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Updated: Dec 30, 2025
Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors
Published on: September 20, 2016
Proficiency Testing of Standardized Samples Shows High Interlaboratory Agreement for Clinical Next Generation
Alissa Keegan1, Julia A Bridge1, Neal I Lindeman1
1From the Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts (Drs Keegan, Lindeman, and Kim); the Departments of Pathology and Microbiology, University of Nebraska Medical Center, Omaha (Dr Bridge); Biostatistics (Mr Long) and Proficiency Testing (Ms Vasalos), ollege of American Pathologists, Northfield, Illinois; the UNC Lineberger Comprehensive Cancer Center (Dr Merker) and the Department of Pathology and Laboratory Medicine (Dr Montgomery), University of North Carolina, Chapel Hill; the Office of the Director, The Joint Pathology Center, Silver Spring, Maryland (Dr Moncur); the Department of Pathology, PierianDx, St Louis, Missouri (Dr Nagarajan); the Department of Pathology and Laboratory Medicine, Strong Memorial Hospital, University of Rochester Medical Center, Rochester, New York (Dr Rothberg); the Department of Hematopathology, MD Anderson Cancer Center, Houston, Texas (Dr Routbort); and the Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland (Dr Xian).
Context.—:
As laboratories increasingly turn from single-analyte testing in hematologic malignancies to next-generation sequencing-based panel testing, there is a corresponding need for proficiency testing to ensure adequate performance of these next-generation sequencing assays for optimal patient care.
Objective.—:
To report the performance of laboratories on proficiency testing from the first 4 College of American Pathologists Next-Generation Sequencing Hematologic Malignancy surveys.
Design.—:
College of American Pathologists proficiency testing results for 36 different engineered variants and/or allele fractions as well as a sample with no pathogenic variants were analyzed for accuracy and associated assay performance characteristics.
Results.—:
The overall sensitivity observed for all variants was 93.5% (2190 of 2341) with 99.8% specificity (22 800 of 22 840). The false-negative rate was 6.5% (151 of 2341), and the largest single cause of these errors was difficulty in identifying variants in the sequence of CEBPA that is rich in cytosines and guanines. False-positive results (0.18%; 40 of 22 840) were most likely the result of preanalytic or postanalytic errors. Interestingly, the variant allele fractions were almost uniformly lower than the engineered fraction (as measured by digital polymerase chain reaction). Extensive troubleshooting identified a multifactorial cause for the low variant allele fractions, a result of an interaction between the linearized nature of the plasmid and the Illumina TruSeq chemistry.
Conclusions.—:
Laboratories demonstrated an overall accuracy of 99.2% (24 990 of 25 181) with 99.8% specificity and 93.5% sensitivity when examining 36 clinically relevant somatic single-nucleotide variants with a variant allele fraction of 10% or greater. The data also highlight an issue with artificial linearized plasmids as survey material for next-generation sequencing.
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