Implementation, Evolution, and Laboratory Performance of Methods-Based Proficiency Testing for Next-Generation Sequencing Detection of Germline Sequence Variants.

CONTEXT.­: Next-generation sequencing (NGS)-based assays are used for diagnosis of diverse inherited disorders. Limited data are available pertaining to interlaboratory analytical performance of these assays. OBJECTIVE.­: To report on the College of American Pathologists (CAP) NGS Germline Program, which is methods based, and explore the evolution in laboratory testing practices. DESIGN.­: Results from the NGS Germline Program from 2016-2020 were analyzed for interlaboratory analytical performance. Self-reported laboratory testing practices were also evaluated. RESULTS.­: From 2016-2020, a total of 297 laboratories participated in at least 1 program mailing. Of the 289 laboratories that provided information on tests offered, 138 (47.8%) offered only panel testing throughout their enrollment, while 35 (12.1%) offered panels and exome testing, 30 (10.4%) offered only exomes, 9 (3.1%) offered only genomes, and 15 (5.2%) offered panels, exomes, and genomes. The remainder (62 laboratories, 21.4%) changed their test offerings during the 2016-2020 timeframe. Considering each genomic position/interval, the median detection percentage at variant positions across the 2016-2020 mailings ranged from 94.3% to 100%, while at reference positions (no variant detected), the median correct response percentage was 100% across all mailings. When considering performance of individual laboratories, 89.5% (136 of 152) to 98.0% (149 of 152) of laboratories successfully met the detection threshold (≥90% of the variants present), while 94.6% (87 of 92) to 100% (163 of 163) of laboratories met the 95% specificity threshold across mailings. CONCLUSIONS.­: Since the inception of this program, laboratories have consistently performed well. The median sensitivity and specificity of detection of sequence variants included in this program (eg, single nucleotide variants, insertions, and deletions) were 100.0%.

Four-Year Laboratory Performance of the First College of American Pathologists In Silico Next-Generation Sequencing Bioinformatics Proficiency Testing Surveys.

CONTEXT.­: In 2016, the College of American Pathologists (CAP) launched the first next-generation sequencing (NGS) in silico bioinformatics proficiency testing survey to evaluate the performance of clinical laboratory bioinformatics pipelines for the detection of oncology-associated variants at varying allele fractions. This survey focused on 2 commonly used oncology panels, the Illumina TruSeq Amplicon Cancer Panel and the Thermo Fisher Ion AmpliSeq Cancer Hotspot v2 Panel. OBJECTIVE.­: To review the analytical performance of laboratories participating in the CAP NGS bioinformatics (NGSB) surveys, comprising NGSB1 for Illumina users and NGSB2 for Thermo Fisher Ion Torrent users, between 2016 and 2019. DESIGN.­: Responses from 78 laboratories were analyzed for accuracy and associated performance characteristics. RESULTS.­: The analytical sensitivity was 90.0% (1901 of 2112) for laboratories using the Illumina platform and 94.8% (2153 of 2272) for Thermo Fisher Ion Torrent users. Variant type and variant allele fraction were significantly associated with performance. False-negative results were seen mostly for multi-nucleotide variants and variants engineered at variant allele fractions of less than 25%. Analytical specificity for all participating laboratories was 99.8% (9303 of 9320). There was no statistically significant association between deletion-insertion length and detection rate. CONCLUSIONS.­: These results demonstrated high analytical sensitivity and specificity, supporting the feasibility and utility of using in silico mutagenized NGS data sets as a supplemental challenge to CAP surveys for oncology-associated variants based on physical samples. This program demonstrates the opportunity and challenges that can guide future surveys inclusive of customized in silico programs.

Tiered Somatic Variant Classification Adoption Has Increased Worldwide With Some Practice Differences Based on Location and Institutional Setting.

CONTEXT.­: The 2017 Association for Molecular Pathology/American Society of Clinical Oncology/College of American Pathologists (CAP) tier classification guideline provides a framework to standardize interpretation and reporting of somatic variants. OBJECTIVE.­: To evaluate the adoption and performance of the 2017 guideline among laboratories performing somatic next-generation sequencing (NGS). DESIGN.­: A survey was distributed to laboratories participating in NGS CAP proficiency testing for solid tumors (NGSST) and hematologic malignancies (NGSHM). RESULTS.­: Worldwide, 64.4% (152 of 236) of NGSST and 66.4% (87 of 131) of NGSHM participants used tier classification systems, of which the 2017 guideline was used by 84.9% (129 of 152) of NGSST and 73.6% (64 of 87) of NGSHM participants. The 2017 guideline was modified by 24.4% (30 of 123) of NGSST and 21.7% (13 of 60) of NGSHM laboratories. Laboratories implementing the 2017 guideline were satisfied or very satisfied (74.2% [89 of 120] NGSST and 69.5% [41 of 59] NGSHM), and the impression of tier classification reproducibility was high (mean of 3.9 [NGSST] and 3.6 [NGSHM] on a 5-point scale). Of nonusers, 35.2% (38 of 108) of NGSST and 39.4% (26 of 66) of NGSHM laboratories were planning implementation. For future guideline revisions, respondents favored including variants to monitor disease (63.9% [78 of 122] NGSST, 80.0% [48 of 60] NGSHM) and germline variants (55.3% [63 of 114] NGSST, 75.0% [45 of 60] NGSHM). Additional subtiers were not favored by academic laboratories compared to nonacademic laboratories (P < .001 NGSST and P = .02 NGSHM). CONCLUSIONS.­: The 2017 guideline has been implemented by more than 50.0% of CAP laboratories. While most laboratories using the 2017 guideline report satisfaction, thoughtful guideline modifications may further enhance the quality, reproducibility, and clinical utility of the 2017 guideline for tiered somatic variant classification.

Laboratory and Clinical Implications of Incidental and Secondary Germline Findings During Tumor Testing.

CONTEXT.­: Next-generation sequencing is a powerful clinical tool for cancer management but can produce incidental/secondary findings that require special consideration. OBJECTIVE.­: To discuss clinical and laboratory issues related to incidental or secondary germline findings in the clinical setting of tumor testing and inform future guidelines in this area. DESIGN.­: A College of American Pathologists workgroup including representation from the American Society of Clinical Oncology, the Association for Molecular Pathology, and the American College of Medical Genetics and Genomics created a review of items that should be considered when developing guidelines for incidental or secondary findings when performing clinical tumor testing. RESULTS.­: Testing recommendations should be cognizant of the differences among anticipated incidental, unanticipated incidental, and secondary findings, and whether normal tissue is also tested. In addition to defining which variants will be reported, robust recommendations must also take into account test design and validation, reimbursement, cost, infrastructure, impact on reflex testing, and maintenance of proficiency. Care providers need to consider the potential of a test to uncover incidental or secondary findings, the recommendation of upfront counseling, the need for consent, the timing of testing and counseling, and that the exact significance of a finding may not be clear. CONCLUSIONS.­: As clinical oncology testing panels have become a mainstay of clinical cancer care, guidelines addressing the unique aspects of incidental and secondary findings in oncology testing are needed. This paper highlights clinical and laboratory considerations with regard to incidental/secondary findings and is a clarion call to create recommendations.

Creation of an Expert Curated Variant List for Clinical Genomic Test Development and Validation: A ClinGen and GeT-RM Collaborative Project.

Modern genomic sequencing tests often interrogate large numbers of genes. Identification of appropriate reference materials for development, validation studies, and quality assurance of these tests poses a significant challenge for laboratories. It is difficult to develop and maintain expert knowledge to identify all variants that must be validated to ensure analytic and clinical validity. Additionally, it is usually not possible to procure appropriate and characterized genomic DNA reference materials containing the number and scope of variants required. To address these challenges, the Centers for Disease Control and Prevention's Genetic Testing Reference Material Program (GeT-RM) has partnered with the Clinical Genome Resource (ClinGen) to develop a publicly available list of expert curated, clinically important variants. ClinGen Variant Curation Expert Panels nominated 546 variants found in 84 disease-associated genes, including common pathogenic and difficult-to-detect variants. Variant types nominated included 346 single nucleotide variants, 104 deletions, 37 copy number variants, 25 duplications, 18 deletion-insertions, 5 inversions, 4 insertions, 2 complex rearrangements, 3 difficult-to-sequence regions, and 2 fusions. This expert-curated variant list is a resource that provides a foundation for designing comprehensive validation studies and for creating in silico reference materials for clinical genomic test development and validation.

A Primer on Chimeric Antigen Receptor T-cell Therapy: What Does It Mean for Pathologists?

CONTEXT.­: Chimeric antigen receptor T-cell (CAR-T) technology has shown great promise in both clinical and preclinical models in mediating potent and specific antitumor activity. With the advent of US Food and Drug Administration-approved CAR-T therapies for B-cell lymphoblastic leukemia and B-cell non-Hodgkin lymphomas, CAR-T therapy is poised to become part of mainstream clinical practice. OBJECTIVE.­: To educate pathologists on CAR-T and chimeric antigen receptor-derived cellular therapy, provide a better understanding of their role in this process, explain important regulatory aspects of CAR-T therapy, and advocate for pathologist involvement in the delivery and monitoring of chimeric antigen receptor-based treatments. Much of the focus of this article addresses US Food and Drug Administration-approved therapies; however, more general issues and future perspectives are considered for therapies in development. DESIGN.­: A CAR-T workgroup, facilitated by the College of American Pathologists Personalized Health Care Committee and consisting of pathologists of various backgrounds, was convened to develop a summary guidance paper for the College of American Pathologists Council on Scientific Affairs. RESULTS.­: The workgroup identified gaps in pathologists' knowledge of CAR-T therapy, including uncertainty in the role of the clinical laboratory in supporting CAR-T therapy. The workgroup considered these issues and summarized the findings to assist pathologists to become stakeholders in CAR-T therapy administration. CONCLUSIONS.­: This manuscript serves to both educate pathologists on CAR-T therapy and serve as a point of initial discussions in areas of CAR-T science, clinical therapy, and regulatory issues as CAR-T therapies continue to be introduced into clinical practice.

Frontline Science: Cxxc5 expression alters cell cycle and myeloid differentiation of mouse hematopoietic stem and progenitor cells.

CXXC5 is a member of the CXXC-type zinc finger epigenetic regulators. Various hematopoietic and nonhematopoietic roles have been assigned to CXXC5. In the present study, the role of Cxxc5 in myelopoiesis was studied using overexpression and short hairpin RNA-mediated knockdown in mouse early stem and progenitor cells defined as Lineage- Sca-1+ c-Kit+ (LSK) cells. Knockdown of Cxxc5 in mouse progenitor cells reduced monocyte and increased granulocyte development in ex vivo culture systems. In addition, ex vivo differentiation and proliferation experiments demonstrated that the expression of Cxxc5 affects the cell cycle in stem/progenitor cells and myeloid cells. Flow cytometry-based analyses revealed that down-regulation of Cxxc5 leads to an increase in the percentage of cells in the S phase, whereas overexpression results in a decrease in the percentage of cells in the S phase. Progenitor cells proliferate more after Cxxc5 knockdown, and RNA sequencing of LSK cells, and single-cell RNA sequencing of differentiating myeloid cells showed up-regulation of genes involved in the regulation of cell cycle after Cxxc5 knockdown. These results provide novel insights into the physiologic function of Cxxc5 during hematopoiesis, and demonstrate for the first time that it plays a role in monocyte development.

Evaluation of Mass Cytometry in the Clinical Laboratory.

BACKGROUND: Mass cytometry can differentiate more channels than conventional flow cytometry. However, for clinical use, standardization and agreement with well-established methods is paramount. We compared mass cytometry to standard clinical flow cytometry. METHODS: Mass and flow cytometry were performed in parallel on peripheral blood samples from 25 healthy individuals. Antibody staining was performed on the same samples at the same time, and analyzed for granulocyte, monocyte, lymphocyte, T, B, NK, CD4 and CD8 percentages. Validation parameters included comparison to flow cytometry, inter- and intra-assay precision and establishment of reference intervals. RESULTS: There was a positive correlation between mass and flow cytometry for the eight populations studied (R2 between 0.26 and 0.97). Slopes of the best-fit lines varied from 0.50 to 1.21 (fluorescence/mass). No significant differences in variance were found (F-test, P > 0.05). However, paired t-tests were significantly different for four of the eight markers (granulocytes, NK cells, T cells and CD4 cells), resulting in different reference intervals. Signal intensities were correlated for monocytes, lymphocytes, T, CD4 and CD8 cells (R2 = 0.41-0.57). The mass cytometry intra-assay precisions were 0.7-8.5% and inter-assay precisions 1.5-13.8%. CONCLUSION: Mass and flow cytometry evaluations of whole blood for major cell populations correlate with similar precision and signal intensity. However, for clinical use, separate reference interval studies are required. Cell population identification should rely on gating strategies that take advantage of the characteristics offered by each method. © 2019 International Clinical Cytometry Society.

Designing and Implementing NGS Tests for Inherited Disorders: A Practical Framework with Step-by-Step Guidance for Clinical Laboratories.

Comprehensive next-generation sequencing (NGS) tests are increasingly used as first-line tests in the evaluation of patients with suspected heritable disease. Despite major technical simplifications, these assays still pose significant challenges for molecular testing laboratories. Existing professional guidelines and recommendations provide a framework for laboratories implementing such tests, but in-depth, concrete guidance is generally not provided. Consequently, there is variability in how laboratories interpret and subsequently implement these regulatory frameworks. To address the need for more detailed guidance, the College of American Pathologists with representation from the Association for Molecular Pathologists assembled a working group to create a practical resource for clinical laboratories. This initial work is focused on variant detection in the setting of inherited disease and provides structured worksheets that guide the user through the entire life cycle of an NGS test, including design, optimization, validation, and quality management with additional guidance for clinical bioinformatics. This resource is designed to be a living document that is publicly available and will be updated with user and expert feedback as the wet bench and bioinformatic landscapes continue to evolve. It is intended to facilitate the standardization of NGS testing across laboratories and therefore to improve patient care.

Novel PLP1 Mutations Identified With Next-Generation Sequencing Expand the Spectrum of PLP1-Associated Leukodystrophy Clinical Phenotypes.

Next-generation sequencing was performed for 2 families with an undiagnosed neurologic disease. Analysis revealed X-linked mutations in the proteolipid protein 1 (PLP1) gene, which is associated with X-linked Pelizaeus-Merzbacher disease and Spastic Paraplegia type 2. In family A, the novel PLP1 missense mutation c.617T>A (p.M206K) was hemizygous in the 2 affected male children and heterozygous in the mother. In family B, the novel de novoPLP1 frameshift mutation c.359_369del (p.G120fs) was hemizygous in the affected male child. Although PLP1 mutations have been reported to cause an increasingly wide range of phenotypes inclusive of the dystonia, spastic paraparesis, motor neuronopathy, and leukodystrophy observed in our patients, atypical features included the cerebrospinal fluid deficiency of neurotransmitter and pterin metabolites and the delayed appearance of myelin abnormalities on neuroimaging studies. Next-generation sequencing studies provided a diagnosis for these families with complex leukodystrophy disease phenotypes, which expanded the spectrum of PLP1-associated leukodystrophy clinical phenotypes.

Ikaros family zinc finger 1 regulates dendritic cell development and function in humans.

Ikaros family zinc finger 1 (IKZF1) is a haematopoietic transcription factor required for mammalian B-cell development. IKZF1 deficiency also reduces plasmacytoid dendritic cell (pDC) numbers in mice, but its effects on human DC development are unknown. Here we show that heterozygous mutation of IKZF1 in human decreases pDC numbers and expands conventional DC1 (cDC1). Lenalidomide, a drug that induces proteosomal degradation of IKZF1, also decreases pDC numbers in vivo, and reduces the ratio of pDC/cDC1 differentiated from progenitor cells in vitro in a dose-dependent manner. In addition, non-classical monocytes are reduced by IKZF1 deficiency in vivo. DC and monocytes from patients with IKZF1 deficiency or lenalidomide-treated cultures secrete less IFN-α, TNF and IL-12. These results indicate that human DC development and function are regulated by IKZF1, providing further insights into the consequences of IKZF1 mutation on immune function and the mechanism of immunomodulation by lenalidomide.

Standards and Guidelines for Validating Next-Generation Sequencing Bioinformatics Pipelines: A Joint Recommendation of the Association for Molecular Pathology and the College of American Pathologists.

Bioinformatics pipelines are an integral component of next-generation sequencing (NGS). Processing raw sequence data to detect genomic alterations has significant impact on disease management and patient care. Because of the lack of published guidance, there is currently a high degree of variability in how members of the global molecular genetics and pathology community establish and validate bioinformatics pipelines. Improperly developed, validated, and/or monitored pipelines may generate inaccurate results that may have negative consequences for patient care. To address this unmet need, the Association of Molecular Pathology, with organizational representation from the College of American Pathologists and the American Medical Informatics Association, has developed a set of 17 best practice consensus recommendations for the validation of clinical NGS bioinformatics pipelines. Recommendations include practical guidance for laboratories regarding NGS bioinformatics pipeline design, development, and operation, with additional emphasis on the role of a properly trained and qualified molecular professional to achieve optimal NGS testing quality.

Development and Validation of Clinical Whole-Exome and Whole-Genome Sequencing for Detection of Germline Variants in Inherited Disease.

CONTEXT: - With the decrease in the cost of sequencing, the clinical testing paradigm has shifted from single gene to gene panel and now whole-exome and whole-genome sequencing. Clinical laboratories are rapidly implementing next-generation sequencing-based whole-exome and whole-genome sequencing. Because a large number of targets are covered by whole-exome and whole-genome sequencing, it is critical that a laboratory perform appropriate validation studies, develop a quality assurance and quality control program, and participate in proficiency testing. OBJECTIVE: - To provide recommendations for whole-exome and whole-genome sequencing assay design, validation, and implementation for the detection of germline variants associated in inherited disorders. DATA SOURCES: - An example of trio sequencing, filtration and annotation of variants, and phenotypic consideration to arrive at clinical diagnosis is discussed. CONCLUSIONS: - It is critical that clinical laboratories planning to implement whole-exome and whole-genome sequencing design and validate the assay to specifications and ensure adequate performance prior to implementation. Test design specifications, including variant filtering and annotation, phenotypic consideration, guidance on consenting options, and reporting of incidental findings, are provided. These are important steps a laboratory must take to validate and implement whole-exome and whole-genome sequencing in a clinical setting for germline variants in inherited disorders.

Viral Pathogen Detection by Metagenomics and Pan-Viral Group Polymerase Chain Reaction in Children With Pneumonia Lacking Identifiable Etiology.

Background: Community-acquired pneumonia (CAP) is a leading cause of pediatric hospitalization. Pathogen identification fails in approximately 20% of children but is critical for optimal treatment and prevention of hospital-acquired infections. We used two broad-spectrum detection strategies to identify pathogens in test-negative children with CAP and asymptomatic controls. Methods: Nasopharyngeal/oropharyngeal (NP/OP) swabs from 70 children <5 years with CAP of unknown etiology and 90 asymptomatic controls were tested by next-generation sequencing (RNA-seq) and pan viral group (PVG) PCR for 19 viral families. Association of viruses with CAP was assessed by adjusted odds ratios (aOR) and 95% confidence intervals controlling for season and age group. Results: RNA-seq/PVG PCR detected previously missed, putative pathogens in 34% of patients. Putative viral pathogens included human parainfluenza virus 4 (aOR 9.3, P = .12), human bocavirus (aOR 9.1, P < .01), Coxsackieviruses (aOR 5.1, P = .09), rhinovirus A (aOR 3.5, P = .34), and rhinovirus C (aOR 2.9, P = .57). RNA-seq was more sensitive for RNA viruses whereas PVG PCR detected more DNA viruses. Conclusions: RNA-seq and PVG PCR identified additional viruses, some known to be pathogenic, in NP/OP specimens from one-third of children hospitalized with CAP without a previously identified etiology. Both broad-range methods could be useful tools in future epidemiologic and diagnostic studies.

Guidelines for Validation of Next-Generation Sequencing-Based Oncology Panels: A Joint Consensus Recommendation of the Association for Molecular Pathology and College of American Pathologists.

Next-generation sequencing (NGS) methods for cancer testing have been rapidly adopted by clinical laboratories. To establish analytical validation best practice guidelines for NGS gene panel testing of somatic variants, a working group was convened by the Association of Molecular Pathology with liaison representation from the College of American Pathologists. These joint consensus recommendations address NGS test development, optimization, and validation, including recommendations on panel content selection and rationale for optimization and familiarization phase conducted before test validation; utilization of reference cell lines and reference materials for evaluation of assay performance; determining of positive percentage agreement and positive predictive value for each variant type; and requirements for minimal depth of coverage and minimum number of samples that should be used to establish test performance characteristics. The recommendations emphasize the role of laboratory director in using an error-based approach that identifies potential sources of errors that may occur throughout the analytical process and addressing these potential errors through test design, method validation, or quality controls so that no harm comes to the patient. The recommendations contained herein are intended to assist clinical laboratories with the validation and ongoing monitoring of NGS testing for detection of somatic variants and to ensure high quality of sequencing results.

Principles and Recommendations for Standardizing the Use of the Next-Generation Sequencing Variant File in Clinical Settings.

A national workgroup convened by the Centers for Disease Control and Prevention identified principles and made recommendations for standardizing the description of sequence data contained within the variant file generated during the course of clinical next-generation sequence analysis for diagnosing human heritable conditions. The specifications for variant files were initially developed to be flexible with regard to content representation to support a variety of research applications. This flexibility permits variation with regard to how sequence findings are described and this depends, in part, on the conventions used. For clinical laboratory testing, this poses a problem because these differences can compromise the capability to compare sequence findings among laboratories to confirm results and to query databases to identify clinically relevant variants. To provide for a more consistent representation of sequence findings described within variant files, the workgroup made several recommendations that considered alignment to a common reference sequence, variant caller settings, use of genomic coordinates, and gene and variant naming conventions. These recommendations were considered with regard to the existing variant file specifications presently used in the clinical setting. Adoption of these recommendations is anticipated to reduce the potential for ambiguity in describing sequence findings and facilitate the sharing of genomic data among clinical laboratories and other entities.

Development and Validation of Targeted Next-Generation Sequencing Panels for Detection of Germline Variants in Inherited Diseases.

CONTEXT: - The number of targeted next-generation sequencing (NGS) panels for genetic diseases offered by clinical laboratories is rapidly increasing. Before an NGS-based test is implemented in a clinical laboratory, appropriate validation studies are needed to determine the performance characteristics of the test. OBJECTIVE: - To provide examples of assay design and validation of targeted NGS gene panels for the detection of germline variants associated with inherited disorders. DATA SOURCES: - The approaches used by 2 clinical laboratories for the development and validation of targeted NGS gene panels are described. Important design and validation considerations are examined. CONCLUSIONS: - Clinical laboratories must validate performance specifications of each test prior to implementation. Test design specifications and validation data are provided, outlining important steps in validation of targeted NGS panels by clinical diagnostic laboratories.