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

Abstract

IntroductionThis study assessed KRAS mutation detection and functional characteristics across 13 distinct technologies and assays available in clinical practice, in a blinded manner.MethodsFive distinct KRAS-mutant cell lines were used to study five clinically relevant KRAS mutations: p.G12C, p.G12D, p.G12V, p.G13D and p.Q61H. 50 cell line admixtures with low (50 and 100) mutant KRAS allele copies at 20%, 10%, 5%, 1% and 0.5% frequency were processed using quantitative PCR (qPCR) (n=3), matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) (n=2), next-generation sequencing (NGS) (n=6), digital PCR (n=1) and Sanger capillary sequencing (n=1) assays. Important performance differences were revealed, particularly assay sensitivity and turnaround time.ResultsOverall 406/728 data points across all 13 technologies were identified correctly. Successful genotyping of admixtures ranged from 0% (Sanger sequencing) to 100% (NGS). 5/6 NGS platforms reported similar allelic frequency for each sample. One NGS assay detected mutations down to a frequency of 0.5% and correctly identified all 56 samples (Oncomine Focus Assay, Thermo Fisher Scientific). One qPCR (Idylla, Biocartis) and MALDI-TOF (UltraSEEK, Agena Bioscience) assay identified 96% (all 100 copies and 23/25 at 50 copies input) and 92% (23/25 at 100 copies and 23/25 at 50 copies input) of samples, respectively. The digital PCR assay (KRAS PrimePCR ddPCR, Bio-Rad Laboratories) identified 60% (100 copies) and 52% (50 copies) of samples correctly. Turnaround time from sample to results ranged from ~2 hours (Idylla CE-IVD) to 2 days (TruSight Tumor 15 and Sentosa CE-IVD), to 2 weeks for certain NGS assays; the level of required expertise ranged from minimal (Idylla CE-IVD) to high for some technologies.DiscussionThis comprehensive parallel assessment used high molecular weight cell line DNA as a model system to address key questions for a laboratory when implementing routine KRAS testing. As most of the technologies are available for additional molecular biomarkers, this study may be informative for other applications.

Rapid clinical mutational testing ofKRAS,BRAFandEGFR: a prospective comparative analysis of the Idylla technique with high-throughput next-generation sequencing

“…We analysed all peer-reviewed literature on the use of the Idylla platform in all tumours in which mutational status is required for treatment of the patient (table 1). 2–25…”

Response: Concerns with conclusions in the article by Sherwood et al ‘Key differences between 13 KRAS mutation detection technologies and their relevance for clinical practice’

“…We thank Dr Karlin-Neumann for his comments 1 on our work 2 and welcome the opportunity to provide clarification on the points raised.…”

“…In summary, our study 2 was carried out in various clinical laboratories (contract), research organisations and in part by assay manufacturers highlighting to laboratories the need for robust local validation. Further, the limitations of the study and the fact that it does not show the superiority of one technology over another are clearly stated and thereby offer a valuable resource regarding the issues faced by diagnostic laboratories.…”

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