EGFR gene mutations are associated with clinical response to EGFR-TKI. Recently available methods of EGFR mutation detection rely on tumor specimens whic" />
The Comparision of Methods for the Detection of EGFR Gene Mutations in Serum of Lung Cancer Patients
|School||Beijing Union Medical College|
|Keywords||Lung cancer serum EGFR gene mutation method comparisionPrimer extension DHPLC EGFR T790M MutationGene sequencing K-ras gene Mutation|
Background:EGFR gene mutation' dissertation">EGFR gene mutations are associated with clinical response to EGFR-TKI. Recently available methods of EGFR mutation detection rely on tumor specimens which are difficult from every patient, especially from advanced lung cancer patients. Studies have showed the consistency of EGFR mutation status between tissue and matched serum DNA. Here we used serum samples instead of tissue to find a more sensitive and accurate method for the detection of EGFR gene mutations.Objective:To find a method with higher sensitivity and accuracy among DHPLC, gene sequencing and Dxs EGFR29mutations Kit for screening EGFR gene mutations in circulating blood of lung cancer patients.Methods:87serum DNA samples from advanced lung cancer patients were selected randomly and analyzed for EGFR exon19and21gene mutations by DHPLC and gene sequencing.49serum samples, which EGFR exon19and21gene mutations had been detected using Dxs EGFR29mutations Kit by Zhou Nan, were also analyzed for the same mutations by DHPLC and gene sequencing.Results:Ini advanced lung cancer patients’ serum samples, mutation rate of EGFR was found to be35.63%(31/87) by DHPLC and4.59%(4/87) by gene sequencing. As a result, DHPLC was more sensitive than gene sequencing for the detection of EGFR mutations (P<0.01). EGFR mutations were not correlated with age, sex and smoking status(P>0.05). The mutation rate of adenocarcinoma was higer than other types of lung cancer, In benign diseases and early-stage lung cancer patients’serum samples, mutations of EGFR were not found by DHPLC, gene sequencing or Dxs EGFR29mutations Kit.Conclusions:Comparing with gene sequencing, DHPLC could serve as a better preliminary screening method for EGFR gene mutations in advanced lung cancer patients’ serum samples. Due to the low percentage of serum mutant DNA, no EGFR mutation was found by the three methods above in early-stage lung cancer patients’ serum. So, a more sensitive method is needed for early-stage lung cancer patients. Objective: To establish a sensitive method to detect EGFR exon20T790M mutation.Methods: We prepared mutant genomic DNA from cell line H1975with EGFR exon20T790M mutation and wild genomic DNA without T790M mutation, amplified the target sequences of mutant and wild EGFR exon20by PCR and extended them with special T790M site primer in primer extension reaction. The extension products were analyzed by DHPLC at80℃respectively。Results: The products, which matched to the mutation site or wild site, could be separated by DHPLC. The peak for ddTTP-extension products corresponding to the T790M mutation site was observed at retention time4.0min, while the peak for ddCTP-extension products corresponding to the wild site was detected at retention time3.5min.Conclusions: The established DHPLC-primer extension assay could be used to detect EGFR exon20T790M mutation site and EGFR exon20wild site simultaneously with high sensitivity. DHPLC-primer extension assay might be a promising method to detect EGFR exon20T790M mutation in circulating blood. Objective: To analyze the feasibility for the detection of K-ras gene mutation using gene sequencing and DHPLC.Methods: Genomic DNA was extracted from cell line sw480containing mutant or wild K-ras exon1codon12. And then, K-ras exon1was amplified by PCR as the target sequence respectively. Mutation amplications were mixed with the same volume of wild type and assayed by DHPLC. Meanwhile, mutant amplications were mixed with wild amplications at sequencial concentrations and assayed by gene sequencing.Results: DHPLC could not distinguish mutant K-ras from wild type K-ras, while gene sequencing could detect the mutant K-ras with low sensitivity (nearly20%).Conclusions: In our lab, DHPLC could not be used to detect K-ras gene mutation, which need be improved. Gene sequencing was accurate but not sensitive enough for the detection of low percentage K-ras mutation in plasma or serum.