Mutant-enriched PCR Combined With Pyrosequencing To Detect KRAS Mutations In Cancer Tissue And Matched Blood In NSCLC Patients

Backgroud and Objective:Many clinical research data have confirmed that the epidermal growth factor receptortyrosine kinase inhibitors（EGFR-TKI) in the treatment of sensitive EGFR mutationNSCLC patients can significantly prolong the progression-free survival and the overallsurvival. But KRAS mutations is associated with primary drug resistance of TKI clearly, sothe detection of KRAS mutations can reduce the risk of TKI targeted therapy. We haveestablished a simple, highly sensitive detection technology that combine mutant-enrichedPCR with pyrosequencing, for KRAS mutations in the cancer tissue and peripheral blood inpatients with NSCLC, which can be used to screen clinical targeted NSCLC patients andprovide the basis for choosing the best drug crowd.Method:We collected43cancer tissue samples and matched blood samples and extracted DNAfrom patients with NSCLC, using the mutant-enriched PCR joint with pyrosequencingtechnology, to detect the carcinoma tissue and matched peripheral blood samples of KRAS,KRAS mutations type Mixed with wild type of KRAS cell line (A549/HCC827) was usedto evaluate the sensitivity of this method. By analyzing the consistency mutation status ofcancer tissue samples and matched blood samples, to establish a kind of detection methodby peripheral means.Results:1. The tissue samples from43patients with NSCLC were tested and we detected4casesof KRAS mutations, which were12codon mutation. Among them,2cases of mutation typeswere AGT, another2cases of mutation types were GAT; Mutation rate was9.3%(4/43).2. Plasma samples from43patients with NSCLC were tested and we detected KRASmutations in3cases, which were12codon mutation. Among them,2cases of mutation types were AGT,1case of mutation types was GAT; Mutation rate was7%(4/43). Theconsistency is75%.3. According to the different proportions of mixed wild type of KRAS HCC827cellline with A549cell lines of KRAS mutations type, when mixing proportion is less than1000:1, we can observe the mutations’ peak, it is indicated the sensitivity of this method is0.1%.Conclusion:1. KRAS mutation exists in advanced NSCLC patients and the KRAS mutation rate intissue samples is9.3%.2. A high consistency of KRAS mutation exists in advanced NSCLC patients betweenin plasma and in tumor tissues. Plasma DNA could be used as a replace fortumor tissueswhen it is hard to obtain the tissues; Before we descide to give EGFR-TKI treament, theKRAS mutation state in peripheral blood can act as a effective reference indicator or not.3. In our study, we have successfullly combined ME-PCR with pyrosequencing in thefirst time. Enrichment-PCR enzyme, introducing a specific restriction enzyme, can makemore KRAS mutations effectively; On this basis, it jointed with pyrosequencing technology,a large sample, high-throughput, accurate detection method has been developed for detectthe KRAS mutations; It also has provided a guidance for NSCLC patients Whether they aresuit for TKI treatment or not, brought the biggest benefit for patients and avoided the wasteof medical resources.