Abstract 744: ctDNA use in molecular diagnostics
Authors: Magliacane Gilda , Grassini Greta , Francaviglia Ilaria , DalCin Elena , Lazzari Chiara , Bulotta Alessandra , Ducceschi Monika , Gregorc Vanesa , Pecciarini Lorenza , Doglioni Claudio , Cangi Maria Giulia 
Published: July 2017

Abstract
The analysis of circulating cell-free tumor DNA (ctDNA), which can be obtained from plasma by non-invasive procedures, is expected to provide useful biomarkers in the management of non-small-cell lung cancer (NSCLC) patients. Indeed, several studies have assessed ctDNA prognostic and predictive value as source of key data for therapeutic targets and drug resistance in carcinoma patients. The expanding number of targeted therapeutics for NSCLC always expects a real-time wider tumor genotyping, and the use of ctDNA as either a complement or an alternative to tumor tissue DNA (ttDNA) could be a valid option for Next-Generation Sequencing (NGS) of key cancer genes.
However, isolation and enrichment of ctDNA is a big challenge because of its high degree of fragmentation and its low concentration against the normally occurring background of cell-free DNA derived from healthy cells. Therefore standardized methods for ctDNA extraction and analysis are crucial aspects in the setting-up of a molecular diagnostic approach.
In this study we aimed to evaluate the ctDNA use for molecular profiling, also analyzing the impact of both pre-analytical and analytical variables on DNA yield and mutation detection.
Matched ttDNA and ctDNA from 30 NSCLC patients were extracted, quantified and quality-controlled, and then investigated by different standard methods (real-time PCR, digital PCR, Mass Spectrometry genotyping) for EGFR, KRAS, BRAF, PIK3CA status. Mutational screening of ctDNA samples by IonTorrent NGS (Oncomine™ Lung cfDNA Assay, ThermoFisher) was also performed. Further, we extended ctDNA evaluation to additional 30 lung cancer patients with no available tumor sample.
We found that cell free DNA concentration in plasma correlated with both stage and number of metastatic sites. Analyzing matched ttDNA and ctDNA by standard methods, we identified mutations in EGFR, KRAS, PIK3CA genes, with an overall concordance of 77%. Interestingly, Oncomine Lung cfDNA assay detected these same mutations with the same allelic frequency of standard methods; mutations in TP53 and ALK genes were also found.
In the 30 patients with no available tumor sample, we found EGFR (10%) and KRAS (7%) mutations on ctDNA by standard methods; NGS analysis is under evaluation.
This study evaluated the use of multiple different methods to detect mutations in NSCLC and showed that ctDNA can be a feasible option for clinical monitoring of lung cancers, including for those patients who cannot undergo invasive diagnostic procedures, due to either comorbidities or absence of biopsable tumor lesions.
Citation Format: Gilda Magliacane, Greta Grassini, Ilaria Francaviglia, Elena DalCin, Chiara Lazzari, Alessandra Bulotta, Monika Ducceschi, Vanesa Gregorc, Lorenza Pecciarini, Claudio Doglioni, Maria Giulia Cangi. ctDNA use in molecular diagnostics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 744. doi:10.1158/1538-7445.AM2017-744