Pepe, Francesco (2018) “Application of Next Generation Technologies in liquid biopsy: focus on Non Small Cell Lung Cancer and Metastatic Colorectal Cancer”. [Tesi di dottorato]

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Background: In the precision medicine era, the increasing request of clinical relevant biomarkers to improve the patients management lead to the need of most biological source. To address this issue, also if tissue represents the gold standard for the assessment of clinical relevant biomarkers mutational status, some alternative approaches, based on the analysis of circulating free DNA (cfDNA) extracted from “liquid biopsies”, are under evaluation. The aims of this thesis were to investigate the role of liquid biopsy in two specific settings: analysis of EGFR mutational status in Non Small Cell Lung Cancer (NSCLC) patients treated with EGFR Tyrosine Kinase Inhibitors (TKIs) and as a screening tool for Colorectal Cancer (CRC) in comparison of Fetal Immunochemical Test (FIT). Methods: Regarding EGFR mutational status assessment in NSCLC patients, the analytical sensitivity of SiRe panel, which covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα) was validated on cell line DNA and cfDNA derived from cancer patients at presentation (n=42), treatment response (n=12) and tumor progression (n=11) were analyzed; all patients had paired tumor tissue and cfDNA previously genotyped with a Taqman-derived assay (TDA). In addition, we tested blood samples prospectively collected from NSCLC patients (n=79) to assess the performance of SiRe in clinical practice. In relation to CRC patients, employing the analytical validated Real Time PCR-based ColoScape assay kit, mutations in the APC, KRAS, BRAF and CTNNB1 genes were assessed on 52 prospectively collected whole-blood samples obtained from FIT+ patients enrolled in the CRC screening program of ASL NAPOLI 3 SUD, using colonoscopy as confirmation. Results: In relation to the analysis of EGFR mutational status in NSCLC patients treated with EGFR TKIs, SiRe showed high analytical performance and a 0.01% lower limit of detection. Regarding the results obtained in the retrospective series, SiRe was able to detect 40 EGFR, 11 KRAS, 1 NRAS and 5 BRAF mutations (96.8% concordance with TDA). In the baseline sample set, SiRe had 100% specificity and 79% sensitivity relative to results obtained on paired tumor tissue. In the prospective series, SiRe detected 8.7% (4/46) of EGFR mutations at baseline and 42.9% (9/21) of EGFR p.T790M in patients at tumor progression. Regarding the application of Real Time PCR based ColoScape assay kit as a screening tool for CRC patients, the assay's sensitivity for advanced adenomas was 53.8% and the specificity was 92.3%. The Positive Predictive Value was 70.0% and negative predictive value was 85.7%. Of note, four of the six positive cases missed by ColoScape had a less than suboptimal DNA input. Had they been ruled out as inadequate, sensitivity would have increased from 53.8% to 69%. Conclusions: In the landscape of EGFR mutated NSCLC patients treated with TKIs, SiRe represents a feasible NGS panel for cfDNA analysis in clinical practice, while in CRC patients setting, ColoScape is a promising tool for screening program aims to evaluate the triage of FIT+ patients.

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