A Novel-Invasive CRC Test Based on Mass Spec of In Vitro Expressed Proteins

DESCRIPTION (provided by applicant): There exists an urgent need to develop an effective, non-invasive method for large-scale colorectal cancer (CRC) screening, 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 56,000 annual deaths caused by CRC. However, current methods for early detection of CRC, which include the fecal occult-blood test (FOBT), a non-invasive fecal DNA test (PreGen-Plus) and endoscopic colorectal examination (colonoscopy) either have nominal effectiveness due to low sensitivity (FOBT, PreGen-Plus) or low compliance due to invasiveness and rigorous preparative requirements (colonoscopy). The goal of this project is to develop a cost-effective, high sensitivity method for scanning protein truncating mutations in the APC gene. These mutations constitute the earliest molecular event in CRC tumorigenesis. However, since only a fraction (<1 %) of mutant APC is present in exfoliated fecal DNA relative to WT, routine techniques like direct sequencing are not applicable. Furthermore, use of hybridization-based assays for targeting the known mutations in APC would require over 1000 probes and still would miss significant de novo mutations. As an alternative, a new method based on mass spectrometry of in vitro expressed proteins (MASSIVE-PRO) provides an attractive basis for CRC screening. During Phase I of this Fast-Track proposal, MASSIVE-PRO will be evaluated using normal and mutant DNA derived from cell lines. A critical goal will be to demonstrate the reliability and sensitivity of assays to detect less than 1 % mutant population that occur throughout the mutation cluster region of the APC gene. During Phase II, MASSIVE-PRO will be evaluated using CRC repositories of stool samples as well as prospective samples obtained from pre-operative CRC patients in collaboration with Dr. Paul Schroy, Director of Clinical Gastroenterological Research at the Boston Medical Center. The results obtained will be validated by full-sequencing on DNA extracted from surgically removed tumors tissue (adenomas and carcinomas). For the purpose of assay validation and eventual FDA approval, AmberGen will work closely with Quest Diagnostics, Inc., which operates the largest network of in vitro diagnostic testing facilities in the U.S.