Cross-platform comparison for the detection of RAS mutations in cfDNA (ddPCR Biorad detection assay, BEAMing assay, and NGS strategy)

// Jessica Garcia 1, 2, 3, 4 , Julien Forestier 5 , Eric Dusserre 1, 3, 4 , Anne-Sophie Wozny 1, 3 , Florence Geiguer 1, 3, 4 , Patrick Merle 6, 7 , Claire Tissot 8 , Carole Ferraro-Peyret 2, 9 , Frederick S. Jones 10 , Daniel L. Edelstein 10 , Valerie Cheynet 4, 11 , Claire Bardel 12, 18 , Gaelle Vilchez 4 , Zhenyu Xu 13 , Pierre Paul Bringuier 2, 9 , Marc Barritault 1, 9 , Karen Brengle-Pesce 4, 11 , Marielle Guillet 14 , Marion Chauvenet 15 , Brigitte Manship 2 , Marie Brevet 3, 9, 16 , Claire Rodriguez-Lafrasse 1, 4 , Valerie Hervieu 9 , Sebastien Couraud 3, 16, 17 , Thomas Walter 2, 5 and Lea Payen 1, 2, 3, 4 1 Laboratoire de Biochimie et Biologie Moleculaire, Groupe Hospitalier Sud, Hospices Civils de Lyon, Lyon, France 2 University of Lyon, Claude Bernard University, Cancer Research Center of Lyon, INSERM U1052, CNRS UMR5286, Lyon, France 3 Hospices Civils de Lyon Cancer institute, CIRculating CANcer (CIRCAN) program, Pierre Benite, France 4 Laboratoire Commun de Recherche Hospices Civils de Lyon – BioMerieux, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, Pierre Benite Cedex, France 5 Service d’oncologie medicale, Hopital E. Herriot, Hospices Civils de Lyon, Lyon, France 6 Service de Pneumologie et oncologie thoracique, CHU G Montpied, Clermont-Ferrand, France 7 Universite d’Auvergne, UMR INSERM 1240, Clermont-Ferrand, France 8 Service de Pneumologie et cancerologie thoracique, CHU de Saint Etienne, Saint Etienne, France 9 Service d’Anatomopathologie, Groupement Hospitalier Est, Hospices Civils de Lyon, Lyon, France 10 Medical Scientific Affairs, Sysmex Inostics, Inc., Mundelein, IL, USA 11 BioMerieux SA medical diagnostic Department, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, Lyon, France 12 Univ Lyon, Universite Lyon 1, CNRS, Laboratoire de Biometrie et Biologie Evolutive UMR5558, Villeurbanne, France 13 SOPHiA GENETICS SA, Headquarters, CH-1025 Saint Sulpice, Switzerland 14 Service de gastro-enterologie, Hopital de la Croix-Rousse, Hospices Civils de Lyon, Lyon 15 Hepatogastroenterologie et oncologie digestive, Groupement hospitalier Sud, Hospices civils de Lyon, Pierre Benite, France 16 EMR 3738 Ciblage Therapeutique en Oncologie, Faculte de medecine Lyon Sud, Universite Lyon 1, Universite de Lyon, Oullins 17 Service de Pneumologie aigue specialisee et cancerologie thoracique, Groupement hospitalier sud, Institut de Cancerologie des Hospices Civils de Lyon, Pierre Benite, France 18 Service de Biostatistique–bioinformatique et plateforme NGS-CHU Lyon, Hospices Civils de Lyon, Lyon, France Correspondence to: Lea Payen, email: lea.payen-gay@chu-lyon.fr Keywords: circulating-free DNA; digital PCR; NGS; liquid biopsy; colon and lung cancer Received: November 03, 2017      Accepted: February 27, 2018      Published: April 20, 2018 ABSTRACT 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.