Comparison of Sanger Sequencing, Pyrosequencing, and Melting Curve Analysis for the Detection of KRAS Mutations

Abstract

Mutations in codons 12 and 13 of the KRAS oncogene are relatively common in colorectal and lung adenocarcinomas. Recent data indicate that these mutations result in resistance to anti-epidermal growth factor receptor therapy. Therefore, we assessed Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS codon 12/13 mutations in formalin-fixed paraffin-embedded samples, including 58 primary and 42 metastatic colorectal adenocarcinomas, 63 primary and 17 metastatic lung adenocarcinomas, and 20 normal colon samples. Of 180 tumor samples, 62.2% were KRAS mutant positive, and 37.8% were negative. Melting curve analysis yielded no false positive or false negative results, but had 10% equivocal calls. Melting curve analysis also resulted in 4 cases with melting curves inconsistent with either wild-type or codon 12/13 mutations. These patterns were generated from samples with double mutants in codons 12/13 and with mutations outside of codons 12/13. Pyrosequencing yielded no false positive or false negative results as well. However, two samples from one patient yielded a pyrogram that was flagged as abnormal, but the mutation subtype could not be determined. Finally, using an electronic cutoff of 10%, Sanger sequencing showed 11.1% false positives and 6.1% false negatives. In our hands, the limit of detection for Sanger sequencing, pyrosequencing, and melting curve analysis was approximately 15 to 20%, 5%, and 10% mutant alleles, respectively.

External Quality Assessment for KRAS Testing Is Needed: Setup of a European Program and Report of the First Joined Regional Quality Assessment Rounds

“…A number of new technologies have also been applied to KRAS testing, such as high-resolution melting analysis and pyrosequencing. The latter has the advantage of higher sensitivity than regular dideoxysequencing [20].…”

Resistance to Anti-EGFR Therapy in Colorectal Cancer: From Heterogeneity to Convergent Evolution

“…Furthermore, the same single lesion can harbor more than one independent clone ( 2 , 57 , 58 ). These observations are particularly relevant when considering that previous studies mainly involved analysis of KRAS exon 2 mutations, and that the most commonly used techniques (Sanger sequencing) have a limit of detection of approximately 15% to 20% ( 59 ). Of interest, it has been shown that more sensitive approaches such as pyrosequencing or digital PCR can increase the detection of mutant RAS alleles, which in turn could translate into the detection of additional refractory patients ( 22 , 57 , 58 , 60 , 61 ).…”

Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies

“…We elected not to Sanger confirm every additional variant despite this approach being the purported gold standard. More than half of our detected variants harbored VAFs <20%, and with published sensitivities of 15% to 20% for variant detection, 34 we reasoned Sanger confirmation was an insensitive method for confirmation. Unfortunately, comprehensive confirmatory sequencing was not practical because of cost and paucity of remaining sample material, highlighted by a discrepant KRAS p.G13C mutation that was not detected by SNaPshot analysis (NSCLC-4).…”