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

Abstract

Background 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. Materials and methods 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. Results 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. Conclusions 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.

Predictive molecular pathology in the time of COVID-19

“…19 In fact, although most cases (95%) were diagnosed by the fully automated approach only, on some occasions, NGS was still carried out to refine uncertain results displaying RT-PCR curves of undetermined interpretation. 20 Moreover, not all clinically relevant biomarkers are testable by Idylla; as an example, automated assays for KIT and PDGFRA gene evaluations have not been developed yet. On the other sides, the disadvantage of the fully automated approach is the higher overall cost for a single patient.…”

Effect of KRAS and NRAS mutations on the prognosis of patients with synchronous metastatic colorectal cancer presenting with liver‑only and lung‑only metastases

“…This suggested that BNA-clamp PCR using Sanger sequencing detects somatic mutations in KRAS, NRAS and BRAF with a high accuracy (42). Gilson et al (43) used DNA pipetted directly in the cartridge of the Idylla system, exhibiting a good sensitivity, specificity, reproducibility and limit of detection, and can be integrated in a laboratory workflow for samples with little tissue without compromising the further complete Table III. Gene mutation status and progression-free survival analysis of patients with metastatic colorectal cancer.…”

“…This suggested that BNA-clamp PCR using Sanger sequencing detects somatic mutations in KRAS, NRAS and BRAF with a high accuracy ( 42 ). Gilson et al ( 43 ) used DNA pipetted directly in the cartridge of the Idylla system, exhibiting a good sensitivity, specificity, reproducibility and limit of detection, and can be integrated in a laboratory workflow for samples with little tissue without compromising the further complete tumor characterization using NGS. In the present study, the ABI PRISM 310 genetic analyzer system and Taqman genotyper v.1.6 software (Applied Biosystems; Thermo Fisher Scientific, Inc.) were used to perform accurate analysis using automated capillary electrophoresis for genotyping.…”

Encorafenib (Braftovi)

“…Gilson et al conducted a study using the Idylla real-time PCR platform to detect BRAF, NRAS, and KRAS mutations in 38 samples from patients with colorectal cancer. 70 All sample mutation statuses were previously confirmed using NGS as a reference. Eight specimens were selected to evaluate the sensitivity and 12 for the specificity for the identification of BRAF mutations using real-time PCR.…”