Screen KRAS Mutations From Plasma Of Cancer Patients

Purpose:KRAS mutations of tumor tissues are predictive marker for EGFR-anitbody treatment in metastistic colorectal cancer patients and EGFR-TKI treatment in NSCLC patients. However, as the development of tumor, the gene status of cancer cells will change and screening gene mutation of primary tumor tissues can’t reflect the gene status of metastistic tumor all the time. There are two purpoes of this study:(1) To choose and estabilish a convenient method to screen KRAS mutations from plasma of cancer patients and compare results with those from tumor tissues; (2) To establish a method that could quantify KRAS mutations from plasma of cancer patients in order to predict the over survival of cancer patients and predict the effect of cancer treatment.Methods:We chose and established COLD-PCR method and PNA-PCR method respectively. Using dilution method, we got the sensitivity of COLD-PCR method and PNA-PCR method.We screened KRAS mutations from plasma of 19 pancreatic cancer patients using COLD-PCR and PNA-PCR method repectively and compared results with thoese from tumor tissues. Then, using PNA-PCR method, we detected plasma KRAS mutations from 100 cases of colorectal cancer and compared results with those from tumor tissues.Results:The sensitivity of COLD-PCR method was 0.5% and its sensitivity increase about 10 fold than regular PCR method. The sensitivity of PNA-PCR was 1:5000 (KRAS mutant type:KRAS wild type) and the PNA could totally suppress the amplification of wild-type DNA and didn’t affect the amplification of KRAS mutant DNA, which means this method has the potential ability to quantify plasma KRAS mutant DNA. There are 16 cases of KRAS mutations from 19 cases of pancreatic cancer tissues. COLD-PCR method could detect 2 cases of KRAS mutations from 19 cases of plasma and the mutation types were the same to those from tumor tissues. However, PNA-PCR method could detect 14 cases of KRAS mutations from these 19 cases of plasma and 13 cases had the identical mutation type with tumor tissues. In total, the coincidence rate of tumor tissues and plasma using PNA-PCR method was 82.4%. There were 49 cases of KRAS mutations from 100 cases colorectal tumor tissues. PNA-PCR method could detect 38 cases of KRAS mutations from plasma specimens. There were 30 specimens that could detect the same KRAS mutant type from both tumor tissues and plasma specimens. There were 17 specimens that could be detected mutations from tumor tissues but not from plasma. There were also 6 specimens that didn’t exist KRAS mutations in tumor tissues but did have mutations in plasma (two of them were silent mutation). In total, the coincidence rate of tumor tissues and plasma was 75.0%.Conclusions:In this study, we established a high sensitivity and specificity PNA-PCR method that could detect plasma KRAS mutations from cancer patients and had potential ability to quanlify plasma KRAS mutant DNA. This method could detect plasma KRAS mutations with high coincidence rate comparing to those from tumor tissues. The PNA-PCR method could even detect KRAS mutations that didn’t exist in tumor tissues, which shows that screening KRAS mutations from plasma may be more significant than from tumor tisssues.