Telomerase activation occurs in about 90% of primary human cancers, making telomerase one of the most widespread cancer markers discovered. Activated telomerase is found in 95% of late-stage breast cancers, 95% of colorectal carcinoma, and 90% of bladder carcinomas. While the mechanism linking telomerase activation to cancer is under intense investigation, it has become evident that telomerase stabilizes tumor cell chromosomes by replenishing TTAGGG repeats at their telomeric ends. With the advent of the Telomeric Repeat Amplification Protocol (TRAP), it is now possible to standardize a highly sensitive test for telomerase. Our objective is to develop a molecular diagnostic test for detecting cancer cells in body fluids. To do this, we have modified the TRAP assay to make it compatible with the clinical laboratory. In the first step of the TRAP assay, the telomerase from cell extracts adds telomeric repeats to the 3' ends of substrate oligonucleotides. The telomerase-generated products are then amplified by a polymerase chain reaction (PCR) which includes dUTP. The products generated vary by six base increments and are resolved by electrophoresis or by hybridization to complimentary sequences. Our TRAP assay can detect fewer than 50 tumor cells, and quantification of the telomerase signal with fluoroimage analyses shows that our TRAP assay is linear, with extracts derived from between 500 and 5000 cells. The inter-assay co-efficient of variation is 12%. Telomerase from tumor cells is readily detected in cell mixtures containing fewer than one 1 % of telomerase-positive cells. Having demonstrated that our assay detects telomerase activity in clinical specimens that contain cancer cells, we are now developing approaches that will further improve test resolution with clinical specimens, such as urine and bladder washings. In addition, we continue to develop new PCR-based modalities for detecting cancer-related DNA mutations in the human K-ras oncogene. Adjunctively, in collaboration with the Microbiology Service, we are applying our non- isotopic PCR-SSCP (single-strand conformation polymorphism) methods toward the identification and characterization of encephalitozoon species. We anticipate that, once validated, our protocols will become routine diagnostic tests.
