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

Abstract

Liquid biopsies have increasingly shown clinical utility. Although next-generation sequencing has been widely used for the detection of somatic mutations from plasma, performance characteristics vary by platform. Therefore, thorough validation is mandatory for clinical use. This study aimed to evaluate the analytical validity of the Oncomine Pan-Cancer Cell-Free Assay. A massively parallel sequencing for the assay was performed using the Ion S5 XL System with Ion 540 kit. The analytical sensitivity and precision were evaluated using pre-characterized reference materials. The specificity was evaluated using plasma from healthy subjects. A comparison with the Cobas EGFR Mutation Test v2 was performed using reference materials and plasma from lung cancer patients. For SNVs and short indels, the analytical sensitivities at variant allele frequencies (VAFs) of 0.1%, 0.5%, and 1% were 50%, 93.4%, and 100% with 20 ng of input, respectively. The overall precision of the true positive variants was 98% at a VAF of 1% with 20 ng input. The assay showed a similar sensitivity to that of the Cobas EGFR Mutation Test v2 at a VAF of 0.5% with 20 ng of input and 100% concordance on clinical samples. The Pan-Cancer Cell-Free Assay can be applied to detect EGFR mutations in advanced lung cancer patients, although follow-up studies will be needed to evaluate the analytical validity for other types of genes and aberrations using clinical samples.

Identification of Potential Genomic Alterations Using Pan-Cancer Cell-Free DNA Next-Generation Sequencing in Patients With Gastric Cancer

“…The CNV ratio call thresholds had been empirically derived by the manufacturer using plasma samples from healthy donors with a normal CNV status [ 21 ]. To pass the bioinformatic QC threshold for CNVs, the CNV ratio for a copy number gain must be >1.15, and called copy number gains were visually reviewed from a copy number plot generated by Ion Reporter [ 22 ]. Assay A does not report copy number loss.…”

Liquid Biopsy and Artificial Intelligence as Tools to Detect Signatures of Colorectal Malignancies: A Modern Approach in Patient’s Stratification

“…Undoubtedly, the main advantage of NGS analysis compared with the targeted approaches is the possibility of investigating a large range of markers reaching up to several hundred genes in a single panel. Also, a single test provides valuable information on single nucleotide polymorphisms, short indels, copy number variations, gene fusions, TMB (tumor mutational burden), or MSI ( 198 ). New NGS panels have been developed to detect the pathological variants associated with cancer at both cfDNA and cfRNA levels (e.g., Oncomine Pan-Cancer Cell-Free Assay - Thermo Fisher, Waltham, MA, USA).…”

Cell-free DNA as a biomarker in cancer

“…That test uses size, density, electrical properties, and immune surface markers (EpCAM+, Cytokeratins+, and CD45-) for selection and has been cleared for breast, colorectal, and prostate cancer to predict outcome [12] . DETECT-A [27] -Thrive (developed into: MCED -Exact Sciences) [121] Early version of CancerSEEK [26] ctDNA Invitae [125,126] (former ArcherDX)…”