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Two multiplex assays that simultaneously identify 22 possible mutation sites in the KRAS, BRAF, NRAS and PIK3CA genes.

ABSTRACT: Recently a number of randomized trials have shown that patients with advanced colorectal cancer do not benefit from therapies targeting the epidermal growth factor receptor when their tumors harbor mutations in the KRAS, BRAF and PIK3CA genes. We developed two multiplex assays that simultaneously screen 22 nucleotides in the KRAS, NRAS, BRAF and PIK3CA genes for mutations. The assays were validated on 294 tumor DNA samples from patients with advanced colorectal cancer. In these samples 119 KRAS codon 12 and 13 mutations had been identified by sequence analysis, 126 tumors were wild-type for KRAS and the analysis failed in 49 of the 294 samples due to poor DNA quality. The two mutation assays detected 130 KRAS mutations, among which were 3 codon 61 mutations, and in addition 32 PIK3CA, 13 BRAF and 6 NRAS mutations. In 19 tumors a KRAS mutation was found together with a mutation in the PIK3CA gene. One tumor was mutant for both PIK3CA and BRAF. In summary, the mutations assays identified 161 tumors with a mutation, 120 were wild-type and the analysis failed in 13. The material cost of the 2 mutation assays was calculated to be 8-fold lower than the cost of sequencing required to obtain the same data. In addition, the mutation assays are less labor intensive. We conclude that the performance of the two multiplex mutation assays was superior to direct sequencing. In addition, these assays are cheaper and easier to interpret. The assays may also be of use for selection of patients with other tumor types.

SUBMITTER: Lurkin I

PROVIDER: S-EPMC2809099 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 (?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 (?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.

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Two multiplex assays that simultaneously identify 22 possible mutation sites in the KRAS, BRAF, NRAS and PIK3CA genes.

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