Performance of the OncomineTM Lung cfDNA Assay for Liquid Biopsy by NGS of NSCLC Patients in Routine Laboratory Practice

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.

Analytical Validation of a Pan-Cancer Panel for Cell-Free Assay for the Detection of EGFR Mutations

“…Thus, reporting from various studies by independent groups may help to determine the performance of the assay. The previous study using the Oncomine Lung Cell-Free DNA Assay, the same platform with the different assay, demonstrated a sensitivity of 81% with <20 ng of cfDNA at a VAF of 0.1%, whereas our data showed a sensitivity of 50% under the same condition [21]. In the report by the manufacturer, the same assay in the present study showed a sensitivity of 80% at a VAF of 0.1% for SNV [22].…”

Evaluation of the Idylla ctEGFR mutation assay to detect EGFR mutations in plasma from patients with non-small cell lung cancers

“…These include the droplet digital PCR (ddPCR) method with an estimated LOD of 0.05% for the exon 19 deletion and L858R mutations and 0.1% for T790M mutations 24 and the OncoBEAM (beads, emulsion, amplification and magnetics) approach with a LOD of nearly 0.02% for T790M mutation 25 . The conventional NGS-based approaches are considered less sensitive, however the implementation of a molecular tagging strategies in NGS technology in the last years allows to significantly reduce the sequencing artifacts and reach sensitivities similar to those observed with the other approaches (up to 0.1%) 26 , 27 .…”

Analytical and clinical validation of a custom 15-gene next-generation sequencing panel for the evaluation of circulating tumor DNA mutations in patients with advanced non-small-cell lung cancer

“…Firefly assay demonstrated superior sensitivity and specificity with a 98.89% detection rate at an allele frequency of 0.2% [ 30 ]. Oncomine ™ Lung cell-free DNA Assay (OLcfA) NGS panel has been shown to be effective in detecting mutations in NSCLC patients with a detection limit of 0.1% [ 31 ]. In this proof-of-concept pilot study, we aim to develop and validate a custom 15-gene ctDNA NGS panel for detecting single nucleotide variants and small indels in lung cancer, with primary focus on EGFR mutation analysis in NSCLC.…”

“…Overall, our custom NGS panel showed comparable performance to the real time PCR based assays (Cobas and Therascreen) and achieved 100% analytical sensitivity and specificity for detecting EGFR mutations with at least 1% allelic frequencies. However, the assay is suboptimal when compared to droplet digital PCR or NGS-based assays which can achieve detection limits of below 1% [ 18 , 31 , 41 ].…”