Search

CfDNA samples from colon (mCRC) and non-small cell lung cancers (NSCLC) (CIRCAN cohort) were compared using three platforms: droplet digital PCR (ddPCR, Biorad); BEAMing/OncoBEAM™-RAS-CRC (Sysmex Inostics); next-generation sequencing (NGS, Illumina), utilizing the 56G oncology panel (Swift Biosciences). Tissue biopsy and time matched cfDNA samples were collected at diagnosis in the mCRC cohort and during 1st progression in the NSCLC cohort. Excellent matches between cfDNA/FFPE mutation profiles were observed. Detection thresholds were between 0.5-1% for cfDNA samples examined using ddPCR and NGS, and 0.03% with BEAMing. This high level of sensitivity enabled the detection of KRAS mutations in 5/19 CRC patients with negative FFPE profiles. In the mCRC cohort, comparison of mutation results obtained by testing FFPE to those obtained by testing cfDNA by ddPCR resulted in 47% sensitivity, 77% specificity, 70% positive predictive value (PPV) and 55% negative predictive value (NPV). For BEAMing, we observed 93% sensitivity, 69% specificity, 78% PPV and 90% NPV. Finally, sensitivity of NGS was 73%, specificity was 77%, PPV 79% and NPV 71%. Our study highlights the complementarity of different diagnostic approaches and variability of results between OncoBEAM™-RAS-CRC and NGS assays. While the NGS assay provided a larger breadth of coverage of the major targetable alterations of 56 genes in one run, its performance for specific alterations was frequently confirmed by ddPCR results., Competing Interests: CONFLICTS OF INTEREST Dr. Cheynet and Dr. Brengel-Pesce are employees of BioMérieux SA. Daniel Edelstein and Frederick Jones are employees of Sysmex Inostics, Inc. Dr. Xu reports to be employed by Sophia Genetics. Dr. Forestier reports personal fees from AMGEN, personal fees from SANOFI, personal fees from MERCK, personal fees from CELGENE, personal fees from ROCHE, personal fees from IPSEN, outside the submitted work. Dr. Bringuier reports personal fees from Astra Zeneca, outside the submitted work. Dr. Brevet reports grants from Astra Zeneca, personal fees from MSD, personal fees from BMS, grants from Pfizer, outside the submitted work. Dr. Garcia and Dr Payen report grants from Sysmex-innostics during the conduct of the study. Dr. Couraud reports grants and non-financial support from Sysmex Innostics, grants and personal fees from Astra Zeneca, grants from Merck, grants and personal fees from BMS, during the conduct of the study; grants and personal fees from Astra Zeneca, grants, personal fees and other from Pfizer, grants, personal fees and other from Roche, grants, personal fees and other from Chugai, grants, personal fees and other from MSD, grants, personal fees and other from Boehringher Ingelheim, grants and personal fees from Lilly, grants and personal fees from Novartis, grants and personal fees from Laidet Medical, grants from Amgen, other from Eformed, outside the submitted work. Other authors have nothing to disclose.

Non invasive somatic detection assays are suitable for repetitive tumor characterization or for detecting the appearance of somatic resistance during lung cancer. Molecular diagnosis based on circulating free DNA (cfDNA) offers the opportunity to track the genomic evolution of the tumor, and was chosen to assess the molecular profile of several EGFR alterations, including deletions in exon 19 (delEX19), the L858R substitution on exon 21 and the EGFR resistance mutation T790M on exon 20. Our study aimed at determining optimal pre-analytical conditions and EGFR mutation detection assays for analyzing cfDNA using the picoliter-droplet digital polymerase chain reaction (ddPCR) assay. Within the framework of the CIRCAN project set-up at the Lyon University Hospital, plasma samples were collected to establish a pre-analytical and analytical workflow of cfDNA analysis. We evaluated all of the steps from blood sampling to mutation detection output, including shipping conditions (4H versus 24H in EDTA tubes), the reproducibility of cfDNA extraction, the specificity/sensitivity of ddPCR (using external controls), and the comparison of different PCR assays for the detection of the three most important EGFR hotspots, which highlighted the increased sensitivity of our in-house primers/probes. Hence, we have described a new protocol facilitating the molecular detection of somatic mutations in cancer patients from liquid biopsies, improving their diagnosis and introducing a less traumatic monitoring system during tumor progression., Competing Interests: CONFLICTS OF INTEREST Sébastien Couraud declares grants, personal fees and non-financial support from Astra Zeneca, Sysmex Innostics, Roche, and Boehringher-Ingelheim in relation with the work under consideration; and grants, personal fees and non-financial support from Pfizer, Chugai, BMS, MSD, Lilly, and Novartis outside the submitted work. Jessica Garcia and Léa Payen declares grants, personal fees and non-financial support from Astra Zeneca, Sysmex Innostics, and BioMérieux in relation with the work under consideration; The other authors have no conflicts of interest to disclose.