease tissues F. AUTSCHBACH, S. EISOLD1, U. HINZ2, S. ZINSER2, M. LINNE-BACHER3, T. GIESE4, T. LO¨FFLER2, M.W. BU¨CHLER2, J. SCHMIDT2

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Pathologisches Institut, Universita¨t Heidelberg 1 Chirurgische Universita¨tsklinik, Universita¨t Rostock 2 Chirurgische Universita¨tsklinik, Universita¨t Heidelberg 3 Abteilung fu¨r Molekulare Pathologie, Universita¨t Heidelberg 4 Institut fu¨r Immunologie, Universita¨t Heidelberg Aims: In view of conflicting results in the literature, we analysed the presence of mycobacterium avium subspecies paratuberculosis (MAP) specific IS900 DNA in a large number of gut samples from individuals with Crohn’ disease (CD), ulcerative colitis (UC) and noninflamed control tissues (no inflammatory bowel disease; nIBD). Methods: The prevalence of MAP DNA in surgically resected tissue specimens (n ¼ 300) was examined using a mechanical-enzymatic disruption technique and nested IS900-specific PCR. CD patients were stratified according to criteria of the Vienna classification and further characteristics to investigate a possible association between MAP specific detection rates and clinical phenotype. Results: The detection rate of IS900 DNA was significantly higher in CD tissues (52%) as compared with UC (2%) or nIBD specimens (5%). In CD, IS900 DNA was detected in samples from diseased small bowel (47%) as well as colon (61%). No firm association between MAP-specific DNA detection rates and clinical phenotypic characteristics could be established. Corticosteroid medication was a factor which negatively influenced IS900 DNA detection rates in CD (po0:01). Conclusions: The presence of MAP-specific IS900 DNA is a predominant feature of CD. Therapeutic intervention against MAP might represent a target for disease mitigation in CD.

358

C4d deposits and increased portal infiltration by macrophages in acute liver allograft rejection

A. DANKOF1, M. SCHMEDING2, L. MORAWIETZ, R. GU¨NTHER1, M. KRUKEMEYER1, B. RUDOLPH1, M. KOCH1, U. NEUMANN2, V. KRENN1 1

Institut fu¨r Pathologie der Charite´, Humboldt-Universita¨t, Berlin 2 Klinik fu¨r Allgemein-, Visceral- und Transplantationschirurgie der Charite´, Campus Virchow Klinikum, Humboldt-Universita¨t, Berlin Aims: Since only very few data exist concerning the role of humoral immunity in human acute liver allograft rejection (ACR) the aim of this study was to clarify if

ACR is associated with depositions of complement split products and an increased infiltration of B-lymphocytes, plasma cells, and macrophages. Methods: Forty five liver biopsy specimens (ACR n ¼ 22; HCV reinfection n ¼ 10; controls n ¼ 13) were analyzed by immunohistochemical and immunofluorescence single and double staining. The average number of CD20+, CD38+ and CD68+ cells per portal tract was established while the presence of C4d and C3d deposits was evaluated semiquantitatively. Results: Comparing cases of ACR and protocol biopsies from patients without histopathological signs of acute rejection (controls), a significantly increased accumulation of CD20+ (p ¼ 0:029) and CD38+ (p ¼ 0:014) cells could be demonstrated in ACR cases . Additionally 50 % of ACR biopsies revealed C4d deposits along portal capillaries, which was associated with a significantly increased portal infiltration by macrophages (p ¼ 0:007). Conclusions: The results support an involvement of a humoral immunresponse in a part of ACR cases.

359 Expression of cytokeratins (CK7, CK20) and mucins (MUC1, MUC2, MUC5AC) in adenocarcinomas of the pancreato-biliary-ampullary region U. DREBBER, M. WIRTZ, H.U. KASPER, S.P. MO¨NIG1, P.M. SCHNEIDER1, A.H. HO¨LSCHER1, H.P. DIENES, S.E. BALDUS Institut fu¨r Pathologie, Universita¨t Ko¨ln 1 Klinik fu¨r Viszeral- und Gefa¨ßchirurgie, Universita¨t Ko¨ln Aims: Adenocarcinomas of the duodenal mucosa, ampulla of Vater, pancreas, and terminal third of the common bile duct are originally defined on topographic grounds. Since diffculties may occur in advanced cases or biopsy diagnostics, we evaluated, if immunohistochemical markers correlate with tumors’ histogenesis. Methods: The study included a series of 63 adenocarcinomas originating from pancreas (n ¼ 39), ampulla of Vater (n ¼ 16), ductus choledochus (n ¼ 6) and duodenum (n ¼ 2). CK7, CK20, MUC1, MUC2 and MUC5AC were visualized immunohistochemically applying the Dako Envision system. Results were scored semiquantitatively (0, 0-5%, 1, 45-35%, 2, 435-65%, 3, 465%) and correlations between antigen expression and tumor localization were analyzed statistically performing x2 tests. Results: While CK7 was expressed in 62 of 63 cases, CK20 was not present in about 45% of the tumors. The level of CK7 (score 3) was significantly higher in pancreatic (90%) compared to ampullary carcinomas

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(56%). CK20 reactivity did not correlate with tumor localization. A varying degree of MUC1 staining was observed in all cases, however, pancreatic carcinomas exhibited score 3 staining in about 87%. MUC2 was non-reactive in 56% of the pancreatic carcinomas, but fewer ampullary cancers (19%). MUC5AC was expressed in 87% of the specimens, exhibiting a significantly stronger positivity in pancreatic carcinomas (74% score 3). Conclusions: Pancreatic carcinomas exhibit higher levels of CK7, MUC1 and MUC5AC compared to tumors of the ampulla, while the latter are characterized by a stronger MUC2 expression. Combined, these markers may help to elucidate the histogenesis of adenocarcinomas of the pancreato-biliary-ampullary region.

360 Heterozygous mutations of tumor suppressor genes

TP53 and p16INK4 can be found in early preneoplastic lesions in chronic pancreatitis suggesting a clonal expansion during progression

E. HEINMO¨LLER1, M. WERTHER1, M. BAUMGART3, M. ZIEMER1 K. HOMAYOUNFAR2, J.FAß2, B.M. GHADIMI3, J. RU¨SCHOFF1 1

Institut fu¨r Pathologie, Klinikum Kassel Klinik fu¨r Allgemein-chirurgie Klinikum Kassel 3 Universita¨t Go¨ttingen 2

Aims: Chronic pancreatitis (CP) is a predisposing disease for pancreatic cancer (PC), however, precise molecular mechanisms of cancer development in the background of CP are ill defined. We have applied laser microdissection to molecular genetic analysis of TP53, p16INK4 and DPC4 genes of pancreatic intraductal lesions (PanIN) in tissues of CP. Methods: Seventy six laser microdissected PanINs (PanIN1A-PanIN3) and normal ducts from formalinfixed paraffin-embedded tissues of a patient suffering from CP were pretreated by whole genome amplification (I-PEP-PCR) followed by microsatellite PCR for LOH analysis. PanINs were analysed for mutations in TP53 and p16INK4 by ABI-sequencing. In addition, immunohistochemical expression analysis of p53, p16INK4 and DPC4 protein was performed. Results: In multiple PanIN-3 lesions, p53 protein overexpression and loss of protein expression for p16INK4 and DPC4 protein was seen. Here, LOH was found at the TP53, p16INK4 and DPC4-locus, sequence analysis showed a mutation of C4T exon 8 bp 14508 of TP53 gene and a splice site mutation G4A bp-12 intron 1 of p16INK4 gene. A heterozygous mutational status

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without LOH was found in some early PanINs (PanIN1A) suggesting the presence of base mutations early in carcinogenesis with subsequent clonal expansion and final gene inactivation by LOH. Conclusions: Heterozygous mutations of tumor suppressor genes TP53 and p16INK4 occur early in CP and are clonally expanded, but final inactivation by LOH occurs later in pancreatic carcinogenesis.

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Alterations of the 3D-architecture of chromosome territories #8 in pancreatic adenocarcinoma S. TIMME, S. STEIN1, T. WIECH, M. SCHURIKE1, M. HAUSMANN, C. CREMER1, M. WERNER, A. WALCH Pathologisches Institut, Universita¨tsklinikum Freiburg 1 Kirchhoff-Institut fu¨r Physik, Universita¨t Heidelberg

Aims: The 3D-architecture of chromosome territories (CTs) has been analyzed especially in cell culture experiments but not in cell nuclei within their natural histological context. We performed 3D-image analysis of the architecture of CT #8 in matched pairs of pancreatic cancer and normal ductal epithelium from histological sections of archival tissues. Methods: A whole chromosome painting probe (CT #8) was hybridized to cell nuclei in a tissue microarray obtained from formalin-fixed, paraffin-embedded blocks of matched pairs (n ¼ 6) from pancreatic cancer and non-neoplastic ductal epithelium. 3D-image data sets were acquired. The cell nuclei were interactively segmented and analyzed using a homemade program package for 3D-image analysis. Several image parameters were compared in neoplastic and non-neoplastic cell nuclei. Results: Cell nuclei of non-neoplastic ductal epithelium revealed a specific distribution of all evaluated parameters of CT#8, without significant differences between the patient samples. In cell nuclei of pancreatic cancer significant changes compared to normal epithelium were found for the roundness (po0:001) and the surface (po0:001) for CT#8, while the radial positioning and the volume revealed no significant differences (p40:05). Conclusions: These results indicate significant changes of the architecture of chromosome territories during malignant transformation of pancreatic cancer which might be correlated to functional changes. The quantitative 3D-approach shown here allows the characterization of higher-order genome architecture and organization as a novel tool in molecular pathology.

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Study Group: Gynecologial Pathology 362

Distinct spatial expression patterns of AP-2a and AP-2c in non-neoplastic human breast and breast cancer

N. FRIEDRICHS, R. JAEGER1, E. PAGGEN, C. RUDLOWSKI2, S. MERKELBACH-BRUSE, H. SCHORLE1, R. BUETTNER Institut fu¨r Pathologie, Universita¨t Bonn 1 Institut fu¨r Entwicklungspathologie, Universita¨t Bonn 2 Abteilung fu¨r Gyna¨kologie, Universita¨t Heidelberg Aims: To better understand the role of AP-2 proteins in mammary neoplasia the analysis of their spatial expression pattern in normal breast and breast cancer is required. Methods: Fifty one specimens of female breast cancer patients and a tissue micro array containing 93 additional female breast cancer cases were stained for AP-2a; AP-2g; estrogen receptor and ErbB-2. In 70 cases survival data was present. Results: In non-neoplastic breast tissue AP-2a was expressed in the glandular cell layer while AP-2g was expressed in the myoepithelial cell layer. Ductal carcinoma in situ revealed strongly AP-2a-positive tumor cells surrounded by AP-2g-positive myoepithelial cells. In invasive carcinoma AP-2a and AP-2g expression was variable. High expression of estrogen receptor and AP-2a showed better survival rates than low expression of these markers. Conclusions: These results for the first time reveal a distinct spatial expression pattern of AP-2a and AP-2g in normal breast and in ductal carcinoma in situ with specific AP-2g expression in myoepithelium. High estrogen receptor and AP-2a expression in invasive breast cancer showed favorable survival rates. AP-2a expression seems to be associated with better prognosis of breast cancer. AP-2g expression has no influence on survival reflecting that myoepithelial cells are not involved in the neoplastic process.

metabolize xenobiotic substances and take part in steroid hormone metabolism. We investigated the expression levels of two groups of metabolizing enzymes, Glutathione S-transferases (GST) and Cytochrome peroxidases (Cyp), in human breast cancers in relation to biological and clinical tumor characteristics. Methods: Paraffine tissue of 356 invasive breast cancers from the University of Bonn (as participant in the Interdisciplinary Study Group on Gene Environment Interaction and Breast Cancer in Germany, GENICA) was analyzed by immunohistochemistry for expression of GSTp, GSTm, GSTa, Cyp1A1, Cyp1A2, Cyp3A4, Cyp1B1, Cyp2E1. Staining results were correlated with T- and N-stages, age of the patients, hormonal receptor status, Her2 and p53, as well as histological tumor typing and grading. Results: Expression of GSTm was found in 48% of the tumors followed by GSTp, Cyp2E1, 3A4 and 1B1 (12–29%). Less than 10% expressed GSTa, Cyp1A1 and 1A2. Expression of Cyp 1B1 and 3A4 was related to poor tumor differentiation (p ¼ 0:07 and 0.04) and lymphatic metastatic spread (p ¼ 0:07). No correlation was found between any of the markers and advanced tumor stages or histological tumor typing. Conclusions: Expression of several metabolizing enzymes in breast cancer cells is maintained even in advanced tumor stages. Enzymes involved in estrogen metabolism (Cyp1B1, 3A4) are possibly associated with poor tumor differentiation. Further follow up data in the course of the GENICA-study will show whether certain marker constellations are related to tumor growth.

364 Epidermal growth factor receptor (EGFR) expression and egfr gene dosage measurement in phyllodes tumour of the breast C. KERSTING1, J. PACKEISEN1, C. LIEDTKE1, B. BRANDT2, P. WU¨LFING3, C.GUSTMANN4, B.HINRICHS4, W. BOECKER1, H. BUERGER1 1

Gerhard-Domagk-Institut fu¨r Pathologie Institut fu¨r Klinische Chemie 3 Klinik fu¨r Frauenheilkunde, WWU Mu¨nster 4 Institut fu¨r Pathologie, Institute fu¨r Pathologie, Ko¨ln-Rodenkirchen und Limburg 2

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Expression of xenobiotic and steroid hormone metabolizing enzymes in human breast cancers S. HAAS1, Y. KO2, H. BRAUCH3, H. P. FISCHER1 1

Institut fu¨r Pathologie, Universita¨t Bonn 2 Johanniter Krankenhaus, Bonn 3 Institut fu¨r Klinische Pharmakologie, Stuttgart

Aims: Biological tumor behaviour of breast cancer is influenced by the indivual potential of tumor cells to

Aims: Phyllodes tumours of the breast are rare, biphasic tumours with a poorly understood pathogenesis. Since egfr mutations belong to the first mutations in breast carcinogenesis, we examined Epidermal growth factor receptor protein expression and amplifications of egfr gene in phyllodes tumours of the female breast.

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Methods: EGFR expression was assessed in 58 phyllodes tumours by means of immunohistochemistry (Novocastra) and tissue micro arrays. egfr amplifications were determined by fluorescence in situ hybridization and quantitative gene dosage PCR for a polymorphic CA repeat sequence in intron 1 of egfr. All data was correlated with pathomorphological dignity. Results: Overexpression of EGFR in stromal cells was detected in 19% of all cases. Only two cases featured EGFR overexpression of epithelial elements. Whole gene amplifications were seen in 15.8% and amplifications of the intron 1 CA repeat in 41.8%. A significant correlation of tumour dignity with EGFR expression and amplifications of the CA repeat was evident, but not with whole gene amplifications. All cases with whole gene amplifications featured EGFR overexpression, whereas only 39% of tumours with CA repeat amplifications did so. Conclusions: EGFR overexpression and amplifications are common findings in phyllodes tumours of the breast and are associated with tumour malignancy. Furthermore, phyllodes tumours turn out to be a helpful model for the investigation of EGFR expression regulation.

365 Selection of house keeping genes for gene expression analysis in human breast cancer by real-time quantitative RT-PCR A. LEBEAU, H. HESSEL, B. MAYER1, U. LO¨HRS Pathologisches Institut der LMU Mu¨nchen 1 Chirurgische Klinik und Poliklinik, Großhadern, LMU Mu¨nchen Aims: Careful normalization is essential when using quantitative RT-PCR to compare mRNA levels between biopsies of different individuals. Generally this involves the use of internal controls, so-called housekeeping genes. However, our former studies showed considerable variation in the expression of distinct housekeeping genes in human breast cancer tissue hindering accurate normalization of the MMP expression levels. Therefore we compared a set of seven housekeeping genes in five breast cancer cell lines and five breast cancer specimens of different individuals to determine which of them can provide the most accurate and biologically relevant results. Methods: Relative expression analysis was performed by using the LightCyclers system (Roche Diagnostics) on five cell lines (T-47D, SK-BR 3, BT-20, Hs 578 T, BT549) and on paraffin sections of five human breast carcinomas. The following housekeeping genes were tested for normalisation of MMP-2 and -3 expression

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results: GAPDH, Cyclophilin B, h-ß2 M, 18S rRNA, PBGD, h-ALAS, UBC. Results: The expression of GAPDH, h-ß2 M, 18S rRNA and PBGD varied markedly between the different breast cancer cell lines and tissue probes, whereas the expression of Cyclophilin B, h-ALAS and UBC was relatively constant in the cell lines as well as the paraffin embedded breast cancer tissue. Conclusions: Our results suggest the use of Cyclophilin B, h-ALAS or UBC alone or in combination as internal control genes for accurate normalization of RT-PCR expression analysis in human breast cancer. Additionally, our data indicate that the reliability of internal standards can be tested in a panel of cell lines that reflects the variability of human tissue. Supported by a grant from the Curt-Bohnewand-Fond (LMU Mu¨nchen).

366 Correlation of intermediate filament expression and in vitro chemosensitivity of primary invasive breast cancer J. PACKEISEN, U. VOGT2, A.V.D. ASSEN3, R.H. KRECH, W. BO¨CKER1, H. BU¨RGER1 Institut fu¨r Pathologie, Klinikum Osnabru¨ck 1 Institut fu¨r Pathologie, Universita¨t Mu¨nster 2 Europa¨ische Laboratorien Assoziation, Ibbenbu¨ren 3 Senologie, Klinikum St. Georg, Georgsmarienhu¨tte Aims: Ex vivo testing of chemosensitivity and its clinical relevance is disputably discussed. It has been considered a relation of cancer chemosensitivity and intermediate filament expression in human tumors such as the association of cytokeratins with markers of prog-nosis and differentiation. The present study explores the correlation of different intermediate filaments to the ex vivo chemosensitivity of the tumor cells in four drug regimes: combination of epirubicin and cyclophosphaminde (EC), mitoxantrone (Mit), combination of paclitaxel and epirubicin (ET), and docetaxel and epirubicin (Edoc). Methods: A group of 725 primary invasiv breast cancer (BC) was investigated. Amoung them 417 patients were node-negativ (N-) and 317 node-positiv (n+). Intermediate filament expression, such as vimentin, CK5, CK14, CK10, CK8/18 and CK19, was characterised by immunohistochemistry on tissue microarrays. The cancer cell cuture were treated with EC, Mit, ET and EDoc, each in six different drug concentrations in modified ATP-CVA assay. AUC values were calculated using trapezoid rules. Chemosensitivity was defined as AUC418000. Results: No significant correlation was found between all studied parameters and chemosensetivity to all drug

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combinations for all BC, independently to nodal status of the patient. Conclusions: Correlation of cytokeratin expression pattern admitted to differentiation in human breast cancer to ex vivo chemosensitivity give no evident hint for its practicability for use in these context.

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Expression of cyclooxygenase-2 (COX-2) in hereditary breast cancer is not correlated with the Her2/neu expression N. BEKTAS1, B. WAPPENSCHMIDT2, K. RHIEM2, R. SCHMUTZLER2, C. WICKENHAUSER1 1

Institut fu¨r Pathologie, Universita¨t zu Ko¨ln Frauenklinik, Universita¨t zu Ko¨ln

2

Aims: We analyzed the expression of COX-2 in relation to the expression of Her2/neu, oestrogen and progestoren receptor as well as the Ki67 proliferation rate in hereditary breast cancer by immunohistochemistry (IHC). Methods: IHC was applied to paraffin embedded specimens of hereditary breast cancer (n ¼ 23) comprising 7 BRCA1, 6 BRCA2 mutations, 2 BRCA1 and 7 BRCA2 unclassified variants (UV’s). A total of 20 sporadic breast carcinomas with a differentiation grade of G2-3 was used as comparative group. In case of Her2/ neu positivity (4 score 1) gene amplification was analyzed performing FISH analysis. Results: In the hereditary breast cancer group COX-2 was expressed in all specimens with variation in quantity and intensity. According to the Immune Reactive Score (IRS) 15 cases were classified as weak, 7 as moderate and 1 as strong. Only one case bearing a BRCA2 mutation was positive for Her2/neu (score 3+) while all patients with BRCA1 mutation were not amplified for this gene. No correlation between COX-2 expression and expression of the oestrogen and progesteron receptor or the Ki67 proliferation rate could be observed in this collective. In the group of the sporadic breast carcinomas strong COX-2 expression correlated with Her2/neu gene amplification. Conclusions: Our data indicate that there is no link between COX-2 and Her2/neu overexpression in hereditary breast cancer as it is described for sporadic breast tumors. The specific pathway leading to COX-2 overexpression in these tumors should therefore be closer analyzed in further studies. COX-2 expression in all cases might point at a potential therapeutic effect of COX inhibitors in therapy of hereditary breast cancer.

368 Stromal fibroblasts in breast tumors support blood monocyte migration

L.A. KUNZ-SCHUGHART, T. SILZLE, M. KADLUBEK, M. KREUTZ1, F HOFSTA¨DTER Institut fu¨r Pathologie 1 Abteilung fu¨r Ha¨matologie und Internistische Onkologie, Universita¨t Regensburg Aims: Tumor-associated macrophages (TAM) represent a major component of the cellular infiltrate in breast tumors. They derive from circulating blood monocytes (MO) by migration and differentiation processes and are characterized by lack of functional activity. Accordingly, the macrophage index in breast carcinomas correlates with poor prognosis. In desmoplastic breast tumors, TAM are predominantly located in the tumorassociated fibroblastic stroma. Thus, the objective of the present study was to gain insight into the mechanism of stromal fibroblasts as sentinel cells modifying blood MO migration. Methods: Cell types: normal and breast tumorderived fibroblasts, MO isolated by counter-current elutriation; culture systems and assays: monolayer cultures, spheroid co-cultures, Boyden chamber; analytical tools: flow cytometry, ELISA, immunohistochemistry. Results: In contrast to normal fibroblasts, fibroblasts from ductal breast carcinomas were excessively infiltrated by a 10–15% blood MO subpopulation. These tumor-derived fibroblasts showed a significantly higher expression of IL-6 which enhanced the release of monocyte chemotactic protein 1 (MCP-1) via an autocrine regulatory pathways. Blockade of MCP-1 in fibroblast–conditioned media reduced MO migration to the basal level in the Boyden-chamber assay but not in a fibroblast-MO spheroid co-culture system. Conclusion: Stromal fibroblasts critically modulate MO migration in tumors. Studies to elucidate the mechanism of action are ongoing. (study supported by the DFG and the State of Bavaria).

369 Be careful with the interpretation of univariate survival curves and of so-called ‘‘independent prognostic factors’’ after application of multivariate Cox models! S. BIESTERFELD Institut fu¨r Pathologie, Universita¨t Mainz Aims: In the past in our group several prognostic studies on breast cancer (and several further types of cancer) have been performed. The present paper deals with some problems that have to be considered for correct and careful interpretation of the results of univariate and multivariate survival analysis.

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Methods and results: After collecting the survival data and the data of variables under investigation, usually a univariate survival analysis is performed as a first step (Kaplan-Meier survival estimation method). Further, to evaluate statistical differences between the resulting actuarial survival curves, the Wilcoxon-Breslow test or the Mantel-Cox test have to be applied. In a second step, frequently a multivariate Cox regression analysis is used in order to evaluate interrelationships between the variables and to identify those ones that may be looked upon as ‘‘independent prognostic factors’’. Unfortunately, frequently the results of survival analysis, especially of Cox regression model, are interpreted as if they really represented a final biological solution and explanation of the clinical problem. In our opinion, however, not only univariate survival curves, but also results from multivariate Cox models should be interpreted very carefully. In several studies we were able to learn step by step about pitfalls in prognostic data interpretation which we would like to present using some comprehensive examples. Conclusion: Univariate and multivariate survival analysis are very important methods in the evaluation of prognostic studies. An exact and correct interpretation of the results of survival analysis, however, needs further careful and critical evaluation, for example by applying curve function models on univariate survival curves or by validation of Cox regression models.

370 Influence of tissue fixation on biomarker expression in breast cancer W. HAEDICKE, M. HOFMANN, A. KAUPISCH, J. RU¨SCHOFF Institut fu¨r Pathologie, Klinikum Kassel Aims: Accuracy has gained central importance in immunohisto-chemistry (IHC) and tissue based molecular biological assays to characterize the expression of biomarkers in tumors, since therapeutic decisions are based on their results. Tissue fixation is one of the major sorces of variation. Therefore, we investigated the influence of tissue fixation on the outcome of IHC, Real Time PCR, FISH and CISH. Methods: We investigated the influence of buffered and non buffered formalin (4% and 10%), Bouin’s fixative, AFA, Prefer, Z-5, PenFix on the expression of the biomarkers Her2/neu (FISH, CISH, Real Time PCR, and immunohistochemistry) Estrogen Receptor, Progesteron Receptor,bcl-2, p53 and MIB1. We used 7 cases of breast carcinoma that were dissected, fixed simultanously with 30 different fixation protocols and compiled on TMAs.

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Results: All Formalin fixation protocols were equally suitable for Her2/Neu CISH, FISH, PCR and IHC. Also the other biomarkers tested showed no significant variations in the different formalin fixation protocols. Boin’s fixative was unsuitable for CISH, FISH and PCR. Alcohol based fixatives increased the sensitivity of Her2/Neu IHC. ER and PR were sinificantly less reactive in tissues fixed afer a delay of 12 hours. MIB1 reactivity was significantly decreased in tissues fixed with alcohol containing fixatives. Conclusions: The antibodies and assays tested showed individual pattern of reactivity on tissues fixed with different proticols, so that pathology labs that perform immunohistocemistry and tiisue based molecular biological assays must control fixation conditions, especially if they perform the assays on tissues from different origins e.g. in multi center studies.

371 The BMPR/SMAD-pathway is involved in dedifferentiation and progression of estrogene receptor positive breast cancer H. BU¨RGER, M. HELMS1, J. PACKEISEN2, C. AUGUST, B. BRANDT1, W. BO¨CKER Institut fu¨r Pathologie 1 Institut fu¨r Klinische Chemie, Universita¨t Mu¨nster 2 Institut fu¨r Pathologie, Klinikum Osnabru¨ck Aims: Estrogene receptor (ER) expression generally is a sign of higher tumour differentiation in invasive breast cancer. However, ER-positive, poorly differentiated carcinomas with a poor clinical outcome exist. In a previous study we could show that these carcinomas are characterized by a specific gene expression pattern. Methods: We are now able to show by means of immunohistochemistry on tissue arrays, CGH analysis and Western Blotting, that the expression of the bone morphogenetic protein receptor IB (BMPR-IB) is the major hallmark in the progression and dedifferentiation of ER- positive breast cancers. Results: Strong expression of BMPR-IB was associated with chromosomal 7p-gains, tumour grade and a poor prognosis. Western Blot analysis revealed that the activation of this receptor is mainly mediated via phosphorylation of SMAD1. An impact of BMPR-IB expression on tumour proliferation (Mib-1) and cytogenetic instability could only be demonstrated in ER positive carcinomas. This pro-proliferative effect was further complemented by a significant anti-apoptotic activity, indicated by XIAP and IAP-2 expression in BMPR-positive carcinomas. Conclusions: Our results show for the first time that the BMPR/SMAD-pathway is activated in breast cancer

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and contributes to breast cancer progression and dedifferentiation in ER positive breast cancer. The definition of this pathway characterizes a new potential target in the molecular-based treatment of invasive breast cancer.

372 Expression of AKT in ovarian cancer A. NOSKE, W. WEICHERT, S. NIESPOREK, I. KOCH, J. SEHOULI1, M. DIETEL, S. HAUPTMANN2, C. DENKERT Institut fu¨r Pathologie, Universita¨t Berlin 1 Institut fu¨r Gyna¨kologie und Geburtshilfe, Universita¨t Berlin 2 Institut fu¨r Pathologie, Universita¨t Halle Aims: The protein kinase family AKT, a major key element of several tumor signaling pathways, is known to be involved in progression of ovarian carcinomas. We aimed to analyze the expression of total and activated AKT as well as the three isoforms in ovarian cancer cell lines and primary ovarian carcinomas. Methods: mRNA level of the AKT isoforms were evaluated by RT-PCR, protein level of total AKT and activated AKT were examined by western blot. To determine the effects on cell proliferation, we inhibited AKT isoforms by RNA interference or treated cancer cells with the PI3K-inhibitor LY29004. Immunohistochemical investigation of total AKT was performed on a set of 45 primary ovarian carcinomas and expression data were compared to clinico-pathological features. Results: We found an over-expression of AKT1 and AKT2 mRNA in ovarian cancer cell lines but only low mRNA level of AKT3. Total AKT was expressed in all ovarian cancer cell lines, whereas expression of phosphorylated AKT was only found in two cancer cell lines. AKT2, but not AKT1 siRNA inhibits cell growth. Inhibition with LY29004 resulted in reduction of cancer cell growth. Increased AKT expression was observed in ovarian carcinomas as compared to surface epithelium of normal ovaries and significantly associated with positive lymph node status (p ¼ 0:002). Conclusions: We found an over-expression of AKT in ovarian carcinomas. Our data indicate that mainly the isoform AKT2 regulates proliferation of ovarian cancer cells suggesting a role for AKT2 in progression of ovarian cancer.

373 Ezrin indicates poor prognosis in ovarian carcinomas M. KO¨BEL, E. GRADHAND, S. HAUPTMANN

Institut fu¨r Pathologie, Universita¨t Halle Aims: Ovarian carcinoma was accountable for more than 2,000 cancer death in Germany in 2002. The high mortality rate is ascribed to late symptoms and diagnosis in FIGO stage III or higher. A crucial point of progression the interaction of tumor cells by its surface CD44 with hyaluronic acid on mesothelium. Since Ezrin, Moesin, and Radixin proteins are in complex with the submembranal tail of CD44, we asked for the role of these proteins in ovarian carcinoma progression. Methods: Paraffin-embedded tissue samples from 160 patients with epithelial ovarian tumors, which were diagnosed at the Institute of Pathology, Martin-LutherUniversity Halle-Wittenberg between 1997 and 2003 were included in this study. Expression of Ezrin and Moesin was investigated by immunohistochemistry and correlated with several clinicopathological parameters. Furthermore, expression of both proteins was inhibited by siRNA in SKOV-3 cells and invasive capacity was studied in the Boyden Chamber Assay. Results: Ezrin and Moesin were overexpressed in a subset of ovarian carcinomas. Only overexpression of Ezrin showed a correlation with overall survival (po0:001), but not with another known risk factor including FIGO stage, residual tumor and histology. Ezrin remained an independent risk factor in multivariate analysis (RR 3.17, CI 1.40 – 7.15, p ¼ 0:005). In vitro, siRNA inhibition of Ezrin and a combined blocking of Ezrin and Moesin reduced significantly Matrigel invasion of SKOV-3 cells. Conclusions: Ezrin is involved in ovarian carcinoma progression and predict prognosis as an independent prognostic factor.

374 Expression of lysophosphatidic acid acyltransferase b (LPAAT-b) in ovarian carcinoma Correlation with tumor grading and prognosis S. NIESPOREK1, C. DENKERT1, W. WEICHERT1, M. KO¨BEL2, A. NOSKE1, J. SEHOULI3, J.W. SINGER4, M. DIETEL1, S. HAUPTMANN2 1

Institut fu¨r Pathologie Charite´ CCM, Universita¨t Berlin 2 Institut fu¨r Pathologie, Universita¨t Halle/Saale 3 Frauenklinik Charite´ CVK, Universita¨t Berlin 4 Cell Therapeutics Inc., Seattle, USA Aims: LPAAT-b is an enzyme involved in lipid biosynthesis whose role in tumor progression has been of emerging interest in the last few years. It may be an interesting new target for chemotherapeutical intervention in patients with malignant tumors.

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Our aim was to evaluate the prognostic role of LPAATb expression and its association with clinicopathological factors. Methods: We investigated the expression of LPAAT-b by RT-PCR and immunohistochemistry in 10 ovarian cell lines as well as in a cohort of 106 ovarian tumors and normal ovaries. Results: LPAAT-b mRNA was found in all cell lines and ovarian tumors examined. Expression of LPAAT-b protein was significantly increased in ovarian carcinomas compared to benign ovarian tissue (p ¼ 0:001). Furthermore, LPAAT-b expression was positively associated with higher tumor grade (p ¼ 0:044), higher mitotic index (po0:0001) and tumor stage (p ¼ 0:032). Expression of LPAAT-b was significantly linked to reduced overall survival time (p ¼ 0:024) as well as to shorter progression-free survival time (p ¼ 0:012) in the subgroup of patients younger than 60 years. Conclusions: Our study shows that LPAAT-b is upregulated in ovarian cancer and is more prevalent in poorly differentiated tumors. In addition, LPAAT-b expression is a predictor of a worse prognosis in patients younger than 60 years. Further studies are needed to investigate if LPAAT-b inhibitors may be a therapeutic option for patients with LPAAT-b overexpressing tumors.

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mation, and size of residual viable tumor. Median follow-up was 48.8 months. Survival analysis was performed using the Kaplan-Meier method and logrank test. Results: Histomorphological response was significantly (p ¼ 0:02) correlated with metabolic response (FDGPET). Patients who achieved a histomorphological response (n ¼ 6) after the 3rd cycle of neoadjuvant CTx had a median overall survival of 43.3 months compared to 25.6 months (p ¼ 0:09) in non-responders (n ¼ 27). In multivariate analysis, the combination of histopathological response with metabolic response allowed the most accurate (p ¼ 0:004) prediction of patient outcome. Conclusions: Metabolic changes (FDG-PET) best predicted patient outcome. Histomorphologic response criteria provided important additional information regarding the response to neoadjuvant CTx in advanced stage ovarian cancer. Additional immunohistological and molecular parameters may further enhance the prognostic accuracy and are currently being analyzed on tissue microarrays.

376 Molecular genetic aberrations in carcinosarcomas of the ovary – A CGH and FISH study

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Comparison between histopathological response criteria and sequential FDG-PET for prediction of survival following neoadjuvant chemotherapy in advanced ovarian cancer S. SASSEN, F. FEND, B. SCHMALFELDT1, N. AVRIL2, W. KUHN3, H. HO¨FLER, J. NA¨HRIG Institut fu¨r Pathologie 1 Frauenklinik u. 2 Nuklearmedizinische Klinik, Technische Universita¨t Mu¨nchen 3 Frauenklinik, Universita¨t Bonn

Aims: We recently reported that sequential positron emission tomography (FDG-PET) accurately predicts survival after neoadjuvant chemotherapy (CTx) of ovarian cancer. The aim of this study was to develop and validate histopathological response criteria from post-treatment specimens. Methods: Thirty three patients with FIGO stage IIIC and IV ovarian cancer underwent platinum-based CTx followed by cytoreductive surgery. Metabolic response was determined by changes in FDG-PET from baseline to the 1st and 3rd cycle of CTx. Tumor specimens were evaluated for histomorphological features suggesting response to CTx such as marked necrosis, apoptosis, stromal fibrosis or reactive inflam-

A. SCHIPF, D. MAYR, U. LO¨HRS, J. DIEBOLD Pathologisches Institut der Universita¨t Mu¨nchen Aims: The present study had two aims: First to investigate molecular aberrations in carcinosarcomas (Malignant Mixed Mullerian Tumors) of the ovary. Second to correlate these findings with the biphasic pattern and to elucidate the possible mode of development of the different tumour components. Methods: Evaluation of molecular genetic changes in a series of 26 carcinosarcomas of the ovary using Comparative Genomic Hybridization (CGH) and Fluorescence in-situ Hybridization (FISH) for 8q24 and 20q13. The different tumour components were identified by HE staining and immunhistochemistry. Results: Numerous aberrations were detected on many chromosomes by CGH analysis. Gains (chromosome 1q, 2p, 8q, 12p, 19q, 20q, X) were observed more frequently than losses (4q, 9q, 13q). Since amplifications on chromosomes 8q and 20q were particularly frequent (about 70%) comparative FISH analyses of the carcinomatous and sarcomatous tumour components were done subsequently. Amplification of znf217 (20q13.2) was seen equally in both tumour components, whereas high level amplification of c-myc (8q24.12) was more prominent in carcinomatous areas than in the sarcomatous tumour component.

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Conclusions: Chromosomal aberrations are frequent in carcinosarcomas of the ovary. Cytogenetic analysis of 26 cases revealed a characteristic pattern showing substantial similarities to high grade ovarian carcinomas. A high level of homology was found between carcinomatous and sarcomatous tumour areas. These findings suggest a monoclonal origin of these tumors. On the molecular genetic level the carcinomatous element appears to be the more unstable part of the tumour.

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Apoptotic mechanisms and the Wnt-signalling pathway are involved in ovarian granulosa cell tumors D. REPPUCCI, S. HU¨RLIMANN1, V. BATOG2, F. BANNWART3, H. MOCH, R. CADUFF Institut fu¨r Pathologie, Universita¨t Zu¨rich 1 Institut fu¨r Pathologie, Kantonsspital Luzern 2 Lab. Anatomie Patologica, R-Brasov 3 Institut fu¨r Pathologie, Stadtspital Triemli, Zu¨rich

Aims: The tumorigenesis of granulosa cell tumors (GCT) is poorly understood. Prognosis of GCT can not be assessed by morphology. The aim of this study was to characterize molecular pathways such as the Wnt-b-catenin pathway together with apoptosis and cell cycle regulators in GCT. Methods: We examined 74 ovarian granulosa cell tumors using the tissue microarray technology (TMA). Expression of E-cadherin, p-cadherin, bcl-2, SMAD2, bcatenin, anti mullerian substance (MIS), p27, Cdc2 and PTEN was determined by immunohistochemistry. Results: Two of the GCT developed metastases (3%). A low mitotic index (o10 mitoses/10HPF) was seen in 57 GCT (77%). Positive expression of b-catenin, bcl-2 and p-cadherin was found in over 90% of GCT. Cdc2 was expressed in 28%, whereas MIS (anti-mullerian substance), SMAD2, and PTEN were detected in 14% of the tumors. p27 and e-cadherin were positive in 10% of the GCT. There was no relationship between these markers and the mitotic index or other morphological parameters. Conclusions: These data suggest an important role of apoptotic mechanisms (bcl-2) and the Wnt-signalling pathway (b-catenin) in GCT.

378 Gene amplification of PIK3CA and p110a protein expression in serous neoplasia of the ovary – correlation with tumor grade and clinical outcome A. STAEBLER, O. BUCHWEITZ1, B. KARBERG, J. BEHM1, P. KUHLMANN, U. NEUBERT, G. KLAPDOR, R. LELLE1, H. SCHMIDT Institut fu¨r Pathologie Frauenklinik, Universita¨t Mu¨nster

1

Aims: In vitro experiments and frequent genomic gains on chromosome 3q26 have implicated PIK3CA encoding the p110a subunit of phosphatidylinositol 3-kinase (PI3 K) as an oncogene in ovarian carcinoma. Our aim is to evaluate this hypothesis in a retrospective clinicopathological study. Methods: Ninety nine cases of serous tumors of the ovary (13 borderline, 10 low-grade and 76 high-grade invasive carcinomas) were analyzed by immunohistochemistry on tissue microarray for expression of p110a; pAkt (target of PI3 K) and proliferation marker Ki67. Chromosomal gains on 3q26 were identified by comparative genomic hybridization (CGH) and correlated with gene amplification of PIK3CA by Taqman PCR. Clinical follow up was available in 87 cases (max. 114 months, median 30 months). Results: Gene amplification of PIK3CA correlated with chromosomal gains on 3q26, tumor grade (no borderline, 1 low-grade carcinoma, 29 high-grade carcinomas) and increased proliferative index, but it had no impact on clinical outcome in carcinomas. Strong and diffuse immunohistochemical stain for p110a was not observed in borderline tumors, but in 3 low-grade and 12 high-grade carcinomas. It was not associated with gene amplification, but it was correlated with expression of pAkt and a shorter disease free survival in patients with invasive carcinomas (mean 14 vs. 46 months, p ¼ 0:015). Conclusions: Gene amplification of PIK3CA is associated with high-grade serous carcinomas of the ovary, but does not show any effect on clinical outcome. However, a significant impact on disease free survival is observed in cases with p110a protein overexpression.

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20th amyloidforum at the 89th annual meeting of the German Society of Pathology, May 18–21, 2005, Wuppertal, Germany

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386 Clinical study and therapy of familial amyloidosis E. HUND Neurologische Klinik, Universita¨t Heidelberg

379 In Memoriam Prof. Dr. W. Thoenes S. STO¨RKEL

387 Diagnostic and therapeutic developments in molecular biology

Institut fu¨r Pathologie, Universita¨t Witten/Herdecke H.-J. SCHMIDT

380 Fibrillary nephropathies

Klinik fu¨r Innere Medizin, Alexander von HumboldtUniversita¨t Berlin

U. HELMCHEN

388 Cornal amyloid in gelatinous drop-like dystrophy: a Institut fu¨r Pathologie, Universita¨t Hamburg

case report W. SAEGER1, C. RO¨CKEN2, W. BO¨CKER3, M. GROPPE4, C. E.UHLIG4

381 Clinical study and therapy of renal amyloidosis M. ZEIER Nierenzentrum, Medizinische Universita¨tsklinik Heidelberg

382 Pathology of cardial Al amyloidosis P. SCHNABEL Institut fu¨r Pathologie, Universita¨t Heidelberg

383 Differential diagnostics of amyloidosis C. RO¨CKEN Institut fu¨r Pathologie, Universita¨t Magdeburg

384 Clinical study and therapy of AL amyloidosis J. PERZ Medizinische Universita¨tsklinik Heidelberg

385 Clinical study and therapy of AA amyloidosis J. ERNST1, H. MICHELS2 1 2

Rheumaklinik Oberammergau Kinderklinik Garmisch-Partenkirchen

1

Institut fu¨r Pathologie, Marienkrankenhaus Hamburg 2 Institut fu¨r Pathologie, Universita¨t Magdeburg 3 Institut fu¨r Pathologie, Universita¨t Mu¨nster 4 Klinik fu¨r Augenheilkunde, Universita¨t Mu¨nster Aims: Gelatinous drop-like corneal dystrophy is a rare familiar disorder characterized by massive subepithelial deposits of amyloid. We present immunohistological and ultrastructural findings of a 25 years old female patient with recurrent deposits since more than 13 years. The sister suffers from similar findings. Methods: Eight specimens from abrasiones of the cornea over a period of 11 years were studied using immunostainings or amyloid A, amyloid TTR, amyloid ß2-microglobulin, k-light chains, l-light chains and lactoferrin. For electron microscopy, apon embedded small specimens of amyloid deposits were used. Results: All specimens showed large hyaline-like deposits in corneal connective tissue under the basement membrane. Some macrophages and capillaries were included. The deposits were positive with Congo red (and weakly with PAS) and presented a strong applegreen birefringence of amyloid-type in polarized light. Immunostainings with amyloid- and light chain- antibodies were negative but lactoferrin was strongly expressed in the deposits. The structures did not change during the long observation period. Electron microscopically, fibrils of amyloid-type measuring approximately 100 A in diameter were found in irregular arrangements. The cells trapped in the deposits were macrophages which appeared to have internalized parts of amyloid fibrils.

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Conclusions: The findings identifying familial corneal amyloidosis containing lactoferrin are in accordance with the literature.

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Secondary amyloidosis as unknown cause for cardiac (AL-type) or renal (AA-type) insufficiency: A bioptical and post mortem analysis Y. BURY, H.P. DIENES, J.W.U. FRIES Institut fu¨r Pathologie, Universita¨t zu Ko¨ln

Aims: Clinical manifestations of secondary amyloidosis are associated as AL- (related to underlying monoclonal light chain production associated with plasmocytoma) or AA-Amyloidosis (related to underlying chronic inflammation). The disease is often recognized (too) late with consequently poor prognosis. Methods: The study evaluated 364 biopsies and 19 sequential autopsies performed on patients with clinical query amyloidosis 1996–2004 at the University of Ko¨ln. The tissue probes were evaluated by light microscopy using hematoxylin-eosin-stained sections as well as

Congo red and immunohistochemistry for Amyloid A, light chain k and l, when amyloidosis was in the differential diagnosis. Results: Of the examinated 364 biopsies 133 cases (36%) were positive; 202 cases (55%) negative and 29 cases (8%) insufficient for diagnosis. Most frequent organ manifestation were kidney (positive for AA in 19/89, for AL in 18/89), liver (positive for AA in 3/46, for AL in 13/46) and heart (positive for AA in 1/19, for AL in 5/ 19). In 15 autopsy cases amyloidosis was a mediate death cause. The leading clinical symptom in the autopsy cases with AA amyloidosis was progressing renal insufficiency without clinical diagnosis of amyloidosis, particularly with regard to patientso45 years. In the group of AL-amyloidosis three patients (o 45 years) died from complications of plasma cell dyscrasia, the others of plasmocytoma. Interestingly, all patients suffered from cardiac insufficiency as death cause, whereas the younger patients clinically presented symptoms for myocarditis. Conclusions: In younger patients with unclear renal or cardiac insufficiency AL- or AA-Amyloidosis should be foreclosed by immunohistochemical methods of bioptical material quantification particularly for Amyloid A.