Next-generation sequencing-based detection of EGFR, KRAS, BRAF, NRAS, PIK3CA, Her-2 and TP53 mutations in patients with non-small cell lung cancer

In recent years, the incidence of non-small cell lung cancer (NSCLC) has become the highest lethal rate of cancer worldwide. Molecular assays of EGFR, KRAS, BRAF, NRAS, PIK3CA and Her-2 are widely used to guide individualized treatment in NSCLC patients. Somatic mutations in 112 NSCLC patients, including 7 oncogenic driver genes, were detected by Iontorrent personal genome machine (PGM). Sanger sequencing was used to test and verify the results of PGM. Apart from uncommon mutations of EGFR, 101 NSCLC specimens were tested by droplet digital PCR (ddPCR). According to NGS results, mutations were detected in EGFR (58/112, 51.79% of tumors), KRAS (10/112, 8.93%), BRAF (2/112, 1.79%), NRAS (2/112, 1.79%), Her-2 (2/112, 1.79%), PIK3CA (6/112, 5.36%) and TP53 (31/112, 27.69%). There were 27 samples without any somatic mutations in all genes while 24 samples harboured mutations in two or more genes. A total of 61 samples had one or more mutations in a single gene. All alterations of 7 genes were presented and the overall detection rate of NGS and Sanger sequencing was determined to be 51.79% (58/112) and 37.50% (42/112), respectively (χ(2)=5.88, P=0.015). Compared with Sanger sequencing, the total sensitivity and specificity of NGS assays was 95.24% (40/42) and 77.14% (54/70), respectively. The overall detection rate of NGS and ddPCR was 45.54% (46/101) and 47.52% (48/101), respectively (χ(2)=0.000598, P=0.98). Compared with ddPCR, the overall sensitivity and specificity of NGS assays was 95.83% (46/48) and 98.11% (52/53), respectively. The findings indicated that the positive mutation rate of EGFR tested by NGS was significantly lower than that by Sanger sequencing, but the difference between ddPCR and NGS was not statistically significant. The high degree of agreement of reportable variants is proposed in both NGS and ddPCR analysis, suggesting the performance of NGS assays in routine clinical detection may be useful in determining the treatment decisions in NSCLC patients.