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  • ctDNA is released by passive mechanisms such as lysis

    2019-08-26

    ctDNA is released by passive mechanisms, such as lysis of apoptotic and necrotic Thapsigargin or digestion of tumor cells by macrophages, and also by active mechanisms. ctDNA shows an enrichment in 150–180 bp fragments, typical of the nucleosomal pattern of DNA fragmentation during apoptosis. Most importantly, ctDNA carries the same somatic alterations as the tumor itself and therefore it can be used to analyze the tumor genotype [14]. Importantly, the results of several studies demonstrated a high concordance rate between mutational EGFR profiles in matched tumor and plasma DNA samples, leading both the US Food and Drug Administration and the European Medicines Agency to approve the clinical use of ctDNA for detection of EGFR mutations in NSCLC patients if no biopsy material is available [15]. In addition, CAP-IASLC have recently updated their guidelines, recommending the use of ctDNA for advanced NSCLC patients with limited tissue samples [16]. Nevertheless, analysis of ctDNA is challenging due to its high fragmentation and its low fraction of the total circulating cell-free DNA (cfDNA), including that of normal cells [17]. Therefore, standardized methods for ctDNA extraction and analysis are crucial aspects in the setting-up of liquid biopsy molecular diagnostic workflow.
    Materials and methods
    Results
    Discussion In this study we report the advantages of the clinical application of ctDNA molecular characterization in NSCLC as a pivotal tool for real-time monitoring of therapy, risk of relapse, resistance mechanisms and identification of therapeutic targets. Although not a replacement for a diagnostic tissue biopsy, the minimal-invasive liquid biopsy is becoming more attractive to classify patients according to their mutational profile [21] and the use of ctDNA extracted from peripheral blood plasma is currently the main source for this application [22]. In particular, we investigated the collection, isolation and analysis of plasma ctDNA either in patients at disease progression after TKI-inhibitor treatment to monitor drug resistance, or in treatment naïve patients with new diagnosis for whom histological specimens were unavailable to identify therapeutic targets. For this latter group of patients, ctDNA analysis showed the presence of EGFR activating mutations in 16% of patients who could then be referred to TKI therapies, allowing them to receive a customized and effective treatment, with a good tolerability profile. Of note, the median age of patients who underwent liquid biopsy at the time of diagnosis was 78 years compared to 66 years in patients whose ctDNA analysis was performed at progressive disease during TKIs. This suggests that liquid biopsy represents a valid method to detect druggable molecular alterations in frail patients who otherwise could not receive treatment because of the lack of ttDNA. We demonstrated the successful use of plasma ctDNA for mutation detection by both standard technologies and NGS, proving the NGS-based liquid biopsy diagnostic role in a clinical setting, following a completely in-house workflow. Several examples of NGS based lung ctDNA genotyping has already been reported [[23], [24], [25]] but routine clinical practice reports using in-house workflows are still limited, and more data are needed in order to expand the use of ctDNA NGS analysis in oncology molecular diagnostics. One of the goals of our study was to generate a robust, reproducible and cost-efficient approach of ctDNA genotyping for our clinical setting, thus providing an alternative choice to outsourcing ctDNA analysis that generally requires long turnaround times. Indeed, the total process time for our NGS assay, from blood sample receipt to result reporting, was as short as 4 days. Furthermore the bioinformatic tools, the standardized protocols, and analysis workflows provided by the Oncomine™ Lung cfDNA assay make data evaluation and interpretation feasible for a diagnostics molecular laboratory to implement in routine clinical practice.