Ovarian cancer is one of the most frequent causes of cancer deaths in women. It was responsible for the deaths of more than 12,000 women last year in the United States. About one in 70 women will develop the disease. One percent of al female deaths in this country are due to ovarian cancer. We are proposing a novel method to identify amplified genes which may be responsible for tumor progression and drug resistance in these patients by targeting extrachromosomal DNA. Extrachromosomal DNA is thought to play a pivotal role in tumorigenesis since amplified oncogenes and drug-resistant genes are often located in extrachromosomal DNA in tumor cells and tumor cell lines. The largest form of extrachromosomal DNA is the double minute chromosome (DM). We now have documented that DMs are frequently found in metaphase spreads from ovarian tumors obtained from fresh biopsy specimens. Therefore, prolonged culture will be avoided since DMs are often lost in vitro. We have developed a strategy to isolate and identify extrachromosomally located (i.e. on DMs amplified oncogenes or drug resistance genes. These amplified oncogenes or drug resistant genes present on DMs can be specifically isolated by chromosome microdissection and the fragments amplified in vitro by polymerase chain reaction (PCR). Initially, we will use PCR-based strategies on DMs to identify the precise region on metaphase chromosomes where the amplified genes of the DMs originate by using gene mapping techniques on normal cell chromosomal preparations. After determining the chromosomal map position of the DM we would be able to determine if we have identified probable new uncharacterized oncogenes and/or drug resistance genes. At that time specific clones could be isolated and their gene sequences ultimately determine. The following specific aims are proposed: 1) Utilize current state-of-the-art chromosome microdissection techniques to determine the incidence of known oncogene amplification on DMs and develop a genetic fingerprinting techniques to characterize DMs. 2) To use chromosome microdissection techniques and PCR amplification to characterize amplified oncogenes and drug-resistant genes in human tumors.
