标准化样本的能力验证显示，基于临床下一代测序的血液系统恶性肿瘤检测在实验室间具有高度一致性，但不同调查材料的变异频率存在差异。

Proficiency Testing of Standardized Samples Shows High Interlaboratory Agreement for Clinical Next Generation Sequencing-Based Hematologic Malignancy Assays With Survey Material-Specific Differences in Variant Frequencies.

作者信息

Keegan Alissa, Bridge Julia A, Lindeman Neal I, Long Thomas A, Merker Jason D, Moncur Joel T, Montgomery Nathan D, Nagarajan Rakesh, Rothberg Paul G, Routbort Mark J, Vasalos Patricia, Xian Rena, Kim Annette S

机构信息

From 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).

出版信息

Arch Pathol Lab Med. 2020 Jan 27. doi: 10.5858/arpa.2019-0352-CP.

DOI:10.5858/arpa.2019-0352-CP

PMID:31986076

Abstract

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 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.

摘要

背景

随着实验室越来越多地从血液系统恶性肿瘤的单分析物检测转向基于新一代测序的panel检测，相应地需要进行能力验证，以确保这些新一代测序检测在为患者提供最佳护理方面具有足够的性能。

目的

报告实验室在美国病理学家学会前4次新一代测序血液系统恶性肿瘤调查中的能力验证表现。

设计

分析了美国病理学家学会对36种不同的工程变体和/或等位基因分数以及一个无致病变体样本的能力验证结果，以评估准确性和相关检测性能特征。

结果

所有变体的总体灵敏度为93.5%（2341个中的2190个），特异性为99.8%（22840个中的22800个）。假阴性率为6.5%（2341个中的151个），这些错误的最大单一原因是在富含胞嘧啶和鸟嘌呤的序列中识别变体存在困难。假阳性结果（0.18%；22840个中的40个）最可能是分析前或分析后错误的结果。有趣的是，变异等位基因分数几乎始终低于工程分数（通过数字聚合酶链反应测量）。广泛的故障排除确定了变异等位基因分数低的多因素原因，这是质粒线性化性质与Illumina TruSeq化学之间相互作用的结果。