KRAS Mutation Analysis by PCR: A Comparison of Two Methods

Abstract

BackgroundKRAS mutation assays are important companion diagnostic tests to guide anti-EGFR antibody treatment of metastatic colorectal cancer. Direct comparison of newer diagnostic methods with existing methods is an important part of validation of any new technique. In this this study, we have compared the Therascreen (Qiagen) ARMS assay with Competitive Allele-Specific TaqMan PCR (castPCR, Life Technologies) to determine equivalence for KRAS mutation analysis.MethodsDNA was extracted by Maxwell (Promega) from 99 colorectal cancers. The ARMS-based Therascreen and a customized castPCR assay were performed according to the manufacturer’s instructions. All assays were performed on either an Applied Biosystems 7500 Fast Dx or a ViiA7 real-time PCR machine (both from Life Technologies). The data were collected and discrepant results re-tested with newly extracted DNA from the same blocks in both assay types.ResultsOf the 99 tumors included, Therascreen showed 62 tumors to be wild-type (WT) for KRAS, while 37 had KRAS mutations on initial testing. CastPCR showed 61 tumors to be wild-type (WT) for KRAS, while 38 had KRAS mutations. Thirteen tumors showed BRAF mutation in castPCR and in one of these there was also a KRAS mutation. The custom castPCR plate included several other KRAS mutations and BRAF V600E, not included in Therascreen, explaining the higher number of mutations detected by castPCR. Re-testing of discrepant results was required in three tumors, all of which then achieved concordance for KRAS. CastPCR assay Ct values were on average 2 cycles lower than Therascreen.ConclusionThere was excellent correlation between the two methods. Although castPCR assay shows lower Ct values than Therascreen, this is unlikely to be clinically significant.

Prognostic value of plasma EGFR ctDNA in NSCLC patients treated with EGFR-TKIs

“…Currently, ARMS PCR has become one of the most important and advanced technologies in the molecular detection of cancer individuals, and its advantages in clinical applications have been widely recognized in industry[17,18]. The detection sensitivity of ARMS method was significantly higher than that of other methods, such as direct sequencing method, which can detect the mutation of 0.1–1.0% gene in samples.…”

Key differences between 13 KRAS mutation detection technologies and their relevance for clinical practice

“…11 Sanger capillary sequencing has long been the gold standard for DNA sequence analysis; however, it does not offer the high sensitivity required to detect somatic mutations at allelic frequencies less than ~20%. 12 Numerous comparisons of technology platforms for EGFR 13 14 and KRAS (mainly in CRC [15][16][17][18][19][20][21][22][23] ) mutation testing conclude that quantitative PCR (qPCR)-based methods offer the sensitivity, tissue economy and turnaround time required by physicians to guide treatment decisions. The advent of next-generation sequencing (NGS) platforms offers pathology laboratories an unparalleled insight into the cancer genome including de novo detection of variants as well as known actionable targets.…”

Detection of EGFR and BRAF mutations by competitive allele-specific TaqMan polymerase chain reaction in lung adenocarcinoma

“…Compared with ARMS, the most commonly used method in clinical practice in China, CastPCR has improved sensitivity and specificity owing to its oligonucleotide blocker that suppresses the wild-type allele, which does not exist in ARMS (28). Previous studies have demonstrated this in multiple types of malignant tumor (22,38,39). Two previous studies used CastPCR technology to validate detected mutation results obtained by NGS (31,40).…”