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.

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.

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.

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.

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.

Novel 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 () gene, which is associated with X-linked Pelizaeus-Merzbacher disease and Spastic Paraplegia type 2. In family A, the novel missense mutation c.

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).

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.

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.

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.

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.

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.

A Model Study of In Silico Proficiency Testing for Clinical Next-Generation Sequencing.

Context: -Most current proficiency testing challenges for next-generation sequencing assays are methods-based proficiency testing surveys that use DNA from characterized reference samples to test both the wet-bench and bioinformatics/dry-bench aspects of the tests. Methods-based proficiency testing surveys are limited by the number and types of mutations that either are naturally present or can be introduced into a single DNA sample.

Objective: -To address these limitations by exploring a model of in silico proficiency testing in which sequence data from a single well-characterized specimen are manipulated electronically.

Taxonomer: an interactive metagenomics analysis portal for universal pathogen detection and host mRNA expression profiling.

Background: High-throughput sequencing enables unbiased profiling of microbial communities, universal pathogen detection, and host response to infectious diseases. However, computation times and algorithmic inaccuracies have hindered adoption.

Results: We present Taxonomer, an ultrafast, web-tool for comprehensive metagenomics data analysis and interactive results visualization.

Design of a Genomics Curriculum: Competencies for Practicing Pathologists.

Context: The field of genomics is rapidly impacting medical care across specialties. To help guide test utilization and interpretation, pathologists must be knowledgeable about genomic techniques and their clinical utility. The technology allowing timely generation of genomic data is relatively new to patient care and the clinical laboratory, and therefore, many currently practicing pathologists have been trained without any molecular or genomics exposure.

Identification of patients with RAG mutations previously diagnosed with common variable immunodeficiency disorders.

Purpose: Combined immunodeficiency (CID) presents a unique challenge to clinicians. Two patients presented with the prior clinical diagnosis of common variable immunodeficiency (CVID) disorder marked by an early age of presentation, opportunistic infections, and persistent lymphopenia. Due to the presence of atypical clinical features, next generation sequencing was applied documenting RAG deficiency in both patients.

Next-generation sequencing of custom amplicons to improve coverage of HaloPlex multigene panels.

Next-generation sequencing (NGS) of multigene panels performed for genetic clinical diagnostics requires 100% coverage of all targeted genes. In the genetic diagnostics laboratory, coverage gaps are typically filled with Sanger sequencing after NGS data are collected and analyzed. Libraries prepared using the hybridization-based custom capture HaloPlex method are covered at ~98% and include gaps in coverage because of the location of the restriction enzyme sites used for fragmentation and differences in the designed and actual library insert size.

College of American Pathologists' laboratory standards for next-generation sequencing clinical tests.

Context: The higher throughput and lower per-base cost of next-generation sequencing (NGS) as compared to Sanger sequencing has led to its rapid adoption in clinical testing. The number of laboratories offering NGS-based tests has also grown considerably in the past few years, despite the fact that specific Clinical Laboratory Improvement Amendments of 1988/College of American Pathologists (CAP) laboratory standards had not yet been developed to regulate this technology.

Objective: To develop a checklist for clinical testing using NGS technology that sets standards for the analytic wet bench process and for bioinformatics or "dry bench" analyses.

A unified test of linkage analysis and rare-variant association for analysis of pedigree sequence data.

High-throughput sequencing of related individuals has become an important tool for studying human disease. However, owing to technical complexity and lack of available tools, most pedigree-based sequencing studies rely on an ad hoc combination of suboptimal analyses. Here we present pedigree-VAAST (pVAAST), a disease-gene identification tool designed for high-throughput sequence data in pedigrees.

Phevor combines multiple biomedical ontologies for accurate identification of disease-causing alleles in single individuals and small nuclear families.

Phevor integrates phenotype, gene function, and disease information with personal genomic data for improved power to identify disease-causing alleles. Phevor works by combining knowledge resident in multiple biomedical ontologies with the outputs of variant-prioritization tools. It does so by using an algorithm that propagates information across and between ontologies.

Methods-based proficiency testing in molecular genetic pathology.

This Perspectives article describes methods-based proficiency testing (MBPT), the benefits and limitations of MBPT, why the time is right for MBPT in molecular diagnostics, and how MBPT for next-generation sequencing is being developed by the College of American Pathologists.