Abstract
Next-generation sequencing (NGS) is a powerful platform for identifying cancer mutations. Routine clinical adoption of NGS requires optimized quality control metrics to ensure accurate results. To assess the robustness of our clinical NGS pipeline, we analyzed the results of 304 solid tumor and hematologic malignancy specimens tested simultaneously by NGS and one or more targeted single-gene tests (EGFR, KRAS, BRAF, NPM1, FLT3, and JAK2). For samples that passed our validated tumor percentage and DNA quality and quantity thresholds, there was perfect concordance between NGS and targeted single-gene tests with the exception of two FLT3 internal tandem duplications that fell below the stringent pre-established reporting threshold but were readily detected by manual inspection. In addition, NGS identified clinically significant mutations not covered by single-gene tests. These findings confirm NGS as a reliable platform for routine clinical use when appropriate quality control metrics, such as tumor percentage and DNA quality cutoffs, are in place. Based on our findings, we suggest a simple workflow that should facilitate adoption of clinical oncologic NGS services at other institutions.
Highlights
The advance of next-generation sequencing (NGS) is a cornerstone of a recent development in molecular pathology, variably referred to as “personalized,” “precision,” or “individualized” medicine
We report the properties of solid and liquid malignancy specimens processed during the first year of clinical oncologic NGS performed within the University of Pennsylvania Health System
We found that when we adhered to two predetermined quality control metrics, i.e., tumor percentage and DNA quantity and quality, we achieved excellent NGS data as determined by virtually perfect concordance between NGS and targeted, single-gene testing
Summary
The advance of next-generation sequencing (NGS) is a cornerstone of a recent development in molecular pathology, variably referred to as “personalized,” “precision,” or “individualized” medicine. Detection of mutations in EGFR, KRAS, BRAF, FLT3, NPM1, and JAK2 is most commonly accomplished by targeted tests that are designed to detect one or at most a small number of mutations in a single gene. With the exception of one commercially-sponsored study [14], in all cases a very limited number of samples was re-examined (range 13–61), often from only a single tissue type While these important contributions confirm the potential usefulness of clinical NGS, they do not address the important question whether a well-validated NGS pipeline performs at an acceptable level in day-to-day clinical practice. Bioinformatics pipeline [26] with pre-determined tumor percentage and DNA quality cutoffs, we achieved excellent NGS data quality as determined by virtually perfect concordance between NGS and targeted single-gene tests for various genes in a large number of solid and liquid malignancy specimens
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