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

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.

Development and validation of a scalable next-generation sequencing system for assessing relevant somatic variants in solid tumors.

Next-generation sequencing (NGS) has enabled genome-wide personalized oncology efforts at centers and companies with the specialty expertise and infrastructure required to identify and prioritize actionable variants. Such approaches are not scalable, preventing widespread adoption. Likewise, most targeted NGS approaches fail to assess key relevant genomic alteration classes. To address these challenges, we predefined the catalog of relevant solid tumor somatic genome variants (gain-of-function or loss-of-function mutations, high-level copy number alterations, and gene fusions) through comprehensive bioinformatics analysis of >700,000 samples. To detect these variants, we developed the Oncomine Comprehensive Panel (OCP), an integrative NGS-based assay [compatible with <20 ng of DNA/RNA from formalin-fixed paraffin-embedded (FFPE) tissues], coupled with an informatics pipeline to specifically identify relevant predefined variants and created a knowledge base of related potential treatments, current practice guidelines, and open clinical trials. We validated OCP using molecular standards and more than 300 FFPE tumor samples, achieving >95% accuracy for KRAS, epidermal growth factor receptor, and BRAF mutation detection as well as for ALK and TMPRSS2:ERG gene fusions. Associating positive variants with potential targeted treatments demonstrated that 6% to 42% of profiled samples (depending on cancer type) harbored alterations beyond routine molecular testing that were associated with approved or guideline-referenced therapies. As a translational research tool, OCP identified adaptive CTNNB1 amplifications/mutations in treated prostate cancers. Through predefining somatic variants in solid tumors and compiling associated potential treatment strategies, OCP represents a simplified, broadly applicable targeted NGS system with the potential to advance precision oncology efforts.

Sensitive multiplex detection of KRAS codons 12 and 13 mutations in paraffin-embedded tissue specimens.

BACKGROUND: Colorectal cancer patients harbouring KRAS mutations in codon 12 or 13 do not benefit from current anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies. Efficient and robust methods are therefore required for routine clinical testing of KRAS mutation status. AIMS: To evaluate a novel multiplex assay for the rapid detection of common KRAS mutations in formalin-fixed paraffin-embedded (FFPE) tissues. METHODS: Genomic DNA was amplified by multiplex PCR using primers targeting the KRAS codon 12/13 region and an internal control gene. PCR products were hybridised on a liquid bead array containing target-specific probes and detected by particle flow cytometry. RESULTS: Analytical performance assessed with plasmid DNA and genomic DNA extracted from cell lines or model FFPE cell line dilutions showed specific detection of seven distinct KRAS mutations with a limit of detection equivalent to 1% tumour. The assay was evaluated at two independent sites with a total of 140 clinical specimens. At site 1, about 45% of the specimens from a set of 86 archived FFPE blocks with unknown KRAS mutation status were found positive for a KRAS mutation. At site 2, each of the seven mutations was detected in at least five independent specimens from a selected set of 54 residual genomic DNAs previously tested with an ARMS/Scorpion laboratory-developed test. CONCLUSIONS: This novel single-well assay is a sensitive tool compatible with the clinical laboratory workflow for the rapid assessment of KRAS mutations in solid tumour specimens. Its performance and multiplex format warrant the development of broader panels including other relevant mutations in the EGFR pathway.

A novel method for the detection, quantitation, and breakpoint cluster region determination of t(15;17) fusion transcripts using a one-step real-time multiplex RT-PCR.

Individuals with acute promyelocytic leukemia (APL) usually express 1 of 3 primary hybrid transcripts associated with a t(15;17). The 3 fusion transcripts are the result of heterogeneous breakpoint cluster regions (bcr) within the promyelocytic leukemia (PML) gene and are denoted bcr1 (long), bcr2 (variant), and bcr3 (short) forms. Many researchers have shown that real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of the involved transcript is a valuable tool for monitoring APL and its treatment. In addition, some research suggests that identification of a specific breakpoint region may be used to predict an individual's likelihood of relapse and possibly their response to all-trans retinoic acid treatment. We describe the first reported 1-step multiplex RT-PCR assay capable of t(15;17) fusion transcript real-time relative quantitation and simultaneous transcript form identification in 2 reactions. This assay uses a novel dual-probe technique to achieve what has required a laborious procedure of 2 or more reactions followed by postamplification analysis. We found a correlation of 100% in detection and breakpoint determination of the long, short, and variant forms with a breakpoint 5' to nucleotide 1709 compared with results from traditional methods.