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BACKGROUND:Cell-free DNA detection is becoming a surrogate assay for tumor genotyping. Biological fluids often content a very low amount of cell-free tumor DNA and assays able to detect very low allele frequency mutant with a few quantities of DNA are required. We evaluated the ability of the fully-automated molecular diagnostics platform Idylla for the detection of KRAS, NRAS and BRAF hotspot mutations in plasma from patients with metastatic colorectal cancer (mCRC). MATERIALS AND METHODS:First, we evaluated the limit of detection of the system using two set of laboratory made samples that mimic mCRC patient plasma, then plasma samples from patients with mCRC were assessed using Idylla system and BEAMing digital PCR technology. RESULTS:Limits of detection of 0.1%, 0.4% and 0.01% for KRAS, NRAS and BRAF respectively have been reached. With our laboratory made samples, sensitivity up to 0.008% has been reached. Among 15 patients' samples tested for KRAS mutation, 2 discrepant results were found between Idylla and BEAMing dPCR. A 100% concordance between the two assays has been found for the detection of NRAS and BRAF mutations in plasma samples. CONCLUSIONS:The Idylla system does not reach as high sensitivity as assays like ddPCR but has an equivalent sensitivity to modified NGS technics with a lower cost and a lower time to results. These data allowed to consider the Idylla system in a routine laboratory workflow for KRAS, NRAS and BRAF mutations detection in plasma.

BACKGROUND:KRAS and NRAS mutations are identified resistance mutations to anti-epidermal growth factor receptor monoclonal antibodies in patients with metastatic colorectal cancer. BRAF status is also routinely assessed for its poor prognosis value. In our institute, next-generation sequencing (NGS) is routinely used for gene-panel mutations detection including KRAS, NRAS and BRAF, but DNA quality is sometimes not sufficient for sequencing. In our routine practice, Idylla platform is used for the analysis of samples that don't reach sufficient quality criteria for NGS assay. METHODS:In this study, data from mCRC samples analyzed from May 2017 to 2018 were retrospectively collected. All samples with a poor DNA quality for sequencing have been assessed using Idylla platform. First, KRAS Idylla assay cartridge has been used for the determination of KRAS mutational status. All KRAS wild-type samples have then been analyzed using NRAS-BRAF assay. Among 669 samples, 67 samples failed the DNA quality control and have been assessed on Idylla KRAS mutation test. RESULTS:Among 67 samples, 50 (75%) samples had a valid result with Idylla KRAS mutation test including 22 carrying a KRAS mutation. For 28 samples, NRAS and BRAF mutational statuses have been assessed using Idylla NRAS-BRAF mutation test. Among 28 samples, 27 (96%) had a valid result including 2 samples bearing a NRAS mutation and 3 samples bearing a BRAF mutation. CONCLUSIONS:Our study shows that an integrated workflow using NGS and Idylla platform allows the determination of KRAS, NRAS and BRAF mutational statuses of 651/669 (97.3%) samples and retrieve 49/67 (73.1%)samples that don't reach DNA quality requirements for NGS.

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.