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* 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. doi: 10.5858/arpa.2021-0384-CP, Next-generation sequencing (NGS) technologies have enabled comprehensive assessment of tumor molecular profiles and revolutionized the clinical testing landscape in oncology. In addition to allowing the assessment of nucleic acid in [...]

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. doi: 10.5858/arpa.2019-0352-CP, The number of clinical laboratories using next-generation sequencing (NGS) methods for the assessment of somatic variants has been growing rapidly. (1,2) This explosion has been driven by the increasing numbers [...]

Context.--Next-generation sequencing-based assays are being increasingly used in the clinical setting for the detection of somatic variants in solid tumors, but limited data are available regarding the interlaboratory performance of these assays. Objective.--To examine proficiency testing data from the initial College of American Pathologists (CAP) Next-Generation Sequencing Solid Tumor survey to report on laboratory performance. Design.--CAP proficiency testing results from 111 laboratories were analyzed for accuracy and associated assay performance characteristics. Results.--The overall accuracy observed for all variants was 98.3%. Rare false-negative results could not be attributed to sequencing platform, selection method, or other assay characteristics. The median and average of the variant allele fractions reported by the laboratories were within 10% of those orthogonally determined by digital polymerase chain reaction for each variant. The median coverage reported at the variant sites ranged from 1922 to 3297. Conclusions.--Laboratories demonstrated an overall accuracy of greater than 98% with high specificity when examining 10 clinically relevant somatic single-nucleotide variants with a variant allele fraction of 15% or greater. These initial data suggest excellent performance, but further ongoing studies are needed to evaluate the performance of lower variant allele fractions and additional variant types. (Arch Pathol Lab Med. 2019;143:463-471; doi: 10.5858/arpa.2018-0336-CP), The past several years have seen the development or expansion of several national and international efforts that aim to accelerate the implementation of precision medicine. (1-4) One area of focus [...]

BackgroundPilocytic Astrocytomas (PAs) are common low-grade central nervous system malignancies for which few recurrent and specific genetic alterations have been identified. In an effort to better understand the molecular biology underlying the pathogenesis of these pediatric brain tumors, we performed higher-order transcriptional network analysis of a large gene expression dataset to identify gene regulatory pathways that are specific to this tumor type, relative to other, more aggressive glial or histologically distinct brain tumours.MethodsRNA derived from frozen human PA tumours was subjected to microarray-based gene expression profiling, using Affymetrix U133Plus2 GeneChip microarrays. This data set was compared to similar data sets previously generated from non-malignant human brain tissue and other brain tumour types, after appropriate normalization.ResultsIn this study, we examined gene expression in 66 PA tumors compared to 15 non-malignant cortical brain tissues, and identified 792 genes that demonstrated consistent differential expression between independent sets of PA and non-malignant specimens. From this entire 792 gene set, we used the previously described PAP tool to assemble a core transcriptional regulatory network composed of 6 transcription factor genes (TFs) and 24 target genes, for a total of 55 interactions. A similar analysis of oligodendroglioma and glioblastoma multiforme (GBM) gene expression data sets identified distinct, but overlapping, networks. Most importantly, comparison of each of the brain tumor type-specific networks revealed a network unique to PA that included repressed expression of ONECUT2, a gene frequently methylated in other tumor types, and 13 other uniquely predicted TF-gene interactions.ConclusionsThese results suggest specific transcriptional pathways that may operate to create the unique molecular phenotype of PA and thus opportunities for corresponding targeted therapeutic intervention. Moreover, this study also demonstrates how integration of gene expression data with TF-gene and TF-TF interaction data is a powerful approach to generating testable hypotheses to better understand cell-type specific genetic programs relevant to cancer.

* Context.--With the increasing integration of molecular alterations into the evaluation of hematologic malignancies (HM), somatic mutation profiling by next-generation sequencing (NGS) has become a common clinical testing strategy. Limited data are available about the characteristics of these assays. Objective.--To describe assay characteristics, specimen requirements, and reporting practices for NGS-based HM testing using College of American Pathologists proficiency testing survey data. Design.--The College of American Pathologists NGS Hematologic Malignancies Survey (NGSHM) results from 78 laboratories were used to determine laboratory practices in NGS-based HM testing. Results.--The majority of laboratories performed tumor-only (88.5% [69 of 78]), targeted sequencing of cancer genes or mutation hotspots (98.7% [77 of 78]); greater than 90% performed testing on fresh bone marrow and peripheral blood. The majority of laboratories reported a 5% lower limit of detection for single-nucleotide variants (73.1% [57 of 78]) and small insertions and deletions (50.6% [39 of 77]). A majority of laboratories used benchtop sequencers and custom enrichment approaches. Conclusions.--This manuscript summarizes the characteristics of clinical NGS-based testing for the detection of somatic variants in HM. These data may be broadly useful to inform laboratory practice and quality management systems, regulation, and oversight of NGS testing, and precision medicine efforts using a data-driven approach. (Arch Pathol Lab Med. 2021;145:1110-1116; doi: 10.5858/arpa.2019-0661-CP, The clinical need for broader identification of somatic variants and the advancement in next-generation sequencing (NGS) technology together have driven the development and incorporation of NGS-based somatic variant testing in [...]

Context.--Detection of acquired variants in cancer is a paradigm of precision medicine, yet little has been reported about clinical laboratory practices across a broad range of laboratories. Objective.--To use College of American Pathologists proficiency testing survey results to report on the results from surveys on next-generation sequencing-based oncology testing practices. Design.--College of American Pathologists proficiency testing survey results from more than 250 laboratories currently performing molecular oncology testing were used to determine laboratory trends in next-generation sequencing-based oncology testing. Results.--These presented data provide key information about the number of laboratories that currently offer or are planning to offer next-generation sequencing-based oncology testing. Furthermore, we present data from 60 laboratories performing next-generation sequencing-based oncology testing regarding specimen requirements and assay characteristics. The findings indicate that most laboratories are performing tumor-only targeted sequencing to detect single-nucleotide variants and small insertions and deletions, using desktop sequencers and predesigned commercial kits. Despite these trends, a diversity of approaches to testing exists. Conclusions.--This information should be useful to further inform a variety of topics, including national discussions involving clinical laboratory quality systems, regulation and oversight of next-generation sequencing-based oncology testing, and precision oncology efforts in a data-driven manner. (Arch Pathol Lab Med. 2017;141:1679-1685; doi: 10.5858/arpa.2016-0542-CP), Clinical testing for somatic or acquired variants in cancer specimens is a common modality of precision, or personalized, medicine. Detection of somatic variants in cancer specimens can be used for [...]

A study was conducted to evaluate whether DNMT3A is recurrently mutated in acute myeloid leukemia samples and whether DNMT3A mutations are associated with poor survival rates. Results indicated that sNMT3A mutations are highly recurrent in such patients and are independently associated with poor outcomes.

Acute promyelocytic leukemia (APL) is characterized by the t(15;17) chromosomal translocation, which results in fusion of the retinoic acid receptor [alpha] (RARA) gene to another gene, most commonly promyelocytic leukemia (PML). The resulting fusion protein, PML-RARA, initiates APL, which is a subtype (M3) of acute myeloid leukemia (AML). In this report, we identify a gene expression signature that is specific to M3 samples; it was not found in other AML subtypes and did not simply represent the normal gene expression pattern of primary promyelocytes. To validate this signature for a large number of genes, we tested a recently developed high throughput digital technology (NanoString nCounter). Nearly all of the genes tested demonstrated highly significant concordance with our microarray data (P < 0.05). The validated gene signature reliably identified M3 samples in 2 other AML datasets, and the validated genes were substantially enriched in our mouse model of APL, but not in a cell line that inducibly expressed PML-RARA. These results demonstrate that nCounter is a highly reproducible, customizable system for mRNA quantification using limited amounts of clinical material, which provides a valuable tool for biomarker measurement in low-abundance patient samples., Introduction Here we describe what is, to our knowledge, the first use of a high-throughput digital system to assay the expression of a large number of genes in primary clinical [...]

Acute myeloid leukaemia is a highly malignant haematopoietic tumour that affects about 13,000 adults in the United States each year. The treatment of this disease has changed little in the [...]

Egr2 is a transcription factor required for peripheral nerve myelination in rodents, and mutations in Egr2 are associated with congenital hypomyelinating neuropathy (CHN) in humans. To further study its role in myelination, we generated mice harboring a hypomorphic Egr2 allele (Egr[2.sup.Lo]) that survive for up to 3 weeks postnatally, a period of active myelination in rodents. These Egr[2.sup.Lo/Lo] mice provided the opportunity to study the molecular effects of Egr2 deficiency on Schwann cell biology, an analysis that was not possible previously, because of the perinatal lethality of Egr2-null mice. Egr[2.sup.Lo/Lo] mice phenocopy CHN, as evidenced by the severe hypomyelination and increased numbers of proliferating Schwann cells of the peripheral nerves. Comparison of sciatic nerve gene expression profiles during development and after crush injury with those of Egr[2.sup.Lo/Lo] Schwann cells revealed that they are developmentally arrested, with down-regulation of myelination-related genes and up-regulation of genes associated with immature and promyelinating Schwann cells. One of the abnormally elevated genes in Egr[2.sup.Lo/Lo] Schwann cells, Sox2, encodes a transcription factor that is crucial for maintenance of neural stem cell pluripotency. Wild-type Schwann cells infected with Sox2 adenovirus or lentivirus inhibited expression of myelination-associated genes (e.g., myelin protein zero; Mpz), and failed to myelinate axons in vitro, but had an enhanced proliferative response to [beta]-neuregulin. The characterization of a mouse model of CHN has provided insight into Schwann cell differentiation and allowed the identification of Sox2 as a negative regulator of myelination.

Context.--Next-generation sequencing (NGS) is revolutionizing the discipline of laboratory medicine, with a deep and direct impact on patient care. Although it empowers clinical laboratories with unprecedented genomic sequencing capability, NGS has brought along obvious and obtrusive informatics challenges. Bioinformatics and clinical informatics are separate disciplines with typically a small degree of overlap, but they have been brought together by the enthusiastic adoption of NGS in clinical laboratories. The result has been a collaborative environment for the development of novel informatics solutions. Sustaining NGS-based testing in a regulated clinical environment requires institutional support to build and maintain a practical, robust, scalable, secure, and cost-effective informatics infrastructure. Objective.--To discuss the novel NGS informatics challenges facing pathology laboratories today and offer solutions and future developments to address these obstacles. Data Sources.--The published literature pertaining to NGS informatics was reviewed. The coauthors, experts in the fields of molecular pathology, precision medicine, and pathology informatics, also contributed their experiences. Conclusions.--The boundary between bioinformatics and clinical informatics has significantly blurred with the introduction of NGS into clinical molecular laboratories. Next-generation sequencing technology and the data derived from these tests, if managed well in the clinical laboratory, will redefine the practice of medicine. In order to sustain this progress, adoption of smart computing technology will be essential. Computational pathologists will be expected to play a major role in rendering diagnostic and theranostic services by leveraging 'Big Data' and modern computing tools. (Arch Pathol Lab Med. 2016;140:958-975; doi: 10.5858/arpa.2015-0507-RA), Next-generation sequencing (NGS) technology, using a massively parallel sequencing paradigm, has markedly altered the landscape of genomic medicine. The high-throughput capabilities of NGS systems have resulted in an exponential accumulation [...]

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. As NGS-based clinical tests are new to diagnostic testing and are of much greater complexity than traditional Sanger sequencing-based tests, there is an urgent need to develop new regulatory standards for laboratories offering these tests. Design.--To develop the necessary regulatory framework for NGS and to facilitate appropriate adoption of this technology for clinical testing, CAP formed a committee in 2011, the NGS Work Group, to deliberate upon the contents to be included in the checklist. Results.--A total of 18 laboratory accreditation checklist requirements for the analytic wet bench process and bioinformatics analysis processes have been included within CAP's molecular pathology checklist (MOL). Conclusions.--This report describes the important issues considered by the CAP committee during the development of the new checklist requirements, which address documentation, validation, quality assurance, confirmatory testing, exception logs, monitoring of upgrades, variant interpretation and reporting, incidental findings, data storage, version traceability, and data transfer confidentiality. (Arch Pathol Lab Med. 2015;139:481-493; doi: 10.5858/arpa.2014-0250-CP), DNA sequencing has evolved from Maxam-Gilbert (1) and Sanger (2, 3) methods in the 1970s to a set of technologies that are collectively referred to as next-generation sequencing (NGS). (4-12) [...]

Abstract Background Pilocytic Astrocytomas (PAs) are common low-grade central nervous system malignancies for which few recurrent and specific genetic alterations have been identified. In an effort to better understand the molecular biology underlying the pathogenesis of these pediatric brain tumors, we performed higher-order transcriptional network analysis of a large gene expression dataset to identify gene regulatory pathways that are specific to this tumor type, relative to other, more aggressive glial or histologically distinct brain tumours. Methods RNA derived from frozen human PA tumours was subjected to microarray-based gene expression profiling, using Affymetrix U133Plus2 GeneChip microarrays. This data set was compared to similar data sets previously generated from non-malignant human brain tissue and other brain tumour types, after appropriate normalization. Results In this study, we examined gene expression in 66 PA tumors compared to 15 non-malignant cortical brain tissues, and identified 792 genes that demonstrated consistent differential expression between independent sets of PA and non-malignant specimens. From this entire 792 gene set, we used the previously described PAP tool to assemble a core transcriptional regulatory network composed of 6 transcription factor genes (TFs) and 24 target genes, for a total of 55 interactions. A similar analysis of oligodendroglioma and glioblastoma multiforme (GBM) gene expression data sets identified distinct, but overlapping, networks. Most importantly, comparison of each of the brain tumor type-specific networks revealed a network unique to PA that included repressed expression of ONECUT2, a gene frequently methylated in other tumor types, and 13 other uniquely predicted TF-gene interactions. Conclusions These results suggest specific transcriptional pathways that may operate to create the unique molecular phenotype of PA and thus opportunities for corresponding targeted therapeutic intervention. Moreover, this study also demonstrates how integration of gene expression data with TF-gene and TF-TF interaction data is a powerful approach to generating testable hypotheses to better understand cell-type specific genetic programs relevant to cancer.