Objectives: Diversity of laboratory-developed tests (LDTs) using next-generation sequencing (NGS) raises concerns about their accuracy for selection of targeted therapies. A working group developed a pilot study of traceable reference samples to measure NGS LDT performance among a cohort of clinical laboratories.
Methods: Human cell lines were engineered via CRISPR/Cas9 and prepared as formalin-fixed, paraffin-embedded cell pellets ("wet" samples) to assess the entire NGS test cycle. In silico mutagenized NGS sequence files ("dry" samples) were used to assess the bioinformatics component of the NGS test cycle. Single and multinucleotide variants (n = 36) of KRAS and NRAS were tested at 5% or 15% variant allele fraction to determine eligibility for therapy with the EGFR inhibitor panitumumab in the setting of metastatic colorectal cancer.
Results: Twenty-one (21/21) laboratories tested wet samples; 19 of 21 analyzed dry samples. Of the laboratories that tested both the wet and dry samples, 7 (37%) of 19 laboratories correctly reported all variants, 3 (16%) of 19 had fewer than five errors, and 9 (47%) of 19 had five or more errors. Most errors were false negatives.
Conclusions: Genetically engineered cell lines and mutagenized sequence files are complementary reference samples for evaluating NGS test performance among clinical laboratories using LDTs. Variable accuracy in detection of genetic variants among some LDTs may identify different patient populations for targeted therapy.
Keywords: Companion diagnostic; Next-generation sequencing; Precision medicine; Reference materials; Test performance.
© American Society for Clinical Pathology, 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.