Somatic mutation detection efficiency in EGFR: A comparison between High Resolution Melting Analysis and Sanger Sequencing

Abstract

Background: The current study focuses on comparing the mutation detection efficiency of High-Resolution Melt Analysis (HRM) with Sanger Sequencing in EGFR gene.Methods: The minor allele frequency of somatic mutations was titrated against total DNA concentration using Sanger sequencing and HRM. Somatic mutation detection efficiency, for exons 19 & 21 of the EGFR gene, was compared in 116 formalin fixed paraffin embedded tumor tissues, after screening 275 tumor tissues by Sanger sequencing.Results: The limit of detection of minor allele fraction of exon 19 mutation was 1% with Sequencing, and 0.25% with HRM, whereas for exon 21 mutation, 0.25% MAF was detected using both methods. Mutation positive samples detected by Sanger sequencing, and wild-type samples detected by HRM were corresponding to each other. About 28% cases in exon 19 and 40% in exon 21, detected as mutated in HRM, was not detected by sequencing. Overall, sensitivity and specificity of HRM were found to be 100% and 67% respectively, and the negative predictive value was 100%, while positive predictive value was 80%.Conclusion: The comparative series study suggests that HRM is a modest initial screening test for somatic mutation detection of EGFR, which must further be confirmed by Sanger sequencing.