Clinical pharmacogenomic testing of KRAS, BRAF and EGFRmutations by high resolution melting analysis and ultra-deep pyrosequencing

Abstract

BackgroundEpidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices.MethodsWe describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform.ResultsWe tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations.ConclusionsHRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

446 Detection of EGFR and KRAS Mutation by Pyrosequencing Analysis in Cytologic Samples of Non-Small Cell Lung Cancer

“…Although, we did not compare pyrosequencing with other methods, pyrosequencing method is sensitive in detecting both EGFR and KRAS mutations. Similar to previous reports, EGFR mutations were prevalent in nonsmokers (65.43%, P < 0.001), whereas KRAS mutations were common in smokers (67.85%, P < 0.001), and the two mutations are mutually exclusive in our study (6)(7)(8)25,29).…”

Ultrasensitive Detection of Unknown Colon Cancer-Initiating Mutations Using the Example of the Adenomatous Polyposis Coli Gene

“…It was reported that LNA-based PCR clamping was able to detect 1% mutated KRAS in the template composite (26). Regarding HRM, KRAS mutations at a ratio of 5% were necessary to be detectable (27,28). A related method, the Digital Melt Curve (DMC) assay, was conducted as a two-step method enriching target genes by sequence-specific capture before combined digital PCR and HRM (29).…”

Molecular analysis of EGFR gene in different types of tumor material from NSCLC patients

“…We may speculate that this patient had greater-than-usual benefit from biological treatment. Although some studies have reported cases of double mutants, they always involved combination one activating mutation and one resistance mutation [35,40]. Double mutants are usually only found in cell lines, especially substitutions in exon 21 (p.L858R) and in exon 20 (p.T790M).…”