158 Abstracts AMPLIFICATION OF 20Q13 LOCUS IN BREAST CANCER: -DEFINING THE MINIMAL REGION. M. Tanned, M. Tirkko- nen 1, T. Stokke 2, A. Kallioniemi 1, R. Karhu 1, M. Heintz 2, C. Collins 2, F. Waldman 2, J.W. Gray 2, J. Isola ~-2, O-P. Kallioniemi ~. 1) Lab. for Cancer Genetics, Tampere University, FIN-33521 Tampere, Finland, and 2) Div. Molecular Cytometry, Dept. Lab. Medicine, Univ. California San Francisco, CA 94143-0808, USA. Comparative genomic hybridization (CGH) has indicated that a major new locus for DNA amplification in breast cancer is 20q13. Based on our CGH data, amplified DNA sequences originating from 20ql 3 are found in 17/83 (21%) of primary breast carcinomas and in 5/15 (33%) of breast cancer cell lines. The gene(s) that are located in the amplicon are currently unknown. In order to study this region in detail and to define the minimal common region of amplification, we physically mapped 150 cosmid clones from a chromosome 20 specific cosmid library by fluorescence in situ hybridization (FISH). Twenty-six cosmids localized to 20q13 were ordered by multi-color pairwise FISH to metaphase chromosomes and interphase nuclei as well as free chromatin. These cosmids were used as FISH probes to study amplification levels in tumor interphase nuclei. Six (35%) of 17 breast cancer cell lines and three (14%) of 15 primary tumors showed 3-15 fold amplification with one or more 20q13-specific cosmids. The size and location of the amplicon varied slightly from one cell line to another. One of the cosmids was consistently amplified in the majority of cases and defined the minimal common region of amplification to appr. 1-2 Mbs. Studies are underway to 1) study amplification and expression levels of known genes that map to this region, 2) further narrow down the region to facilitate positional cloning efforts and 3) define the frequency and prognostic value of this amplification in larger series of clinical specimens. CYTOGENETIC FINDINGS IN 152 COLORECTAL ADENOCARCINOMAS - CORRELATION WITH CLINICOPATHOLOGIC FEATURES. Georqia Bardi, Bertil Johansson, Nikos Pandis, Nils Mandahl, Elisabeth Bak-Jensen, Claus Fenger, Felix Mitelman, and Sverre Heim, Departments of Clinical Genetics and Pathology, Lund University Hospital, S-22185, Sweden and Departments of Medical Genetics and Pathology, Odense University and University Hospital, Winsl~wparken 15, Odense C, Denmark. Cytogenetie analysis of short-term cultures from 152 colorectal carcinomas revealed clonal chromosome aberrations in 115 tumors and only normal karyotypes in the remaining 37. In the abnormal group, 48 tumors had only numerical changes, whereas 67 had at least one structural rearrangement with or without concomitant numerical changes. The most common gains were of chromosomes 7, 13, 20, and 16; the most common losses were of chromosomes y, 18, 14, 15, 4, and 21. The most common structural rearrangements affected chromosomes i, 8, 17, 13, and 7. The chromosome bands most frequently involved in structural changes were 8q10, 13q10, 17pll, 17q10, ip22, llq13, 19p13, and 20p13. The recurring abnormalities most often affecting these bands were i(8)(ql0), i(13)(ql0), del(17)(pll), and i(17)(ql0), leading to losses of 8p and 17p but gains of 8q, 13q, and 17q. Whereas good candidate tumor suppressor genes exist to explain the importance of -18 and 17p-, the molecular consequences of the other chromosomal rearrangements remain unknown. When the cytogenetic data were correlated with clinicopathologic parameters, statistically significant correlations between the karyotype and tumor site as well as tumor grade were found. Rectal carcinomas more often had abnormal karyotypes than tumors of other sites. The poorly differentiated carcinomas were generally those with the more massive chromosomal rearrangements. GENETIC HETEROGENEITY IN COLONIC ADJACENT ADENOMAS AND NORMAL MUCOSA WITH IN SITU HYBRIDISATION. CARCINOMAS, AS DETECTED J. Herberqs, A. de Bruine, J.-W. Arends, F. Ramaekers, A. Hopman. Dept. Pathology and Dept. Molecular Cell Biology & Genetics, Univ. Limburg, P.O. BOX 616, 6200 MD Maastricht, The Netherlands. We performed fluorescence in situ hybridisation (ISH) on cell suspensions from colonic carcinomas, adjacent adenomas and normal tissue, with repetitive centromeric probes for the chromosomes i, 7, 17, 18, X and Y to detect numerical chromosomal aberrations. The most occurring numerical chromosomal aberration in both carcinomas and adenomas was a trisomy or tetrasomy for chromosome 7 (60%). In adjacent normal mucosa no numerical aberration for chromosome 7 was detected. In transition from adenomas to carcinomas we detected an increase in the number of numerical aberrations in 87% of the cases. We found in 3 cases (30%) the same numerical aberrations in the adenoma as in the carcinoma, while extra aberrations were detected in the carcinoma. In 6 cases (60%) numerical chromosome aberrations were only found in the carcinomas. In normal mucosa, adjacent to a carcinoma, only in 2 cases (20%) a numerical aberration, namely an extra chromosome X was found. To investigate genetic heterogeneity in the carcinomas we performed on frozen sections combined immunocytochemistry (ICC) with antibodies against cytoskeletal markers (cytokeratines, lamines) and ISH with the centromeric probes. To solve the problem of the interpretation of the ISH signals in overlapping epithelial nuclei, we combined ICC using anti-lamin B2 (LN43, a nuclear membrane marker) with ISH. THE MULTIPLE ENDOCRINE NEOPLASIA TYPE 2 SYNDROMES AND HIRSCHSPRUNG DISEASE ARE DUE TO DIFFERENT MUTATIONS IN THE RECEPTOR TYROSINE KINASE RET. Bruce Ponder, Lois Mulligan, Charis Eng, Patrick Edery I, Stanislas Lyonnet I, Darrin Smith, Alan Tunnacliffe, John Kwok. CRC Human Cancer Genetics Research Group, University of Cambridge, Cambridge, UK and IHopital des Enfants-Malades, INSERM U- 12, Paris, France. Multiple endocrine neoplasia type 2 (MEN 2) is a dominantly inherited syndrome which comprises tumours of thyroid C cells, adrenal medulla and parathyroid, and developmental abnormalities (usually overgrowth) of the autonomic ganglion plexuses of the gut. There are 3 clinical varieties of MEN 2, which differ in the pattern of tissues involved. Hirschsprung disease is a common developmental abnormality in which there is absence (rather than overgrowth) of the gut autonomic ganglion cells. The ret protooncogene is a receptor tyrosine kinase, expressed during development in the tissues which are involved in MEN 2 and Hirschsprungs disease. In 68/70 families with MEN 2A (thyroid, adrenal and parathyroid involvement) and in 6/7 families with F-MTC (familial thyroid C cell tumours only), we found mutations in the cysteine-rich region of the extracellular domain of ret leading to substitution of eys by another amino acid. The site of mutation and the precise amino acid substitution correlate with the spectrum of tissues involved in each family. In MEN 2B (thyroid, adrenal but not parathyroid tumours, overgrowth of gut ganglion cells) 26/28 mutations were identical, leading to a met -> thr substitution in the catalytic core of the tk domain of ret. In 6/19 Hirschsprungs families so far, there are missense or stop mutations in the extracellular domain of ret, predicted to lead to a truncated protein. The data so far suggest that MEN 2 mutations cause inappropriate activity of ret, while Hirschsprung mutations are associated with loss of activity.