The advent of next-generation sequencing (NGS) technologies had seen rapid adoption by clinical laboratories. Herein, we report the clinical validation data of a laboratory developed test-APEX Tissue (MiRXES), which enables simultaneous detection of single-nucleotide variants (SNVs), insertions/deletions (InDels), copy number amplifications and fusions through both DNA and RNA sequencing.
A total of 65 formalin-fixed and paraffin-embedded tissue samples were collected from the Diagnostic Molecular Oncology Center (DMOC) at the National University Hospital, Singapore. Orthogonal tests including Sanger sequencing, polymerase chain reaction, immunohistochemistry and fluorescent in situ hybridisation were performed. The concordance of the variants identified with the APEX Tissue NGS panel was assessed with reference to the standard-of-care, single-gene test methods.
The sensitivity and specificity for the variant type calling were 94.4% [34/36] and 100.0% [352/352] for SNVs, 100.0% [16/16] and 100.0% [88/88] for InDels, 100.0% [4/4] and 100.0% [2/2] for amplifications, 100.0% [11/11] and 100.0% [32/32] for fusions respectively. The overall concordance rate was 99.6%. Two false negative results were attributed to insufficient and/or poor quality of DNA extracted from the archival samples dated >2 years ago. All samples achieved a minimum of 95% target regions with ≥ 500x coverage and a mean sequencing depth of 4,800x. The inter-operator reproducibility, inter- and intra-run repeatability, were all reported at 100%. The list of variants identified by the NGS panel for the studied cohort was summarized in the table with the number and percentage of patients tabulated (n, %).
Incidence rate of genetic variants identified by APEX tissue NGS Panel
Gene name Non-small cell lung cancer (31, 48%) Colorectal cancer (21, 32%) Gastrointestinal stromal tumor (7, 11%) Breast cancer (5, 8%) Cancer of unknown primary (1, 1%) ALK 4, 13% 1, 100% AR 1,5% BRAF 6, 29% CTNNB1 3, 10% EGFR 20, 64% FGFR1 3, 60% FGFR3 2, 10% FGFR4 2, 10% GNAS 1, 5% HER2 1, 3% 4, 80% HER3 1,5% KIT 7, 100% KRAS 1, 3% 13, 62% MET 1, 5% NRAS 2, 10% PIK3CA 1, 3% 9, 43% 1, 20% RAF1 2, 10% RET 1, 3% 1, 5% ROS1 6, 19% TP53 7, 22% 7, 33% 1, 20%
The 50-gene APEX Tissue hotspot NGS panel represents a robust assay with a high overall concordance rate of 99.6% when compared with the standard-of-care, single-gene molecular tests.
The authors.
MiRXES Pte Ltd.
S. Teo, Y.L. Toh, C.K. Tan: Non-Financial Interests, Institutional, Full or part-time Employment: MiRXES. All other authors have declared no conflicts of interest.