BACKGROUND: Epidermal 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.
METHODS: We 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.
RESULTS: We 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.
CONCLUSIONS: HRM 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.