Molecular Test for Inherited Mutations in Breast Cancer

DESCRIPTION (provided by applicant): Breast cancer is the most common malignancy which occurs in women and the second leading cause of cancer death. While the majority of breast cancers are sporadic (e.g. not inherited), approximately 10% are due to inherited mutations in the BRCA1 and BRCA2 genes leading to a lifetime risk of 85% for breast cancer and 40-65% for ovarian cancer. Importantly, recent progress in understanding the molecular basis for inherited breast and ovarian cancer has led to the development of candidate drugs (PARP inhibitors) which for the first time offer the hope of preventing and treating cancer in women carrying defective BRCA1/2 genes. There is also increasing evidence that pre-surgical genetic screening of breast cancer patients helps assist in deciding on therapeutic options. Therefore, it is becoming increasingly important to be able to identify carriers of BRCA1/2 mutations. However, current breast cancer susceptibility gene testing which involves DNA sequencing is prohibitively expensive in many cases. The objective of this project is to develop a cost-effective technology to screen for mutations in the BRCA1/2 genes. Two related methods have been successfully tested in Phase I, both based on in vitro expression of peptides from overlapping segments of PCR amplified BRCA1/2 genomic DNA and mRNA. One approach utilizes an ELISA-based protein truncation test (ELISA-PTT) to detect chain-truncations which constitute 95% of all deleterious mutations in BRCA1/2. A second approach, utilizes mass spectrometry of in vitro expressed proteins (MASSIVE-PRO) to scan for all possible mutations, including missense mutations. In both cases, studies on normal and mutant bearing cell-lines and a limited number of patient samples reveals the tests are highly effective scanning techniques with a sensitivity approaching DNA sequencing. During Phase II, we propose to further develop and extensively evaluate these novel BRCA1/2 assays. The ELISA-PTT chain-truncation assay developed in Phase I will be improved to cover the entire BRCA1/2 coding region using single-step PCR reactions. The test will utilize a combination of DNA and mRNA extracted from blood. The MASSIVE-PRO assay will also be extended to scan for both chain truncations and missense mutations in both genes. Advanced technology based on several innovations, which can significantly reduce the number of PCR, and cell-free reactions will be evaluated. The new assay will be extensively evaluated in collaboration with Dr. Jessica K. Booker, Associate Director of the Molecular Diagnostics Laboratory at the University of North Carolina School Of Medicine (UNCSM) using a repository of 250 pre-validated genomic DNA and mRNA samples from patients screened for hereditary breast cancer. Clinical a-testing will be performed during the third year in both Dr. Booker's laboratory and at Quest Diagnostics, Inc. Breast cancer is the most common malignancy which occurs in women and the second leading cause of cancer death. While the majority of breast cancers are not inherited, over 20,000 cases per year in the U.S. are due to inherited mutations in two specific genes leading to a lifetime risk of 85% for breast cancer and 40- 65% for ovarian cancer. Importantly, a new generation of drugs are being developed which target hereditary breasted cancer. However, current breast cancer susceptibility gene testing is often prohibitively expensive for patients and insurance companies. The objective of this project is to develop a new, cost-effective technology to screen for mutations indicative of hereditary breast cancer. A new approach to detect these breast cancer mutations developed by AmberGen will be extensively tested during this project. This test promises to be rapid, highly sensitive, low-cost and easy to perform in a typical clinical laboratory setting. If successful, the test will enable more extensive screening and consequently increased surveillance and early treatment for hereditary breast cancer.