Abstract 5580: Identification and monitoring of somatic mutations in circulating cell-free DNA by next-generation sequencing in patients with lung adenocarcinoma
Authors: Francaviglia Ilaria , Magliacane Gilda , Grassini Greta , Girlando Salvatore , Medicina Daniela , Lazzari Chiara , Bulotta Alessandra , Pecciarini Lorenza , Doglioni Claudio , Cangi Maria Giulia 
Published: August 2018

Abstract
The analysis of circulating cell-free tumor DNA (ctDNA), which can be obtained from plasma by non-invasive procedures, was proven useful to provide biomarkers in the management of non-small-cell lung cancer (NSCLC) patients. Several studies assessed ctDNA prognostic and predictive value as source of key data for therapeutic target selection and drug resistance in such patients. The purpose of the present study was to compare the assessment of NSCLC common hot spot mutations in ctDNA samples using the NGS Oncomine cfDNA Lung Cancer assay (ThermoFisher) to the standard clinical tests (i.e. real-time quantitative PCR and droplet digital PCR), performed in both FFPE tumor and ctDNA samples. In particular we aimed to evaluate the feasibility and accuracy of this assay for (1) the detection of EGFR/KRAS mutations in 50 patients with newly diagnosed lung cancer and no possibility to obtain tissue samples and (2) the identification of anti-EGFR treatment acquired resistance mutations in 50 NSCLC EGFR-mutated patients with disease progression. The Oncomine cfDNA Lung assay is a multiplexed sequencing test, designed to detect 150 hotspot mutations in 11 lung cancer-related genes (ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, and TP53) with a limit of detection as low as 0.1%. We analyzed a total of 100 ctDNAs which were sequenced using the Ion S5 system. Libraries were templated using the Ion Chef and multiplexed as 8 libraries on a 520 chip. Data analysis was performed in Torrent Suite using the variant Caller plugin. The total process time, from ctDNA isolation to result reporting, was as short as 4 days, supporting a workflow where blood samples are received early on day 1 and final variant calls are available on day 4. ctDNA NGS analysis for the newly diagnosed patients with no available tumor sample identified KRAS mutations (7%) and more importantly, targetable EGFR mutations (10%). For the patients with progression disease, EGFR acquired resistance mutations were found in 78% of the cases. Overall, the most frequently mutated genes were EGFR (85%), TP53 (26%), PIK3CA (18%), ALK (10%), and MET (8%). Taken the EGFR mutation detected by routine methods as the gold standard, the concordance with EGFR variants detected by NGS was 97%. Interestingly, the NGS approach detected the same EGFR mutations with the same allelic frequency of standard methods. These preliminary data confirm the potential of the Oncomine cfDNA lung assay for plasma genotyping which allows both noninvasive multiplexed detection of targetable genomic alterations and monitoring of acquired resistance mutations in lung cancer.
Citation Format: Ilaria Francaviglia, Gilda Magliacane, Greta Grassini, Salvatore Girlando, Daniela Medicina, Chiara Lazzari, Alessandra Bulotta, Lorenza Pecciarini, Claudio Doglioni, Maria Giulia Cangi. Identification and monitoring of somatic mutations in circulating cell-free DNA by next-generation sequencing in patients with lung adenocarcinoma [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 5580.