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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).
Proficiency testing for next-generation sequencing (NGS) in hematologic malignancies shows high accuracy (99.2%). Challenges remain in detecting variants in GC-rich regions and with artificial plasmid DNA, impacting variant allele fractions.
Area of Science:
- Clinical diagnostics
- Molecular pathology
- Genomic medicine
Background:
- Laboratories are transitioning to next-generation sequencing (NGS) panel testing for hematologic malignancies.
- Proficiency testing is crucial for ensuring the accuracy and reliability of these NGS assays.
- Accurate diagnostic testing is essential for optimal patient care in hematologic malignancies.
Purpose of the Study:
- To evaluate the performance of laboratories participating in proficiency testing for NGS-based hematologic malignancy testing.
- To identify challenges and areas for improvement in NGS assay performance for hematologic malignancies.
Main Methods:
- Analysis of College of American Pathologists (CAP) proficiency testing data from four surveys.
- Evaluation of laboratory performance on 36 engineered variants and a negative sample.
- Assessment of accuracy, sensitivity, specificity, and variant allele fraction detection.
Main Results:
- Overall accuracy was 99.2% with 99.8% specificity and 93.5% sensitivity for clinically relevant variants.
- False-negative results (6.5%) were often associated with variants in GC-rich sequences (e.g., CEBPA).
- Lower-than-expected variant allele fractions were observed, linked to linearized plasmid material and Illumina TruSeq chemistry.
Conclusions:
- Laboratories demonstrate high overall accuracy in NGS-based hematologic malignancy testing.
- Challenges exist with variant detection in GC-rich regions and with the use of artificial linearized plasmids in proficiency testing materials.
- Further optimization of NGS assays and proficiency testing materials is needed to address identified performance limitations.
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