Abstract 940: Analytical and clinical validation of the Idylla™ ctKRAS and ctNRAS-BRAF liquid biopsy tests identifies mCRC patient groups with high and low ctDNA shedding
Authors: Jacobs Bart , Claes Bart , Pomella Valentina , Tejpar Sabine , Bachet Jean-Baptiste , Laurent-Puig Pierre , Maertens Geert , Sablon Erwin 
Published: August 2018

Abstract
Background: Ras mutation analysis is required for the administration of anti-EGFR therapies to mCRC patients. Incorporating ctDNA testing in routine diagnostics allows rapid detection of baseline RAS mutation status from a single blood draw. We validated ctDNA analysis of 21 KRAS, 18 NRAS and 4 BRAF ctDNA mutations using 2 ml of plasma on the Idylla™ platform. The tests were performed using the Idylla™ ctKRAS and ctNRAS-BRAF Mutation Tests, comprising all required reagents for fully automated and integrated ctDNA analysis from plasma with a turnaround time of approximately 2hrs.
Methods: Analytical validation was performed using EDTA plasma samples containing fragmented DNA containing mutant sequences of interest. For both assays the LOD was calculated using regression models. Reproducibility studies were conducted using the Acrometrix™ Idylla™ ctRAS Verification Panel consisting of 4 KRAS mutations, 4 NRAS mutations and 1 BRAF mutation. During between-laboratory reproducibility studies, the panel of reference samples was tested at three different sites using two different Idylla™ instruments. Between-lot reproducibility was assessed on three different lots. For clinical validation, pre-treatment plasma samples from 203 mCRC patients enrolled in the prospective multicenter RASANC study (NCT02502656) were retrospectively assessed. Proper informed consent and statistical analysis were included. As a comparator test, NGS analysis was performed on ctDNA according to Pécuchet et al. (2016). In addition, tissue RAS/BRAF results were available from standard of care testing.
Results: The between-lab reproducibility for the 4 mutations evaluated with the Idylla™ ctKRAS Mutation Test is with 95% confidence at least 98.7% Idylla™ and is with 95% confidence above 98.7% for the 5 mutations evaluated with the Idylla™ ctNRAS-BRAF Mutation Test. The between-lot reproducibility for the evaluated mutations is with 95% confidence at least 98.9% for the ctKRAS Test and 98.5% for the ctNRAS-BRAF Test. In the clinical study, overall RAS agreement between Idylla™ and NGS on plasma was 90.0% with a sensitivity of 89.5% and specificity of 90.4%. RAS concordance between FFPE tissue and plasma was 78.9% in the whole study population. Low amounts of cfDNA in the sample, absence of liver metastases and primary tumor resection were associated with discordant results. In the population with liver metastases showing sufficient cfDNA, overall RAS agreement between plasma and tissue RAS status was 92.5% with a sensitivity of 93.2% and specificity of 90.9%.
Conclusions: The Idylla™ ctKRAS and ctNRAS-BRAF Mutation Tests provide a sensitive, reliable and fast solution for RAS-BRAF ctDNA testing. Results confirm that RAS-BRAF mutation status can be adequately determined using plasma from metastatic colorectal cancer patients with liver metastases.
Citation Format: Bart Jacobs, Bart Claes, Valentina Pomella, Sabine Tejpar, Jean-Baptiste Bachet, Pierre Laurent-Puig, Geert Maertens, Erwin Sablon. Analytical and clinical validation of the Idylla™ ctKRAS and ctNRAS-BRAF liquid biopsy tests identifies mCRC patient groups with high and low ctDNA shedding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 940.