Analytical validation of a 1021-gene NGS panel for comprehensive tumor profiling in FFPE and liquid biopsy samples.

Abstract

e14629 Background: In the era of targeted therapy and immunotherapy, accurate molecular profiling through next-generation sequencing (NGS) is essential for the personalization of cancer treatment. This study assesses the analytical performance of a CE-IVD 1021-gene NGS panel (GenePlus) in identifying tumor biomarkers from FFPE tissues and plasma-derived cfDNA, while also ensuring clinical utility across various sample types. Methods: The NGS assay evaluated 1021 tumor-associated genes, comprising 38 genes for fusions, with a thorough analysis of SNVs, Indels, CNVs, MSI, and TMB. The analytical validation evaluated the sensitivity, specificity and reproducibility of the NGS panel using both reference standards and clinical samples. All type of variants such as SNVs, indels, CNV and gene rearrangements are included in the analysis. Additionally gene signatures such as microsatellite instability (MSI), and tumor mutational burden (TMB) were assessed. Clinical validation was conducted on 1,368 solid tumor from metastatic cancer cases that were referred for molecular profiling, ensuring a minimum tumor cell content (TCC) of 20%. Parallel molecular profiling of tissue and plasma was conducted for 10 cases to evaluate the efficacy of liquid biopsy. Results: The assay exhibited 100% sensitivity and specificity for SNVs, Indels, fusions and CNVs) at 2% variant allele frequency (VAF) in formalin-fixed paraffin-embedded (FFPE) samples and 0.5% VAF in cell-free DNA (cfDNA) samples. Clinical and reference samples demonstrated high reproducibility and inter-assay concordance (N = 50 and N = 6, respectively). The mean sequencing depth exceeded 500× for FFPE and 2000× for cfDNA, facilitating reliable detection. Moreover, NGS profiling was successfully conducted in 98.32% of the 1368 cases, revealing actionable alterations in 64.98% of patients. On-label biomarkers for targeted therapies were identified in 12.57%, which increased to 20.15% when including biomarkers for immunotherapy. MSI-high and TMB-high constituted 1.19% and 9.22%, respectively. In paired tissue and plasma samples, 70% of alterations detected in tissue were also observed in cfDNA. In certain instances, reduced concordance was linked to treatment effects that diminished ctDNA levels. Conclusions: The 1021-gene NGS panel exhibited high sensitivity, specificity, and reproducibility, thereby establishing it as a reliable instrument for molecular profiling in clinical settings. The applicability to both FFPE and liquid biopsy samples enhances its clinical utility, especially in instances of insufficient tissue.