) This project aims to develop two new technologies for detection of point mutations associated with colorectal cancer and breast cancer. We will combine the polymerase chain reaction (PCR), restriction endonuclease digestion reaction (RE), and the ligase detection reaction (LDR) to detect K-ras and p53 mutations in clinical samples from cancer patients.
Our specific aims are: (1) to develop a multiplex PCR/LDR method to screen rapidly and simultaneously for multiple mutant alleles at a sensitivity of 1 cancer cell in 100 - 1,000 normal cells, and (ii) to develop a highly sensitive PCR/RE/LDR method to identify specific mutant alleles at a sensitivity of 1 cancer cell in 100,000 - 1,000,000 normal cells. Ligation primers will be designed and tested to optimize ligation fidelity and compatibility for multiplexing. Mutant Tth DNA ligases and nucleotide analogue containing primers will be tested to improve PCR/LDR sensitivity and fidelity. For PCR/RE/LDR, a general method to selectively introduce restriction sites into the PCR product derived from the wild- type allele will be applied and tested using the K-ras and p53 genes. Multi-pairing and non-pairing nucleotide analogues as well as proofreading polymerases will be tested for their effect on the efficiency and specificity of the restriction site conversions. We intend to use clinical samples to demonstrate the feasibility and potential clinical relevance of mutation detection in K-ras and p53 using PCR/LDR and PCR/RE/LDR in colorectal and breast cancer. For colon cancer, we will evaluate multiplex PCR/LDR as a method for quantitative mutation detection in K-ras and p53 mutations as molecular markers of disseminated cancer cells in tissues at risk for occult micrometastases (lymph nodes, pelvic washings, and bone marrow aspirates). In addition, PCR/LDR and PCR/RE/LDR will be evaluated as diagnostic tests for colorecal cancer by screening stool and colonic lavage samples for K-ras mutations.
|Khan, Sajid A; Zeng, Zhaoshi; Shia, Jinru et al. (2017) EGFR Gene Amplification and KRAS Mutation Predict Response to Combination Targeted Therapy in Metastatic Colorectal Cancer. Pathol Oncol Res 23:673-677|
|Khan, Sajid A; Morris, Melinda; Idrees, Kamran et al. (2016) Colorectal cancer in the very young: a comparative study of tumor markers, pathology and survival in early onset and adult onset patients. J Pediatr Surg 51:1812-1817|
|Cheng, Yu-Wei; Pincas, Hanna; Huang, Jianmin et al. (2014) High incidence of LRAT promoter hypermethylation in colorectal cancer correlates with tumor stage. Med Oncol 31:254|
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|Dharmasiri, Udara; Njoroge, Samuel K; Witek, Ma?gorzata A et al. (2011) High-throughput selection, enumeration, electrokinetic manipulation, and molecular profiling of low-abundance circulating tumor cells using a microfluidic system. Anal Chem 83:2301-9|
|Nash, Garrett M; Gimbel, Mark; Cohen, Alfred M et al. (2010) KRAS mutation and microsatellite instability: two genetic markers of early tumor development that influence the prognosis of colorectal cancer. Ann Surg Oncol 17:416-24|
|Bacolod, Manny D; Barany, Francis (2010) Gene dysregulations driven by somatic copy number aberrations-biological and clinical implications in colon tumors: a paper from the 2009 William Beaumont Hospital Symposium on Molecular Pathology. J Mol Diagn 12:552-61|
|Cheng, Yu-Wei; Idrees, Kamran; Shattock, Richard et al. (2010) Loss of imprinting and marked gene elevation are 2 forms of aberrant IGF2 expression in colorectal cancer. Int J Cancer 127:568-77|
|Pingle, Maneesh; Rundell, Mark; Das, Sanchita et al. (2010) PCR/LDR/universal array platforms for the diagnosis of infectious disease. Methods Mol Biol 632:141-57|
|Nash, Garrett M; Gimbel, Mark; Shia, Jinru et al. (2010) KRAS mutation correlates with accelerated metastatic progression in patients with colorectal liver metastases. Ann Surg Oncol 17:572-8|
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