Analytical Validation of the Next-Generation Sequencing Assay for a Nationwide Signal-Finding Clinical Trial

Abstract

The National Cancer InstituteeMolecular Analysis for Therapy Choice (NCI-MATCH) trial is a national signal-finding precision medicine study that relies on genomic assays to screen and enroll patients with relapsed or refractory cancer after standard treatments. We report the analytical validation processes for the next-generation sequencing (NGS) assay that was tailored for regulatory compliant use in the trial. The Oncomine Cancer Panel assay and the Personal Genome Machine were used in four networked laboratories accredited for the Clinical Laboratory Improvement Amendments. Using formalin-fixed paraffin-embedded clinical specimens and cell lines, we found that the assay achieved overall sensitivity of 96.98% for 265 known mutations and 99.99% specificity. High reproducibility in detecting all reportable variants was observed, with a 99.99% mean interoperator pairwise concordance across the four laboratories. The limit of detection for each variant type was 2.8% for single-nucleotide variants, 10.5% for insertion/deletions, 6.8% for large insertion/deletions (gap !4 bp), and four copies for gene amplification. The assay system from biopsy collection through reporting was tested and found to be fully fit for purpose. Our results indicate that the NCI-MATCH NGS assay met the criteria for the intended clinical use and that high reproducibility of a complex NGS assay is achievable across multiple clinical laboratories. Our validation approaches can serve as a template for development and validation of other NGS assays for precision medicine. (J Mol Diagn 2017, 19: 313e327; http://dx

The long tail of molecular alterations in non-small cell lung cancer: a single-institution experience of next-generation sequencing in clinical molecular diagnostics

“…This relatively high success rate was related to the standardised preanalytical and analytical procedures, including formalin fixation (10% neutral buffered formalin for 6–48 hours), case selection and automated analytical steps (ie, Ion Chef for the template preparation) 27. As a CE-IVD marked NGS assay, the Oncomine Solid Tumour kit enables multiplexed sequencing of FFPE tumour samples with the accuracy (overall sensitivity and specificity of 96.98% and 99.99%, respectively) required by a clinical diagnostic laboratory 28. Recently, the analytical sensitivity of this panel was further validated by a ring study that confirmed the sensitivity reported by the manufacturer 29.…”

Future Clinical Trials

“…As the National Cancer Institute launched the NCI-MATCH trial 33–35 , an ongoing screening effort of 795 samples (according to the interim analysis at http://ecog-acrin.org/nci-match-eay131/interim-analysis) identified 9% mutations matching one of the 10 arms of the study. To a large extent influenced by the study drug/gene “menu”, finding the matched mutation was as likely as 18% in melanoma, brain tumors and lymphomas, 13% in colorectal and breast cancers vs. no matching in all screened so far patients with pancreatic adenocarcinoma, head and neck, endometrial, and small cell carcinoma.…”

Precision Medicine Requires Precision Laboratories

“…The approach to analytic validation of the targeted NGS assay used in the NCI-MPACT trial was recently reported in this journal [7]. It is clear that findings from validation of the NCI-MPACT assay were helpful to Lih et al in their approach to the NCI-Molecular Analysis for Therapy Choice (MATCH) Trial which presented an even broader challenge for the laboratories [1]. …”

“…Although it is clear that assay validation is essential for clinical implementation, it could be questioned what each contributes to the literature. To place the study of Lih et al [1] in the appropriate context, some of the more recent validation studies with closest similarity to that of Lih et al will be reviewed.…”

“…The report by Lih et al in this issue of The Journal of Molecular Diagnostics presents the latest chapter in this story [1]. Unlike the earlier low and medium throughput technologies, the massively parallel sequencing by NGS offers the opportunity for simultaneous targeting of different genetic alterations that include single nucleotide variants (SNVs), insertions/deletions (indels), copy number variations (CNVs), and gene fusions.…”