Abstract 604
Background
This study assessed, for the first time, KRAS mutation detection and functional characteristics across 12 distinct platforms and chemistries available in clinical practice.
Methods
5 distinct KRAS-mutant cell lines (MIA PACA-2, PANC-1, MDA-MB231, SW620 and NCI-H460) were obtained from AstraZeneca, Alderley Park Cell Bank, and 5 clinically relevant KRAS mutations were studied: p.G12C, p.G12D and 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 analysed using qPCR (n = 3), digital PCR (n = 1), capillary sequencing (n = 1), NGS (n = 5) and MALDI-TOF (n = 2) assays.
Results
Important performance differences were revealed which could impact patient treatment decisions, particularly sensitivity, regulatory status (e.g. CE-IVD) and turnaround time. Only 384/672 data points across all 12 methods were identified correctly. Successful genotyping of admixtures ranged from 0% (Sanger sequencing) to 100% (NGS). 4/5 NGS platforms reported similar allelic frequency for each sample. Of these, one was able to detect mutations down to a frequency of 0.1% and correctly identify all 56 samples (Oncomine™ Focus Assay, Thermo Fisher Scientific). A qPCR near impact device (Idylla™, Biocartis) and MALDI-TOF (UltraSEEK™, Agena Bioscience) accurately identified 96% (all 100 copies & 23/25 50 copies input) and 93% (23/25 100 copies & 23/25 50 copies input) of samples, respectively. Conversely, the digital PCR assay (KRAS PrimePCR™ ddPCR™, Bio-Rad Laboratories, Inc.) was non-specific, identifying the wrong mutation in 8 different mutation/allele frequency combinations. Turnaround time from clinical sample to result ranged from ∼2 hours (Idylla™ CE-IVD) to 1 day (cobas® CE-IVD) to >1 week for most NGS assays, while the level of required laboratory expertise ranged from minimal (Idylla™ CE-IVD) to high (NGS platforms).
Conclusions
This comprehensive parallel assessment used high molecular weight cell-line DNA as a model to address key questions for a clinical laboratory when implementing routine KRAS testing. As most of the technologies are available for other molecular biomarkers, results may be informative for other diagnostic functions.
Clinical trial identification
N/A
Legal entity responsible for the study
AstraZeneca
Funding
AstraZeneca
Disclosure
J.L. Sherwood, H. Brown, A. Kohlman: Employee of and shareholder in AstraZeneca London. A. Schreieck: institution received financial support for molecular analysis from AstraZeneca. B. Claes: Reports a patent pending for isolation of nucleic acids. B. Agrawal: Employee of Vela Diagnostics.A.O.H. Nygren: Employee of and shareholder in Agena Bioscience. All other authors have declared no conflicts of interest.