Evaluation of oncogene amplification in intact and truncated cell nuclei of gastro-esophageal cancer cell lines by DNA in situ hybridisation

Adenocarcinoma arising around the gastro-esophageal junction (GEJ) is a hig hly malignant form of cancer. Its incidence is rising sharply. The study of oncogenes in these carcinomas may give information concerning treatment an d prognosis. In the present study, the fluorescence in situ hybridisation ( FISH) technique was optimised for genetic characterisation of oncogenes in archival cancer specimens. Three cell lines derived from GEJ adenocarcinoma s were investigated, i.e. JROECL 19, JROECL 33 and OACM5.1C, both in fresh and paraffin-embedded preparations. Furthermore, paraffin-embedded material of three xenografts was studied, i.e. JROECL 19, JROECL 33, and OACM4.1X, We focussed on the oncogenes MYC and HER2/neu, since they are frequently in volved in intestinal cancers. Firstly, our results indicate that it is feas ible to detect oncogene-specific probes with the FISH technique in formalin -fixed, paraffin-embedded material. Secondly, it appeared that the optimal section thickness for analysis was 2 mum. This thickness resulted in minima l nuclear overlap, which facilitates counting of FISH spots. Due to the tru ncation phenomenon, however, the sensitivity of the technique is less than FISH on intact nuclei. Importantly, (high level) oncogene amplifications we re easily recognised in 2 mum thick sections. Finally, counting of the indi vidual copy number of the MYC and HER2/neu oncogenes was feasible enabling an arbitrary assessment of low- and high-level amplification. In conclusion , FISH is an accurate technique for detecting amplification of oncogenes in paraffin-embedded patient material.