Journal Article

Evaluation of KRAS, NRAS and BRAF hotspot mutations detection for patients with metastatic colorectal cancer using direct DNA pipetting in a fully-automated platform and Next-Generation Sequencing for laboratory workflow optimisation

Dubouis, Ludovic, Husson, Marie, Rouyer, Marie, Demange, Jessica, Perceau, Marie, Leroux, Agnès, Merlin, Jean-Louis, Harlé, Alexandre, Franczak, Claire, Gilson, Pauline

2019

Overview

Assessment of KRAS, NRAS (RAS) and BRAF mutations is a standard in the management of patients with metastatic colorectal cancer (mCRC). Mutations could be assessed using next-generation sequencing (NGS) or real-time PCR-based assays. Times to results are 1 to 2 weeks for NGS and 1 to 3 days for real-time PCR-based assays. Using NGS can delay first-line treatment commencement and using PCR-based assays is limited by the number of possible analysed targets. The Idylla system is a real-time PCR cartridge-based assay, able to analyse hotspots mutations using one section of FFPE tumour tissue sample. To combine short delays and analysis of a large gene-panel, we propose here a laboratory workflow combining the Idylla system and NGS and compatible with FFPE samples with low tissue quantity. In this study we evaluated and validated the Idylla system for the analysis of RAS and BRAF mutations by pipetting directly DNA in the cartridge instead of FFPE section as recommended by the manufacturer. DNA extracted from 29 FFPE samples from mCRC patients with NGS-characterized RAS and BRAF mutations were tested with the Idylla KRAS and the Idylla NRAS-BRAF mutation tests to assess sensitivity, specificity, reproducibility and limit of detection of each test. A 100% concordance was found between NGS and Idylla results for the determination of KRAS (12/12), NRAS (12/12) and BRAF (11/11) mutations with a sensitivity and a specificity of 100%. The system showed a good reproducibility with CV inferior to 3%. LOD was reached with 2.5 ng of DNA for KRAS and NRAS mutations and 5 ng of DNA for BRAF mutations. The analysis of RAS and BRAF mutations using DNA pipetted directly in the cartridge of the Idylla system showed a good sensitivity, specificity, reproducibility and LOD, and can be integrated in a laboratory workflow for samples with few tissue without compromising a further complete tumour characterization using NGS.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Aged / Aged, 80 and over / Assaying / Automation / Bioassay / Biology and Life Sciences / Cancer / Cancer genetics / Cancer metastasis / Cancer patients / Cancer therapies / Care and treatment / Colorectal cancer / Colorectal carcinoma / Colorectal Neoplasms - genetics / Colorectal Neoplasms - pathology / Deoxyribonucleic acid / DNA / DNA Mutational Analysis / DNA sequencing / DNA, Neoplasm - genetics / Female / Gene mutation / Gene sequencing / Genes / Genetic aspects / Genetic research / Health aspects / High-Throughput Nucleotide Sequencing - methods / Humans / Identification and classification / K-Ras protein / Life Sciences / Limit of Detection / Male / Medical prognosis / Medicine and Health Sciences / Metastases / Metastasis / Methods / Mutation / Mutation - genetics / Mutation hot spots / Neoplasm Metastasis / Next-generation sequencing / Optimization / Patients / Polymerase chain reaction / Quality / raf Kinases - genetics / Real time / Reproducibility / Reproducibility of Results / Research and analysis methods / Sensitivity / Sensitivity analysis / Sensitivity and Specificity / Tissues / Tumors / Workflow / Workflow software