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Context.— Clinical testing for tumor cell-free DNA (cfDNA) has evolved rapidly, but no practice guidelines exist. Objective.— To summarize cfDNA laboratory practices based on self-reporting and assess preanalytical, analytical, and postanalytical trends that may influence the quality, accuracy, and consistency of cfDNA testing. Design.— Data were derived from the College of American Pathologists cfDNA proficiency testing program submitted by 101 participating laboratories from 2018 to 2019. Results.— Most laboratories performing clinical circulating tumor DNA testing are commercial/nonhospital (71.2%; 72 of 101) and international (77.2%; 78 of 101) laboratories. Commercial laboratories had higher monthly test volumes than hospital-based laboratories (median, 36 versus 7–8) and tended to have larger gene panels (median, 50 versus 11 genes) when panel-based testing was offered. The main clinical indications include therapy selection and treatment/disease monitoring. Plasma is the most commonly accepted specimen, which is predominantly collected in cell-stabilizing tubes. Equal proportions of laboratories use next-generation sequencing (NGS) and non-NGS methods to assess key genes, including EGFR, BRAF, KRAS, NRAS, and IDH1. Most laboratories reported a lower limit of detection (LLOD) of 0.5%, variant allele frequency or less, which did not differ by method, NGS or non-NGS, except for EGFR. Sixty-five percent (17 of 26) of laboratories using the US Food and Drug Administration (FDA)-approved non-NGS EGFR assay report analytical sensitivities higher than 0.5%, as compared to 15% (16 of 104) of laboratories using an alternative NGS or non-NGS method. There is also a wider range in LLODs obtained for the FDA-approved EGFR assay than nonapproved assays. Conclusions.— These results highlight emerging practice trends and serve as a foundation to initiate future practice recommendations.

OBJECTIVE: Black women suffer a higher mortality from endometrial cancer (EC) than White women. Potential biological causes for this disparity include a higher prevalence of obesity and more lethal histologic/molecular subtypes. We hypothesize that another biological factor driving this racial disparity could be the EC microbiome. METHODS: Banked tumor specimens of postmenopausal, Black and White women undergoing hysterectomy for early stage endometrioid EC were identified. The microbiota of the tumors were characterized by bacterial 16S rRNA sequencing. The microbial component of endometrioid ECs in The Cancer Genome Atlas (TCGA) database were assessed for comparison. RESULTS: 95 early stage ECs were evaluated: 23 Black (24%) and 72 White (76%). Microbial diversity was increased (p

The basis for acquired resistance to JAK inhibition in patients with JAK2-driven hematologic malignancies is not well understood. We report a patient with a myeloproliferative neoplasm (MPN) with a BCR activator of RhoGEF and GTPase (BCR)–JAK2 fusion with initial hematologic response to ruxolitinib who rapidly developed B-lymphoid blast transformation. We analyzed pre-ruxolitinib and blast transformation samples using genome sequencing, DNA mate-pair sequencing (MPseq), RNA sequencing (RNA-seq), and chromosomal microarray to characterize possible mechanisms of resistance. No resistance mutations in the BCR-JAK2 fusion gene or transcript were identified, and fusion transcript expression levels remained stable. However, at the time of blast transformation, MPseq detected a new IKZF1 copy-number loss, which is predicted to result in loss of normal IKZF1 protein translation. RNA-seq revealed significant upregulation of genes negatively regulated by IKZF1, including IL7R and CRLF2. Disease progression was also characterized by adaptation to an activated B-cell receptor (BCR)–like signaling phenotype, with marked upregulation of genes such as CD79A, CD79B, IGLL1, VPREB1, BLNK, ZAP70, RAG1, and RAG2. In summary, IKZF1 deletion and a switch from cytokine dependence to activated BCR-like signaling phenotype represent putative mechanisms of ruxolitinib resistance in this case, recapitulating preclinical data on resistance to JAK inhibition in CRLF2-rearranged Philadelphia chromosome-like acute lymphoblastic leukemia.

PURPOSE: Several professional societies have published guidelines for the clinical interpretation of somatic variants, which specifically address diagnostic, prognostic, and therapeutic implications. While these guidelines for the clinical interpretation of variants include data types that may be used to determine the oncogenicity of a variant (e.g., population frequency, functional and in silico data or somatic frequency), they do not provide a direct, systematic, and comprehensive set of standards and rules to classify the oncogenicity of a somatic variant. This insufficient guidance leads to inconsistent classification of rare somatic variants in cancer, generates variability in their clinical interpretation and importantly impacts patient care. Therefore, it’s essential to address this unmet need. METHODS: Clinical Genome Resource (ClinGen) Somatic Cancer Clinical Domain Working Group and ClinGen Germline/Somatic Variant Subcommittee, the Cancer Genomics Consortium (CGC), and the Variant Interpretation for Cancer Consortium (VICC) used a consensus approach to develop a Standard Operating Procedure (SOP) for the classification of oncogenicity of somatic variants. RESULTS: This comprehensive SOP has been developed to improve consistency in somatic variant classification, and has been validated on 94 somatic variants in ten common cancer-related genes. CONCLUSION: The comprehensive SOP is now available for classification of oncogenicity of a somatic variant.

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.

Context.— The College of American Pathologists (CAP), a laboratory accreditation organization with deemed status under the Clinical Laboratories Improvement Amendments of 1988 administers accreditation checklists. Checklists are used by laboratories to ensure regulatory compliance. Peer-level laboratory professionals audit laboratory records during inspections to assess compliance. Objective.— To identify the most frequently cited deficiencies for molecular oncology laboratories undergoing CAP accreditation inspections and describe laboratory improvement opportunities. Design.— The CAP Molecular Oncology Committee (MOC), which is involved in maintaining the Molecular Pathology checklist, reviewed data and inspector comments associated with the most frequently observed citations related to molecular oncology testing from laboratories inspected by the CAP during a 2-year period (2018–2020). Results.— Of 422 molecular oncology laboratories that underwent accreditation inspections, 159 (37.7%) were not cited for any molecular oncology–related deficiencies. For the All Common (COM) and Molecular Pathology checklists, there were 364 and 305 deficiencies, corresponding to compliance rates of 98.8% and 99.6%, respectively. The most frequently cited deficiencies are described. The COM checklist deficiencies were associated most often with the analytic testing phase; the MOL checklist deficiencies were more evenly distributed across the preanalytic, analytic, and postanalytic phases of testing. Conclusions.— Molecular oncology laboratories demonstrated excellent compliance with practices that support high-quality results for patients and the health care providers who use those test results in patient management. This review includes a critical assessment of opportunities for laboratories to improve compliance and molecular oncology testing quality.

Purpose Biallelic hypomorphic variants in PPA2, encoding the mitochondrial inorganic pyrophosphatase 2 protein, have been recently identified in individuals presenting with sudden cardiac death, occasionally triggered by alcohol intake or a viral infection. Here we report 20 new families harboring PPA2 variants. Methods Synthesis of clinical and molecular data concerning 34 individuals harboring five previously reported PPA2 variants and 12 novel variants, 11 of which were functionally characterized. Results Among the 34 individuals, only 6 remain alive. Twenty-three died before the age of 2 years while five died between 14 and 16 years. Within these 28 cases, 15 died of sudden cardiac arrest and 13 of acute heart failure. One case was diagnosed prenatally with cardiomyopathy. Four teenagers drank alcohol before sudden cardiac arrest. Progressive neurological signs were observed in 2/6 surviving individuals. For 11 variants, recombinant PPA2 enzyme activities were significantly decreased and sensitive to temperature, compared to wild-type PPA2 enzyme activity. Conclusion We expand the clinical and mutational spectrum associated with PPA2 dysfunction. Heart failure and sudden cardiac arrest occur at various ages with inter- and intrafamilial phenotypic variability, and presentation can include progressive neurological disease. Alcohol intake can trigger cardiac arrest and should be strictly avoided.

It is estimated that 350 million individuals worldwide suffer from rare diseases, which are predominantly caused by mutation in a single gene1. The current molecular diagnostic rate is estimated at 50%, with whole-exome sequencing (WES) among the most successful approaches2–5. For patients in whom WES is uninformative, RNA sequencing (RNA-seq) has shown diagnostic utility in specific tissues and diseases6–8. This includes muscle biopsies from patients with undiagnosed rare muscle disorders6,9, and cultured fibroblasts from patients with mitochondrial disorders7. However, for many individuals, biopsies are not performed for clinical care, and tissues are difficult to access. We sought to assess the utility of RNA-seq from blood as a diagnostic tool for rare diseases of different pathophysiologies. We generated whole-blood RNA-seq from 94 individuals with undiagnosed rare diseases spanning 16 diverse disease categories. We developed a robust approach to compare data from these individuals with large sets of RNA-seq data for controls (n = 1,594 unrelated controls and n = 49 family members) and demonstrated the impacts of expression, splicing, gene and variant filtering strategies on disease gene identification. Across our cohort, we observed that RNA-seq yields a 7.5% diagnostic rate, and an additional 16.7% with improved candidate gene resolution. A diagnostic tool based on blood RNA-seq is shown to identify causal genes and variants linked to clinical phenotypes in individuals with rare diseases for which whole-exome genetic sequencing was uninformative.

Context.—The performance of laboratory testing has recently come under increased scrutiny as part of important and ongoing debates on regulation and reimbursement. To address this critical issue, this study compares the performance of assay methods, using either commercial kits or assays designed and implemented by single laboratories (“home brews”), including next-generation sequencing methods, on proficiency testing provided by the College of American Pathologists Molecular Oncology Committee.Objective.—To compare the performance of different assay methods on College of American Pathologists proficiency testing for variant analysis of 3 common oncology analytes: BRAF, EGFR, and KRAS.Design.—There were 6897 total responses across 35 different proficiency testing samples interrogating 13 different variants as well as wild-type sequences for BRAF, EGFR, and KRAS. Performance was analyzed by test method, kit manufacturer, variants tested, and preanalytic and postanalytic practices.Results.—Of 26 reported commercial kits, 23 achieved greater than 95% accuracy. Laboratory-developed tests with no kit specified demonstrated 96.8% or greater accuracy across all 3 analytes (1123 [96.8%] acceptable of 1160 total responses for BRAF; 848 [97.5%] acceptable of 870 total responses for EGFR; 942 [97.0%] acceptable of 971 total responses for KRAS). Next-generation sequencing platforms (summed across all analytes and 2 platforms) demonstrated 99.4% accuracy for these analytes (165 [99.4%] acceptable of 166 total next-generation sequencing responses). Slight differences in performance were noted among select commercial assays, dependent upon the particular design and specificity of the assay. Wide differences were noted in the lower limits of neoplastic cellularity laboratories accepted for testing.Conclusions.—These data demonstrate the high degree of accuracy and comparable performance across all laboratories, regardless of methodology. However, care must be taken in understanding the diagnostic specificity and reported analytic sensitivity of individual methods.

Context.— There has been a rapid expansion of next-generation sequencing (NGS)–based assays for the detection of somatic variants in solid tumors. However, limited data are available regarding the comparative performance of NGS and non-NGS assays using standardized samples across a large number of laboratories. Objective.— To compare the performance of NGS and non-NGS assays using well-characterized proficiency testing samples provided by the College of American Pathologists (CAP) Molecular Oncology Committee. A secondary goal was to compare the use of preanalytic and postanalytic practices. Design.— A total of 17 343 responses were obtained from participants in the BRAF, EGFR, KRAS, and the Multigene Tumor Panel surveys across 84 different proficiency testing samples interrogating 16 variants and 3 wild-type sequences. Performance and preanalytic/postanalytic practices were analyzed by method. Results.— While both NGS and non-NGS achieved an acceptable response rate of greater than 95%, the overall performance of NGS methods was significantly better than that of non-NGS methods for the identification of variants in BRAF (overall 97.8% versus 95.6% acceptable responses, P = .001) and EGFR (overall 98.5% versus 97.3%, P = .01) and was similar for KRAS (overall 98.8% and 97.6%, P = .10). There were specific variant differences, but in all discrepant cases, NGS methods outperformed non-NGS methods. NGS laboratories also more consistently used preanalytic and postanalytic practices suggested by the CAP checklist requirements than non-NGS laboratories. Conclusions.— The overall analytic performance of both methods was excellent. For specific BRAF and EGFR variants, NGS outperformed non-NGS methods and NGS laboratories report superior adherence to suggested laboratory practices.

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.

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.

Contains fulltext : 218305.pdf (Publisher’s version ) (Closed access) BACKGROUND: The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative. METHODS: We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology. RESULTS: Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor beta signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory. CONCLUSIONS: Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor beta signaling and hippocampal function.

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.

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.

Investigations aimed at defining the 3D configuration of eukaryotic chromosomes have consistently encountered an endogenous population of chromosome-derived circular genomic DNA, referred to as extrachromosomal circular DNA (eccDNA). While the production, distribution, and activities of eccDNAs remain understudied, eccDNA formation from specific regions of the linear genome has profound consequences on the regulatory and coding capabilities for these regions. Here, we define eccDNA distributions in Caenorhabditis elegans and in three human cell types, utilizing a set of DNA topology-dependent approaches for enrichment and characterization. The use of parallel biophysical, enzymatic, and informatic approaches provides a comprehensive profiling of eccDNA robust to isolation and analysis methodology. Results in human and nematode systems provide quantitative analysis of the eccDNA loci at both unique and repetitive regions. Our studies converge on and support a consistent picture, in which endogenous genomic DNA circles are present in normal physiological states, and in which the circles come from both coding and noncoding genomic regions. Prominent among the coding regions generating DNA circles are several genes known to produce a diversity of protein isoforms, with mucin proteins and titin as specific examples.

Patients with advanced systemic mastocytosis (SM) (e.g. aggressive SM (ASM), SM with an associated hematologic neoplasm (SM-AHN) and mast cell leukemia (MCL)) have limited treatment options and exhibit reduced survival. Midostaurin is an oral multikinase inhibitor that inhibits D816V-mutated KIT, a primary driver of SM pathogenesis. We conducted a phase II trial of midostaurin 100 mg twice daily, administered as 28-day cycles, in 26 patients (ASM, n=3; SM-AHN, n= 17; MCL, n=6) with at least one sign of organ damage. During the first 12 cycles, the overall response rate was 69% (major/partial response: 50/19%) with clinical benefit in all advanced SM variants. With ongoing therapy, 2 patients achieved a complete remission of their SM. Midostaurin produced a ⩾50% reduction in bone marrow mast cell burden and serum tryptase level in 68% and 46% of patients, respectively. Median overall survival for the entire cohort was 40 months, and 18.5 months for MCL patients. Low-grade gastrointestinal side effects were common and manageable with antiemetics. The most frequent grade 3/4 nonhematologic and hematologic toxicities were asymptomatic hyperlipasemia (15%) and anemia (12%). With median follow-up of 10 years, no unexpected toxicities emerged. These data establish the durable activity and tolerability of midostaurin in advanced SM.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a malignant primary cutaneous T-cell lymphoma that is challenging to distinguish from other neoplastic and reactive panniculitides. In an attempt to identify somatic variants in SPTCL that may be diagnostically or therapeutically relevant, we performed both exome sequencing on paired tumor-normal samples and targeted sequencing of hematolymphoid-malignancy-associated genes on tumor biopsies. Exome sequencing was performed on skin biopsies from 4 cases of skin-limited SPTCL, 1 case of peripheral T-cell lymphoma, not otherwise specified with secondary involvement of the panniculus, and 2 cases of lupus panniculitis. This approach detected between 1 and 13 high-confidence somatic variants that were predicted to result in a protein alteration per case. Variants of interest identified include 1 missense mutation in ARID1B in 1 case of SPTCL. To detect variants that were present at a lower level, we used a more sensitive targeted panel to sequence 41 hematolymphoid-malignancy-associated genes. The targeted panel was applied to 2 of the biopsies that were evaluated by whole exome sequencing as well as 5 additional biopsies. Potentially pathogenic variants were identified in KMT2D and PLCG1 among others, but no gene was altered in >2 of the 7 cases sequenced. One variant that was notably absent from the cases sequences is RHOA G17V. Further work will be required to further elucidate the genetic abnormalities that lead to this rare lymphoma.

Context.— Detection of high-risk human papillomavirus (HR-HPV) in squamous cell carcinoma is important for classification and prognostication. In situ hybridization (ISH) is a commonly used HR-HPV–specific test that targets viral RNA or DNA. The College of American Pathologists (CAP) provides proficiency testing for laboratories performing HR-HPV ISH. Objective.— To compare the analytical performance of RNA- and DNA-based ISH methods on CAP HR-HPV proficiency tests. Design.— Data from the 2016–2018 CAP HPV ISH proficiency testing surveys were reviewed. These surveys consist of well-characterized samples with known status for HR-HPV, including 1 to 2 copies, 50 to 100 copies, 300 to 500 copies, and no copies of HR-HPV per cell. Results.— Ninety-five participants submitted 1268 survey results from 20 cores. Overall, RNA ISH had a significantly higher percentage of correct responses than DNA ISH: 97.4% (450 of 462) versus 80.6% (650 of 806) (P < .001). This disparity appears to be the consequence of a superior sensitivity of RNA ISH compared to DNA ISH for samples with 1 to 2 and with 50 to 100 copies of HR-HPV per cell: 95.2% (120 of 126) versus 53.8% (129 of 240), P < .001, respectively, and 100% (89 of 89) versus 76.3% (119 of 156), P < .001, respectively. Conclusions.— An assessment of CAP HR-HPV proficiency test performance indicates that RNA ISH shows significantly higher accuracy than DNA ISH owing to higher analytical sensitivity of RNA ISH in tumors with low (1–2 copies per cell) to intermediate (50–100 copies per cell) HR-HPV viral copy numbers. These data support the use of RNA over DNA ISH in clinical laboratories that perform HR-HPV testing as part of their testing algorithms.

The performance of laboratory testing has recently come under increased scrutiny as part of important and ongoing debates on regulation and reimbursement. To address this critical issue, this study compares the performance of assay methods, using either commercial kits or assays designed and implemented by single laboratories ("home brews"), including next-generation sequencing methods, on proficiency testing provided by the College of American Pathologists Molecular Oncology Committee.To compare the performance of different assay methods on College of American Pathologists proficiency testing for variant analysis of 3 common oncology analytes:There were 6897 total responses across 35 different proficiency testing samples interrogating 13 different variants as well as wild-type sequences forOf 26 reported commercial kits, 23 achieved greater than 95% accuracy. Laboratory-developed tests with no kit specified demonstrated 96.8% or greater accuracy across all 3 analytes (1123 [96.8%] acceptable of 1160 total responses forThese data demonstrate the high degree of accuracy and comparable performance across all laboratories, regardless of methodology. However, care must be taken in understanding the diagnostic specificity and reported analytic sensitivity of individual methods.

There has been a rapid expansion of next-generation sequencing (NGS)-based assays for the detection of somatic variants in solid tumors. However, limited data are available regarding the comparative performance of NGS and non-NGS assays using standardized samples across a large number of laboratories.To compare the performance of NGS and non-NGS assays using well-characterized proficiency testing samples provided by the College of American Pathologists (CAP) Molecular Oncology Committee. A secondary goal was to compare the use of preanalytic and postanalytic practices.A total of 17 343 responses were obtained from participants in theWhile both NGS and non-NGS achieved an acceptable response rate of greater than 95%, the overall performance of NGS methods was significantly better than that of non-NGS methods for the identification of variants inThe overall analytic performance of both methods was excellent. For specific

The Clinical Genome (ClinGen) Resource is a US National Human Genome Research Institute (NHGRI)-funded program dedicated to building an expert curated and freely available central resource that defines the clinical relevance of genes and variants for use in precision medicine. Teams of experts in various clinical domains come together as working groups within ClinGen to facilitate the interpretation, annotation and utilization of genes-variants for clinical application. Somatic Cancer is one such ClinGen Clinical Domain Working Group (CDWG) that consists of over 90 members worldwide, including clinicians, clinical laboratory diagnosticians, genomic scientists and bioinformaticians. Members of this CDWG identify high priority somatic variants in different cancer types that require expert curation and consensus in their clinical interpretation. In order to accurately implement practice guidelines/standards for variant interpretation, the Somatic Cancer CDWG recently established a somatic Variant Curation Expert Panel (VCEP) approval process. Based on their interest and clinical domain expertise, a subset of the CDWG members formed somatic VCEPs to perform authoritative curation on the shortlisted genes-somatic variants within the context of a disease, therapeutic indication or biological pathway. Expert curation within these VCEPs is performed by utilizing guidelines such as those recommended by AMP/ASCO/CAP (Li et al. 2017) and the Somatic Cancer CDWG (Ritter et al. 2016). Wherever necessary, the somatic VCEPs will develop: 1) gene- and disease-specific modifications to address gaps in existing variant assessment guidelines, 2) quantitative approaches for variant interpretation, and 3) implement standardized protocols for annotating somatic variants in genes for a specific disease, drug or biological pathway. Furthermore, ClinGen recently formed the Cancer Variant Interpretation (CVI) committee to provide support, review and feedback on the provisional somatic variant interpretation proposals developed by the VCEPs. The CVI provides somatic VCEPs with a preliminary approval before the final approval by the ClinGen CDWG oversight committee and ultimately ‘expert panel' status in ClinVar, an NCBI-maintained database of clinically relevant gene variants. NTRK fusions in cancer is the first Somatic VCEP going through the ClinGen Somatic Expert Panel approval process. Alterations in the FGFR pathway in GU cancers is the second somatic VCEP under consideration. The Somatic CDWG uses the CIViC (Clinical Interpretation of Variants in Cancer) platform for curation of somatic variants. To date, the CDWG has curated 268 evidence items relating to cancer variants in CIViC, 6 assertions, and 33 evidence source suggestions. The ultimate goal of the Somatic CDWG is to enhance the usability, dissemination and implementation of cancer somatic changes in the ClinGen resource and other cancer variant knowledgebases. Citation Format: Shruti Rao, Deborah Ritter, Arpad Danos, Gordana Raca, Angshumoy Roy, Kilannin Krysiak, Wan-Hsin Lin, Erica Barnell, Matthew McCoy, Beth Pitel, Dmitriy Sonkin, Jue Wang, Seyed Ali Hosseini, Shamini Selvarajah, Ian King, Rashmi Kanagal-Shamana, Xinjie Xu, Jeremy L. Warner, Funda Meric-Bernstam, Jason D. Merker, Marilyn Li, Alex H. Wagner, Malachi Griffith, Obi L. Griffith, Shashikant Kulkarni, Subha Madhavan. ClinGen somatic cancer working group: Disseminating standardized cancer molecular diagnostic data and evidence through global collaboration and expert curation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3215.

e13681 Background: Cell-free tumor DNA or circulating tumor DNA tests are increasingly used in clinical care to detect somatic mutations from solid tumors. However, data on laboratory performance characteristics using standardized samples is limited. Methods: Well-characterized reference materials were used for the College of American Pathologists (CAP) cell-free tumor DNA proficiency testing surveys, which consisted of stabilized DNA fragmented to simulate cell-free DNA in a synthetic plasma matrix. For the 2018A, 2018B, 2019A and 2019B surveys, laboratories tested for hotspot mutations (single and dinucleotide sequence changes) in EGFR, BRAF, KRAS, NRAS, and IDH1 at variant allele fractions ranging from 0.1% - 1.0%. As per CAP proficiency testing standards, results were scored according to the known mutation(s) engineered at designated variant allele fractions in each PT sample. Nine laboratories were excluded from analysis because they provided incomplete results. Statistical significance was calculated using a multivariate logistic regression model. Results: In 2018 and 2019, 101 laboratories submitted survey results for at least one proficiency testing mailing. There were 5088 total proficiency testing responses for EGFR, BRAF, KRAS, NRAS, and IDH1 mutations across 12 different samples. For the 3585 responses submitted for BRAF, KRAS, NRAS, and IDH1, sensitivity ranged from 94.6 – 100%, while specificity exceeded 99%. There were no significant differences in performance between analytical methodologies for BRAF, KRAS, NRAS, and IDH1 mutations. Performance characteristics for EGFR mutations among the 1503 responses showed a combined sensitivity of 87.1% and specificity of 98.7%. For laboratories detecting mutations in EGFR, next-generation sequencing methods exhibited a sensitivity (true positivity) of 95.7% while the sensitivity of non-NGS methods was lower at 81.7% ( P= 0.02). Conclusions: These findings demonstrate high sensitivity and specificity for clinical laboratories performing cell-free tumor DNA tests. For EGFR mutations, NGS outperformed non-NGS methods. These data suggest excellent overall agreement among laboratories performing clinical cell-free tumor DNA tests. Further investigation across variant allele fractions and additional variant types is warranted.

RNA sequencing (RNA-seq) is a complementary approach for Mendelian disease diagnosis for patients in whom exome-sequencing is not informative. For both rare neuromuscular and mitochondrial disorders, its application has improved diagnostic rates. However, the generalizability of this approach to diverse Mendelian diseases has yet to be evaluated. We sequenced whole blood RNA from 56 cases with undiagnosed rare diseases spanning 11 diverse disease categories to evaluate the general application of RNA-seq to Mendelian disease diagnosis. We developed a robust approach to compare rare disease cases to existing large sets of RNA-seq controls (N=1,594 external and N=31 family-based controls) and demonstrated the substantial impacts of gene and variant filtering strategies on disease gene identification when combined with RNA-seq. Across our cohort, we observed that RNA-seq yields a 8.5% diagnostic rate. These diagnoses included diseases where blood would not intuitively reflect evidence of disease. We identified RARS2 as an under-expression outlier containing compound heterozygous pathogenic variants for an individual exhibiting profound global developmental delay, seizures, microcephaly, hypotonia, and progressive scoliosis. We also identified a new splicing junction in KCTD7 for an individual with global developmental delay, loss of milestones, tremors and seizures. Our study provides a broad evaluation of blood RNA-seq for the diagnosis of rare disease.

Introduction Myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) is a rare disease in the 2016 revised World Health Organization (WHO) classification. Diagnostic criteria include the following: persistent thrombocytosis (>450 × 109 /L) with clustering of atypical megakaryocytes, refractory anemia, dyserythropoiesis with ring sideroblasts, and the presence of the spliceosome factor 3b subunit (SF3B1) mutation. It is unclear if anemia should be a required criterion for this diagnosis as cases which show all other features of MDS/MPN-RS-T but without anemia exist. Methods We searched for borderline cases of MDS/MPN-RS-T in which refractory anemia was absent at diagnosis in two major academic institutes. Results Three cases without anemia were identified. These cases all showed other classic morphologic and clinical features of MDS/MPN-RS-T, including thrombocytosis, atypical megakaryocytes with clustering, and characteristic SF3B1 and JAK2 V617F mutations. Conclusion Given these findings, the requirement of refractory anemia as a diagnostic criterion for MDS/MPN-RS-T should be re-evaluated. Removal of refractory anemia as a diagnostic criterion would incorporate current borderline cases and extend the spectrum of this disorder.

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.

Purpose Sixty-75% of individuals with rare and undiagnosed phenotypes remain undiagnosed after whole exome sequencing (ES). With standard ES reanalysis resolving 10–15% of the ES negatives, further approaches are necessary to maximize diagnoses in these individuals. Methods In 38 ES negative patients an individualized genomic-phenotypic approach was employed utilizing: A) Phenotyping; B) Reanalyses of FASTQ files, with innovative bioinformatics; C) Targeted molecular testing; D) Whole genome sequencing (WGS) and E) Conferring of clinical diagnoses when pathognomonic clinical findings occurred. Results Certain and Highly Likely diagnoses were made in 18/38 (47%) individuals, including identifying two new developmental disorders. The majority of diagnoses (>70%) were due to our bioinformatics, phenotyping and targeted testing identifying variants that were undetected or not prioritized on prior ES. WGS diagnosed 3/18 individuals, with structural variants not amenable to ES. Additionally, Tentative diagnoses were made in three (8%) and in five individuals (13%) candidate genes were identified. Overall, diagnoses/potential leads were identified in 26/38 (68%). Conclusions Our comprehensive approach to ES negatives maximizes the ES and clinical data for both diagnoses and candidate gene identification, without WGS in the majority. This iterative approach is cost-effective and is pertinent to the current conundrum of ES negatives.

Purpose.—Clinical use of analytical tests to assess genomic variants in circulating tumor DNA (ctDNA) is increasing. This joint review from the American Society of Clinical Oncology and the College of American Pathologists summarizes current information about clinical ctDNA assays and provides a framework for future research.Methods.—An Expert Panel conducted a literature review on the use of ctDNA assays for solid tumors, including preanalytical variables, analytical validity, interpretation and reporting, and clinical validity and utility.Results.—The literature search identified 1338 references. Of those, 390, plus 31 references supplied by the Expert Panel, were selected for full-text review. There were 77 articles selected for inclusion.Conclusions.—The evidence indicates that testing for ctDNA is optimally performed on plasma collected in cell stabilization or EDTA tubes, with EDTA tubes processed within 6 hours of collection. Some ctDNA assays have demonstrated clinical validity and utility with certain types of advanced cancer; however, there is insufficient evidence of clinical validity and utility for the majority of ctDNA assays in advanced cancer. Evidence shows discordance between the results of ctDNA assays and genotyping tumor specimens, and supports tumor tissue genotyping to confirm undetected results from ctDNA tests. There is no evidence of clinical utility and little evidence of clinical validity of ctDNA assays in early-stage cancer, treatment monitoring, or residual disease detection. There is no evidence of clinical validity or clinical utility to suggest that ctDNA assays are useful for cancer screening, outside of a clinical trial. Given the rapid pace of research, reevaluation of the literature will shortly be required, along with the development of tools and guidance for clinical practice.

Systemic mastocytosis (SM) is a highly heterogeneous disease with indolent and aggressive forms, with the mechanisms leading to malignant transformation still remaining to be elucidated. Here, we investigated the presence and frequency of genetic variants in 34 SM patients with multilineal KIT D816V mutations. Initial screening was performed by targeted sequencing of 410 genes in DNA extracted from purified bone marrow cells and hair from 12 patients with nonadvanced SM and 8 patients with advanced SM, followed by whole-genome sequencing (WGS) in 4 cases. Somatic mutations were further investigated in another 14 patients with advanced SM. Despite the fact that no common mutation other than KIT D816V was found in WGS analyses, targeted next-generation sequencing identified 67 nonsynonymous genetic variants involving 39 genes. Half of the mutations were somatic (mostly multilineal), whereas the other half were germline variants. The presence of ≥1 multilineal somatic mutation involving genes other than KIT D816V, ≥3 germline variants, and ≥1 multilineal mutation in the SRSF2, ASXL1, RUNX1, and/or EZH2 genes (S/A/R/E genes), in addition to skin lesions, splenomegaly, thrombocytopenia, low hemoglobin levels, and increased alkaline phosphatase and β2-microglobulin serum levels, were associated with a poorer patient outcome. However, the presence of ≥1 multilineal mutation, particularly involving S/A/R/E genes, was the only independent predictor for progression-free survival and overall survival in our cohort.

We assessed the frequency and clinicopathologic significance of 19 genes currently identified as significantly mutated in myeloid neoplasms, RUNX1, ASXL1, TET2, CEBPA, IDH1, IDH2, DNMT3A, FLT3, NPM1, TP53, NRAS, EZH2, CBL, U2AF1, SF3B1, SRSF2, JAK2, CSF3R, and SETBP1, across 93 cases of acute myeloid leukemia (AML) using capture target enrichment and next-generation sequencing. Of these cases, 79% showed at least one nonsynonymous mutation, and cases of AML with recurrent genetic abnormalities showed a lower frequency of mutations versus AML with myelodysplasia-related changes (P

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.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.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).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.

Importance The debate about the role of the Food and Drug Administration (FDA) in the regulation of laboratory-developed tests (LDTs) has focused attention on the analytical performance of all clinical laboratory testing. This study provides data comparing the performance of LDTs and FDA-approved companion diagnostics (FDA-CDs) in proficiency testing (PT) provided by the College of American Pathologists Molecular Oncology Committee. Objective To compare the analytical performance of LDTs and FDA-CDs on well-characterized PT samples and to compare the practice characteristics of laboratories using these assays. Design, Setting, and Participants This comparison of PT responses examines the performance of laboratories participating in the College of American Pathologists PT for 3 oncology analytes for which both FDA-CDs and LDTs are used: BRAF , EGFR , and KRAS . A total of 6897 PT responses were included: BRAF (n = 2524; 14 PT samples), EGFR (n = 2216; 11 PT samples), and KRAS (n = 2157, 10 PT samples). US Food and Drug Administration companion diagnostics and LDTs are compared for both accuracy and preanalytic practices of the laboratories. Main Outcomes and Measures As per the College of American Pathologists PT standards, results were scored and the percentages of acceptable responses for each analyte were compared. These were also broken down by the specific variants tested, by kit manufacturer for laboratories using commercial reagents, and by preanalytic practices. Results From analysis of 6897 PT responses, this study demonstrates that both LDTs and FDA-CDs have excellent performance overall, with both test types exceeding 97% accuracy for all 3 genes ( BRAF , EGFR , and KRAS ) combined. Rare variant-specific differences did not consistently favor LDTs or FDA-CDs. Additionally, more than 60% of participants using an FDA-CD reported adapting their assay from the approved procedure to allow for a greater breadth of sample types, minimum tumor content, and instrumentation, changing the classification of their assay from FDA-CD to LDT. Conclusions This study demonstrates the high degree of accuracy and comparable performance of both LDTs and FDA-CDs for 3 oncology analytes. More significantly, the majority of laboratories using FDA-CDs have modified the scope of their assay to allow for more clinical practice variety, rendering them LDTs. These findings support both the excellent and equivalent performance of both LDTs and FDA-CDs in clinical diagnostic testing.

Investigations aimed at defining the 3-D configuration of eukaryotic chromosomes have consistently encountered an endogenous population of chromosome-derived circular genomic DNA, referred to as extrachromosomal circular DNA (eccDNA). While the production, distribution, and activities of eccDNAs remain understudied, eccDNA formation from specific regions of the linear genome has profound consequences on the regulatory and coding capabilities for these regions. High-throughput sequencing has only recently made extensive genomic mapping of eccDNA sequences possible and had yet to be applied using a rigorous approach that distinguishes ascertainment bias from true enrichment. Here, we define eccDNA distribution, utilizing a set of unbiased topology-dependent approaches for enrichment and characterization. We use parallel biophysical, enzymatic, and informatic approaches to obtain a comprehensive profiling of eccDNA inC. elegansand in three human cell types, where eccDNAs were previously uncharacterized. We also provide quantitative analysis of the eccDNA loci at both unique and repetitive regions. Our studies converge on and support a consistent picture in which endogenous genomic DNA circles are present in normal physiological DNA metabolism, and in which the circles come from both coding and noncoding genomic regions. Prominent among the coding regions generating DNA circles are several genes known to produce a diversity of protein isoforms, with mucin proteins and titin as specific examples.

FLT3 fusions are associated with myeloid and lymphoid neoplasms with eosinophilia. We describe a patient presenting with clinicopathologic features of both chronic eosinophilic leukemia, not otherwise specified (CEL, NOS) and systemic mastocytosis (SM). The bone marrow demonstrated a myeloproliferative neoplasm with eosinophilia and aggregates of atypical mast cells. Cytogenetic analysis revealed a t(13;14)(q12;q32), which was subsequently molecularly characterized as a novel TRIP11-FLT3 rearrangement. A KIT D816V mutation was also identified. The patient rapidly transformed to T-lymphoblastic leukemia/lymphoma and expired shortly after diagnosis. This is the fifth FLT3 fusion gene described in the literature; the presence of both myeloid and lymphoid neoplasms implicates involvement of an early hematopoietic progenitor by rearranged FLT3. We suggest that leukemias and lymphomas with FLT3 fusion genes exhibit similar clinicopathologic features to, and should be included in, the WHO category of "Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1, or with PCM1-JAK2."

PurposeCurrent clinical genomics assays primarily utilize short-read sequencing (SRS), but SRS has limited ability to evaluate repetitive regions and structural variants. Long-read sequencing (LRS) has complementary strengths, and we aimed to determine whether LRS could offer a means to identify overlooked genetic variation in patients undiagnosed by SRS.MethodsWe performed low-coverage genome LRS to identify structural variants in a patient who presented with multiple neoplasia and cardiac myxomata, in whom the results of targeted clinical testing and genome SRS were negative.ResultsThis LRS approach yielded 6,971 deletions and 6,821 insertions 50 bp. Filtering for variants that are absent in an unrelated control and overlap a disease gene coding exon identified three deletions and three insertions. One of these, a heterozygous 2,184 bp deletion, overlaps the first coding exon of PRKAR1A, which is implicated in autosomal dominant Carney complex. RNA sequencing demonstrated decreased PRKAR1A expression. The deletion was classified as pathogenic based on guidelines for interpretation of sequence variants.ConclusionThis first successful application of genome LRS to identify a pathogenic variant in a patient suggests that LRS has significant potential for the identification of disease-causing structural variation. Larger studies will ultimately be required to evaluate the potential clinical utility of LRS.

The tremendous increase in DNA sequencing capacity arising from the commercialization of “next generation” instruments has opened the door to innumerable routes of investigation in basic and translational medical science. It enables very large data sets to be gathered, whose interpretation and conversion into useful knowledge is only beginning. A challenge for modern healthcare systems and academic medical centers is to apply these new methods for the diagnosis of disease and the management of patient care without unnecessary delay, but also with appropriate evaluation of the quality of data and interpretation, as well as the clinical value of the insights gained. Most critically, the standards applied for evaluating these new laboratory data and ensuring that the results and their significance are clearly communicated to patients and their caregivers should be at least as rigorous as those applied to other kinds of medical tests. Here, we present an overview of conceptual and practical issues to be considered in planning for the integration of genomic methods or, in principle, any other type of “omics” testing into clinical care.

Current clinical genomics assays primarily utilize short-read sequencing (SRS), which offers high throughput, high base accuracy, and low cost per base. SRS has, however, limited ability to evaluate tandem repeats, regions with high [GC] or [AT] content, highly polymorphic regions, highly paralogous regions, and large-scale structural variants. Long-read sequencing (LRS) has complementary strengths and offers a means to discover overlooked genetic variation in patients undiagnosed by SRS. To evaluate LRS, we selected a patient who presented with multiple neoplasia and cardiac myxomata suggestive of Carney complex for whom targeted clinical gene testing and whole genome SRS were negative. Low coverage whole genome LRS was performed on the PacBio Sequel system and structural variants were called, yielding 6,971 deletions and 6,821 insertions > 50bp. Filtering for variants that are absent in an unrelated control and that overlap a coding exon of a disease gene identified three deletions and three insertions. One of these, a heterozygous 2,184 bp deletion, overlaps the first coding exon of PRKAR1A, which is implicated in autosomal dominant Carney complex. This variant was confirmed by Sanger sequencing and was classified as pathogenic using standard criteria for the interpretation of sequence variants. This first successful application of whole genome LRS to identify a pathogenic variant suggests that LRS has significant potential to identify disease-causing structural variation. We recommend larger studies to evaluate the diagnostic yield of LRS, and the development of a comprehensive catalog of common human structural variation to support future studies.

Pediatric-type follicular lymphoma and pediatric marginal zone lymphoma are two of the rarest B-cell lymphomas. These lymphomas occur predominantly in the pediatric population and show features distinct from their more common counterparts in adults: adult-type follicular lymphoma and adult-type nodal marginal zone lymphoma. Here we report a detailed whole-exome deep sequencing analysis of a cohort of pediatric-type follicular lymphomas and pediatric marginal zone lymphomas. This analysis revealed a recurrent somatic variant encoding p.Lys66Arg in the transcription factor interferon regulatory factor 8 (IRF8) in 3 of 6 cases (50%) of pediatric-type follicular lymphoma. This specific point mutation was not detected in pediatric marginal zone lymphoma or in adult-type follicular lymphoma. Additional somatic point mutations in pediatric-type follicular lymphoma were observed in genes involved in transcription, intracellular signaling, and cell proliferation. In pediatric marginal zone lymphoma, no recurrent mutation was identified; however, somatic point mutations were observed in genes involved in cellular adhesion, cytokine regulatory elements, and cellular proliferation. A somatic variant in AMOTL1, a recurrently mutated gene in splenic marginal zone lymphoma, was also identified in a case of pediatric marginal zone lymphoma. The overall non-synonymous mutational burden was low in both pediatric-type follicular lymphoma and pediatric marginal zone lymphoma (4.6 mutations per exome). Altogether, these findings support a distinctive genetic basis for pediatric-type follicular lymphoma and pediatric marginal zone lymphoma when compared with adult subtypes and to one another. Moreover, identification of a recurrent point mutation in IRF8 provides insight into a potential driver mutation in the pathogenesis of pediatric-type follicular lymphoma with implications for novel diagnostic or therapeutic strategies.

Rare genetic variants are abundant in humans yet their functional effects are often unknown and challenging to predict. The Genotype-Tissue Expression (GTEx) project provides a unique opportunity to identify the functional impact of rare variants through combined analyses of whole genomes and multi-tissue RNA-sequencing data. Here, we identify gene expression outliers, or individuals with extreme expression levels, across 44 human tissues, and characterize the contribution of rare variation to these large changes in expression. We find 58% of underexpression and 28% of overexpression outliers have underlying rare variants compared with 9% of non-outliers. Large expression effects are enriched for proximal loss-of-function, splicing, and structural variants, particularly variants near the TSS and at evolutionarily conserved sites. Known disease genes have expression outliers, underscoring that rare variants can contribute to genetic disease risk. To prioritize functional rare regulatory variants, we develop RIVER, a Bayesian approach that integrates RNA and whole genome sequencing data from the same individual. RIVER predicts functional variants significantly better than models using genomic annotations alone, and is an extensible tool for personal genome interpretation. Overall, we demonstrate that rare variants contribute to large gene expression changes across tissues with potential health consequences, and provide an integrative method for interpreting rare variants in individual genomes.

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. Design.—DNA from the College of American Pathologists reference genome was enriched using the Illumina TruSeq and Life Technologies AmpliSeq panels and sequenced on the MiSeq and Ion Torrent platforms, respectively. The resulting data were mutagenized in silico and 26 variants, including single-nucleotide variants, deletions, and dinucleotide substitutions, were added at variant allele fractions (VAFs) from 10% to 50%. Participating clinical laboratories downloaded these files and analyzed them using their clinical bioinformatics pipelines. Results.—Laboratories using the AmpliSeq/Ion Torrent and/or the TruSeq/MiSeq participated in the 2 surveys. On average, laboratories identified 24.6 of 26 variants (95%) overall and 21.4 of 22 variants (97%) with VAFs greater than 15%. No false-positive calls were reported. The most frequently missed variants were single-nucleotide variants with VAFs less than 15%. Across both challenges, reported VAF concordance was excellent, with less than 1% median absolute difference between the simulated VAF and mean reported VAF. Conclusions.—The results indicate that in silico proficiency testing is a feasible approach for methods-based proficiency testing, and demonstrate that the sensitivity and specificity of current next-generation sequencing bioinformatics across clinical laboratories are high.

Mature T-cell and natural killer (NK)-cell neoplasms account for a small proportion of non-Hodgkin lymphomas (NHL). Despite the fact that they are relatively uncommon, T-cell lymphoproliferations often are submitted to the clinical molecular laboratory for testing. The diagnostic need for molecular testing is because T cells do not have a definitive immunophenotypic marker of clonality, like kappa and lambda antigen receptor immunophenotyping in B cells, so molecular methods can be used. A variety of molecular assays are available to evaluate T-cell receptor (TR) gene rearrangements, structural and numeric chromosomal abnormalities, and viral sequences associated with mature T- and NK-cell neoplasms. However, many of these molecular tests, especially PCR-based TR gene rearrangement testing, have important caveats that must be considered when interpreting the assays. Therefore, molecular results must be considered in the context of available clinical information, histology, immunophenotype, and other laboratory data for proper interpretation and clinical use.

Mast cell (MC) leukemia (MCL) is a subtype of systemic mastocytosis (SM) defined by the World Health Organization as ⩾ 20% of MCs in the bone marrow (BM) aspirate, with (leukemic variant) or without (aleukemic variant) ⩾ 10% of MCs in peripheral blood (PB). The European/American Consensus Group on Mastocytosis has recently proposed a new subclassification of MCL that distinguishes acute vs chronic MCL based on the presence vs absence of organ damage, respectively.

Individual variation in the Ig germline gene repertoire leads to individual differences in the combinatorial diversity of the Ab repertoire, but the study of such variation has been problematic. The application of high-throughput DNA sequencing to the study of rearranged Ig genes now makes this possible. The sequencing of thousands of VDJ rearrangements from an individual, either from genomic DNA or expressed mRNA, should allow their germline IGHV, IGHD, and IGHJ repertoires to be inferred. In addition, where previously mere glimpses of diversity could be gained from sequencing studies, new large data sets should allow the rearrangement frequency of different genes and alleles to be seen with clarity. We analyzed the DNA of 108,210 human IgH chain rearrangements from 12 individuals and determined their individual IGH genotypes. The number of reportedly functional IGHV genes and allelic variants ranged from 45 to 60, principally because of variable levels of gene heterozygosity, and included 14 previously unreported IGHV polymorphisms. New polymorphisms of the IGHD3-16 and IGHJ6 genes were also seen. At heterozygous loci, remarkably different rearrangement frequencies were seen for the various IGHV alleles, and these frequencies were consistent between individuals. The specific alleles that make up an individual's Ig genotype may therefore be critical in shaping the combinatorial repertoire. The extent of genotypic variation between individuals is highlighted by an individual with aplastic anemia who appears to lack six contiguous IGHD genes on both chromosomes. These deletions significantly alter the potential expressed IGH repertoire, and possibly immune function, in this individual.

Non-Hodgkin lymphomas (NHLs) represent a diverse group of hematopoietic neoplasms. Morphology and immunophenotypic characterization are the cornerstone of the diagnosis and classification of NHLs; however, cytogenetic and molecular studies are being increasingly used to provide further information about classification and prognosis. In addition, these molecular techniques are being used for the detection of minimal residual or recurrent disease after the patient has undergone treatment. This review will discuss the major genetic changes associated with NHLs and describe the methods currently used in the clinical laboratory for cytogenetic and molecular detection of these abnormalities.