Abstract
Targeted next-generation sequencing (NGS) based on molecular tagging technology allowed considerable improvement in the approaches of cell-free DNA (cfDNA) analysis. Previously, we demonstrated the feasibility of the OncomineTM Lung cell-free DNA Assay (OLcfA) NGS panel when applied on plasma samples of post-tyrosine kinase inhibitors (TKIs) non-small cell lung cancer (NSCLC) patients. Here, we explored in detail the coverage metrics and variant calling of the assay and highlighted strengths and challenges by analyzing 92 plasma samples collected from a routine cohort of 76 NSCLC patients. First, performance of OLcfA was assessed using Horizon HD780 reference standards and sensitivity and specificity of 92.5% and 100% reported, respectively. The OLcfA was consequently evaluated in our plasma cohort and NGS technically successful in all 92 sequenced libraries. We demonstrated that initial cfDNA amount correlated positively with library yields (p < 0.0001) and sequencing performance (p < 0.0001). In addition, 0.1% limit of detection could be achieved even when < 10 ng cfDNA was employed. In contrast, the cfDNA amount seems to not affect the EGFR mutational status (p = 0.16). This study demonstrated an optimal performance of the OLcfA on routine plasma samples from NSCLC patients and supports its application in the liquid biopsy practice for cfDNA investigation in precision medicine laboratories.
Highlights
The determination of the mutational status of cell-free DNA is becoming a fundamental and attractive tool in the liquid biopsy field of cancer patients [1,2,3]
We showed that the next-generation sequencing (NGS) results for the search of Thr790Met mutation were comparable with those obtained with a digital polymerase chain reaction (PCR) approach, achieving a 100%
We tried to focus on the main advantages as well as potential drawbacks of the OncomineTM Lung cell-free DNA Assay (OLcfA) application on a routine non-small cell lung cancer (NSCLC) clinical setting
Summary
The determination of the mutational status of cell-free DNA (cfDNA) is becoming a fundamental and attractive tool in the liquid biopsy field of cancer patients [1,2,3]. Sci. 2020, 10, 2895 circulating tumor DNA (ctDNA) derived from tumor cells from normal cells can be challenging [4]. Since molecular findings by genomic characterization of ctDNA are able to drive treatment decisions, clinical laboratories have been adopting highly sensitive and accurate assays for a precise identification of different DNA alterations. In this scenario, next-generation sequencing (NGS) approaches provide interesting application choices to deal with rare ctDNA molecules diluted within cfDNA and are rapidly gaining traction as a valid assay for cancer
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