High Sensitivity Molecular Test for Colorectal Cancer

DESCRIPTION (provided by applicant): There exists an urgent need to develop an effective, non-invasive method of detecting colorectal cancer (CRC), the second leading cause of cancer deaths in the U.S. Such non-invasive testing, if instituted for a large segment of the population, could result in a dramatic reduction in the approximately 55,000 annual deaths caused by CRC. However, current methods for early detection of CRC, which include the fecal occult-blood test (FOBT) and endoscopic colorectal examination (colonoscopy), either have nominal effectiveness due to low sensitivity (FOBT) or very low compliance due to rigorous preparative requirements (colonoscopy). The principal objective of this project is to develop a cost-effective and high throughput method for detection of molecular markers of CRC from fecal DNA. CRC assays on fecal DNA must also be highly sensitive, since mutations characteristic of CRC are likely to be present in concentrations relative to WT of less than 1%. Chain truncating mutations in the APC gene have the highest genetic correlation (approximately 80%) known for CRC. However, full-sequencing of the APC gene is prohibitively expensive due to its large size (8.5 Kb) and lacks sufficient sensitivity, while targeting the known mutations would require over 1000 probes and still miss significant de novo mutations. As an alternative, a newly developed ELISA-based protein truncation test (ELISA-PTT) will be evaluated. In contrast to conventional protein truncation tests, ELISA-PTT eliminates the need for electrophoresis and radioactivity, making it cost-effective and suitable for high throughput applications. ELISA-PTT will be combined with digital PCR and subractive techniques in order to detect chain-truncating mutants at concentrations as low as 0.4% present in DNA extracted from stool. Preliminary studies demonstrate AmberGen's ability to routinely isolate fecal DNA from small stool samples (>2mg). During Phase I, this new approach will be further evaluated using CRC repositories of stool samples as well as new samples obtained from pre-operative CRC patients in collaboration with Dr. Paul C. Schroy, Director of Clinical Research for the Section of Gastroenterology at Boston Medical Center. Dr. Tim Heeren, Professor of Biostatistics at the Boston University School of Public Health, will assist in statistical analysis of data. The results obtained with these new technologies will be validated using full-sequencing of DNA extracted from surgically removed tumors tissue. During Phase II, an optimized non-invasive assay system will be evaluated for CRC population screening.