Assessment of EGFR mutations in plasma from advanced NSCLC patients by modified cell-free DNA extraction method and blocker PCR assays.

e23115 Background: Epidermal growth factor receptor (EGFR) mutation testing in plasma cell-free DNA (cfDNA) for lung cancer patients is an emerging clinical tool.However,the sensitivity of plasma testing,compared with tissue testing, is not high enough. We aimed to investigate the likelihood of improving the plasma testing sensitivity by increase the cell-free DNA(cfDNA )yields using a modified extraction method and blocker PCR. Methods: Blood samples were collected from 106 patients without EGFR-TKI treatment and 127 patients with acquired EGFR-TKI resistance.CfDNA was extracted from plasma by the modified extraction method and mutations were identified by blocker PCR.The yields of circulating tumor DNA (ctDNA) and cfDNA from 106 patients were quantified by standard curve.Next generation sequencing (NGS)was employed to verify the reliability of blocker PCR. Meanwhile,paired tumor tissues were examined by amplification refractory mutation system (ARMS). Results: The novel extraction method can extract small fragment less than 100bp.The yield of total cfDNA of 106 plasma was mostly between4 000and 200,000 copies per milliliter.With regard to exon 19 deletions(E19-DELs) , the ctDNA proportion of 85% samples (17/20)was from 0.1% to 5% . As for L858R mutations, the fraction of ctDNA of 84.6%(11/13)cases was calculated with the range of 0.01% to 0.1%.For 106 patients,the overall concordance rate,sensitivity and specificity between plasma and tumor tissue was 99.06%[k = 0.97; 95% confidence interval (CI), 0.87-0.99; P < 0.0001],94.87%(95% CI, 83.11%-98.58%) and 100 % (95% CI,94.58%-100%).Among the 127patients,40.16%(51/127)T790M was detected in the plasma, 78.44%(40/51) of which coexists with an EGFR activating mutation. Conclusions: The modified extraction method can improve ctDNA yields and thus improve the sensitivity of liquid biopsy.The blocker PCR offers the possibility of detecting mutations in cfDNA with high concordance with tumor tissues.