Comparison of BEAMing assays and competitive approaches in the detection of main alteration of RAS in circulating DNA of non small-cell lung cancer (NSCLC) and metastatic colon cancer. Manuscript 2016

e23056 Background: A number of RAS mutations confer resistance to anti-EGFR therapies routinely used in the treatment of colon cancer. The objective of this study was to evaluate the pertinence of analyzing circulating-free plasma DNA (cfDNA) as an indicator of the mutational status of a tumor, in order to use liquid biopsies instead of invasive and painful tumor biopsies during tumor progression. Methods: A cohort of 24 lung and 25 colon cancer patients was constituted in the Hospices Civils of Lyon. Liquid biopsy plasma samples were collected at diagnosis (colon cancer) and during tumor progression (lung cancer) for the purpose of the current study. KRAS and NRAS somatic alterations were quantified using three different technologies: the Droplet Digital polymerase chain reaction (ddPCR) from BioRad, the BEAMing Digital PCR from Sysmex Inostics, and the NGS NextSeq 500 by Illumina with the Accel-Amplicon 56G Oncology Panel from SWIFT BIOSCIENCES. Results: We observed a high level of sensitivity and specificity with the BEAMing technology, which provided us with excellent matches, around 96% and 73%, between solid and liquid biopsies taken at diagnosis (colon cancer) or during tumor progression (lung cancer), respectively. Indeed, when examining cfDNA from patients displaying one of the KRAS or NRAS mutations, 11 of the 13 mutations were confirmed using this technology, whereas only 5-6 matched the initial NGS status, using the two other technologies. The detection threshold was estimated at 1% for samples containing at least 0.8 ngctDNA/µL for the multiplex screening ddPCR from BioRad and for the 56G Oncology Panel from SWIFT BIOSCIENCES. The threshold was lower, at 0.03%, in samples containing only 0.25 ng ctDNA/µL for the BEAMing technology, which includes a PCR pre-amplification step. Conclusions: The advantage of the Illumina NGS technology is the larger coverage of longer gene regions, and thus the detection of more genetic mutations. Finally, the BEAMing technology enabled us to follow the appearance and disappearance of somatic alterations, with a very high level of sensitivity.