Identification and characterization of a 19q12 amplicon in esophageal adenocarcinomas reveals Cyclin E as the best candidate gene for this amplicon

Genomic DNA amplification in tumors is frequently associated with an increa sed gene copy number of oncogenes or other cancer-related genes. We have us ed a two-dimensional whole-genome scanning technique to identify gene ampli fication events in esophageal adenocarcinomas. A multicopy genomic fragment from a tumor two-dimensional gel was cloned, and genomic amplification enc ompassing this fragment was confirmed by Southern blot analysis. The corres ponding DNA sequence was matched by BLAST to a BAC contig, which allowed th e use of electronic-PCR to localize this amplicon to 19q12. Sequence tagged site-amplification mapping, an approach recently implemented in our labora tory (Lin, L. et al. Cancer Res., 60: 1341-1347, 2000), was used to charact erize the amplicon, Genomic DNA from 65 esophageal and 11 gastric cardia ad enocarcinomas were investigated for 19q12 amplification using quantitative PCR at 11 sequence tagged site markers neighboring the cloned fragment. The amplicon was narrowed from >8 cM to a minimal critical region spanning <0. 8 cM, between D19S919 and D19S882. This region includes the cyclin E gene. Fourteen expressed sequence tags (ESTs) covering the minimal region were th en assayed for potential gene overexpression using quantitative reverse tra nscription-PCR. Seven of the selected ESTs were found to be both amplified and overexpressed, Among these seven ESTs, cyclin E showed the highest freq uency of gene amplification and overexpression In the tumors examined, whic h allowed us to finalize the core-amplified region to <300 kb. These result s indicate that cyclin E Is the likely target gene selected by the amplific ation event at 19q12. The fact that cyclin E overexpression was found only in the amplified tumors examined indicates that gene amplification underlie s the cyclin E gene overexpression. Our study represents the first extensiv e analysis of the 19q12 amplicon, and is the first to physically map the co re-amplified domain to a region of <300 kb that includes cyclin E. Amplific ation of 19q12 was found neither in the 28 esophageal squamous cancers nor in the 39 lung adenocarcinomas examined but was observed in 13.8% of esopha geal and 9.1% of gastric cardia adenocarcinomas.