Endocrinopathology

Endocrinopathology

276 . Oral Communications 248 CYTOKERATIN AND NEUROFILAMENT PROTEIN IMMUNOPATTERNS IN MERKEL CELL CARCINOMA AND ANAPLASTIC SMALL CELL CARCINOMA OF TH...

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276 . Oral Communications

248 CYTOKERATIN AND NEUROFILAMENT PROTEIN IMMUNOPATTERNS IN MERKEL CELL CARCINOMA AND ANAPLASTIC SMALL CELL CARCINOMA OF THE LUNG U. Schmidt, L.-D. Leder Pathology, University hospital, Hufelandstr. 55, D-45122 Essen Aims: This study, after presenting clinicopathologic data on 82 Merkel cell carcinomas (MCCs), focuses on the major problem of differential diagnosis, namely the distinction between MCC and anaplastic small cell carcinoma of the lung (SCCL). Cytokeratin (Ck) and Neurofilament (Nf) protein imrnunostainingwas performed and the pattens were compared of the MCCs with 18 genuine seCL. Results: The bulk of tumours (61/82) was seen in consultation. 46 patients were females, 36 were males. The patients median age was 72 years with a range from 41 to 90 years. The most common location was head and neck (28), followed by arms (18), trunk (13) and legs (12). In 10 patients tumours coexisted with haematological malignancies (7 small cell lymphocytic lymphomas, 1 Hodgkin's disease, 1 chronic myeloid leukemia, 1 idiopathic myelofibrosis). Histopathologic analysis identified 2 phenotypes: The one comprising the majority of tumours (64/82) stood out with highly characteristic features such as uniform round nuclei, a finely dispersed hypodense chromatin, inconspicuous nucleoli and numerous mitoses; it therefore was considered prototypic. Less frequent were tumours with a small cellular phenotype indistinguishable from SCCL in routine Haematoxylin-Eosin slides (18/82). With a Ck cocktail (Pan-ek: Cks 1-8, 10, 13-16, 19), most (51/64) prototypic tumours,and more important, 16/18 cases of the small cell variant revealed focal paranuclear positivities; 8 prototypic cases and 1 small cellular tumor stained diffusely positive. Ck20 staining was positive with 45/58 MCCs analysed. Conversely, 20/22 SCCL exhibited diffuse cytoplasmic reactivities after Pan Ck staining and 18/18 were Ck20 negative. Furthermore, 30/46 MCCs including 8/12 of the small cell variant expressed Nf protein, but none of the seCL did so. Conclusions: There exist two MCC variants. HE histology features of prototypic MCCs are nearly pathognomonic. The small cell variant of MCC, however, has to be distinguished from seCL. It differs from Iretastatic SCCL in that focal paranuclear Ck positivities are seen. Additionally, coexpression of Ck20 and Nf excludes metastatic SCCL reliably.

Oral Communication: Neuro/Endocrinopathology 249 NEUN - A USEFUL NEURONAL MARKER IN DIAGNOSTIC mSTOPATHOLOGY H.K. WOLF*, R. BUSLEI**, R. SCHMIDT-KASTNER***, P.K. SCHMIDT-KASTNER***, T. PIETSCH**' O.D. WIESTLER**' I. BLUMCKE** Institut fur Pathologie, Universitat Mainz*; Institut fiir Neuropathologie, Universitat Bonn**; Neuroscience Research Institute, University of Ottawa, Ottawa, Ontario, Canada*** Aims: The monoclonal antibody A60 specifically recognizes the DNA binding neuron-specific protein NeuN which is present in most neuronal cell types of vertebrates. Our goal was to examine the potential use of NeuN as a neuronal marker in diagnostic histopathology. Methods: We examined NeuN expression in a wide range of formalin-fixed and paraffin-embedded human surgical and autopsy specimens from the central and peripheral nervous system including 9 gangliogliomas and 14 dysembryoplastic neuroepithelial tumors, one ganglioneuroma and one dysplastic cerebellar gangliocytoma. Results: Following microwave antigen retrieval, almost all neuronal populations revealed strong immunoreactivity for NeuN in nuclei, perikarya and some proximal neuronal processes while more distal axon cylinders and dendritic ramifications were not stained. The stain greatly enhanced the gray matter architecture. NeuN immunoreactivity was not detected in Purkinje cells, most neurons of the internal nuclear layer of the retina and in sympathetic chain ganglia. The neuronal component of all glioneuronal lesions showed marked immunoreactivity for NeuN. There was no staining of non-neuronal structures. Conclusions: NeuN immunoreactivity is a sensitive and specific neuronal marker in formalin-fixed and paraffin-embedded tissues and may be useful in diagnostic histopathology.

250 Vimentin-Immunocytochemistry of Normal Human Follicular and C Cells of the Thyroid Gland 1

C. Langner (a.G.), Ch. GraB, J. Vogel , O. Basten (a.G.), O. Klinge Institute fOr Pathologie, Stadtische Kliniken Kassel und Siegburg 1

Aims: Vimentin-immunoreactivity in epithelial neoplasms is usually considered to represent metaplastic change, in some cases corresponding to mesenchymal differentiation seen at light and ultrastructural level or to tumour anaplasia. While thyroid tumours are known to express vimentin, conflicting data exist concerning vimentin content of normal follicular cells; furthermore vimentin content of normal C cells has never been referred to before. Methods: Paraffin-embedded material of 30 non-neoplastic thyroid glands (normal gland, diffuse and nodular hyperplasia, including toxic goitre) was examined applying a double immunostaining technique using monoclonal antibodies directed against vimentin and calcitonin. Results: Follicular cells show vimentin-immunoreactivity in nearly all glands, except few cases of toxic goitre. Usually only a small number of cells are stained, the reaction mainly confined to the basal parts of the cytoplasm. Most C cells do not contain vimentin, but there are unequivocally double stained cells in some cases. Conclusions: Demonstration of vimentin in normal human follicular and C cells provides evidence that vimentin-immunoreactivity of epithelial thyroid tumours is not the result of metaplastic change, but represents maintenance of the intermediate filament pattern of their cells of origin. The failure to detect vimentin in follicular cells of some cases of toxic goitre may indicate a relationship between vimentin content and the functional state of the cells.

251 OCULOPHARYNGEAL MYOPATHY WITH ASSOCIATED NEUROGENIC CHANGES IN THREE GENERATIONS OF A FAMILY S. Kellermann (a.G)*, P. Baum (a.G.)**, J. Ziegan (a.G.)***, A. Wagner (a.G.)**, R. Schober* *Institut fur Pathologie, Abteilung fUr Neuropathologie, **Klinik fUr Neurologie, Universitiit Leipzig und ***Institut fur Pathologie, Stiidtisches Klinikum Leipzig-SUdost Oculopharyngeal muscular dystrophy (OPMD) is a rare, autosomal-dominant, usually late onset disorder that affects extraocular eye muscles, pharyngeal muscles and even the musculature of the trunk and limb. The differential diagnosis includes ocular myasthenia gravis, late-onset Kearns-Sayre syndrome, and neurogenic oculopharyngeallesions such as spinal muscular atrophy. We here report clinical, electrophysiological, and biopsy studies in a family with a history of neuromuscular disorders involving 6 members over 3 generations. Grandmother, father and two brothers of the father became ill with the same symptoms as the presented patients and died from neuromuscular disease, consistant with an autosomal-dominant pedigree. The biopsied patients - brother and sister of the third generation- both showed the classical clinical symptoms of OPMD. The 45y old sister had a 6 years history of bilateral ptosis, progressive dysphagia and limb-girdle weakness. Electrophysiology, surprisingly, revealed different findings in different muscular regions with predominating neurogenic as well as myopathic changes. Two biopsies were perfonned in a 1 year interval, the first one showing only borderline changes. The second biopsy displayed typical myopathic changes with rimmed vacuoles, disseminated fiber atrophies and characteristic ultrastructural findings. Additional neurogenic changes could be distinguished: clear fiber-type grouping, small dark angulated fibers and groups of atrophic fibers. The 47y old brother, in contrast, showed the typical features of the disease without any neurogenic changes. In agreement with few comparable reports we conclude that there is an etiologically relevant neurogenic component in OPMD, and there are possibly different morphological phenotypes of OPMD even in members of one affected family. These may represent different stages in a dynamic disease process that includes de- and reinnervation in addition to the predominating myopathic changes.

Neuro/Endocrinopathology . 277

252 MEN-l GENE MUTATIONS IN SPORADIC NEUROENDOCRINE AND ADRENOCORTICAL TUMORS B. Goetz, S. Muletta, R.R. de Krijger*, K. Riitimann, EJ.M. Speel, P. Saremaslani, J. Roth, Ph.U. Heitz and P. Komminoth Departments. of Pathology, Division of Cell & Molecular Pathology, University of Ziirich, Switzerland and *Rotterdam, The Netherlands.

254 CLONAL ANALYSIS OF BENIGN AND MALIGNANT, SPORADIC PANCREATIC ENDOCRINE TUMORS A. Perren, J. Roth, S. Muletta, P. Saremaslani, EJ.M. Speel, Ph.U. Heitz, P. Komminoth Department of Pathology, University of Ziirich, Switzerland

Sporadic neuroendocrine tumors (NET) and adrenocortical lesions frequently exhibit loss of heterozygosity (LOH) at chromosome IIql3, the locus of the recently identified tumor suppressor gene MEN/N, which is mutated in the germline DNA of patients with multiple endocrine neoplasia type I (MEN-I). In order to investigate the role of the MEN-I (MENIN) gene in these tumors, we screened DNA extracted from a total of 47 specimens, including 8 adrenocortical tumors (3 adenomas, 5 carcinomas), 27 pancreatic endocrine tumors (8 insulinomas, 5 VIPomas, 5 gastrinomas, I glucagonoma, 8 non-secreting tumors) and 12 NET (4 lung, 2 stomach, 2 duodenum, 2 ileum, 2 metastases) for somatic mutations in the 9 coding exons of the MEN-I gene by PCR-based, non-isotopic SSCP analysis and heteroduplex MOE gel electrophoresis followed by direct cycle sequencing of PCR products from samples with aberrant band patterns. We identified a total of 6 somatic MEN-l gene mutations (6/47; 12.7%) and 4 polymorphism (RI71Q twice, L432L, 0418D). None of the adrenocortical tumors contained a mutation. Two NET of the lung harbored a somatic missense mutation in exon 3 (DI72V) and an insertional mutation in exon 2, respectively. One gastrinoma of the duodenum exhibited a nonsense mutation in exon 3 (Q209X). One VlPoma contained a nonsense mutation in exon 9 (Q450X), 1 gastrinoma of the pancreas a 14 bp deletion in exon 2 (454deI14) and 1 insulinoma a 7 bp deletion in exon 10 (779IdeI7). The remaining NET showed inconspicuous band patterns in all examined exons. Our results indicate that (I) alterations of the MEN-I gene are involved in the tumorigenesis of a subset of sporadic NET of the lung (214; 50%), gastrointestinal tract (1/8; 12.5%) and pancreas (3/27; 11%) but are probably not involved in the formation of adrenocortical tumors; (2) gene alterations not only involve somatic stop codon mutations but also missense and insertional mutations; and (3) that mutations appear not to be clustered in a particular part of the MEN-I gene.

Pancreatic endocrine tumors (PET) are neoplasms arising from cells which are "phenotypically" neuroendocrine and thus share the broad spectrum markers for neuroendocrine differentiation. PET frequently display a marked phenotypic heterogeneity in terms of growth pattern and production of hormones. It is unclear whether this morphological heterogeneity is caused by the proliferation of different cell clones or by variable differentiation of one clonal tumor cell population. We have therefore analyzed 34 sporadic PET of female patients by means of a PCR-mediated clonality analysis assay which is based on the inactivation patterns of the polymorphic X-linked genes encoding the androgen receptor (AR) and the phosphoglycerate kinase (PGK-I) proteins. Tumor and non-tumorous tissues of each patient was isolated by microdissection from tissue sections of paraffm-embedded specimens. Twenty of the patients were heterozygous for the AR microsatellite region or Bst XI polymorphic site of the PGK-I gene, permitting analysis of clonality. Pretreatment of the DNA with the methylation-sensitive restriction endonuclease Hpa II blocked amplification of one of the two AR or PGK-l alleles in a total of 7 of 20 PET (35%), indicating a clonal pattern of X~hro~os?me inactivation. One additional tumor exhibited a oligoclonal mactIvation pattern and two further PET a loss of heterozygosity (LOH) at the AR locus, which is indicative for monoclonality. In 10 PET (50%), Hpa II pretreatment had no effect, indicating a random pattern of X-chromosome !nactivat.ion an~ polyclon~l cellular composition. When comparing mformative bemgn and malIgnant PET, 5n (71%) benign PET showed a polyclonal and 8/13 (61 %) malignant tumors a monoclonal (5), oligoclonal (I) or LOH (2) pattern. The monoclonal composition of PET was not associated with a particular growth pattern, proliferation index or immunohistochemical expression pattern. Our findings suggest, that PET might initially represent poly- or oligocl,?nal neopl~tic lesions which are eventually outgrown by a more ~ggre.sslve clone WI~ growth advantage which is potentially giving rise to IDvaslon and metastatIc spread.

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CLASSIFICATION OF HUMAN OLIGODENDROGLIOMAS WITH NEURAL NETWORKS AND EUCLIDEAN BASED FEATURE SELECTION (EUBAFES) M. Scherf(a.G)I, JR. Iglesias-Rozas2 I GSF-Forschungszentrum fur Umwelt und Gesundheit, Medis Institut. Neuherberg 2 Pathologisches Institut- Katharinenhospital. Stuttgart Aims: The correctness of the diagnosis of brain tumours depends on the specialist experience, while the examination of histological features is influenced by subjectivity of the descriptive terminology as well as the exhaustion-factor of the pathologist during examination. The aim of our research is to show the possibility of an effective, automated classification of human gliomas by neural Networks and the Euclidean based feature selection approach EUBAFES. Methods: 115 typical oligodendrogliomas (WHO grade TI) 174 oliogastrozytomas ( WHO grade TI) and 141 malignant oligodendrogliomas (WHO grade III) were surveyed. 50 histological characteristics were examined from each tumor. Two classification tasks were carried out: differentiation between benignant (grade ll)and malignant (grade III) oligodendrogliomas, and classification between the three tumor types. To reduce the complexity of the classification task we applied EUBAFES to select only those features which are important for the diagnostic task. Neural networks with radial bases functions were used as classifiers. Results: EUBAFES- selected six resp. five relevant features for the three-resp. the two-class problem. We obtained a classification accuracy of 76% (three-class problem) and 87% (two-class problem). Using all characteristics we only obtained 66% rep. 79"10. The results will be compared and discussed wit other neural Network approaches (I). Conclusions: Neural Networks are powerful tools for the histological classification of gliomas as well as the evaluation of the WHO grade of malignity. Due to the application of EUBAFES we were able to select relevant histological features and to improve the classifier performance.

THE EXPRESSION OF MATRIX METALLOPROTEINASES IN MALIGNANT NERVOUS SYSTEM TUMOR CELL LINES IS INVERSELY CORRELATED WIm PROLIFERAnON l 2 M. Thier , E. Roeb , B. Breue~, J. Weis3 lInstitut fur Neuropathologie and 2Medizinische Klinik III, UniversitiitskJinilrum der RWTH, Aachen, Germany; Pathologisches Institut der Universitiit Bern, Switzerland Aims: The proliferative and infiltrative activities are major determinants of the prognosis of brain tumors. Still, the molecular basis of brain tumor cell migration is largely unknown. As a first step to elucidate the patterns oftissue invasion by malignant nervous system tumor cells, the expression of bioactive matrix-metalloproteinases (MMPs) in established tumor cell lines was analyzed. Methods: Supernatants of two neuronal (SK-N-SH, SK-N-MC) and four glial (U138MG, U373MG, AI72, C6) cell lines and of two pluripotent cell lines with facultative neuronal and glial differentiation (P19 and NT2) were examined by gelatine zymography (10 % polyacrylamide copolymerized with I mglml gelatine). Zones of enzymatic activity were subsequently visualized by Coomassie blue staining. The bands were analyzed densitometrically (Image Master, Pharmacia). The proliferation rates of the tumor cells were determined by two independent methods, the measurement of population doubling times and the analysis of BrdU incorporation. Results: Most cell lines produced gelatinase A (MMP-2); only few lines expressed gelatinase B (MMP-9) at low levels. Expression of bioactive MMPs varied from barely existent (p19 and C6 cells) to strong (U138MG and SK-N-SH). There was no difference in expression between the glial and neuronal lines in general. Interestingly, for the cell lines with high MMP expression levels, low population doubling times and BrdU incorporation frequencies were recorded, and vice versa. Dis~ussion: These data indicate that the proliferative activity and the po_ tential to degrade extracellular matrix are inversely correlated in these tumor cell lines. They are compatible with the hypothesis that MMP expression is downregulated in highly proliferative tumor cells.

278 . Oral Communications

Oral Communications: Soft Tissue Tumors

258 Intracellular distribution of P-catenin in soft tissue tumors

256 GENETIC CHANGES IN THE FffiROBLASTIC STROMA OF INVASIVE HUMAN COLON AND BREAST CANCERS N. Wernert, K. LOcherbach (a. G.) Institute of Pathology, University of Bonn, P.O.Box 2120, 53011 Bonn, FRG Aims: The aim of this study was to find out wether genetic changes in invasive human colon and breast carcinomas are restricted to tumor cells or can also occur in the fibroblastic stroma which is generally considered a non-neoplastic, tumor cell-induced component. Methods: 20 invasive human colon and 4 breast carcinomas have been microdissected into epithelial and fibroblastic stromal components. Loss of heterozygosity (LOH) of loci DI85549 (I8q, DCC-region), DI8560 (I8q21, near DCC), D185477 (I8q22) and 0135317 (I3q22-q31) was analysed in the colon cancers and of loci 0175579 (17q, BRCA-l telomeric), 0 16S539 (I6q24-qter, near BBC I), 0165398 (I6q22, near Ecadherin) and 0185477 (I8q22) within the breast cancers employing PCRamplification of polymorphic microsatellite DNA. Exons 5 and 8 of the p53 gene were screened for mutations by 55CP and exon 8 also by direct sequenzing within the 20 colon carcinomas. Results: LOH or allelic imbalances at at least one locus were found in tumor areas in 57 % of the colon carcinomas and in 3 of the 4 breast cancers. They were present in stroma areas in 52% of the colon carcinomas and in 2 of 4 breast cancers. Shifts in 55CP screening of exons 5 and 8 of p53 were found in 27% of tumor areas and in 15% of stroma areas of the colon carcinomas. Sequenzing of exon 8 revealed 3 mutations within tumor components (codon 278, CCT ... CTT, Pro ... Leu, codon 284, ACA'" ATA, Thr'" De and codon 299 ,CTG'" CTT, Leu, silent mutation) and 2 mutations within the stroma (codon 285, GAG'" AAG, Glu ... Lys, Codon 283, cae ... CGT, Arg, silent mutation). Conclusions: Our results show that genetic changes in invasive human colon and breast cancers are not restricted to neoplastic cells but can also occur within the fibroblastic stroma. Possible interpretations of these findings include epithelial-mesenchymal transitions or independent, maybe carcinogen-induced stromal changes. The biological implications of these results await further clarification.

257 Loss of heterozygosity of the retinoblastoma (RBI) gene in lipomas of a retinoblastoma patient H. Rieder " D. Lohmann', B. Poensgen " B. Fritz " M. Asian " D. Drohm', F.J. 5trombach Angersbach', H. Rehder' 1 MZ Humangenetik, Universitaet Marburg; , Institut f. Humangenetik, Universitaet der GSH Essen; 1 Diakonie-Hospital, Marburg; Germany. Aims: An increased frequency of lipomas has been reported in patients with hereditary retinoblastoma (RB). This suggests that, like in RB tumors and RB associated sarcomas, a constitutive oncogenic RB I mutation and loss of the normal RB I allele may contribute to the molecular pathogenesis in lipomas of RB patients. We, therefore, investigated somatic loss of the RBI gene in lipoma samples of a 27 year old patient with sporadic bilateral RB treated by enucleation of both eyes at the age of I year and presenting with multiple lipomas in young adulthood. Methods: Cytogenetic, fluorescence in situ hybridization (FISH) and loss of heterozygosity (LOH) analyses of the RB I locus were performed on three different lipomas after short term cultivation. Constitutive RB I mutations were investigated on peripheral blood cell DNA using heteroduplex analyses, single strand conformation polymorphisms analyses, and direct sequencing. Results: In two of three lipomas a deletion of chromosome 13q was the sole karyotype abnormality, del(I3)(qI2-13q31) and del(13)(qI2-13qI4), respectively. In both tumors FISH and LOH studies demonstrated loss of the same allele of the Rbi gene. Chromosome and RBI FISH analyses on peripheral blood lymphocytes as well on skin fibroblasts revealed a normal constitutional karyotype with a normal RB I signal distribution. Extensive molecular screening failed to disclose a small constitutive RB I mutation. Conclusions: It is generally accepted that all patients with sporadic bilateral RB are heterozygous for a constitutive oncogenic RB I mutation. But, as in our case, due to the complexity of the RBI gene, mutations still may escape detection despite extensive genetic screening procedures. On the other hand, recurrent LOH of the identical RB I allele in two different lipomas of our patient further substantiates that a predisposing RB I gene mutation may causally be involved in the development of this second primary neoplasm.

C. KUhnen!, P. Herter, H.u. Steinau3, O. Muller, K.-M. Muller' I: Institute of Pathology, University Hospital Bergrnannsheil, Bochum 2: Max-Planck-Institute of Molecular Physiology, Dortmund 3: Department of plastic and hand surgery -bum centre-, University Hospital Bergmannsheil, Bochum Aims: 13-Catenin originally known as an intracellular mediator of epithelial cell-to-cell adhesion is also involved in signal transduction processes. Since its important function in colorectal carcinogenesis has recently been recognized, the aim of our study was to investigate whether a similar intracellular distribution of 13-catenin can be detected in sarcomas and sarcoma-like lesions. Methods: 45 soft tissue tumors were examined by immunohistochemistry. Results: Different types of (3-catenin-distribution were observed: A more continuous localization at the cell membrane was evident especially in epithelioid-like sarcomas. In contrast only focal staining of cell membranes could be found in different tumors as well. Furtheron increased cyloplasmatic (3catenin levels were detected in various types of tumors. Conclusions: A nuclear and intracellular accumulation of (3catenin has been observed in progression of colorectal tumors. The findings of increased levels of (3-catenin in soft tissue tumors may indicate a similar important function in the pathogenesis of these neoplasias.

259 Iron Deposits, Cell Types, and Proliferating Cell Populations in Pigmented Villnodular Synovitis (PVNS) of the Knee T. Aigner,a.G.; S. Oehler, a.G.; H. G. Fassbender*; G. Niedobitek, T. Kirchner Institute of Pathology, University ErIangen-Niirnberg * Zentrum fur Rheuma-Pathologie (WHO-Center), Mainz Aims: Pigmented villonodular synovitis (PVNS) of the knee is a tumorlike process of uncertain nature. The aim of our study was to characterise the .prevalent inflammory cells, the proliferating cell populations, and the iron deposit distribution in PVNS. Methods: Twelve· cases of PVNS of the knee as well as 6 normal controls were analysed histochemically for iron deposits, immunohistochemically for the distribution of vascular structures and inflammatory cell populations. Collagen type I collagen expressing fibroblastic cells were identified by in situ hybridization. The proliferative cell compartment was identified using MIB-I staining and confirmed by double-labeling experiments. Results: There was no correlation between intra- or extracellular iron deposits and proliferation, giant cell formation, vascularity, number of macrophages, and foam cell formation. In contrast, significant levels of iron deposits were associated with strongly type I collagen mRNA expresssing fibroblastic cells. No significant increase of lymphocytes or granulocytes was seen in PVNS compared to other areas of the synovial specimens of the same patients not affected by PVNS. The identification of the proliferative cell compartments showed that, besides fibroblastic cells, numerous CD68 positive macrophages were Ki-67 positive. CD68 positive foam cells, iron-loaded macrophages and fibroblasts, and CD68 positive giant cells were, however, consistently negative for the Ki-67 antigen. Conclusions: PVNS is not an inflammatory process involving chronic or acute inflammatory cellular infiltrates. Rather it appears to originate from the interplay of proliferating macrophages and fibroblasts, both activated directly or indirectly by an excessive iron load. Giant cells probably develop by fusion of CD68-positive histiocytes.

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"Benign metastasizing leiomyoma" ? A case report without evidence for imbalanced genetic aberrations.

leiomyomas with subsequent development of multifocal extrauterine smooth muscle tumors, most often located in the lung. It remains unclear whether this evolves from a morphological innocent appearing low grade sarcoma or from proliferation of multifocal but autochtonous cellular foci. Frequently, and in part recurrent karyotypic abnormalties were described for leiomyomas and leiomyosarcomas. Thus, we looked for imbalanced genetic aberrations in a case of a "benign metastasizing leiomyoma" by means of comparative genomic hybridisation (CGH). Methods: A 46 year old female developed multiple bilateral IWlg nodules 4 years after hysterectomy. Fine needle biopsy of one nodule and subsequent resection of three lung nodules were performed. These and the hysterectomy specimen were investigated by conventional histology, immWlohistochemistry and CGH using standard protocols. Results: Revision of the hysterectomy specimen revealed multiple leiomyomas without any evidence for malignancy. Lung nodules were composed of benign appearing smooth muscle cells with epithelial lined cleft like spaces. Leiomyomata of the uterus and the lung showed a reactivity against actin, desmin, estrogen- and progesteron receptor antigens. DNA analysis by CGH revealed a normal karyotype without evidence for an imbalanced loss or gain of DNA. Conclusion: Recurrent cytogenetic alterations are common in uterine leiomyomas, most often del (7)(ql1.2-22q31-32) and t(12;14) (qI4-15; q23-24). Leiomyosarcomas display diverse karyotypic abnormalties, most often involving chromosomes 1,7,13 and 14. Thus, the missing karyotypic imbalance in the presented case favors a multifocal smooth muscle proliferation with a pathogenesis which is different from usualleiomyomas as well as leiomyosarcomas.

PS3 MUTATIONS IN SARCOMAS H. Taubert1, A. Meye 1, P. Wiirl2, M. Bache3, H. Schmidt l , F.-W. Rath 1, lInstitut fUr Pathologie, 2 Klinik fUr Allgemeinchirurgie, 3K1inik und Poliklinik fUr Strahlentherapie, Martin-Luther-Universitat Halle-Wittenb. Aims: Overview of p53 mutations in sarcomas should enable to evaluate role of p53 alterations in sarcoma tumorigenesis. Methods: PCR-SSCP-Sequencing analysis for p53 in soft tissue sarcomas and and exploitation of a p53 mutational data base. Results: We have described investigation of 145 soft tissues sarcoma (STS) patients for the presence of p53 mutations and their prognostic relevance. At combining our results with the data from the p53 mutational database (Hainaut et al. 1997), altogether more than 200 entries for sarcomas were available. In sarcomas two groups with different p53 mutational frequencies can be distinguished; a group with a low incidence of mutations «5%) and a second group with a moderate to high incidence of mutations(>5%). The first group includes synovial sarcoma, fibrosarcoma and neuroblastoma, whereas the second group consists of the other STS and bone sarcomas. For all sarcomas a mean frequency of 17.1 % p53 mutations was calculated. The mutations are mostly point mutations (83%) including missense mutations (80%) and nonsense mutations (3%), followed by deletions (11%), insertions (4%) and splice mutations (2%). Majority of point mutations represent G:C to A:T transitions (55.9%), followed by G:C to T:A transversions (17.5%) and A:T to G:C transitions (10.9%). Almost half of the G:C to A:T transitions (50.8%) occurred at CpG sites, rather suggesting endogenous than exogenous mechanisms as mutation trigger. Most striking investigation was that in STS only patients with non-frameshift mutations showed a considerably poorer prognosis than patients without p53 mutations whereas in patients with frameshift mutations prognosis seemed not to be affected. Conclusions: P53 mutational status at considering localization, kind, and allelic state appears as important factor for diagnosis and prognosis in sarcomas and will come certainly into focus at future therapy.

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ALVEOLAR RHABDOMYOSARCOMA PRESENTING AS ACUTE LEUKEMIA

DIFFERENTIAL NUCLEAR EXPRESSION OF 10-67: IS IT IMPORTANT IN ADULT SOFT TISSUE SARCOMAS? U. Rohr, M. Heinzinger, R. Parwaresch*, R.M. Bohle Institute fur Pathologie, Universitat Kiel* und Giel3en

L. Tietze, K. Giinther, S. Merkelbach-Bruse, S. Handt Institute of Pathology, RWTH, Aachen, Germany Aim: There are rare cases of histologically benign appearing uterine

PETRA REINECKE!, C.D.GERHARZ\ K_P.TIllELE2, U.JANIG3, HE. GABBERT I Institute oj Pathology' and Department oj Hematology, Oncology and Clinical Immunolggy', Heinrich-Heine-Untversity, Diisseldoif, Institute oj Paidopathology, Christian-Albrechts-Untversity, Kiel, Germany

Case report: Rhabdomyosarcoma is the most common soft tissue sarcoma in adolescence and childhood, which manifests by the locally destructive growth of the primary tumor or its metastases. We report on a 29-year-old man with an alveolar rhabdomyosarcoma presenting with an unusual leukemia-like picture. On admission, the patient suffered from diffuse bone pain and renal insufficiency. Peripheral blood analysis showed anaemia, thrombocythaemia and blast-like cells. A bone marrow aspirate revealed extensive infiltration by atypical blast-like cells which were interpreted as acute lymphoblastic leukemia. Although confirmation of this diagnosis by immunophenotyping did not succeed, the chemotherapy was started immediately and led to partial remission. Histologic analysis of a bone marrow biopsy from the iliac crest, however, revealed an extensive solid tumor with alveolar spaces, lined by primitive round cells with positive PAS-reaction in the cytoplasm ImmW10staining demonstrated a positive reaction of the tumor cells for vimentin, destnin and smooth-muscle-actin. Chromosomal analysis showed a t(2;13) translocation typical for alveolar rhabdomyosarcoma. Although muhiple lytic lesions of the skeletal system became evident during the fiuther clinical course, the site of origin of the primary tumor could not be defined retrospectively. Conclusion: Rhabdomyosarcoma should be included in the differential diagnosis of systemic diseases with extensive bone marrow infiltration by tumour cells that could otherwise be misinterpreted as a haernatologic malignancy.

Aims: As many other nuclear markers, e.g. steroid receptors, Ki-67 epitopes are differentially expressed in tumor cell nuclei. It is unclear whether this phenomenon represents tumor cell heterogeneity, different stages of the cell cycle or a biological phenomenon with prognostic impact. Methods: We analysed 104 primary ASTS [leiomyosarcomas(LMS), liposarcomas(LPS), malignant fibrous histiocytomas(MFH), MPNST, synovial sarcomas(SS)], formalin-fixed, paraffin-embedded, by APAAP immunohistochemistry (IRC), antigen retrieval techniques and two antibodies (MIB-I, Ki-S-5) against formalin-resistant Ki-67 epitopes. Ki67 expression was evaluated in 10 high power fields according to Coindre by 4 indexes: a) A-index: sum of all (weak, moderate, and strong stained) Ki-67+ nuclei /1000 tumour cells, b) IDl-index: sum of allstrong stained Ki-67+ nuclei/1000 tumour cells, c) ID2-index: sum of all moderate and strong stained Ki-67+ nuclei/IOOO tumour cells, and d) the weighed Rindex: sum of strong stained nuclei x3, moderate stained nuclei x2, and weak stained nuclei xl. Prognostic impact was analysed by Kaplan-Meier and logrank statistics with respect to survival. Results: Quantitative Ki-67 expression did not vary significantly if determined by MIB-I or Ki-S-5. Compared to the 101-, 102- and Rindex, the A-index turned out to be the strongest prognostic parameter within the whole group of ASTS as well as within each single sarcoma type investigated. At a cut off level of 100 (equivalent to 10% of tumour cells stained) significant (p::S0,05) correlations between A-index and overall survival existed in LMS, LPS, MFH, SS, while a trend to significance (p~0,06) was observed in MPNST. The A-index also correlated significantly with the event free survival in LMS and LPS, a trend to significance (p<0,07) was observed in SS and MPNST. Conclusion: Quantitative evaluation of all three expression levels (weak, moderate, and strong Ki-67 staining) is necessary to obtain the most comprehensive prognostic informations of proliferation markers in primary ASTS.

280 . Oral Communications

264 DNA cytometry in soft tissue tumors - diagnostic and predictive aspects R. Bollmann', H.U. Steinau 2 (a.G.), A. Bosse3; Institut f. Pathologie Bonn' u. RWTH Aachen 3" Bergmannsheil Bochum - Klinik filr Plastische Chirurgie2 The aim of our study was to determine whether DNA cytometry is of additional value in assessing the dignity of soft tissue tumors and whether correlation between recorded DNA parameters and histopathological grading can be found. Methods: In a retrospective study air dried cytological imprint samples of I 14 non-infantile soft tissue tumors were analysed by DNA cytometry. Per case at least 150 nuclears were measured (Cydok, FA. Hilgers, Kllnigswinter). The results were compared with classical morphological parameters. Results: DNA aneuploidy has a sensitivity of 94 % and a specifity of 82 %: 50 of 53 malignant soft tissue tumors were DNA aneuploid; from 6 I nonsarcomatous soft tissue tumors 1I were found to be aneuploid. These lesions were classified as intermediate dignity. No aneuploid tumor could be found among 30 histologically benign soft tissue tumors. The positive predictive value of DNA cytometry was 82 %: of 61 aneuploid soft tissue tumors I I were non-sarcomatous tumors with intermediate dignity. The negative predictive value of DNA cytometry was 97 %: from 53 non-aneuploid soft tissue tumors only three were found to be sarcomas. Malignant soft tissue tumors demonstrate a significantly higher DNA index (01) and DNA grade of malignancy (DNA MG) than benign lesions. Our results show that DNA ploidy may serve as an additional parameter in problematic histological dignity assessments of soft tissue tumors. Conclusions: The histopathological grading levels of sarcomas show significantly different 01 and DNA MG, so that DNA cytometry enables substantiated grading of soft tissue.

Oral Communications: Uropathology

266 Polyoma Virus Infection in Human and Primate Renal Transplants: Not only CMV is associated with distinct morphological changes and allograft deterioration V. Nickeleit*. I. Binet*, F. Guda/*. P. Dalquen*. H. H. Hirsch*. G. Thiel*, R.B. Colvin**. and M. J. Mihatsch* University of BASEL, SWITZERLAND*; and Harvard Medical School, BOSTON, USA** Background: Polyoma yiruses (PV, mainly of the BK type) have rarely been observed in human and primate KIcrney grafts. However, within a few months, we encountered an 'outbreak' of 6 such cases in human allograft recipients, all of which under heavy immunosuppression (mainly tacrolimusIFK506). All patients had abundant PV infected cells (i.e. 'decoy cells') in the urine. These 6 cases compare with 13 CMV infections seen in more than 10 years. Aim: To characterize histology and outcome associated with PV infection. Methods: A) 6 human allograft recipients showed graft infection by BK virus [7±1 months (and I case 24 mths) post transplantation; confirmed by IHC; PeR; and EM). Several renal allograft biopsies (mean: 5±1; all between 12/1/96 and 911197) in each patient were studied. Graft function (creatinine) and outcome (nephrectomy) were recorded. B) In comparison, the histology and mc in 6 PV infected primate renal allograft recipients was analyzed. Results: (A, Humans): Cytology: BK virus infected epithelial cells showed marked nuclear enlargement and polymorphism. The intranuclear viral inclusion bodies were irregular (pale eosinophilic) without a well developed halo. Nucleoli were absent. Many nuclei had a homogenous 'ground-glass' or smudgy appearance. His/ology: BK virus associated changes were found in epithelial cells from the urothelial umbrella cell layer to parietal epithelial cells along Bowman's capsule, most frequently in tubular cells. Early changes were primarily detected in the medulla in scattered collecting ducts. Later on, also the renal cortex was involved. Often, a mononuclear cell infiltrate and tubulitis was present in the interstitium. However, frequently a discrepancy was noted between the large extent of the interstitial infiltrate and only few infected tubular cells. Within weeks, interstitial fibrosis developed, often marked around virally affected tubuli. Outcome: Creatinine levels deteriorated over 7 months from 291±178 to 484 ± 326 IlmoIII with 2 graft losses (total of 5 patients). (B Primates: In addition, histology in primates showed: infection of native kidneys WIt and I without nephritis), infection of the ureter and allograft (with fulminant vascular rejection), infection of mesenchymal cells. Conclusion: Patients under tacrolimus with 'decoy cells' in urine cytology and deteriorating renal function might have a PV infection of their grafts. Histological changes are characteristic and indicate poor prognosis. The pathobiology of the interstitial infiltrate (i.e. cellular rejection vs. 'secondary nephritis') is undetermined, however, appears to be rejection in primate· allograft models. These observations are crucial for patient management.

265 PS3 OVEREXPRESSION AS INDEPENDENT PROGNOSTIC MARKER IN SOFT TISSUE SARCOMAS IS ANTIBODY-DEPENDENT P. Wiid (a.G.);H. Taubert (a.G.),*A. Meye (a.G.), D. Berger (a.G.), *H.-J. Holzhausen~H. Schmidt (a.G.), R. Hinze~F .•W. Rath Department of General Surgery and Institute of Pathology, Martin-Luther-University Halle-Wittenberg, Halle/Saale, Germany Aims: The role ofp53 detection by immunohistochemistry as independent prognostic marker in soft tissue sarcomas (STS) has not been clarifyed completely. Methods: 198 primary STS of 6 entities with 15 known p53 mutations embedded in paraffin were investigated for p53 overexpression with 5 different p53 antibodies. The results of staining were correlated with the clinical outcome of all 198 patients Results: The used antibodies had different positive staining rates (CM-I 61.1%; Pab240 61.1%; Pabl801 62.6%; 00-158.2%; 00-7 36.2%) without differences between the entities. In a multivariate cox regression modell (adjustment to stage, entity, localisation and therapy) only DO-I, 00-7 and Pabl801 showed a correlation to survival in a different grade. CM-I and Pab240 had a univariate but not a multivariate correlation. Conclusion: p53 overexpression is of importance for STS. Using antibodies with an epitope in the N-terminal region of the p53 protein (DO-I, 00-7, Pab 180 I) the immunohistochemistry is an independent prognostic marker for STS.

267 Atrophic and hyperplastic human renal tubular epithelia express the embryonal gene PAX-2 in contrast to renal cell carcinoma H.-J. Gltlne', U. Rothenpiele~ and E.F. Grllne' 'Pathologisches Institut, Universit~t Malburg 2Abteilung fOr Innere Medizin, Ludwig-Maximilians-Universitiit. MUnchen The paired-box gene family (PAX) plays a crucial role in the development of the nervous system and urogenital trael in mammals. Studies in PAX-2 knockout mice suggest that PAX-2 is necessary for the genesis of the ureter and metanephric kidney. PAX-2 mRNA and protein are downregulated in differentiated epithelium of the adult kidney. Tissue regeneration and reparation can be charaelerized by reexpression of developmental-control genes. Own experiments in mice and rat have shown an expression of PAX-2 in regenerating prOXimal tubular epithelia after acute renal failure. lt was now studied whether PAX-2 mRNA and protein are expressed in chronically damaged and hyperplastic tubular epithelia of the human kidney. Regular kidney and acquired polycystic kidney disease (APCD) (nephrectomy specimens each n=3), renal cell carcinoma (n=10), degenerative renal diseases (benign nephrosclerosis n=5, diabetic nephropathy n=4), immunologic glomerular disease (lgA nephritis n=6, membranoproliferative glomerulonephritis n=5) and chronic interstitial nephritis (n=3 renal biopsies) were analyzed. In situ hybridization of PAX-2 mRNA was done on fonnaldehyde-fixed sections with digoxigenin-Iabeled RNA transcripts. Immunhistology for PAX-2 proein was done by the APAAP and immunogoldsilver enhancement techniques with a polyclonal rabbit antibody after protease or microwave treatment of sections. In nonnal human adult kidney only medullary and cortical collecting tubules and medullary distal tubules showed PAX-2 expression. In APCD, epithelia, lining cysts, and covering papillae were strongly positive for PAX-2 mRNA and protein. Chronically damaged proximal tubular epithelia exhibited an irregular expression of PAX-2 mRNA and protein irrespective of the undertying disease. PAX-2 label was often cytoplasmic instead of solely nuclear. In areas of fragmented tubules interstitial fibroblastlike cells also showed PAX-2 protein expression. Renal celt carcinomas were mostly negative for PAX-2 protein (n=8) or had a faint spotty positivity in tubular fonnations of carcinoma celts (n=2). Thus, PAX-2 is not only reexpressed in reversibly damaged regenerating epithelia but also in chronic tubular lesions and chronic proliferating benign lesions, perhaps indicating an ongoing but vain differentiation process in chronic damage. The mesenchymal expression of PAX-2 points to a mesenchymal-epithelial conversion ability of PAX-2. PAX-2 does not seem to be important in renal carcinogenesis.

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PATTERNS OF GENOMIC ALTERATIONS IN CLEAR CELL RENAL CARCINOMAS F. Jiang (a.G.)*, J. Richter (a.G.)*, L. Bubendorf (a.G.)*, A. Schaffer***, R. Desper***, Th. Gasser (a.G.)**, G. Sauter*, H. Moch*, M.J. Mihatsch* Institute for Pathology* and Urologic Clinics**, University of Basel, Switzerland, National Human Genome Research Institute***, NIH, Bethesda, USA Aims: A number of tumor suppressor genes have been implicated in renal cell carcinoma (RCC) initiation and progression. Loss of chromosome 3p is regarded as an early event in development of clear cell RCC. However, the genes involved in tumor progression are not well characterized. Comparative Genomic Hybridization (CGH) allows detection of DNAsequence copy number gains and losses in large sets of archival tumor tissue. These alterations are of interest since they may pinpoint the location of unknown oncogenes and tumor suppressor genes. Methods: 116 clear cell RCC were analyzed by CGH. DNA was extracted from microdissected archival tumor tissue. The tumors were staged according to VICe. A chi-square test was applied to analyze which chromosomal changes occur together an unexpected number of times. A significance level of < 0.00062 after applying a Bonferroni's correction was assessed to be significant at the overall nominal level of 0.05. Results: In all tumors, deletions were most prevalent at 3p (61%), 4q (50%), 6q (40%), 9p (35%), 13q (37%), and Xq (21%). Gains of chromosomal material were most frequently seen at chromosome 17 (20%), 5q, 9q (14% each), and Xp (17%). There were no high level amplifications. There were no statistical differences in the prevalence of specific chromosomal changes in stage 2 and stage 3 tumors. Interestingely, there were groups of alterations that were found to occur a disproportionate number of times. Chromosome 13q losses occurred frequently together with losses of 4q, 6q, 9p and gains of 17q (p<0.OOOO14). Another group of chromosomal alterations included combinations of chromosome 5q, 2q, Ip and l2q losses (<0.00015). Conclusions: These data show that stage 2 and 3 clear cell RCC are genetically highly complex. Since there are significant associations between specific chromosomal alterations, the results provide evidence that a loss of multiple tumor suppressor genes in preferred combinations may be required for progression of RCC.

Metastatic clear cell renal cell carcinoma (RCC); a cytogenetic differential approach B. K. Amo-Takyi, S. Handt, B. Gunawan, H.-G. Hollweg, L. FUzesi Institut fllr Pathologie, Medizinische Fakultit, RWTH Aachen Aim: To trace the origins of two clear cell tumours in a 70-year-old Caucasian woman; one in the thyroid gland, and the other in the skin, 16 and 20 years respectively after tumour nephrectomy abroad. It was to find out whether cytogenetics could be used for a conclusive diagnosis between primary clear cell thyroid carcinoma and its cutaneous metastasis on one hand, and thyroid and cutaneous metastases ofRCC on the other hand. Methods: Paraffin sections of the previously formalin-fixed thyroid tumour, and the fresh cutaneous tumour were stained with HE and PAS. Samples of both tumours were also prepared for electron microscopy. Immunohistochemistry with antibodies against thyroglobulin, pancytokeratin and keratin subtypes 7, 8, 18, and 19, chromogranin, calcitonin, CEA, vimentin and epithelial membrane were performed. 5-6 flm sections of both tumours were analyzed with a-satellite probes of chromosomes 3, 7 and 17 using chromosomal in situ hybridization (CISH). The cutaneous tumour was also cultured and analyzed cytogenetically. Results: The thyroid tumour displayed some follicle-like structures that stained positive with both PAS and thyroglobulin, giving evidence of possibly entrapped thyroid follicles in metastatic RCC. The cutaneous tumour was negative for both PAS and thyroglobulin. There was no evidence of neurosecretory granules in the tumours. Classical cytogenetical analysis of the cultured cutaneous tumour cells revealed monosomies 3 and 14, well-known specific primary and secondary aberrations respectively in clear cell RCC; and hitherto not reported in thyroid carcinomas. Targeted CISH of both tumours revealed monosomy 3, indicating a cytogenetical correlation between them. There was no evidence of typical chromosomal aberrations for thyroid carcinomas like structural changes on 10q, structural rearrangements or translocations involving chromosome 7. Conclusion: Although neither histological sections, nor paraffin blocks of the original nephrectomy specimen abroad were available for review, the original tumour was on record there as clear cell RCC. Therefore the two tumors' renal origin was confirmed.

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Comparative cytogenetic investigations in renal ceO tumors H. Burger (a. G.), R. Simon (a.G.), T. Dijkhuizen (a.G.)**, C. Barkhausen (a.G.), O. Brinkmann (a.G.)*, E. van den Berg (a.G.)**, St. St6rkel***, W. BOeker, L. Hertle (a.G.)*, H.J.Terpe ~har~-DomaJdc-Institute of Pathology and *Department.of Urology Umverslty of Munster, Germany, **Department of Medical GenetICS, University of Groningen, Gromngen Netherlands. ***Institute of Pathology, University ofWitten-Herdecke, Wuppertal, Germany.

CYCLIN Dl EXPRESSION IN RENAL CELL CARCINOMA H. Moch*, U. Wagner (a.G.)*, A. Dastoor (a. G.)*, Th, Gasser (a.G.)**, G. Sauter*, MJ. Mihatsch*; Insitute for Pathology, University of Basel; Switzerland Aims: Cyclin Dl is a cell-cycle regulator and candidate proto-oncogene implicated in the pathogenesis of numerous tumor types. However, no studies have examined the role of cyclin D I in renal cell carcinoma (RCC). The aims of this study were to characterize the incidence and prognostic value of cyclinD I overexpression in renal cell carcinomas (RCC). Methods: Protein expression of cyclinD 1 was evaluated by immunohistochemistry using the P2DllFli antibody (Novocastra, Newcastle, UK) in 204 RCC, Any cells showing nuclear immunoreactivity were scored as positive. CyclinDl expression was determined in tumor areas with the highest density of cyclinDl positive cells. Clinical follow-up data were available in 135 tumors. The prognostic value of cyclinDl expression was estimated by the KaplanMeier statistic. A cox regression analysis was used to test for independent prognostic information. Results: By immunhistochemistry, 78% of RCC showed evidence of nuclear cyclinD 1 expression. There. was a heterogenous expression of cyclinDI, ranging from few to about 80% positive cells within the tumors. 20% positive cells was used as a cutpoint to define groups with low and high cyclinD I expression. High cyclinD I expression was detected in 50% of clear cell RCC, but in only 3 of 25 papillary RCC and not in chromophobe RCC (n=6). Tumors with high cyclinDl expression were found more frequently in low grade tumors (p=O.04) and low stage tumors (p
Aims; Renal cell tumors can be subdivided into distinct enlJlJes by morphological and cytogenetical criteria. The most frequent aberrations involve chromosomes I, 3p, 7, 17 and Y. Furthermore several genetical alterations associated with tumor progression have been described. Our investigation discusses cytogenetic alterations using a panel of cytogenetic methods in comparison to morphology and cytology. Methods: 101 renal cell tumors (clear cell n=67, chromophilic n=18, chromophobe n=3, Duct-Bellini n=5 carcinomas and oncytomas n=8) were investigated by conventional cytogenetic, comparative genomic hybridization (CGH) and fluorescence-in-situ hybridization (FISH), using DNA probes for centromer 1, 3, 7,17, Y and band 3p21. Results: l.Cytogenetic aberrations that are known to be associated with morphology and progression detected by CGH and FISH were confirmed by classical cytogenetics 2. Furthermore CGH can give evidence for unbalanced translocations involving chromosome 3 (t3;5, t3;6) frequently found in clear cell carcinomas. 3. FISH analysis provides further informations of tumour heterogenity and ploidy. 4. For verification and quantification of CGH-profiles another data from FISH are helpful. 5 Additional, cytogenetic aberrations could not be detected using these methods. Conclusions: CGH and FISH are powerful tools to detect almost all chromosomal aberrations important for carcinogenesis and progression of renal cell tumors. In further clinical-pathological studies all relevant cytogenetic data can be obtained using these methods.

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Expression of matrix metaUoproteinue 2 aDd 9 and their inhibiton in renal cell carcinoma B. Hernmerlein (a.G.), A Kugler (a.G.):P. Thelen*(a.G.), R:-H. Ringert* (a.G.), H -J. Radzun, Zentrurn Pathologie, Abt. Pathologie und*Zentrurn Chirurgie, Abt. Urologie, Universitit GOttingen Aims: The purpose of the present study was to analyze the expression and balance of MMP-2 and -9 and their inhibitors TIMP-I and -2, in order to yield more information about factors influencing spread and metastasis of renal cell carcinomas. Methods: Total RNA of 17 renal cell carcinomas, 6 tumour-free renal tissues, and 5 primary cell cultures was prepared and integrity of RNA was proved by RT-PCR of Interleukin-Ia. First strand eDNA synthesis ofMMP-2, MMP-9, TIMP-I and -2 was performed using Ilig of total RNA, MULV reverse transcriptase and genespecific primers. PCR was carried out in a final volume of 100 III with a three step temperature profile. PCR cycles, primer concentration, and annealing temperature were optimized to yield maximum efficiency of amplification. PCR products were compared using an external Low DNA Mass Standard, a video densitometer, and computer analysis system (E.A.S.Y. Win 32, Herolab). For comparison the total MMP-2 and -9 and ofTIMP-l and2 expression was used. Data were compared with the grade of differentiation and tumour stage. Results: I. Compared with tumour-free tissues MMP expression was 2 times higher in locally confined carcinomas and 4 times higher in advanced tumours. 2. No difference was found concerning the expression ofTIMPs. 3. The MMP:TIMP ratio is significantly increased in advanced tumours and grade 3 carcinomas in comparison to locally confined, grade 2 tumours or tumour-free tissue. This is due to the different expression levels ofMMP. 4. 'As shown in primary cell culture carcinoma cells are able to produce TIMPs in addition to MMPs. Conclusions: MMP expression charscterizes aggressively growing renal cell carcinomas. Tumour cells are also capable of producing TIMPs. A pharmacological induction of TIMP-expression may lead to a more balanced MMP:TIMP ratio and may reduce the risk of spread and metastasis.

INDUCTION OF CD95-MEDIATED APOPTOSIS IN HUMAN RENAL CARCINOMA CELL LINES U.Ramp, E.Ebel, K.Jaquet, M.Dejosez, H.E.Gabbert, C.D.Gerharz Institut fur Pathologie, Moorenstr. 5, 40225 DUsseldorf Aims: Human renal cell carcinoma (RCC) is known to be resistant against conventional chemotherapy and irradation, which might be due to defects in apoptotic pathways. Since the CD95 (APO-ifFas) system has been shown to be involved in apoptosis induced by chemotherapy, we analyzed the expression ofCOO5 receptor and ligand as well as the inducibility of CD95-dependent apoptosis in 26 newly established human RCC cell lines. Methods and results: I. As shown by RT-PCR, all 26 RCC cell lines expressed CD95-receptor rnRNA and 22 RCC cell lines exhibited the corresponding CD95-ligand. 2. Exposure to the CD95 agonistic antibody CHI 1 (500 nglml) resulted in a 7 - 31 fold increase of apoptosis in 5 out of26 RCCs, as determined by light microscopic counting. 3. MIT-assay revealed that induction of apoptosis by CHI I antibody was accompanied by a significant reduction of cell number (p<0.05; 66 ± 3 % of the control in the most responsive cell line). 4. Pretreatment of CHI I-responsive cell lines with IFN-gamma (100 U/m1) resulted in a marked (9 - 45 fold) augmentation of apoptosis and decrease of cell number (p<0.05; 10 ± 4 % of the control in the most responsive cell line), whereas exposure to IFNgamma alone exhibited only minor effects. 5. IFN-gamrna pretreatment could overcome CHI I-resistance of3 other RCCs resulting in a marked (2 - 12 fold) induction of apoptosis and reduction ofcell number (p<0.05; 66 ± 2 % of the control in the most responsive cell line), whereas exposure to IFN-gamma alone exhibited only minor effects. 6. 2 out of 8 apoptosis-inducible RCCs exhibited a p53 point mutation as revealed by DNA sequencing. 7. Northern blot analysis of positive (bax) and negative (bcl-2 and Bag-I) regulators ofapoptosis downstream the CD95 (APO1fF as) system revealed no correlation to the inducibility of apoptosis by CHI I-antibody. Conclusions: CD95 mediated induction ofapoptosis is possible in a significant proportion ofhurnan RCCs. Induction ofapoptosis in p53mutated RCC cell lines suggests the existence ofp53-independent pathways for CD95-triggered apoptosis.

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APOPTOSIS IN RENAL CELL CARCINOMA: IN·SITU END LABELLING, LIGHT MICROSCOPIC ASSESSMENT AND ULTRASTRUCTURE Andrea Tannapfel, Ch. Wittekind Inst. of Pathology, University of Leipzig AIMS: To compare in situ end labelling (ISEL) of apoptosis in renal cell carcinoma and suurounding non-neoplastic renal tissue with quantitative and semiquantitative assessment of HE stains and ultrastructUral observations, t1iis study was performed. MATERIALS AND METHODS: In situ end labelling was performed of paraffm-embedded material of 25 renal cell carcinomas and in corresponding non-neoplastic renal tissue of the patients. The number of ISEL positive cells were evaluated using an Image Analysing System (CYRUS, Zeiss, Germany). The results obtained were correlated With the extent of apoptosis in Hematoxylin and Eosin stained sections and with ultrastructural, electron microscopy observations. RESULTS: ISEL-~ositive cells were found in all tumors examined to various extents Without a spatial predominance within the tumors (median: 4.9+/- 2.1 [1-151). We observed a significant correlation between the number of cells Identified with ISEL and that observed by HE stain (median: 3.2 +/- 1.5 (1-14], ~ < 0.01). In normal, non-neoplastic kidney, few ~lomerular panetal epithelial cells and distal tubular cells were apoptotlc by means ofISEL and electron microscopy (median: 0.52 +/- 0.36 L0-2]). In the peritumorous tissue, a higher frequency of apoptotic cells was observed, in most cases accompanied by a dense inflammatory peritumorous infiltrate. Renal cells undergoing apoptosis showed uniform ISEL staining within their nucleus with a surrounding "halo" or peripheral nuclar membrane staining. The membrane staining pattern correlated ultrastructurally with early a~optosis. Some apoptotic cells and apoptotic bodies, clearly identified WItii electron microscopy, were unlabelled. Necrosis within the tumors exhibited a weakly non-specific staining pattern, but were easily distinguishable from apoptotic cells by light mICrOscopy. CONCLUSIONS: Our results indicated, that ISEL-technique is a useful adjunct for assessing apoptosis if used in conjunction with standard morpho!o¢cal,methods. Iri renal tissue ~h~re apoptosis is difficult to assess by Itght mIcroscopy, ISEL can help m IdentIfymg early stages of apopto-

PROGNOSTIC SIGNIFICANCE OF CHROMOSOMAL ABERRATIONS IN SUPERFICIAL BLADDER CANCER M.Neuhaus (a.G.), H.Moch (a.G.), U.Wagner (a.G.),3U.Schmid, 4R.Maurer,Z'f.c.Gasser (a.G.), M.J.Mihatsch, G.Sauter. Institute of Pathology and 2Urologic Clinics. University hospital Basel,3Institutes of pathology of the Cantonal nospital St.Gallen and the 4Triemli hospital Zurich, Switzerland.

SIS.

Backeround: A disturbed cellular DNA content is of potential diagnostic and prognostic significance in urinary bladder cancer. ~ To evaluate the prognostic significance of specific chromosomal aberrations in superficial bladder cancer, formalin fixed tissues of 105 tumors (67 pTa, 38 pTJ) were examined by fluorescence in situ hybridization (FISH). FISH allows visualization and quantitation of chromosomes on a cell by cell level. Centromere probes for the chromosomes Y, I, and 17 were used. The median follow up period of our patients was 58 months. ~ A polysomy 17 was found in 40%, a polysomy I in 46% of tumors. There was a strong association between polysomies of the chromosomes I and 17 (p
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Genetic alterations of chromosome 8 in transitional cell carcinoma of the bladder investigated by CGH, FISH and double differential PCR (ddPCR) R. Simon (a.G.), A. Beckmann (a.G.)", H. Buerger (a.G.), W. Boecker, L. Hertle (a.G.)"", B. Brandt (a.G.)", H.-J. Terpe Gerhard-Damagk-Institute of Pathology, "Institute of Laboratory Medicine and ""Department of Urology, University of Miinster, Germany Aims: Genetic alterations of chromosome 8 frequently affect a variety of solid tumors including bladder cancer. Recently, frequent gains of 8q, and 8q2l-q22 in particular, give evidence for a novel candidate oncogene site. It was the aim of our study to get further insight into chromosome 8q sequence changes. Methods: A set of 78 paraffin embedded bladder cancer samples (pTa n=20, pTi n=26, pT2-pT4 n=32; Gl n=3, G2 n=37, G3 n",,38) was investigated by comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH) analysis of centromeres 7, 8 and the MYC locus. Gene dosage estimation of MYC was performed by double differential PCR (ddPCR) using primers located inside ofthe coding regions of the MYC gene and the reference genes 13globin and S002. Digital image analysis after gel electrophoresis was performed using Scanpack 1.0 (Biometra). Results: 1. 51% (40/78) of the tumors investigated showed abnormalities of chromosome 8. 2. Sequence copy number changes and polyploidy of chromosome 8 were found to be associated with both tumor stage and grade. 3. In all cases, 8p was affected by losses while 8q showed gains of chromosomal material. 4. Losses of 8p effected either the whole short arm or 8p II-pter. Gains of 8q were seen as well for the whole long ann, 8cen-q22, 8q1O-q22, 8qlO-qter or 8q22-qter. In four cases, CGH revealed amplifications at 8q21-q22 and in two cases at 8q24. 5. Losses of 8p material without simultaneous gains of 8q were found in pTa and pTi grade 2 tumors only, whereas gains of 8q without losses of 8p were detected only in grade 2 and mainly grade 3 pTi and pT2-pT4 tumors. 6. FISH and ddPCR analysis revealed that the MYC oncogene showed mainly low level copy gains. Conclusions: Our data give evidence that gains of 8q material are probably due to the formation of differently sized isochromosomes [i (8q)] rather than oncogene activation.

PROGNOSTIC SIGNIFICANCE OF CYCLIN Dl EXPRESSION IN SUPERFICIAL BLADDER CANCER. U.Wagner (a.G.), K.SUess (a.G.), H.Moch (a.G.),3U.Schmid (a.G.)!R.Maurer (a.G.),2T.C.Gasser (a.G.), MJ.Mihatsch, G.Sauter. Institute of Pathology and 2Uroiogic Clinics, University hospital Basel,3Institutes of pathology of the Cantonal hospital St.Gallen and the 4Triemli hospital ZUrich, Switzerland.

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EVIDENCE OF POLYCLONAL ORIGIN OF BLADDER CANCER DETERMINED BY DUAL COLOUR FLUORESCENCE IN SITU HYBRIDIZATION. A. Hartmann', U. Roessner', M. Kriegrnaw, F. Hofstaedter', R. Knuechel, 'Institute of Pathology, University of Regensburg, and Dept. ofUrology, Ludwig Maximilian University Munich, Gennany Aims: Papillary bladder tumors biopsied during photodynamic diagnosis with 5-aminolevulinic acid were used to assess clonality of early tumor stages, and to judge on the prevalence of 9 p versus 9 q deletions in comparison to changes of the p53 gene locus. Methods: An overall of 53 biopsies of 10 patients were used to microdissect urothelium from 20llm frozen sections. Fluorescence in situ hybridization was done with a dual colour staining technique of biotinylated centromeric probes of chromosome 9 and 17 and the digoxigenin labeled gene locus probes FACC (chromosome 9q22), CDKl2 (chromosome 9p21) and p53 (chromosome 17p). Signals from a minimum of ISO and a maximum of350 cells were counted. Results: Tumor stages were 31 pTa G1, 20 pTa G2, and 2 pTl G3 tumors, number of multiple tumors ranged from 2-6, and recurrencies were observed between 4 and 32 months. As expected deletion and monosomies on chromosome 9 prevailed (75%) with less than 10 % p53 deletions in the tumors investigated. Interestingly, 9p deletions were found more frequently than 9q deletions. While some of the differentiated tumors showed no genetic changes in respect to the 3 gene loci investigated, and the majority of patients had clonal tumors, there were 3 patients with distinctly different genetic changes in multiple tumors, indicating 2 seperate and independently recurring clones. Conclusions: Although bladder cancer seems to be a clonal disease in the majority of cases our data indicate the possibility of polyclonal origin of papillary bladder cancer. The role of chromosome 9 to determine clonality is emphasized, and 9p deletion as the earliest event in the development of bladder cancer is suggested.

Backeround; The biological behavior of urinary bladder neoplasms is determined by a malfunction of multiple tumor suppressor genes and oncogenes many of which may have a role in cell cycle regulation. Cyclin D I is involved in cell cyele control, probably playing a role in progressing cells through G1 phase. Studies have suggested that Cyclin Dl expression may be relevant in bladder cancer, however, the prognostic significance of Cyclin D I expression is unknown. ~ Cyclin Dl expression was evaluated by immunohistochemistry in 392 bladder carcinomas (P2DIIFII). Proliferative activity was determined by counting the Ki67 labeling index (MIB 1). Follow up information was available in 337 patients with superficial bladder tumors (stages pTalpTl). ~ Cyclin 0 I overexpression was linked to low stage and grade. A cyelin Dl positivity was found in 119 of 234 pTa, 49 of 119 pTl, and in 8 of 39 pT2-4 carcinomas (p--Q.OOI2). Cyclin Dl was detectable in 53 of 92 grade 1,89 of 188 grade 2 but only in 34 of 112 grade 3 carcinomas (p=O.0003). Cyclin Dl positivity was also linked to papillary growth pattem and was detectable in 172 of 355 papillary but only in 4 of 37 solid tumors (p
FREQUENT AMPLIFICATION OF THE CYCLIN 01 LOCUS ON CHROMOSOME l1q13 IN HIGH GRADE BLADDER CANCER R. Knuechel, A. Hartmann, M. Wallinger, F. Hofstaedter, Institute of Pathology, University of Regensburg, Germany. Aims: CyclinDl (COl) plays an important role in the control of the Gl- restriction point of the cell cycle. We recently reported overexpression of CD1 in 65% of papillary superficial bladder carcinomas. In this study we evaluated the frequency of COl gene amplification in bladder cancer in correlation to the protein overexpression assessed by immunohistochemistry. Methods: An overall of 36 bladder cancer biopsies were used to microdissect urothelium from 20llm frozen or paraffin sections. Fluorescence in situ hybridization was done with a dual colour staining technique of a directly labeled spectrum green centromeric probe of chromosome 11 and a spectrum orange labeled gene locus probe (CDl-11q13, Vysis). Signals from a average of 200 cells were counted. Immunohistochemistry was done with a three step immunoperoxidase technique using a mouse anti human monoclonal antibody (DCS6, Progen). Results: Overall, an amplification of the CDI gene locus with a gene/centromer- ratio>1.4 was found in 10 of the 36 bladder cancers (27.7%). All amplified tumors showed an overexpression of the COl protein. Whereas only 2 of 21 well differentiated tumors (pTalIGi/2) showed an amplification, 8 of 15 undifferentiated G3 tumors were amplified. Interestingly, 2 of3 pTaG3 ~d both investigated pTlG3 tumors were amplified. The majority of pTaGI-2 tumors with overexpression of CDI protein showed no amplification of the gene. The majority of amplified tumors showed a high level of amplification with both a intra- and extrachromosomal localization. Conclusions: Amplification of the cyelin 01 locus at the llq13 amplicon can be found in approximately half of the high grade bladder cancers, whereas the frequent overexpression of the CD1 protein in well differentiated tumors is only rarely due to an gene amplification.

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PATTERNS OF CHROMOSOMAL IMBALANCES IN ADVANCED URINARY BLADDER CANCER. J. Richter (a.G.), L. Beffa (a.G.), F. Jiang (a.G.), U. Wagner (a.G.), T. Gasser* (a.G.), U. Wagner (a.G.), H. Moch, M. Mihatsch, G. Sauter. Institute of Pathology and Urologic Clinics*, University of Basel, Switzerland. Background: Progression of urinary bladder cancer is driven by a malfunction of specific genes. However, many of the genes involved in this process are yet unknown. To identify these genes, cytogenetic changes are of interest since they may pinpoint the location of yet unknown oncogenes and tumor suppressor genes. Design: To learn more about the cytogenetic changes that are linked to advanced bladder cancer we analyzed 90 bladder carcinomas by comparative genomic hybridization (CGH). The series included 37 papillary pTI (minimally invasive), 24 papillary pT2-4 (deeply invasive), and 29 solid pT2-4 carcinomas. CGH allows to screen for all relative DNA copy number gains and losses present in a tumor. . ~ In all tumors, deletions were most prevalent at 8p (33%), 9p (30%), lip, (24%) 9q (23%), 6q- (22%), and Ilq- (22%). Gains of chromosomal material were most frequently seen at 8q (38%), Iq (38%), 20q (29%), l7q (29%), and 5p (24%). Thirty high level amplifications were seen at 12 different sites including Iq22-24, 3p24, 5pI4-15, 6p22, 8q21-22, IOp13-14, IIq13, 12q15, 13q33-34, 17q21, 18pII, 20q13 and Xp2I. There was no difference in the number of aberrations per tumor (mean 7.5) between carcinomas having different stages, grades and growth patterns. Deletions of 5q, 6q, and 15q as well as gains of 5p and Xq were significantly more frequent in pT2-4 than in pTI carcinomas. These loci may harbor genes contributing to the further progression of bladder tumors having already the potential of invasive tumor growth. Deletions of 9q were significantly linked to papillary tumor growth (p<0.05). Interestingly there were groups of alterations that frequently occurred together such as 9p- and II q13+, 20q+ and II q13+ or 17q+, Iq+ and 3p+ or Ilq-. These loci might carry genes that interact with each other in specific molecular pathways. Conclusion: These data show that advanced bladder carcinomas are highly complex on a genetic level. Distinct genetic differences between the stages pTI and pT2-4 may allow an improved molecular staging of these tumors, potentially on the basis of cytologic specimens.

THE CLINICAL SIGNIFICANCE OF INCIDENTAL PROSTATE CANCER (PTlalb). Lukas BUbendorf,*Jorg Gysin, Guido Sauter, Holger Moch;"Thornas C. Gasser and MJ. Mihatsch, Institute of Pathology and"Urologic Clinics, University hospital Basel, Switzerland.

281 DETECTION OF MORPHOLOGICAL DIFFERENCES BETWEEN NUCLEI OF THE BENIGN NODULAR HYPERPLASIA, PRENEOPLASIAS AND CANCER OF THE PROSTATE BY HIGH RESOLUTION NUCLEAR IMAGE ANALYSIS

AIMS: Incidental prostate cancer is found in 16% of transurethral resections and transvesical enucleations from patients with presumed benign prostatic hyperplasia, and shows variable biological behaviour. There is still no consensus about the histological criteria to best define pTIa and pTIb stage as a mean to guide therapy decisions. We therefore sought to investigate the relationship of histologic features of incidental prostate cancer with long term prognosis. METHODS: The influence of the histological features of incidental prostate cancers from 186 transurethral resections and 25 transvesical enucleations on progression, overall, relative and tumor-specific survival was analyzed (mean follow-up 5.5±3.5 years). RESULTS: The histologic grade (Gleason), the percentage of chips involved, the presence of perineural invasion, and luminal cristalloids were strongly interrelated, significant predictors for all clinical endpoints, whereas the absolute number of involved chips provided no significant prognostic information. Among the tumors with ~% of the chips involved, histologic grade allowed the stratification of two additional groups with excellent and unfavorable long-term prognosis, respectively. The overall survival after 6 years was 62% in low grade tumors (Gleason score 2-6) but only 18% in high grade tumors (Gleason score 7-10). Interestingly, a high patient age (>76 years) at the time of diagnosis proved to be a strong and independent predictor of early progression and tumor associated death. CONCLUSIONS: Histological features can provide useful information to predict prognosis as a help to guide therapy decisions in incidental prostate cancer. The combined evaluation of the percentage of chips involved and the histologic grade (Gleason) appears most useful to separate stage pTIa tumors with low aggressive potential from stage pTIb tumors with high aggressive potential.

Oral Communications: Gastroenteropathology

283 EXPRESSION OF CARBOHYDRATE CORE ANTIGENS IN ESOPHAGEAL SQUAMOUS CELL CARCINOMAS

U. Flucke (a.G.), S.E. Baldus, T.K. Zirbes, V. Koch (a.G.), K. Schmitz (a.G.), I. von Both (a.G.), S.P. Monig (a.G.)*, W. SchrOder (a.G.)*, J. Thiele, A.H. Holscher (a.G.)*, H.P. Dienes

K. Friedrich (a.G.), V. Dimmer (a.G.), G. Haroske, W. Meyer (a.G.), A Rothert (a.G.), F. Theissig (a.G.), K.D. Kunze Institute of Pathology; University ofTechnology Dresden

Institutfiir Pathologie, *Klinikfiir Chirurgie, Universitiit zu K61n, 50924 K61n

Aim: The study was designed to detect differences between the nuclear populations of the benign nodular hyperplasia (BPH), the preneoplasias and the prostate cancer. Further points of interest were changes in the nuclear morphology of the prostate cancer in correlation to clinicopathological features. Methods: Paraffin sections from 53 radical prostatectomy specimens were stained according to the Feulgen method. 200 nuclei each from the cancer, the preneoplasias and the benign nodular hyperplasia were measured by a high resolution image cylometry workstation. From each nucleus 135 nuclear features derived from the segmented extinction image were computed on a MicroVAX 4000 computer. Results: The univariate and multivariate statistical analysis of the nuclear populations of the BPH, the prostatic inintraepithelial neoplasia (pIN) and the prostate cancers reveal differences in features of the nuclear size and shape, the chromatin amount and the chromatin distribution between these nuclear populations. The comparison of the BPH and the PIN on one hand and the PIN and the cancer on the other hand shows also differences especially in features of the chromatin distribution and amount. The nuclei of cancers with lymph node metastases and cancers with capsular penetration, respectively exhibit a higher irregularity especially in features of the chromatin distribution than their counterparts without these unfavorable prognostic criteria. Conclusions: The processes of malignant transformation and tumor progression, reflected by the clinicopathological features, are associated with changes in the nuclear morphology, especially with the chromatin amount and distribution. These changes are detectable by the high resolution image analysis.

Aims: Tumor-associated carbohydrate antigens which are involved in cell-cell adhesion are discussed to exert a possible impact on prognosis in human cancer. During the last years, several authors demostrated an expression of blood group and sialosyl-Tn antigens in squamous cell carcinomas of the esophagus. Methods: We investigated formalin-fixed and paraffin-embedded specimens from 86 patients with esophageal squamous cell carcinoma using an ABC-peroxidase assay and various monoclonal antibodies (mabs): Mab A78-G/A7 detects the Thomsen-Friedenreich disaccharide (TF), whereas mab BW835 reacts with TF disaccharide only if bound to MUCI mucin peptide core. Peanut agglutinin (PNA) also recognizing TF antigen exhibits cross-reactivities with related carbohydrate structures. Mab B72.3 is directed against sialosyl-Tn antigen. Results: 42 specimens (48.8 %) showed BW835 reactivity, whereas only 34 (39.5 %) exhibited A78-G/A7 staining. On the other hand, PNA-binding carbohydrates were expressed by 84 tumors (97.7 %). Sialosyl-Tn antigen identified by mab B72.3 was detectable in 42 cases (48.8 %). Ifthe staining patterns were analyzed in relation to TNM stages or grading, no significant correlations were obtained. Univariate survival analysis according to Kaplan-Meier did not reveal a prognostic impact of any of the antigens under study. Conclusions: TF as well as sialosyl-Tn antigen detected by mabs are expressed in ca. 50 % ofthe esophageal squamous cell carcinomas. The strong PNA staining may be explained by the cross-reactivity with related carbohydrate antigens. These structures may be in involved in adhesion phenomena, however, their clinicopathological value is obviously limited.

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MOLECULAR MIMICRY IS NOT RESPONSIBLE FOR THE FORMATION OF ANTIGASTRIC AUTOANTIBODIES IN H. PYLORI GASTRITIS G. Faller (a. G.), H. Steininger, Th. Kirchner Institute of Pathology, University of Erlangen-NUrnberg

EXPRESSION AND MUTATION ANALYSIS OF THE TRANSFORMING GROWTH FACTOR-j3 RECEPTOR TYPE II AND MICROSATELLITE INSTABILITY IN GASTRIC CANCER H.-C. Wirtz, W. MUlier, M. Scheven, T. Noguchi, H.E. Gabbert, Institut fur Pathologie, Heinrich-Heine-Universitat DUsseldorf Aims: Recently, it has been reported that TGF j3 receptor type II (Tj3II) is mutated frequently in carcinomas of the mutator phenotype. In the present study the protein expression and mutation analysis of Tj3II was compared with the mutator phenotype and the prognosis in gastric cancer. Methods: Tj3II was analyzed immunohistochemically in 135 curatively resected paraffin embedded gastric carcinomas. At least 5 different microsatellite markers were analyzed in 130 of 135 microdissected gastric carcinomas. Mutator phenotype was accepted when microsatellite instability was detected in at least 30 % of the investigated loci. Sequence analysis of gDNA was performed within the poly(A) sequence of the Tj3II exon 3. Results: Tj3II immunoreactivity could be shown in 77 out of 135 (57.0%) gastric carcinomas. Mutations of the Tj3II exon 3 were observed in 5 out of 110 (4.5%) carcinomas investigated. Mutator phenotype could be verified in 14 of 130 (10.8%) carcinomas investigated. 4 of the 5 (80%) carcinomas with mutation of Tj3II were carcinomas of the mutator phenotype. Tj3II immunoreactivity could be found in 10 of 14 (71.4%) carcinomas of the mutator phenotype and in 4 of 5 (80%) carcinomas with mutations of Tj3I!. The mean survival time of Tj3II positive carcinomas versus Tj3II negative carcinomas was 2.83±2.58 years versus 2.4±2.36 years. Conclusions: In the present study it could be shown that Tj3II protein expression is preserved in the majority of gastric carcinomas with mutator phenotype. Furthermore, mutations within the Tj3II gene were correlated significantly with a mutator phenotype. In conclusion the TGF j3 signal transduction pathway plays a role also in gastric carcinomas of the mutator phenotype, but the functional consequences of Tj3II mutations still have to be elucidated.

Background/aims: Recent studies have shown that autoantibodies against canalicular structures within human parietal cells occur in a considerable proportion of H. pylori infected patients and that these autoantibodies are associated with gastric mucosa atrophy. It has been suggested that molecular mimicry between H. pylori and the host, in particular between Lewis X and Y antigens expressed in the LPS and on gastric epithelial cells, represents a relevant pathomechanism leading to antigastric autoimmunity in H. pylori gastritis. The aim of this study was to prove this concept by means of absorption assays using H. pylori strains posi tive for Lewis X and/or Y. Methods: 14 H. pylori infected patients were included in this study. H. pylori was determined by histology and serology. Using immunohistochemistry, antigastric autoantibodies reacting against canaliculi within human parietal cells were detectable in all sera. To analyse the possible role of molecular mimicry, all sera were absorbed to H. pylori Iysates containing Lewis antigens, as well as to E. coli and PBS as controls. Effective absorption was tested in an ELISA. To detect antigastric autoantibodies after absorption, the immunohistochemical procedure was performed again. Results: After absorption of the sera to H. pylori Iysates, the immunoreactivity of all sera fell below the cut value. In contrast, no decrease in anti-H. pylori reactivity was seen in the controls. Despite effective preabsorption, anticanalicular autoantibodies were still detectable. Conclusion: Molecular mimicry between H. pylori and the host, in particular on the level of Lewis antigens, does not seem to be responsible for the formation of antigastric autoantibodies in chronic H. pylori gastritis.

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GASTROINTESTINAL FIBROID POLYPS REPRESENT HYPERPLASIAS OF CD34-POSITIVE GIST-CELLS. F. Borchard, P. Wille Institut fUr Pathologie, Heinrich-HeineUniversitat, DUsseldorf

MDM2 GENE AMPLIFICATION IN GASTRIC CARCINOMASCORRELATION WITH MDM2 PROTEIN EXPRESSION AND CLINICOPATHOLOGIC FACTORS T. GUnther l , R. Schneider-Stock!, M. Pross 2, M. Ebert 3, H.-U. Kasper!, l A. Roessner !lnstitut fur Pathologie, 2Klinik fur Chirurgie, 3Klinik flir Gastroenterologie und Hepatologie; Otto-von-Guericke Universitat, Magdeburg Aims: This investigation aims at determining the frequency of MDM2 amplification in 43 patients with gastric carcinoma. The results obtained by genetic examinations are correlated with the immunohistologic evidence ofMDM2 and clinicopathologic data. Methods: We investigated 43 gastric carcinomas. The mean age of the patients was 63.6 years (33-81 years). Tumors were classified according to LAUREN (2 I intestinal type, 18 diffuse type; 4 carcinomas had mixed differentiation). For staging, the pTNM classification was used. MDM2 protein was detected with Clone 1810 (Novocastra, UK), applying the PAP technique. Tumor DNA was isolated by phenol chloroform extraction. IOllg were digested using restriction endonuclease EcoR!, separated on agarose gels, and transferred to a nylon membrane. Filters were hybridized with a human 404bp long MDM2-cDNA fragment, applying a non-radioactive labeling and detection technique. Assessment of Southern Blots was made densitometrically. Results: MDM2 amplification (more than three-fold) manifested in 13 tumors (30%). Seven carcinomas (16%) showed only a weak amplification (one to three-fold). A high portion of MDM2 gene amplification was seen in carcinomas with a diffuse growth pattern, in grade ill tumors of the intestinal type, and in nodal-positive tumors. MDM2 gene amplification correlated significantly with MDM2 protein expression. Conclusions: The up-regulation of MDM2 oncogene in association with the inactivation of tumor suppressor genes seems to play a role in the carcinogenesis of poorly differentiated gastric carcinomas, particularly in those of the diffuse type.

Aims: The histogenesis of gastrointestinal fibroid polyps (GIFP) is uncertain: it was formerly attributed to proliferations of mesenchymal cells of neural, histiocytic or endothelial origin. This stUdy was designed in order to study the histogenesis of GIFP by immunohistochemistry (IHC). Methods: 11 GIFP (9 gastric and 2 colonic) were investigated by IHC using 9 markers for various kinds of differentiation: (CD34, desmin, alpha-sm1, KP1, PGM1(CD68), MAC387, FVIII, CD31 and 8100. Results: In fibroid polyps there was a proliferation of submucosal perivascular mesenchymal cells consistantly positive for QBEnd (CD34), while neuronal (8100), myogenic (alpha-sm1, desmin), histiocytic (MAC387, KP1 and PGM1, partly corresponding to CD 68) and true endothelial markers (CD 31) were negative. Conclusions: Gastrointestinal fibroid polyps represent reactive proliferations of CD34-positive perivascular GI mesenchymal cells. According to our studies, the stem cells of these mesenchymal hyperplastic perivascular may be the putative progenitors of the recently detected true GI8Ttumors with no association to myogenic and neurogenic differentiation.

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Therapeutical effects of the apoptosis-inducing human monoclonal antibody SC-l in treatment of gastric adenocarcinoma

Tumor grade of ductal adenocarcinoma of the pancreas: its prognostic significance and reproducibility of assessment in comparison to immunocytochemical detection of proliferation activity J.Liittges, S.Schemm, I.Vogel, M.Pacena, G.Kloppel Institut fur Pathologie, Universitat Kiel

Vollmers, H.P.fa), Krenn,V.(a), Timmermann,W.(b), mert,B.(b), Zimmermann,U.(c), HenseI,F.(a), Hermann,R.(a), Thiede,A.(b) and Miiller-Hermelink, H.K.(a). Inst.f.Pathologie (a), Chirurgische Klinik (b), Inst.f.Biotechnologie (c), Universitiit Wiirzburg, Wiirzburg, Germany Background:The human monoclonal antibody SCot (IgM), isolated from a patient with a diffuse type adenocarcinoma of the stomach, inhibits stomach cancer growth in vitro and in vivo by inducing tumor-ceU specific apoptosis. Methods:For a clinical phase vn study eight patients with poorly differentiated adenocarcinoma received purified SCot antibody intraveneously prior to gastrectomy. Tumors and Iymphnodes were investigated immunohistochemicaUy for apoptosis-induction and morphologicaUy for antibody-induced regression. Results:Neither during nor after infusion the patients develop serious complications or toxic crossreactivity symptoms. Liver enzymes showed no significant changes induced by antibody infusion and aU patients are stiu alive five months after the start of the study. All primary tumors showed a significant increase of apoptotic tumor ceUs, and histologicaUy, tumor regression was observed characterized by a decreased ceUular density of carcinoma ceUs, an inflammatory infJItration of granulocytes and macrophages and the occurence of pycnotic nuclei. Normal tissues like liver etc. was not affected by the apoptotic activity. Conclusion:Specific induction of apoptosis might become a most promising approach in cancer therapy by isolation of human monoclonal antibodies against tumor-specific receptors directly from cancer patients.

Aims: Tumor grade has been shown an independent prognostic factor of the ductal adenocarcinoma of the pancreas. Using the defined criteria of the WHO (1996) its reproducibility was tested in regard to intra- and interobserver variance. Grade was also correlated to immunocytochemically determined proliferative activity, in order to find out wether the latter method is more objective. Methods: 55 cases of ductal adenocarcinoma of the panceas head (33 women, 22 men; mean age 59.9 ys) were histologically graded on H&E stained section with the evaluation of 60 HPF per case by two different observers. Corresponding slides were immunocytochemically stained with the MoAb Ki-S5, a nuclear proliferation marker. The percentage of positive nuclei and the staining intensity were evaluated. Results: There were 12 grade I, 13 grade II and 30 grade III tumors with a significant longer survival for grade I and 2. Intraobserver variance of the grading concerned 10% of the cases, which however differed only in one grade. The immunocytochemically assessed proliferation activity correlated well in the cases ofgrade 2 and 3, but weakly to grade I. Conclusion: Using standardized criteria tumor grade is an independent prognostic factor and can be reproduced reliably. Additional immunocytochemical stainings determining the proliferative activity are useful and faster to evaluate.

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K-RAS MUTATION AND PREVALANCE OF DUCT LESIONS IN THE NORMAL UNSELECTED PANCREAS A. Reinecke-LUthge, B. Mol1mann, M. Menke, J. LUttges, G.K1oppel Institut fUr Pathologie, Universitat Kiel

T~nascin as Potential ~ool for Identifying Cc:!lagen?us Colitis S. Muller, a. G.; D. Neurelter, a. G.; G. Verbeke, T. Aigner, a.G.; T. Kirchner Institute of Pathology, University Erlangen-Niirnberg • Institute of Pathology, Mannheim, University of Heidelberg

Aims: Recent molecular studies suggest that many duct lesions of the pancreas classified as "hyperplasia" or "metaplasia" might be potential precursor lesions of pancreatic ductal adenocarcinoma. K-ras mutations are known to occur in duct lesions as well as carcinomas. This study examines the type, prevalance, distribution and K-ras codon 12 mutation rate of duct lesions within the normal pancreas. Methods: Autopsy pancreases from 99 subjects (age range: 6 - 92) were screened for the presence of mucinous hypertrophy (MHT), adenomatoid hyperplasia (AH), ductal papillary hyperplasia with (DPH+) or without mild atypia (DPH-), and squamous metaplasia (SQM). In 38 pancreases cells of duct lesions, normal ducts and acinar tissue were microdissected and tested for K-ras codon 12 mutations. Mutations were detected by PCR amplification of the exon I of the K-ras gene combined with denaturinggradient-gel-electrophoreses (DGGE) and analyzed by sequencing. Results: The various types of duct lesions occur equally distributed in the pancreas. They are more common beyond the age of 40. MH was present in 55.2 %, DPH- in 20.3 %, DPH+ in 7,8 %, and SQM in 33.7 % of all pancreases. K-ras mutations were found in II different duct lesions (9 MHT, I DPH+, I SQM) from 9/38 pancreases (24 %). GAT and GTT mutations were most common (80%). Normal duct epithelium and acinar cells lacked K-ras mutations. Conclusions: K-ras codon 12 mutation occurs in the "normal" pancreas and is detetced in various types of duct lesions. Its presence does not inevitably leed to carcinoma, but certain K-ras mutation patterns may facilitate carcinogenesis in some duct lesions.

Aims: The diagnosis of collagenous colitis (cq depends on the histological-histochemical identification of characteristic subepithelial collagenous band-like structures. Recently, we have shown that collagen type VI and tenascin staining ig characteristic of the mucosa in CC. In this study, we tried to evaluate whether immunodetection of collagen type VI or tenascin is a potential tool for identification of collagenous colitis. Methods:. Histochemical and immunohistochemical analysis was performed on normal mucosas biopsies (43) as well as specimens showing collagenous colitis (21 including II borderline cases), Crohn's disease (28), ulcerative colitis (14), ischemic colitis (6), infectious colitis (6), and "so-called" chronic non-specific colitis (10). Statistical evaluation was done by the Chi-Quadrat-test and the Fisher's exact test. Results:. A specific evaluation scheme for type VI collagen and tenascin staining was established and tested in order to distinguish collagenous colitis from a) normal colonic specimens and b) specimens showing other acute or chronic inflammatory large bowel disease. Immunohistochemical detection of subepithelial tenascin was shown to be clearly positive in all cases with histologically characteristic collagenous colitis (p
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292 D-CATENIN EXPRESSION IN COLONIC POLYPS OF PEUTZJEGHERS (PJ) AND JUVENILE-POLYPOSIS (JP) SYNDROMES W. BACK!; C. HEUBNER (a.G.)I, S. LOFF (a.G.)2, D. JENNE (aGl Ipathol. Institut und 2Kinderchirurg. Klinik, Klinikum Mannheim, Fakultat fur Klinische Medizin del' Universitat Heidelberg, 3Max-Planck-Institut fur Psychiatrie, Abt. Neuroimmunologie, Martinsried Aims: Like adenomatous-polyposis-coli (APC)-syndrome PJ and JP syndromes are autosomal dominant genetic disorders and have an increased risk for intestinal cancer. l3-catenin is a key component of interepithelial adhesive junctions and is upregulated in colonic adenomas and in APC. We asked whether the expression of l3-catenin is also altered in hamartomatous polyposis syndromes. Methods: Paraffin and resin sections from PJ polyps (4 families), JP polyps (2 families), solitary juvenile polyps (n=7), hyperplastic polyps (n=IO), adenomatous polyps (n=IO), APC polyps (2 families) and normal colon mucosa were used to perform immunohistochemistry for the expression of l3-catenin, proliferation markers (Ki 67 and PCNA), APCgene product and cyclooxygenase 2 (COX2). Evaluation was semiquantitative using an immunohistological scoring system. Results: In contrast to normal colon mucosa PJ polyps, JP and solitary juvenile polyps,which all lack APC mutations, show enhanced 13catenin staining in the basal cytoplasm of deep crypt infoldings. This correlates to the irregularly expanded proliferative compartments. Moreover in basal crypt portions some epithelial cell nuclei show a weak nuclear localization of l3-catenin. In PJ and juvenile polyps COX 2 expression ist elevated in only individual epithelial cells. Conclusions: Genetic defects in the regulation of cytosolic B-catenin levels are most likely implicated in the development of adenomas as well as in the formation of hamartomatous polyps. Although the specific defects in PJ and JP syndromes have not yet been elucidated, our data indicate that all three preneoplastic syndromes have defects in a common signalling pathway that results in an increased proliferative capacity of intestinal epithelial cells.

294 CHROMOSOMAL GAINS AND WSSES IN ULCERATIVE COLITIS-RELATED CARCINOMA Daniela E. Aust l ,2 (a.G.), R.F. Willenbucher l (a. G.), G.B. Baretton2, 2 F.M. Waldman t (a. G.) and U. Uihrs tCancer Center, University of California, San Francisco 2pathologisches Institut del' Ludwig-Maximilians-Universitat, Munchen Background/Aims: Patients with longstanding ulcerative colitis (UC) are at high risk for development of colorectal cancer. Preliminary data suggest that chromosomal alterations are present in the UC-related cancer progression pathway. The most common of these alterations may identify chromosomal regions important for neoplastic progression. The aim of this study was to determine the most common chromosomal gains and losses in UC-related carcinomas. Methods: 20 cases of UC-related carcinoma (UICC stages II to IV) were selected. Comparative genomic hybridization (CGH) was performed in duplicate for each sample using DOP-PCR amplification after microdissection. Results: CGH showed chromosomal alterations in all but one sample. The most frequently lost regions were 18q (65%), 5q (60%), 8p (50%), l7p (40%) and all of chromosome 4 (40%). The most frequently gained regions were 8q (65%) and 20q (45%). The average number of changes was 8.2 per case. Conclusions: CGH identified alterations that may be important for UC-related carcinogenesis. The most frequently lost regions are known to contain the folllowing tumor suppressor genes: 18q (DCC, DPC4, JVI8-1), 5q (APC) and 17p (p53). The most frequently gained regions were chromosome 8q (the site of c-myc) and chromosome 20q (also frequently gained in breast and sporadic colon cancer). Although arising in an inflammatory field, UC-related carcinogenesis appears to involve many of the same loci important in sporadic colon cancer.

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EXPRESSION OF P2I (WAFIICIPl) IN COLORECTAL CARCINOMAS: CORRELATION WITH P53 STATUS, TNM STAGING AND PROGNOSIS T.K. Zirbes, S.E. Baldus, S. Nolden (a.G.), J. Lorenzen, S.T. Shafizadeh (a.G.), D. Kunze (a.G.), J. Thiele, H.P. Dienes

CORRELATION OF THE EXPRESSION OF THOMSENFRIEDENREICH ANTIGEN WITH TNM CLASSIFICATION AND PROGNOSIS IN COLORECTAL CARCINOMA S.E. Baldus, T.K. Zirbes, D. Kunze (a.G.), S.T. Shafizadeh (a.G.), S. Nolden (a.G.), F.-G. Hanisch (a.G.)*, J. Thiele, H.P. Dienes

InstitutfUr Pathologie, Universitiit zu K61n, 50924 K6ln

Institute fUr Pathologie und *Biochemie, Universitiit zu K61n. 50924 K6ln

Aims: The protein p2l (WAFIICIPl) is a cyclin-dependent kinase (Cdk) inhibitor induced by the tumor suppressor p53. In contrast to the multitude of studies regarding p53 over-expression and mutations, little infonnation is available regarding p2l expression in colorectal cancer. In the present study, we investigated its relationship with TNM stage and prognosis. Methods: We analyzed 188 patients with complete 5-year follow-up regarding survival. Fonnalin-fixed and paraffin-embedded specimens from the primary tumors were stained by the catalyzed reporter deposition-(CARD)-technique including modifications ofMerz et al. (Lab. Invest. B 149-156, 1995) using monoclonal antibodies directed against p21 (Calbiochem) and p53 (BioGenex). Results: 127 cases (67.6 %) were evaluated as p21 positive. The over-expression of p53 antigen did not show a significant correlation with p2l reactivity. Furthermore, the detection ofp2l was not associated with TNM staging. However, p21 negative patients exerted a lower survival probability. This result was significant (p < 0.05) in an univariate analysis (Kaplan-Meier). Conclusions: p21 (WAFIICIP1) antigen is expressed in the majority of colorectal carcinomas as detected by the very sensitive CARD technique. This p53-related antigen which is involved in the regulation of apoptosis may have important regulatory implications regarding the biology of colorectal cancer. According to our results, a lack of p21 expression is a marker of an unfavourable prognosis.

Aims: Thomsen-Friedenreich (TF) antigen is a well known carcinoma-associated antigen, which may be involved in the process of tumor dissemination, i.e. liver metastasis may be mediated by the so-called "asialoglycoprotein" receptor. In the present study, we evaluated possible correlations ofTF expression with TNM staging and prognosis of colorectal carcinomas, using reagents with different fine-specificities. Methods: In this study, 264 patients with colorectal carcinoma and complete 5-year survival data were included. Specimens fixed in formalin and paraffin-embedded were investigated by ABC-peroxidase immunohistochemistry. In addition to peanut agglutinin (PNA), two monoclonal antibodies (mabs) were included: mab A78-G/A7, specifically reacting with TF disaccharide and mab BW835, which detects TF antigen only ifbound to the MUCI peptide core. Results: Whereas 96.2 % of the specimens were PNA-reactive, only 64.0% and 58.3 %, respectively, expressed A78-G/A7 and BW835 epitopes. BW835 staining increased significantly with rising TNM stages (p < 0.005). Additionally, and in contrast to A78-G/A7, BW835 reactivity was much lower in mucinous/colloidal subtypes compared to tubular/ papillary adenocarcinomas. Regarding prognosis, expression of all antigens showed an decrease of median survival associated with an increase of the tumor area expressing these antigens. This difference was significant in the case of A78-G/A7 (p < 0.05) in an univariate analysis. Conclusions: According to our data, the expression ofTF antigen indicates a progredient course of colorectal cancer. It has to be emphasized that mabs should be preferred for detection, since PNA has a broad binding specificity. On the other hand, variations in the fine-specificity of mabs is reflected by different staining patterns.

288 . Oral Communications

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Time course of nuclear and extranuclear events of apoptotic cell death in alcoholic steatohepatitis Schutz A. (a.G.)I, Tannapfel, A. I, Halm, U.(a.Gl, Sommerer, F. (a.G.)I, Mossner J. (a.Gl, Wittekind, Ch. 1 Institute of Pathologyl , Department of Internal Med. 112, Univ. of Leipzig Aims: In alcoholic steatohepatitis an increased frequency of apoptosis is observed. These cells are visualized by means of their morphology and by performing ISEL (in situ end labeling)-technique. We performed double immunofluorescence labeling to investigate the temporary relatiohship of nuclear and extranuclear events of apoptosis to signals, which are known inducers of the apoptotic process. For this purpose we employed Annexin V-FITC, labeled nucleotides and antibodies against FAS/APOI. Material and Methods: Fresh frozen tissue of eight patients with alcoholic steatohepatitis was available. In the first step of fluorescencelabeling we visualized the FAS/APOI immunoreactivity, while employing mouseanti-FAS and CY3 tagged goat anti-mouse-IgG, effecting a red fluorescence signal. In the second step we demonstrated the exposure of phosphatidylserine, using Annexin V-FITC, causing a green fluorescence. In the second part, we combined the visualization of F ASIAPO I with the detection of DNA cleavage using the ONCOR apo-kit with a green fluorescence signal. Controls were performed on normal liver tissue. Results: Apoptotic cells were found in an increased number in tissue of steatohepatitis with a predominant localization in the pericentral region. Double immunoreactive cells, exhibiting a fluorescence signal for FASIAPO I and phosphatidylserine and for FASIAPO I and for fragmented DNA were found in varying numbers especially in areas close to inflammatory infiltrates. Summary: Our results indicated, that in alcoholic steatohepatitis both extranuclear and nuclear- events of apoptosis occur in a tight time interval. Both are related to signal induction by FAS/APOI-ligand (FAS/L). This could stress the idea that a common interleukin-I ~-converting enzyme (ICE) family depending and FAS/FAS/L interaction involving pathway leads to nuclear and extranuclear events that are the specific features of apoptotic cell death.

EVALUATION OF D·ll AS MARKER FOR THE DIAGNOSIS OF HEPATOCELLULAR CARCINOMA G. Cathomas, P. Saremaslani, L.M. Terracciano*, S SchrOder**, Ph.U. Heitz, P. Komminoth Department of Pathology, University of ZUrich, Switzerland *Insitute for Pathology, University of Basel, Switzerland **Institute for Patholgy, Hamburg, Germany Aims: The differential diagnosis of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) or metastatic carcinoma of the liver may be difficult. Immunohistochemistry is a useful tool to differentiate these tumors, however, a panel of antibodies is usually necessary. The monoclonal antibody D-I I, usually used for typing adrenocortical tumors, was produced using a human liver membrane protein and shows strong cytoplasmatic reactivity against hepatocytes. The aim of this study was the evaluation of the D-II antibody as an immunohistochemical marker for HCC. Methods: Twentynine routinely processed, formalin-fixed, paraffinembedded liver biopsies and one liver wedge resection fragment were analyzed by immunohistochemistry for the D-II antibody. Results: All 14 HCC showed reactivity for the 0-11 antibody. In 10/14 a strong cytoplasmatic signal was observed in most tumor cells whereas in four biopsies only a subset of tumor cells showed a positive signal. Adjacent liver cells always revealed a strong reactivity. In three tumors with histological features of a combined hepatocellular and cholangiocellular carcinoma (HCC-CC), one showed a strong reactivity for 0-11 wheareas two did nol. In contrast, immunostaining for 0-11 was negative in 13 metastatic tumors including 6 adeno- and 2 neuroendocrine carcinomas, one amelanotic malignant melanoma and a transitional cell carcinoma. Conclusions: Our data show that the 0-11 antibody is highly reactive in HCC but negative in CC or metastatic tumors of the liver making 0-11 a useful tool in the daily diagnosis of HCC. Interestingly, a variable staining pattern has been observed in the mixed HCC-CC, indicating that these tumors may represent a heterogenous entity.

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Expression of p21wAF1/CIP1 in Primary Liver Tumors is Associated with Enhanced Apoptosis

Loss of cellular retinol-binding protein-l (CRBP) in stellate cells and myofibroblasts is a hallmark of hepatocellular carcinoma (HCC). Annette Schmitt-Graff, H. Muller, M Bochaton-Piallat,* G.Benzonana; G. Gabbiani* Institute fiIr Pathologie der Universitaten Freiburg und Genf* Aims: CRBP-I is a key protein involved in retinoid metabolism. This study focuses on the distribution of CRBP-I in HCC and in non-neoplastic liver tissue. Methods: Six normal organ donor livers and 12 HCCs were immunohistochemically evaluated for the expression of CRBP-l. To characterize CRBP-I positive cells double immunolabelling was performed using antibodies to a-smooth muscle actin, desmin, CD3I, CD34 and C068 (PGM I). A computer-aided image analysis (KS 400, Zeiss) was done. Results: CRBP-I was found to be distributed predominantly in the cytoplasm of stellate and myofibroblastic cells and some endothelial and vascular smooth muscle cells in the normal liver and the parenchyma surrounding HCCs. The cytoplasm, but not the nucleus of non-neoplastic hepatocytes was faintly immunostained. In HCCs, CRBP-I was abnormally localized in the nuclei of scattered tumor cells. In all cases ofHCC, CRBP-l was largely absent in stellate cells and myofibroblasts as confirmed by image analysis. Conclusions: The loss ofCRBP-1 in stellate cells and myofibroblasts and the abnormal nuclear expression in some tumor cells suggest profound disturbances of the sequestration and metabolism of retinol and the complex stromal-epithelial interactions in HCe. Since retinoids are considered to exert anti-proliferative and anti-tumor effects and to influence cellular differentiation and gene expression, the modulation CRBP-I may playa role in tumor development and progression and become a useful marker in the diagnosis of HCC.

S. Duensing (a.G.), P. Flemming, N. Ihara** (a.G.), Anette Duensing (a.G.), K. Oldhafer* (a.G.), and A. Georgii.

Pathologisches InstitUl und *Abteilung fUr Abdominal- und Transplantationschirurgie, Medizinische Hochschule Hannover, D30625 Hannover, Germany and **University of Hiroshima, 1-2-3 Kasumi, Minami-Ku, Hiroshima City, Japan. Aims: p21 WAFI/CIPI is a cyclin-dependent kinase (CDK) inhibitor which has been suggested as a critical downstream effector of p53. However, the definite role of p21 WAFI/CIPI for the induction of cell cycle arrest versus apoptotic cell death is still discussed controversially. The potential relevance of 21 WAFIiCIPI for the induction of apoptotic cell death in primary liver tumors was investigated in our study. Methods: A total of 41 patients (25 male, 16 female, median age 58.5 years, range, 9 to 87 years) who underwent surgery for primary liver tumors (hepatocellular adenomas, HCA, n=18; hepatocellular carcinomas, HCC, n=23) was analyzed. Formalin-fixed, paraffinembedded archieval tissue specimens were evaluated for nuclear p21 WAFIICIPI immunostaining using the monoclonal antibody EAIO. Apoptosis was analyzed employing DNA in situ nick end labelling. Reliability of the latter assay was confirmed by electrophoresis of extracted tumor DNA resulting in characteristic DNA laddering. Results: Positive staining for p21 WAFI/CIPI was detected in six of twentythree HCC (26~, whereas normal liver tissue and all HCA remained negative. p21 W I/CIPI positive HCC exhibited a significantly higher incidence of apoptotic cells ~median percentage 5%, range, 0.1-12%) when compared to p21 WAFI/C PI negative HCC (median percentage of apoptotic cells 0.1%, range, 0-5%; p:O;O.016) and adenomas (median percentage of apoptotic cells 0%, range 0-5%; p:O;O.003). Conclusions: The significant correlation between expression of p21 WAFl/CIPI and apoptosis in primary liver tumors may suggest maintained but ineffective growth control mechanisms even in less differentiated tumors.

Hematopathology . 289

Working Group: Hematopathology

300 CHANGE OF MORPHOLOGICAL COMPLICATIONS AFTER BMT AND PBSCT T. Friedrich·, G. Wahn (a.G.)·, W. Helbig (a.G.)", M.Kubel (a.G.)··, W. Ponisch (a.G.)", G.Vogelsang (a.G.)·" ·Institut fur Pathologie, ..Abteilung fur Hamatologie der Klinik II fur Innere Medizin der Universitiit Leipzig, "·JHMC Baltimore, USA Aims: Bone marrow transplantation (BMT) and Peripheral Stem Cell Transplantation (PBSCT) are accompanied with a spectrum of typical complications. We analyzed the shift of these main problems after introduction of main new therapeutic schedules within a period of 30 years. Methods: We reviewed clinical and paraclinical data and main morphological findings from 485 autopsy cases after BMT and PBSCT in association to basic disease, conditioning regimen, kind of transplantation, and others. Results: In the first phase (1967-1986) after conditioning with single dose whole body irradiation without lung shielding a predominance of severe toxic lung injuries, early and severe therapy-resistant acute GvhD with profound immune deficiency, and hemorrhagic diathesis was the typical setting. After the introduction of fractionated whole body irradiation with lung shielding, a better CMV-prophylaxis and a more sufficient supportive hemotherapy (1987-1991) these complications were mainly solved. The onset of acute GvhD was prolonged, late acute and chronic GvhD became a problem. PBSCT instead of BMT (since 1992) was accompanied with a quicker hematological reconstitution. Recurrence of basic disease remained a constant problem over the years. We found an interesting inverse constellation between the two BMT centers, with more toxic organ complications and a lower relapse rate in JHMC and an opposite situation in the BMT center Leipzig. Conclusions: We found an unequivocal shift in prevalence, severity, and time ofonset of main morphological complications.

301 MYELOFIBROSIS - THE MOST IMPORTANT PROGNOSTIC FACTOR IN MYELODYSPLASIA WITHOUT EXCESS OF BLASTS G. Busche, W. Wilczak (a.G.), H. Maschek, A. Ganser (a.G.)~H. Choritz (a.G.),

A. Georgii. Pathologisches Institut und*xlinik liir Hlimatolol!ie und Onkologie der Medizinischen Hochschule Hannover Background: Myelofibrosis is regarded as a typical complication of chronic myeloproliferative disorders, but only few authors considered the possible occurence in myelodysplastc syndromes (MDS). Methods: Bone marrow biopsies from a total of718 patients with primary MDS were evaluated for myelofibrosis applying Gomori silver impregnation from semthin sections (3 I!m) after plastic embedding without decalcification. Results: Myelofibrosis was detected within bone marrow from 121 patients (I7.4 %). Fibrosis was associated with dysplastic features of megakaryopoiesis as well as with a more severe anemia, a higher frequency of splenomegaly and karyotype anomalies. No correlation was detected with FAB classes of MDS as well as with blast count, dysplastic features ofgranulopoiesis and erythropoiesis. Follow up analysis did not reveal a significant associaton with leukemic transformation, however the survival was highly significantly reduced by fibrosis (median: 9.8 vs. 18.2 months; p < .000001) mainly due to bone marrow insufficiency, and myelofibrosis turned out to be the most important poor prognostic factor in MDS without excess of blasts (p < .0004; multivariate analysis). Conclusions: Myelofibrosis is a typical complication of MDS with poor prognosis mainly due to bone marrow insufficiency rather than to leukemic transformation. The poor prognosis of this complication justifies the separation of a "fibrotic variant" of MDS which should be considered in therapeutic strategies.

302 CML WITH BONE MARRow FEATURES SIMILAR TO CMML EXTRACTED BY HIsTOMORPHOMETRY - A CMML RELATED DISORDER RATHER THAN A CHRONIC MYELOPROLIFERATION G. Biische, J. Popp (a.G.), S. Baer-Henney (a.G.), J. Schlue (a.G.), H. Maschek, B. Heinze (a.G.), R. Hehlrnann (a.G.), H. Ansari (a.G.), T. Buhr, A. Georgii and the German eML Study Group Pathologisches Institut der Medizinischen Hochschule Hannover Background: CML without t(9;22) resp. bcr-abl gene is considered as a heterogeneous group which may largely be accounted for by myeloproliferative variant of CMML. Because of overlapping peripheral blood features, bone marrow findings may provide significant diagnostic tools in order to detect CMML mimicking CML by peripheral blood findings. Methods: Bone marrow biopsies (BMB) from reference patients with CMML (n = 33) or Ph-positive CML (n = 10 I) were evaluated for the volume ratio of fatty tissue as well as to the numerical densities of basophils, eosinophils and storing histiocytes applying semthin sections (3 I!m) after plastic embedding without decalcification. A non-linear regression model was developed based on these histomorphometric parameters in order to extract patients with myeloproliferative variant of CMML, and the practicability of this model was tested by applying to BMB from 354 patients of a CML therapy study. Results: Highly significant differences were detected between CMML and CML with respect to the volume ratio of fatty tissue as well as to the numerical densities of basophils, eosinophils and storing histiocytes. A nonlinear regression model based on these histomorphometric parameters correctly classified 100/101 (99 %) ofCML and 32 / 33 (97 %) ofCMML patients. By applying this model to diagnostic bone marrow biopsies from 354 patients recruited as CML in a multicenter therapy study including 32 Phnegative patients, 19 (5 %) patients were extracted by bone marrow constellation as "CMML". Peripheral blood findings largely resembled CML, however, bone marrow features, age and survival distributions were highly significantly different to CML and almoust identic with the CMML control group (n = 33). Conclusions: Leukemia with peripheral blood findings of CML, but with bone marrow features similar to CMML should be considered as a CMML related disorder with a rather rapid course, poor survival as well as an age distribution indistinguishable from CMML.

303 PROGNOSTIC IMPACT OF CELL PROLIFERATION (pCNAINDEX) AND APOPTOSIS IN IDIOPATmC (pRIMARY) OSTEOMYELOFIBROSIS (IMF) H.M. Kvasnicka, J. Thiele, C. Regn, V. DieW· (a.G.), R. Fischer Institut fUr Pathologie und ·Medizinische Klinik I der Universitlit zu Koln, Joseph-Stelzmann-Str. 9, D-50924 Koln Aims: Prognostic classification of patients with idiopathic (primary) osteomyelofibrosis (IMF) is generally based on static clinical and haematological parameters derived from heterogenous and small patient populations. Furthennore, the prognostic impact of dynamic disease features like incidence of apoptosis and proliferative activity has been usually neglected in this context. Methods: To detennine dynamic parameters of predictive value a total of ISO patients (81 females and 69 males, median age 66 yrs.) with the unequivocally established diagnosis of IMF and a representative bone marrow biopsy on admission before any therapy was analyzed in a consecutive study over a period of20 years. Median survival time was 59 months. For the demonstration of apoptosis the in-situ end-labelling technique was applied and proliferative activity was assessed by the monoclonal antibody PCIO directed against proliferating cell nuclear antigen (PCNA). To characterize the dynamics of haematopoiesis an index was calculated consisting of the ratio between PCNA-positive nuclei and the apoptotic cell fraction. Results: Multivariate survival analysis (CART-analysis) revealed a substantial improvement of prognostic efficiency by inclusion of dynamic disease features in risk classification especially in intennediate risk cases, whereas fiber density failed to disclose a significant prognostic value. Conclusions: Our results suggest that dynamics of haematopoietic cell turnover exert a predictive property in IMF independently of bone marrow fibrosis and that generalization of the disease process, i.e. extramedullary haernatopoiesis is compatible with an unfavourable survival.

290 . Working Group

304 ANALYSING TIlE BINDING CAI'ACITY OF PERIPHERAL BLOOD CD34+ CELLS TO HEMOPOIETIC STROMA: A COMPARATIVE STUDY ON P. VERA AND

306 Cloning of a partial cDNA encoding the human sinus lining ceO specific antigen Ki-M9

NORMAL PROGENITORS BY CYTOADHESION ASSA YS

Claudia Wickenhauser (a. G.), 1. Thiele. Semra Frimpong (a.G) Beate Schmitz (a. G.), R. Fischer Institut fur Pathologie, Universitat zu Kaln, Joseph-Stelzmann-Slr. 9, 050924 Koln Aims: In former studies we analysed the expression ofVLA-4, VLA-5, IL3Rand c-kit of CD34+ progenitors and found a higher and stronger membrane associated c-kit expression on blood progenitors of patients with Polycythemia vera (P. vera) Based on these results we developed a semiquantitiative cytoadhesion assay and analysed the interaction of hemopoietic fibroblasts and highly enriched C034' progenitors. Methods: Peripheral blood C034' progenitors from peripheral blood of healthy donors (n=5) and patients with P. vera (n=5) were seeded onto nearconfluent cultivated fibroblasts from the bone marrow of two healthy donors. After adding of neutralizing antibodies to VLA-4, VLA-5, IL-3R and c-kit cell proliferation was analysed in a time - dependent manner. Moreover, an microadhesion assay with surface coated polystyrene plates was applied to measure the binding intensity of progenitors to adhesion molecule ligands Rl'suItS: In cocultures, adding of neutralizing antibodies to IL-3R and c-kit as well as blocking of surface - bound adhesion molecules on C034' cells caused progenitor cell death in P. vera and normal progenitors. This feature is in keeping with an essential influence of these receptors on the proliferative activity of hemopoietic progenitors. In microadhesion assays, a strong binding capacity of C034+ progenitors to all adhesion molecules tested could be detected. Conclusions: C034' progenitors from P. vera and normal peripheral blood demonstrate a membrane - bound expression for VLA-4, VLA-5, c-kit and IL-3R. In addition, These cells adhere to ligand bound polystyren plates. In cocultures with bone marrow fibroblasts neutralizing antibodies directed against IL-3R and c-kit generate cell death of P. vera as well as normal progenitors.

P. MIDDEL (A.G.)·, B.W. PATIERSON (A.G.)·, F. ZSCHUNKE(A.G.)*, H.-H. WACKER", M.R. PARWARESCH", H.-I. RADZUN* *Department of Pathology, University of Gottingen and "Institute of Hematopathology, University ofKiel Aims: The monoclonal antibody Ki-M9 recognizes an antigen, which is expressed by human sinus ling cells (SLC) of the lymph nodes and follicular dendritic cells (FOC). To get further insight into the function of this antigen we cloned the encoding cDNA for the Ki-M9 antigen. Methods: A human spleen A.gtll expression library was screened with the mAb Ki-M9 by means of immunohistochemistry. Positive clones were selected, isolated and purified. The longest insert was subcloned to facilitate sequencing and expression of the recombinant protein. For in situ hybridization (ISH) a 270 bp fragment was subcloned in pBlueScript II KS+ and run off transcripts were generated using Digixigenin-II-dUTP and T7- or TJ-polimerase for sense and antisense cRNA-probes, respectively. Results: Sequencing of the cDNA demonstrated a 1650 bp fragment, which consisted of one open reading frame encoding 552 amino acids. Western blot analysis of the recombinant MBP-Ki-M9 fusion protein showed a 100 kD protein (MBP 42 kD, Ki-M9 58 kD). ISH signals were detected exclusively in SLC and FOC in lymphatic tissue. Condusion: We have cloned a 1650 bp fragment of the cDNA encoding the Ki-M9 antigen, which is expressed exclusively by SLC and FDC. Analysis of the deduced amino acid sequence demonstrated the characteristics of a membrane associated protein. The nature and function of the Ki-M9 antigen is unknown due to its FDC and SLC restricted expression the Ki-M9 antigen might function in antigen presentation during humoral immune response.

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Expansion of Megakaryocytic Precursors in Philadelphiapositive Chronic Myeloproliferative Diseases Anette Duensing (a.G.), S. Duensing (a.G.), A. Kreft, G. BUsche, and A. Georgii. I, Medizinische Pathologisches Institut, Carl-Neuberg-StrafJe Hochschule Hannover. D-30625 Hannover, Germany. Aim: Abnormal megakaryopoiesis represents a basic aspect of chronic myeloproliferative diseases (CMPD) and serves as a diagnostic feature between chronic myelogenous leukemia (CML) with small, hypolobulated megakaryocytes (MK), and polycythemia vera (PV), primary thrombocythemia (PTH), and chronic megakaryocytic granulocytic myelosis (CMGM), respectively, with large, predominantly hyperlobulated MK. We analyzed whether quantitative differences of MK precursors characterized by the c-mptICD34+ phenotype are associated with different entities of CMPD. Methods: Bone marrow aspirates were obtained from twenty patients with CMPD (CML, n=10; PV, n=3; PTH, n=4; CMGM, n=3) and analyzed for c-mptICD34+ megakaryocytic precursors employing double immunofluorescence staining. Samples obtained from eight patients lacking histopathological alterations in routine examination were used as control. Results: Mononuclear c-mptICD34+ bone marrow cells exceeding the physiologic level (<:: 1%) were detected in six of nine patients with Ph+ CML and one of four patients with PTH, whereas all patients with PV or CMGM lacked increased numbers of megakaryocytic precursors as did one patient with Ph- CML. The correlation between Ph+ disease and increased numbers of megakaryocytic progenitors in the bone marrow 2 was statistically sigificant (pS:0.046, X -test). Conclusions: Expansion of c-mptICD34+ MK precursors in Ph+ CMPD in contrast to non-elevated levels in Ph- disease may suggest that abnormal megakaryopoiesis in CMPD is not uniformly regulated. These findings may correlate with the typical morphology of MK in Ph+ CML with an immature appearing phenotype.

THE CYTOGENETIC PROFILE OF FOLLICULAR NONHODGKIN'S LYMPHOMA G.Ott (a.G.), T.Katzenberger (a.G.), A.Rosenwald (a.G.), J.Kalla (a.G.), Th.Rudiger (a.G.), J.G. Muller, M.Michaela Ott (a.G.), and H.K.MullerHermelink Pathologisches Institut der Universitlit, Wurzburg AIMS: To define the genetic constitution of follicular lymphoma (FL) in grades I-III as classified according to the REAL classification. METHODS: Cytogenetic investigations were performed in 124 cases of FL. Analyzable metaphases were found in 90 cases and clonal aberrations were detected in 83 lymphomas. RESULTS: The occurrence of the t(14;18) showed a progressive decline with 93%, 77% and 57% in grades I, II, and III, respectively. Vice versa, chromosomal losses in 6q and gains of chromosomes '7 and X augmented with grade, being in the range of 25%, II %, and 0%, respectively, in grade I, 20%, 30% and 13% in grade II and of 52%, 38% and 28% in grade III. A strong correlation was found between loss of genetic material in 17p, the site of the p53 gene, and secondary/simultaneous transformation in 4/6 (66%) cases. On the cytogenetic level, grade III lymphomas could be subdivided in two groups: one t(l4;18)+ group with an additional17p- in 30% of cases, and a t(l4;18)- group with cytogenetic characteristics of large B-ceillymphomas, most notably a t(8;14) in one case and a t(3;14) in two cases. Interestingly, FL with a predominant diffuse growth pattern were negative for the t(l4; 18), but all showed rearrangements of 1p36. CONCLUSIONS: On the cytogenetic level, there seems to be a continuous spectrum of progression in grades I-III of follicular lymphoma with characteristical aberrations being unevenly distributed. The t(l4;18) constitutes a hallmark ofFL of low grades, but is absent in diffuse variants and in a substantial portion of high grade lesions. On the basis of cytogenetics, grade III lymphomas constitute a heterogeneous group of tumours. Cytogenetic investigations, therefore, may be important in the delineation of different biological subgroups.

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PHYSICAL DELETIONS OF PS3 ARE FOUND IN AGGRESSIVE BAND T-CELL NEOPLASMS AND ARE FREQUENT IN TRANSFORMED FOLLICULAR LYMPHOMAS A.Rosenwald (a.G.), G.Ott (a.G.), T.Katzenberger (a.G.), I.Kalla (a.G.), Th.RUdiger (a.G.), Katharina Peters (a.G.), M.Michaela Ott (a.G.), and H.K.MUller-Hennelink Pathologisches Institut der Universitlit, WUrzburg AIMS: The characterization of p53 deletion patterns in various subgroups of Non-Hodgkin's lymphoma by FISH and comparison to fmdings in classical cytogenetics and p53 immunohistochemistry. METHODS: 120 B-NHL (34 FL IJII, 14 low grade MALT, 4 MZL, 10 FL III, 22 high grade MALT, 36 DLCL) and 23 T-NHL were investigated for p53 allelic loss by bicolour FISH. In most of the cases, classical cytogenetics and p53 immunostaining were also perfonned. RESULTS: In B-cell neoplasms, p53 deletions were found in 1/52 (2%) low grade lymphomas, 3/22 (14%) high grade MALT, 6/10 (60%), FL III and 8/36 (22%) DLCL. In high grade lesions (FL III and DLCL), there was a tendency for t(l4;18) positive cases to be deleted for p53 (8/11, 73%) in contrast to t(14;18) negative cases (6/35, 17%). In addition, p53 deletions were associated with secondary/simultaneous transfonned lymphomas (5/6 cases; 83%). p53 deletions in T-cell NHL were encountered in 3/23 cases. Nearly all cases which were deleted for the gene also showed overexpression of p53 in immunohistochemistry; however, p53 expression was also found in a considerable proportion of non-deleted cases. CONCLUSIONS: In keeping with reports in the literature, p53 alterations were observed in aggressive B- and T-cell NHL. However, previous studies paid attention to p53 mutations rather than the allelic loss, which seems to be the crucial step for full p53 inactivation in many cases. We observed a high rate of p53 deletions in transfonned and de novo large cell t(14;18)-positive lymphomas, thus indicating the important role of p53 deletion both in the progression of FL as well as in primary transfonnation of genninal centre lymphomas. p53-sequencing studies in selected cases are currently under way in order to detennine the partial or complete inactivation status of p53 and its correlation to expression in immunohistochemistry.

ISOLATION AND CHARACTERIZATION OF A NOVEL HUMAN GENE, INDUCED IN B-LYMPHOCYTES BY LATENT EPSTEINBARR VIRUS (EBV) INFECTION C.S. Verbeke, M.P. Birkenbach* Pathologisches Institut, Fakultllt flIr Klinische Medizin Mannheim der Universitllt Heidelberg 'Department of Pathology, Kovler Viral Oncology Laboratories, The University of Chicago Aims: To elucidate the molecular mechanisms of so-called immortalization ofB-celis upon latent EBV infection, a novel human gene EBI4 (!illV-Induced 1) was isolated and characterized on DNA-, RNA-, and protein level. Methods: Isolation of the EBV-induced gene was performed by subtractive hybridization of an EBV-infected (BL4l ~ vs. an EBV -nega~ve cell line (BL4IIB95-8), subsequent cDNA-screenmg, and sequenc.mg. FISH was used for chromosomal mapping. Transient and stable cell lmes were established. RNA-expression was analyzed by multiple-tissue Northern blotting, in vivo and in vitro expression on protein level by Western blotting and immunofluorescence, using two different polyclonal anti-EBI4 antibodies. By immunoprecipitation and experiments with cytochalasin, brefeldin A and cWoroquin intracellular interactions and metabolization were analyzed. Human and murine homologue genes were identified by genebank- and cDNA-screening. Results: EBI4 is a type II glycoprotein of 38kD, expressed in activated, CD40L or CD30 positive T- and B-lymphocytes. Neoplastic cells of Hodgkin and non-Hodgkin lymphoma are EBI4 negative, however, reactive T cells do express EBI4. In vitro expression of EBI4 is associated with poor cell survival. EBI4 forms homodimers and is associated with the cytoskeletton. Expression of the protein on the cell membrane is followed by extracellular cleavage o~ a 3 ~ peptide and ra~id intra~llular degradation. The EBI4 gene IS localized on 2q36. EBI4 IS evoluatlOnary higWy conserved and belongs to an emerging family of human and murine homologous genes. Conclusions: EBI4 is a cell membrane receptor protein which functions as an activation antigen on T and B lymphocytes and seems to have an apoptosis-inducing effect as well. EBI4 belongs to a family of highly conserved human and murine genes. Analogies with CDIOO and the TNFfamily request further investigation.

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MARGINAL ZONE LYMPHOMAS (MZL) OF DIFFERENT SITES EXHIBIT DIFFERENT GENETIC ABNORMALITIES

M. Michaela Ott (a. G.), A. Rosenwald.(a. G.),T. Katzenberger (a. G. ), J. Kalla (a. G. ), G. Ott (a. G.) and H. K. MOlier-Hermelink. Pathologisches Institut der Universitiit WOrzburg Aims: MZL is a rather new entity among non-Hodgkin's lymphomas of B-eell lineage. Low-grade lymphomas of the mucosa-associated lymphoid tissues (MALT) exhibit a marginal zone pattern as well as the former monocytoid B-cell lymphomas of the lymph node, the tonsil and the spleen, which have now been designated as MZL. Since these tumors do not only share morphologic but also immunophenotypic characteristics, we tried to evaluate whether the origin at different sites might be associated with distinct genetic abnormalities. Methods: The karyotypes of 19 splenic MZL were compared to those of 7 MZL with nodal onset and 20 cases of low-grade B-eeillymphoma of MALT. Deletions of the p53 gene as well as of the 013525 locus on chromosome 13 were studied by bicolour FISH in 14 splenic MZL, in 5 cases of nodal MZL and in 13 cases of low-grade lymphomas of MALT. Results: Generally, splenic and nodal MZL showed more complex karyotypic changes as low-grade lymphomas of MALT. While the chromosomal translocation t(11;18)(q21;q21) was detected as the most common genetic abnonmality in low-grade lymphomas of MALT (frequency in cases with abnormal karyotypes: 53%), none of the MZL of either nodal or splenic origin exhibited this chromosomal change. Trisomy 3 was present in two lymphomas of MALT (15%) and in 4 splenic MZL (26.7%) but not in nodal MZL. Breaks and/or losses at 7q22-32 were a frequent finding in splenic MZL (20%), but were not detected in nodal MZL or MALT-type lymphomas. A deletion of p53 was present in four cases of splenic MZL (29%), while no case of low-grade lymphoma of MALT or of nodal MZL showed this molecular change. No deletions of 0135:25 were encountered. Conclusions: Despite their striking similarity regarding morpholgy and immunophenotype, MZL show distinct genetic differences according to the site of their primary onset. Our findings point to differences in the mechanism of tumourigenesis in these lymphomas and may support the hypothesis of nodal MZL being distinct from low-grade lymphoma of MALT or a MZL of the spleen.

NUCLEAR ARCHflF.CJURE IN APoPfOSIS AND MUMMIFICAnON IN HOOOKIN CEll.. LINES J. Lorenzen, Sabine Wuttke (a. G.) , Karen Hell (a. G.) lnstitut ftir Pathologie, Universitlit Koln Aims: Previous studies have established that the neoplastic cells of Hodgkin's disease, i. e. Hodgkin- and Reed-Sternberg- (HRS-) cells, frequently display features of a cell death that differs from classical apoptosis. This fonne fruste of apoptosis, the mummification, can be characterized by the lack of intemucleosomal DNA-fragmentation, lack of chromatin condensation and the preservation of antigenicity. In the search for the underlying molecular events we have investigated the nuclear architecture in apoptosis and mummification. Materials & Methods: Apoptosis and mummification was studied in an established model system using the Hodgkin cell line HDLM2. Immunofluorescent studies were performed to detect the distribution of spliceosomal proteins, lamin, nuclear pore complexes, and PCNA amongst other matrix-associated proteins. DNA fragmentation was shown by application of the TUNEL technique. Furthermore, we applied fluorescent in-situ hybridisation for snRNP U I and U2 as well as for nuclear poly-adenylated RNA and chromosomes 8, 14/22 and 18. Relevant samples were evaluated by use of scanning confocal microscopy. Results: Having defined the distribution of subnuclear domains in the log-phase Hodgkin cell line, we found significant changes during apoptosis induced by CD95-stimulation. Spliceosomal complexes, nuclear pores and struclural matrix proteins condense and frequently are extruded into the cytoplasmic space. Similar changes occur during mummification and thus are alleast partially independent of intemucleosomal DNA-fragmenlation. A variety of caspase inhibitors was found to interfere with this subnuclear re-organisation during cell death. Conclusions: One of the primary manifestations of cell death is the reorganisation of nuclear chromatin structure. Our data indicate, that the topology of subnuclear domains reflects molecular events involved in apoptosis. Hence, using this approach we can disclose the differences between apoptosis and mummification on the molecular level and hope to gain insights into the pathology of Hodgkin's disease.

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IMMUNOHISTOCHEMICALANALYSIS OF CD25ANTIGEN EXPRESSION IN PARAFFIN-EMBEDDED TISSUE OF HODGKIN'S DISEASE Karen Hell (a. G.), P. Borchmann (a. G.)*, J. Lorenzen, K. RothfuB (a. G.)*, A. Engert (a. G.)* Institut fur Pathologie, Universitiit Koln, *Medizinische K1inik I, Universitlit Koln Aims: The role of the cytokine interleukin 2 (IL-2), a growth factor for Band T-Iymphocytes and natural killer cells, in Hodgkin's disease (HD) is still unclear and only little is known about interleukin 2 receptor (IL-2R) expression in Hodgkin and Reed Sternberg (HRS) cells. A sensitive immunohistochemical staining method was applied to evaluate IL-2R (CD25) expression on paraffin sections. We investigated biopsies of first diagnosis and relapse of HD to look for the loss of CD25 activation. Materials & Methods: We have determined the expression of IL2R in HRS cells in paraffin sections by immunohistochemistry using antigen retrieval protocolls and signal amplification by reporter-deposition (immunomax method). 35 cases of HD at first diagnosis and 18 cases of HD at first diagnosis and relapse were investigated. The positive HRS cells were evaluated as follows: negative (-, 0%), occasional (+,1-10%), weak (++, >10-30%), moderate (+++, >30-70%), strong (++++, >70%). Statistical analysis were performed by Pearson's chi-square test. Results: At first time of diagnosis of HD in 25 from 35 cases the HRS cells expressed the CD25 antigen in the following distribution: 3 occasionally positive cases, 6 weakly positive cases, 8 moderate and 8 strong positive cases. No correlation between the subtype of HD and the stage of disease could be shown. In the 18 cases with biopsies from first diagnosis and relapse HRS cells were negative for CD25 in 4 cases and positive in 14 cases (+=3, +++=5, ++++=6) at first biopsie. At relapse in 2 cases HRS cells did not express CD25 and in 16 cases they were positive (+=3, ++=3, +++=5, ++++=5). Conclusions: Using for immunohistochemistry the enhanced immunomax method it is possible to stain IL-2R on paraffin sections and to investigate archive material. Analysis of intraindividual positivity of HRS cell for CD25 expression in first biopsies and relapse showed a clear trend towards mantaining the initial CD25 expression (p=O,024). These data suggest that independence of paracrine IL-2 stimulation seems not to be a major factor in relapse in HD.

Clinical impact of Grading the Nodular Sclerosing Hodgkin's Disease under modem therapy of the German Hodgkin Study Group R. von Wasielewski', A. Georgii l , R. Fischer, ML. Hansmann, K. Hllbne~, J. Franklin4, V. Diehl4 . Pathologische Institute der Medizinischen HochschulelUniversitliten:. IHannover, 2Koln, 3Frankfurt, 4Klinik I fiIr Innere Medizin der Universitlit Koln Background: Significant differences in clinical outcome between grade 1 and 2 ofND.HD could not be reconfirmed by several groups as originally reported by the British authors in 1989-1992. Especially the use of modem therapeutical approaches has been held responsible for these discrepancies. However, this topic seems to be of intrerest since NS.HD represents with more than 60% the largest histopathological group ofHO. Aims: Grading NS.HD histomorphologically into the two subcategories and correlating clinical data and follow-up with this grading. Material and Methods: Biopsies from 1,902 NS.HD (64.9%) of all cases of the German Hodgkin Study Group, (GHSG) were graded according to the British criteria into 1,553 grade 1 (81.6%) and 349 grade 2 (18.3%) by consensus diagnosis of 4 histopathologists (R.F., ML.H., K.H., A.G.). Only pretherapy biopsies from patients of the GHSG receiving standardized therapies according to their stage of disease were included. Results: At the early and intermediate stages, no differences in clinical outcome was observed between NS 1 and NS2. In advanced stages, significant differences in freedom from treatment failure (p=O.007) and survival (p=0.0001) were detected. In NS2, hemoglobin and the number of lymphocytes were significantly lower, both factors known to be of prognoctic relevance. A multivariant analysis confirmt that grading HD histomorphologically into NSI and NS2 is an independent prognostic factor in HD. Conclusions: The prognostic significance of the grading of NS.HD does not disappear even under modem therapies. Interestingly this significance is based mainly on differences among the advanced stages of disease. The worse clinical outcome ofNS2 patients is not explained by more frequent relapses after complete remission (NSI 9.5% versus NS2 10.3%) but by differences in the progression rale (NSI 5.3% versus NS2 13.3%).

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STRATEGIES TO DIFFERENTIATE BETWEEN CLASSICAL HODGKIN'S LYMPHOMA, LYMPHOCYTE PREDOMINANT HODGKIN'S DISEASE AND T-CELL RICH B-CELL LYMPHOMA Th. RUdiger, G. Ott, M.M. Ott, S. Miiller-Deubert and H. K. Miiller-Hennelink; Institute of Pathology, WlIrzburg, Gennany Aims: The comet diagnosis of cases at the interface between classical Hodgkin's lymphoma, Iymphocyte-predominant Hodgkin's disease and T-cell ri~h B-celllymphoma comprises an important problem in hematopathology. Inter-observer reliability is poor suggesting that the diagnostic criteria need to be improved. Methods: We investigated 197 cases (155 classical Hodgkin's lymphomas, 32 T-cell rich B-cell lymphomas and 10 paragranulomas) by paraffin immunohistochemistry. Special interest was given to cases with a B-cell phenotype oftumor cells. Apart from the immunoreactivity ofthe tumor cells the composition of the inflammatory background was noted. Results: Classical Hodgkin's lymphoma was positive for C030 in 95% of cases, for CO15 in 75% and for C020 in 22%. They expressed vimentin in more than 95% of the cases. T-cell rich B-celllymphoma expressed vimentin in only 6%. C030 and COl5 were negative in all cases. The tumor cells stained for C020 in 100% of cases. The reactive inflammatory infikrate in both classical Hodgkin's lymphoma and T-cell rich B-cell lymphoma was rich in small lymphocytes expressing TIA I but CDS7-positive cells or B-Iymphocytes were rarely observed. In contrast, in paragranuloma C057-positive cells and small B-Iymphocytes predominated the background infiltrate; TIA-I expression was detected in few cells. L&H cells were negative for C030 and COl5 but expressed C020 in all cases. Conclusions: In those classical Hodgkin's lymphomas that are negative for C030 and CO15 and positive for C020 the vimentin positivity of the tumor cells together with many reactive TIA-I positive cells gives a clue towards the correct diagnosis. In T-cell rich B-celllymphoma the tumor cells express C020 but are negative for vimentin, C030 and CO15. Many of the reactive cells are positive for TIA I, few C057-positive cells are encountered. The distinctive immunophenotype of lymphocyte predominant Hodgkin's disease (paragranuloma) comprises tumor cells that are positive for C020, often for J-ehain and the epithelial membrane antigen (EMA). They are negative for C030 and COI5.1n the reactive background CD57-positive cells outnumber the TIA-I positive cells. This scheme should allow a reliable classi· fication of cases into the respective category. Its prognostic relevance needs to be further investigated.

PATTERN ANALYSIS OF PCR PRODUCTS FROM LYMPHOPROLIFERATIVE LESIONS M. Sperling l , G. L. Selders (a. G.)2, K. 00nhuijsen 1 I Institut fiIr Pathologie, Stlitisches Klinikum Braunschweig 2 Perkin Elmer, Applied Biosystems GmbH, Weiterstadt Aims: PCR amplification of rearranged immunoglobulin heavy chain (lgH) gene CDR3 junctions is an important method for determination of clonality in lymphoproliferative .disorders. The PCR products are commonly analysed in ethidium bromide or silver stained gels. DNA fragment analysis of PCR products with an automated sequencer shows more detailed banding patterns. We tried to fmd an objectiv cut off level between monoclonal and oligo- or polyclonal B-cell proliferations by calculating ratios between different peak levels of the analysed fragments. Methods: Formalin fixed, paraffin embedded tissue of ten hyperplastic tonsils, seven gastric biopsies with chronic gastritis and six B-cell NHL were processed according to Wright and Manos (1990). A semi-nested PCR according to Wan et al. (1990) was done with a primer (Fr3A) which was labled with the fluorescent dye 6-FAM. The PCR products were run on an ABI PRISM™ 310 Genetic Analyser and analysed with the GeneScan™ analysis 2.1 programm. Ratios of peak levels were calculated between the highest and the third highest peak (HTP) as well as between the second highest and the' third highest peak (STP). Results: In all samples of hyperplastic tonsils HTP as well as STP ratios vary between 1.0 and 1.7. In all gastric biopsies both ratios vary between 1.0 and 2.0. In the NHL samples HTP varies between 7.8 to 72.4. The STP ratios of five of the NHL samples vary between 1.0 and 1.8. In one NHL sample STP was 13.1 indicating a rearrangement of IgH genes in both alleles. Conclusions: Fragment analysis of PCR products with calculation of peak level ratios helps to differentiate between monoclonal and oligo- or polyclonal B-cell proliferations. From our results we propose that one or two peaks should be at least five times higher then all other peaks in a DNA fragment analysis to assume monoclonality.

Gynecopathology . 293

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TRANSCRIPTION PATTERN OF NEW ISOLATED GENES, HIGHLY TRANSCRIBED IN ALCL LYMPHOMA INA BETTINGER (a.G.), H. MERZ (a.G.), A. C. FELLER Institut fUr Pathologie, Medizinische Universitat zu Lubeck, Lubeck Aims: Our aim is to characterize the transcription of new isolated genes from the ALCL cell line L82 in various nonnal and neoplastic tissue and to find new diagnostic markers, distinguished between ALCL and Hodgkin's Lymphoma, Methods: By RT-PCR we examined the transcription of one known (lectin) and four unknown genes, which were isolated from a subtractive ALCL cDNA library. Therefore total RNA was isolated from snape frozen tissues, derived from patients with different Hodgkin's and NonHodgkin's lymphoma. The RNA was reverse transcribed with oligo-dTPrimer and amplificated with the gene-specific sense and antisense primers. The specifity of the amplified products were detennined in a southern blot with digoxigenin-Iabeled internal cDNA-fragments. Results: As examined so far, the lectins seems not to transcribed in low malignant lymphomas, including Hodgkin's lymphoma. This result confinned the transcription pattern found in HD cell lines. The transcription of one unknown gene is restricted to ALCL and was also not found in nonnal tissues or in carcinomas. The two other genes were highly transcribed in ALCL cell lines and only moderatly in HD cell lines. Conclusion: We were able to determine the transcription pattern of known and unknown genes, highly and differentially transcribed in ALCL cell lines. One gene is restricted to ALCL and therefore may be important for the pathogenesis and diagnosis.

CARCINOMA OF THE FALLOPIAN TUBE D.Schmidt*, K.Dudzik*, U.Hahn#,G.Dallenbach-Hellweg* *Institute of Pathology, A2,2, Mannheim, #Women's Hospital, University of Heidelberg Aims: Although several large studies have been published on fallopian tube carcinoma, this tumor is generally thought to be rare, and its clinicopathological features are ill-defined. The present study was carried out to assess these features in a large number of cases. Methods: We reevaluated all cases with a diagnosis of either fallopian tube carcinoma or metastatic ovarian carcinoma for age, histological subtype, tumor grade, stage (new TNM system) and localization. Immunohistochemistry for steroid honnone recptors (ER, PR), for p53, and for growth fraction (Ki-S5) were detennined. Clinical follow-up. Results: We studied 59 patients with a mean age of61.7 yrs.(range: 39 to 83 yrs.). Most cases corresponded to the serous type (42/59). Less frequent types were: endometrioid (8/59), transitional (3/59), mucinous (2/59), secretory (1/59), lymphoepithelial (1/59), and malignant mixed Mullerian (1/59).51/59 were carcinomas of high grade (grade 3 or 4). 28/59 presented in advanced stage (stage III). Low-stage carcinomas were frequently associated with hydrosalpinx. We often found in situ carcinoma which was not a prerequisit for diagnosis of tubal carcinoma. 25 cases were located on the right side, 26 on the left side, and 8 were bilateral. More tumors were positive for progesteron receptors than for estrogen receptors. More than 70 % of the cases were strongly positive for p53. The growth fraction of the tumors correlated with the histological subtype and the tumor grade. Most patients died of disease. Conclusions: Carcinoma of the fallopian tube is not as rare as frequently thought. The expression of steroid honnone receptors and of p53 is a common finding, so that no prognostic groups can be distinguished based on these features.The prognosis is generally poor.

Working Group: Gynecopathology

317 Heinz Pickartz

Institut fUr Pathologie am Evangelischen Waldkrankenhaus, Berlin-Spandau Das serose periloneale Karzinom der Frau

Peritoneale Formationen einer mit einem serasen Ovarialkarzinom identischen Neoplasie werden als serase Karzinome des Peritoneum bezeichnet Sind diese Tumoren Mesotheliome oder "ektope" Ovarialtumoren? Wir hatten Gelegenhei~ 8 serase Karzinome des weiblichen Peritoneum zu untersuchen. Alle Tumoren exprimierten Zytokeratin 19 (20 - 100% der Tumorzellen markiert), das epitheltypische Gykoprotein Ber-EP4 (80 bis 100%), die Tumormarker CA 125 und CA 19.9. Vimentin fand sich in drei Karzinomen in etwa 30% der Zellen. Orei Tumoren exprimierten den Ostrogenrezeptor (bis 80"10 der ZeUen positiv), zwei von ihnen zusatzlieh den Progesteronrezeptor (40"/0 positive Zellen). In keinem Tumor fand sieh CEA Die Wachstumsfraktion der Twnoren schwankte zwischen 20 und 80% (Ki-67 oder MIB I). In zwei Tumoren war fokal die plazentare alkalische Phosphatase nachweisbar, ein Tumor besaJl einzelne Riesenzellen vom synzytialen Typ mit kratliger Markiernng bei Nachweis von Il-HCG. Das immunhistologisehe Markerprotil ist ebenso wie das konventionelle histologisehe Bild mit einem serasem Karzinom des Ovars in Einklang zu bringen. Trotz der groBen Ahnliehkeit zwischen epithelialen Mesotheliomen und serasen Karzinomen des Ovars sprechen insbesondere die starke Expression von Ber-EP4 und die Expression der Stroidrezeptoren gegen die Mesotheliom-Natur dieser Tumoren. Die Frage naeh dec Matrix der serasen Karzinome des wcibliehen Peritoneum ist im EinzelfaU Dicht zu beantworten. Systematische Untersuchungen des weibliehen Peritoneum haben in einem hohen Prozentsatz (uber 40%) glandulare Strukturen nachgewiesen, welche wegen ihrer Ahnlichkeit mit dem Epithel der Tuben als EndosaJpingiose bezeichnet werden. Die Herde bieten teilweise strukturelle Ahnlichkeit mit Endometriose-Herden, jedoch olUle Zeichen fur eine menstnllltions-iihnJiche Funktion. Die Epithelien besitzen ein gleichartiges immnnhistologisches Protil wie die Tubenschleimhant Endosalpingiose-Herde werden anch in Assoziation mit serasen "Implantaten" gefunden, was einerseits als Hinweis dafUr dienen kann, daB ein Teil der "Implantate" autochthone Uisionen darsteUt und andererseits isolierte serase Karzinome des Peritoneum auf dem Boden einer Endosalpingiose entstanden sein k6nnen. Die Hiiutigkeit des ser6sen Karzinoms des Peritoneum wird in der Literatur mit 5 bis 10% der scrasen Karzinome i:les Ovars bezi/Tert, die Prognose wird mit einer mittleren Uberlebenszeit von 19 Monaten angeben und ist damit schlechter als die des scrasen Ovarialkarzilloms (mUZ: 31 Monate).

319 PRIMARY MALIGNANT TUMORS OF THE FALLOPIAN TUBE: ADENOCARCINOMAS, MALIGNANT MIXED MOLLERIAN TUMORS AND LEIOMYOSARCOMA L,·C. Horn*, Cornelia Werschnik**, K, Bilek** *Institute of Pathology, **Department of Obstertics & Gynecology, Leipzig University Aims: Primary malignant tumors of the Fallopian tubes are extremely rare and correct diagnosis is rarely made before operation. The purpose of the study was to evaluate the distinct morphologic pattern and prognostic factors by histology, Methods: 35 primary malignant tumors of the Fallopian tube occured during a period of 34 years. The histological slides were reexamined for tumor differentiation, nuclear grade and restaging. The clinical reports were examined for follow up, Results: These 35 primary malignant tumors included 30 primary adenocarcinomas (Ae), 4 malignant mixed MiiIlerian tumors (MMMT) and one leiomyosarcoma. All patients were postmenopausal with a median age of 58.6 years (42-77 years). 76.5% of the patients were between 50-70 years of age. 70% of AC were poorly differentiated and most of them presented in advanced stage of disease (FIoo stage ill or IV). 44.1% of the patients died .of disease with a median recidive free survival for all stages of 44.4 months. Four cases showed liver metastases. There was no difference in disease free and overall survival for AC vs. MMMTs. The only prognostic factor Was FIGO stage. Tumor type, nuclear grading, DNA-parameter as well detection of c-erbB-2 have no prognostic impact. Conclusions: Primary malignant tumors of the Fallopian tube, including AC, MMMTs and leiomyosarcomas are rare tumors, These tumors should only be diagnosed histologically if there is a clear relation to tubal structures, even adjacent carcinoma in situ of the tubal mucosa, with main tumor in the Fallopian tube. Adjuvant chemotherapy in pure AC should be based on a cis-platinium regimen, in cases with sarcomatous component anthracyclines should be added.