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

James L Sherwood; Helen Brown; Alessandro Rettino; Amelie Schreieck; Graeme Clark; Bart Claes; Bhuwnesh Agrawal; Ria Chaston; Benjamin S G Kong; Paul Choppa; Anders O H Nygren; Ina L Deras; Alexander Kohlmann

doi:10.1136/esmoopen-2017-000235

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

ESMO Open. 2017 Sep 28;2(4):e000235. doi: 10.1136/esmoopen-2017-000235. eCollection 2017.

Authors

Affiliations

¹ Precision Medicine and Genomics, Innovative Medicines and Early Development Biotech, AstraZeneca, Cambridge, UK.
² West Midlands Regional Genetics Laboratory, Birmingham Women's NHS Foundation Trust, Birmingham, UK.
³ IMGM Laboratories, Martinsried, Germany.
⁴ Department of Medical Genetics, University of Cambridge, Cambridge Biomedical Research Campus, Cambridge, UK.
⁵ Biocartis NV, Mechelen, Belgium.
⁶ Vela Diagnostics, Singapore.
⁷ NewGene, Newcastle upon Tyne, UK.
⁸ Thermo Fisher Scientific, Clinical Sequencing Division, West Sacramento‎, California, UK.
⁹ Agena Bioscience, San Diego, California, USA.
¹⁰ Illumina, San Diego, California, USA.

Abstract

Introduction: This study assessed KRAS mutation detection and functional characteristics across 13 distinct technologies and assays available in clinical practice, in a blinded manner.

Methods: Five 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.

Results: Overall 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.

Discussion: This 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.

Keywords: EGFR mutation; MALDITOF; NGS; NSCLC; ddPCR; platform comparison; qPCR.