Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing

Abstract

Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10-5). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples.

Prevalence of BRAF gene mutation in samples of primary and metastatic colorectal cancer: A meta‐analysis

“…Of them, mutation was seen in six samples (Sakai et al, 2015). No evidence of BRAF gene mutation was observed in the study conducted by Naghibalhossaini on 110 tumour samples (Aparicio et al, 2014).…”

“…Investigation of BRAF gene mutation among 100 colorectal tumour samples was carried out by Sakai et al in 2015. Of them, mutation was seen in six samples (Sakai et al, ). No evidence of BRAF gene mutation was observed in the study conducted by Naghibalhossaini on 110 tumour samples (Aparicio et al, ).…”

Randomized study of FOLFIRI plus either panitumumab or bevacizumab for wild‐type KRAS colorectal cancer‐WJOG 6210G

“…Total nucleic acid content was purified from 0.5 to 1.0 mL serum using QIAamp Circulating Nucleic Acid kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The sensitivity assays were performed with genomic DNA extracted from cultured cell lines and healthy human blood samples as described previously …”

“…The sensitivity assays were performed with genomic DNA extracted from cultured cell lines and healthy human blood samples as described previously. (20) Mutant alleles of KRAS and NRAS exon 2 (codons 12 and 13), exon 3 (codons 59, 61,117 and 146), and BRAF exon 15 (codon 600) were assessed. (20,21) Multiplex polymerase chain reaction (PCR) containing pooled primers for KRAS, NRAS, and BRAF was performed with the Complete PCR Reagent set (Sequenom, Japan), followed by the preparation of a barcoded DNA library using the Ion Plus Fragment Library kit (Thermo Fisher Scientific, Japan) and IonXpress barcode adaptors (Thermo Fisher Scientific, Japan).…”

Can detection of Braf p.V600E mutation be improved? Comparison of allele specific multiplex sequencing to present tests

“…[4,5] In attempting to meet these objectives, methods with significantly increased sensitivities and specificities have been developed, including allele-specific PCR. [6][7][8][9][10][11][12][13] Although allele-specific PCR and ASMS both use allelespecific primers, they are two quite different methods. In allele-specific PCR, allele-specific primers are used in the amplification step.…”