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 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 rigorous preparative requirements (colonoscopy). The principal objective of this project is to develop a cost-effective, high sensitivity, non-invasive method for scanning protein truncating mutations in the APC gene from fecal DNA as part of a large-scale population CRC screening test. Such mutations constitute the earliest molecular event in CRC tumorigenesis. During Phase I, a new method based on mass spectrometry of in vitro expressed proteins (MASSIVE-PRO) was developed and evaluated for detection of chain truncating mutations in the APC gene using cell-line as well as a few patient-derived DNA samples (stool and tissue). All of the goals of the Phase I project were achieved including: i) Demonstration of the ability to scan the complete MCR region of the APC gene, ii) Ability to achieve high sensitivity (~1%) to detect chain truncating mutations, iii) Ability to detect mutations using DNA isolated from the stool of CRC patients. Significant improvements in the MASSIVE-PRO technology beyond the Phase I goals were also achieved including: i) Ability to scan missense mutations using the K-Ras gene as a model from cell-line, tumor and stool DNA; ii) Enhanced ability to perform multiplexed MASSIVE-PRO using MS-MS sequencing. During Phase II, research will shift to a thorough evaluation of MASSIVE-PRO on DNA derived from fecal samples including prospective samples obtained from pre-operative CRC patients in collaboration with Dr. Paul Schroy, Director of Clinical Research for the Section of Gastroenterology at the Boston Medical Center. A critical goal will be to reliably detect less than a 1% level of chain truncating APC mutations relative to wild-type protein. Enhanced sensitivity will also be obtained by utilizing MASSIVE-PRO to scan for missense mutations in K-Ras and other genes associated with early CRC tumorigenesis. The results obtained from MASSIVE-PRO will be validated by full-sequencing on DNA extracted from surgically removed matched tumor tissue. For the purpose of developing a CRC population screen which meets the requirements of a clinical laboratory environment and for 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. All results will be statistically analyzed in collaboration with Prof. Josie Dupuis, Associate Professor of Biostatistics, Boston University School of Public Health. 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 (150,000 new cases and 56,000 deaths). Current methods for early detection of CRC include the fecal occult blood test (FOBT) and endoscopic colorectal examination (colonoscopy) but these methods are of limited effectiveness due to either their sensitivity or cost. The objective of this project is to develop a novel non-invasive molecular test based on mass spectrometry of in vitro expressed proteins to screen for mutations in various genes involved in early development of CRC. This test promises to be rapid, highly sensitive and low-cost. If successful, the test will enable more extensive screening and consequently increased surveillance and early treatment of colorectal cancer. ? ? ?
