Clinical Value Study Of Cell Free DNA In Colorectal Cancer

Monoclonal antibodies (MoABs) targeting the epidermal growth factor receptor (EGFR) has improved the overall outcome of metastatic colorectal cancer (mCRC),but along with specific toxicity profiles and major costs. So predictive and prognostic biomarkers are urgent for these drugs. Among them, KRAS and BRAF mutations have been successfully been identified as responsible for tumor resistance to treatment. In general the testing of tissue from the primary tumor has been accepted as a basis for treatment of mCRC. However, studies show us gene mutations vary along with treatment and tumor heterogeneity determine that the absence of detectable mutations in the primary tumor cannot formally exclude the presence of mutant metastatic disease. Furthermore, either primary or metastatic tumor tissue is rarely available for testing due to practical and ethical reasons. So alternative methods for mutational testing could become of great clinical value.The presence of cell free DNA (cfDNA) was identified more than60years ago, and base studies have suggested plasma cfDNA in cancer was significantly higher than healthy donors, and there were tumour specific genetic alterations such as KRAS or BRAF mutations in plasma. Also, the concentration of cfDNA is believed to play a predictive and prognostic role in different cancers, including colorectal cancer. However, cfDNA has not been translated into clinical practice till the last decade because of several methodological factors such as the methods of cfDNA isolation or quantitation, and downstream mutation detection method. Here we have optimized the method of cfDNA isolation and quantitation analysis. Newly develope a highly sensitive ARMS-based quantitative PCR with a LNA blocker (ARMS-qPCR(LNA)) to analyze plasma KRAS or BRAF mutations in colorectal cancer.Part one. The diagnosis and prognostic value of quantitative estimates of cfDNA in Colorectal cancerPurpose:To determine the predictive and prognostic value of quantitative estimates of cfDNA in Coloretal cancer.Methods:The study included178patients at different stages of CRC and56healthy donors. The plasma cfDNA was isolated and accurately quantified. Receiver operating characteristic curve (ROC) was applied to analyze the diagnosis value of cfDNA level; clinically follow up the patients of stage IV to determine the prognostic value of cfDNA.Results:Association was observed among plasma DNA level and various clinico-pathological features such as histologic grading, and TNM stage. ROC analysis showed cfDNA level had no diagnosis value for patients of stage â… -â…¢. Survival analysis confirmed the prognostic importance of cfDNA. High levels were clear indicators of a poor OS.Conclusion:This study shows that the baseline cfDNA levels in plasma of patients with CRC has no diagnosis value, but is an effective prognosis biomarker.Part two. KRAF and BRAF mutation detection in plasma from Patients with Colorectal cancerPurpose:To determine whether plasma cfDNA is a viable alternative approach for KRAS and BRAF mutation testing in colorectal cancer. Methods:The study included165CRC patients. An ARMS-based quantitative PCR with a LNA blocker (ARMS-qPCR(LNA)) was developed to asses the KRAS and BRAF mutation in preoperative plasma and paired tumor tissue. Firstly intra-tumoral heterogeneity analysis was used to determine whether it is a robust mutation detection method for the detection of low-abundance BRAF and KRAS mutations.Results:The newly built ARMS-qPCR(LNA) is a robust mutation detection method to escape the most false negative caused by intra-tumoral heterogeneity. The accordance of plasma and tumor mutation are strongly correlated to TNM stage and cfDNA level. The majority of KRAS and BRAF mutations detected in tumors were also found in plasma(21/25(84%)) in patients of stage IV.Conclusion:KRAS and BRAF analysis in plasma cfDNA is only a viable alternative to tissue analysis in mCRC(stage IV). Quantitative levels of cfDNA and plasma mutation positive are strongly correlated and hold promise of clinical application.