1 1 8 A b s t r a c t s

AN INVESTIGATION OF GENETIC ALTERATIONS IN GASTRIC CANCER

INTERPHASE CYTOGENETICS OF GASTRIC ADENOCARCINOMAS

R. Sud t, IC. TalbotL JDA. Delhanty ~

Human Genetics Group, The Galton Laboratory, University College London, Wolfson House, 4 Stephenson Way, London, NW1 2HE

Department of Pathology, Northwick Park and St Mark's NHS Trust, Warlord Road, Harrow, HA1 3UJ

D. Garaano, M. Galli, M. Matucci, D. Palli, A. Parenti, L. Pecciarini, T. Rubeca, P. Turco

Centro per Io Studio e la Prevenzione Oncologica, Firenze, Italy

The genetic changes that are involved in the development and progression of gastric cancer are not well characterised. We investigated a series of twenty- six gastric cancers with corresponding normal tissue for alterations of the APC, MCC, DCC, and TP53, tumour suppressor genes, and for microsatellite instability. We examined exons 6, 8, I 1, 14, and the 5' half of exon 15 of the APC gene for mutations by SSCP and heteroduplex analysis. In addition we used the protein truncation test (PTT) to screen the mutation cluster region of APC for chain-terminating mutations. SSCP and heteroduplex analysis was used to screen the entire coding region of the TP53 gene. Loss of heterozygosity (LOH) analysis of TP53, APC, MCC and DCC was also carried out.

Mutation of the APC gene was detected in 4% (1/26) of gastric cancers. LOH of APC was observed in 8% of informative cases, of MCC in 10% of cases, and of DCC in 4.6% of cases. Microsatellite instability was investigated at fifteen dinucleotide repeat loci. This was also an infrequent event, occurring in one tumour only. LOH was detected at microsatellite loci on chromosomes 14q, 22q, and 2p in the region of the hMSH2 gene. LOH at 22q was most frequent (22% of informative cases). We found alterations of the TP53 gene to be the most frequent genetic change in gastric cancer:, mutations were identified in 31% (8/26) of tumours and LOH in 29% of tumours.

DELETION MAPPING OF LONG ARM OF CHROMOSOME 6 IN GASTRIC CARCINOMA

B. Carvalho*, R. Seruca ÷, F. Gartner ÷, I. VeigaL F. Carneiro*

*IPATIMUP - Instituto de Patologia e Imunologia Molecular da Universidade do Porto *IPO - Instituto Portugu~s de Oncologia do Porto

There are only a few cytogenetic studies in gastric cancer and no consistent specific abnormalities have been described so far. Studies with standard cytogenetic tecniques generally yielded poor results because of frequent necrosis, contamination of fibroblasts and normal ce l l s , and fungine infections.

In situ hybridization (ISH) using chromosome specific centromeric a- satellite DNA probes provides an alternative for the rapid detection of numerical abnormalities in interphase nuclei of tumors.(l) In the present study we investigated numerical chromosomal abnormalities in fresh surgical specimens and in cultured cells.of gastric adenocarcinomas.

ISH analysis was performed on 20 primary gastric cancers using four centromeric a-satellite DNA probes derived from chromosomes 7, 8, 9, X. Molecular hybridization and detection of the hybridized signals were performed according to the manifacturer's protocol (Oncor, Gaithesburg, MD, USA) using an immunocytochemical method with HRP-streptavidin complex and 3, 3 diamirlobenzidine (DAB) as chromogen substrate. For each case 500 intact interphase nuclei were scored for the number of the hybridization signals using a Zeiss photomicroscope.

Preliminary results showed in one patient monosomy X, in another one trisomy 7 and in two patients monosomy 7.

Numerical abnormalities of chrom. 7 were frequently described not only in gastric cancer but also in several other tumor types. The significance of these anomalies, in particularly of trisomy 7, is still debated, because they were also frequently observed in nonneoplastic tissue. Monosomy of chromosome X was also reported, even if less frequently, in gastric cancer.

In our opinion, in situ hybridization can be regarded as an useful tool to confirm suspected numerical aberration or to extend the analysis to patients with hyperdiploid or complex karyotypes.

References (1) Rao P. at al Diagn. Mol. Pathol. 1993 2 (4): 264-268.

In a previous study, our group has identified two distinct regions of deletion at the long arm of chromosome 6 in a series of 51 gastric carcinomas (1). These two regions appear to be involved in the early stages of gastric carcinogenesis. The first region of deletion is interstitial, located between 6q16.3-q21 and 6q22.3-q23.1 (frequency of LOH of 50%) and the second one is terminal, distal to 6q23-q24 (frequency of LOH of 37%).

For further characterization of the first region of deletion we used six highly polymorphic microsatellite markers within this region.

Using these six markers we examined the pattern of allelic loss in 6 primary gastric carcinomas that showed LOH in the first region of deletion and retention at more proximal or distal loci in 6q. Additionally, we analyzed 6 more gastric tumours and respective derived xenografts and cell lines.

Seventy five percent of the cases were informative for at least one of the markers used. Allelic imbalance was found in 4 of the 9 informative tumours (44%).

With this analysis we delimited the region of deletion between the D6S268 and ARG 1 to D6S268 and D6S261 with an average of distance of 5.2 CM.

References (1) Queimado Le t al Genes Chromosom Cancer 1995; 14: 28-34.