The 2016 revision of the World Health Organization classification of lymphoid neoplasms

We describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.

The somatic genomic landscape of glioblastoma

Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer: A Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists

Background The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and resulting COVID-19 pandemic present important diagnostic challenges. Several diagnostic strategies are available to identify current infection, rule out infection, identify people in need of care escalation, or to test for past infection and immune response. Serology tests to detect the presence of antibodies to SARS-CoV-2 aim to identify previous SARS-CoV-2 infection, and may help to confirm the presence of current infection. Objectives To assess the diagnostic accuracy of antibody tests to determine if a person presenting in the community or in primary or secondary care has SARS-CoV-2 infection, or has previously had SARS-CoV-2 infection, and the accuracy of antibody tests for use in seroprevalence surveys. Search methods We undertook electronic searches in the Cochrane COVID-19 Study Register and the COVID-19 Living Evidence Database from the University of Bern, which is updated daily with published articles from PubMed and Embase and with preprints from medRxiv and bioRxiv. In addition, we checked repositories of COVID-19 publications. We did not apply any language restrictions. We conducted searches for this review iteration up to 27 April 2020. Selection criteria We included test accuracy studies of any design that evaluated antibody tests (including enzyme-linked immunosorbent assays, chemiluminescence immunoassays, and lateral flow assays) in people suspected of current or previous SARS-CoV-2 infection, or where tests were used to screen for infection. We also included studies of people either known to have, or not to have SARS-CoV-2 infection. We included all reference standards to define the presence or absence of SARS-CoV-2 (including reverse transcription polymerase chain reaction tests (RT-PCR) and clinical diagnostic criteria). Data collection and analysis We assessed possible bias and applicability of the studies using the QUADAS-2 tool. We extracted 2x2 contingency table data and present sensitivity and specificity for each antibody (or combination of antibodies) using paired forest plots. We pooled data using random-effects logistic regression where appropriate, stratifying by time since post-symptom onset. We tabulated available data by test manufacturer. We have presented uncertainty in estimates of sensitivity and specificity using 95% confidence intervals (CIs). Main results We included 57 publications reporting on a total of 54 study cohorts with 15,976 samples, of which 8526 were from cases of SARS-CoV-2 infection. Studies were conducted in Asia (n = 38), Europe (n = 15), and the USA and China (n = 1). We identified data from 25 commercial tests and numerous in-house assays, a small fraction of the 279 antibody assays listed by the Foundation for Innovative Diagnostics. More than half (n = 28) of the studies included were only available as preprints. We had concerns about risk of bias and applicability. Common issues were use of multi-group designs (n = 29), inclusion of only COVID-19 cases (n = 19), lack of blinding of the index test (n = 49) and reference standard (n = 29), differential verification (n = 22), and the lack of clarity about participant numbers, characteristics and study exclusions (n = 47). Most studies (n = 44) only included people hospitalised due to suspected or confirmed COVID-19 infection. There were no studies exclusively in asymptomatic participants. Two-thirds of the studies (n = 33) defined COVID-19 cases based on RT-PCR results alone, ignoring the potential for false-negative RT-PCR results. We observed evidence of selective publication of study findings through omission of the identity of tests (n = 5). We observed substantial heterogeneity in sensitivities of IgA, IgM and IgG antibodies, or combinations thereof, for results aggregated across different time periods post-symptom onset (range 0% to 100% for all target antibodies). We thus based the main results of the review on the 38 studies that stratified results by time since symptom onset. The numbers of individuals contributing data within each study each week are small and are usually not based on tracking the same groups of patients over time. Pooled results for IgG, IgM, IgA, total antibodies and IgG/IgM all showed low sensitivity during the first week since onset of symptoms (all less than 30.1%), rising in the second week and reaching their highest values in the third week. The combination of IgG/IgM had a sensitivity of 30.1% (95% CI 21.4 to 40.7) for 1 to 7 days, 72.2% (95% CI 63.5 to 79.5) for 8 to 14 days, 91.4% (95% CI 87.0 to 94.4) for 15 to 21 days. Estimates of accuracy beyond three weeks are based on smaller sample sizes and fewer studies. For 21 to 35 days, pooled sensitivities for IgG/IgM were 96.0% (95% CI 90.6 to 98.3). There are insufficient studies to estimate sensitivity of tests beyond 35 days post-symptom onset. Summary specificities (provided in 35 studies) exceeded 98% for all target antibodies with confidence intervals no more than 2 percentage points wide. False-positive results were more common where COVID-19 had been suspected and ruled out, but numbers were small and the difference was within the range expected by chance. Assuming a prevalence of 50%, a value considered possible in healthcare workers who have suffered respiratory symptoms, we would anticipate that 43 (28 to 65) would be missed and 7 (3 to 14) would be falsely positive in 1000 people undergoing IgG/IgM testing at days 15 to 21 post-symptom onset. At a prevalence of 20%, a likely value in surveys in high-risk settings, 17 (11 to 26) would be missed per 1000 people tested and 10 (5 to 22) would be falsely positive. At a lower prevalence of 5%, a likely value in national surveys, 4 (3 to 7) would be missed per 1000 tested, and 12 (6 to 27) would be falsely positive. Analyses showed small differences in sensitivity between assay type, but methodological concerns and sparse data prevent comparisons between test brands. Authors' conclusions The sensitivity of antibody tests is too low in the first week since symptom onset to have a primary role for the diagnosis of COVID-19, but they may still have a role complementing other testing in individuals presenting later, when RT-PCR tests are negative, or are not done. Antibody tests are likely to have a useful role for detecting previous SARS-CoV-2 infection if used 15 or more days after the onset of symptoms. However, the duration of antibody rises is currently unknown, and we found very little data beyond 35 days post-symptom onset. We are therefore uncertain about the utility of these tests for seroprevalence surveys for public health management purposes. Concerns about high risk of bias and applicability make it likely that the accuracy of tests when used in clinical care will be lower than reported in the included studies. Sensitivity has mainly been evaluated in hospitalised patients, so it is unclear whether the tests are able to detect lower antibody levels likely seen with milder and asymptomatic COVID-19 disease. The design, execution and reporting of studies of the accuracy of COVID-19 tests requires considerable improvement. Studies must report data on sensitivity disaggregated by time since onset of symptoms. COVID-19-positive cases who are RT-PCR-negative should be included as well as those confirmed RT-PCR, in accordance with the World Health Organization (WHO) and China National Health Commission of the People's Republic of China (CDC) case definitions. We were only able to obtain data from a small proportion of available tests, and action is needed to ensure that all results of test evaluations are available in the public domain to prevent selective reporting. This is a fast-moving field and we plan ongoing updates of this living systematic review.

Antibody tests for identification of current and past infection with SARS‐CoV‐2

Context.— Next-generation sequencing (NGS) is a technology being used by many laboratories to test for inherited disorders and tumor mutations. This technology is new for many practicing pathologists, who may not be familiar with the uses, methodology, and limitations of NGS. Objective.— To familiarize pathologists with several aspects of NGS, including current and expanding uses; methodology including wet bench aspects, bioinformatics, and interpretation; validation and proficiency; limitations; and issues related to the integration of NGS data into patient care. Data Sources.— The review is based on peer-reviewed literature and personal experience using NGS in a clinical setting at a major academic center. Conclusions.— The clinical applications of NGS will increase as the technology, bioinformatics, and resources evolve to address the limitations and improve quality of results. The challenge for clinical laboratories is to ensure testing is clinically relevant, cost-effective, and can be integrated int...

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Review of Clinical Next-Generation Sequencing.

Epstein-Barr virus (EBV)-positive mucocutaneous ulcer (EBV MCU) is a B-cell lymphoproliferative disorder occurring in elderly or iatrogenic immunocompromised patients. It has not been reported in solid organ transplant recipients. We observed 7 patients with EBV MCU in a cohort of 70 transplant recipients with EBV posttransplant lymphoproliferative disorder (PTLD). Transplants included: 5 renal, 1 heart, and 1 lung. Median patient age was 61; 5 were male. EBV MCU was observed in oral mucosa in 4 and gastrointestinal tract in 3. Duration of immunosuppressive therapy before EBV MCU was 0.6 to 13 years. Ulcers were undermined by inflammatory cells and polymorphic or monomorphic large cell lymphoproliferation. Reed-Sternberg-like cells were present in 5/7. Large B cells were CD20, CD30, and EBV-encoded RNA positive in all cases. Diagnosis in 3 recent patients was EBV MCU; 4 patients diagnosed before familiarity with EBV MCU were classified as monomorphic large cell (n=3) and polymorphic (n=1) PTLD. None of the patients had EBV DNA in their blood (<1000 copies/mL) at diagnosis or follow-up versus 35/44 transplant patients with systemic PTLD (P<0.001). All lesions resolved with reduced immunosuppression (7/7), change in immunosuppression (2/7), and rituximab (3/7). Five patients are living: 4 healthy, 1 awaiting second renal transplant. Two patients died 3 and 5 years after resolution of EBV MCU. No patient recurred with EBV MCU or other PTLDs. EBV MCU mimics more aggressive categories of PTLD but lacks EBV DNA in blood, which may be a useful distinguishing feature. Lesions are likely to resolve with conservative management. Awareness of EBV MCU in the posttransplant setting is necessary for appropriate diagnosis and treatment.

EBV-positive mucocutaneous ulcer in organ transplant recipients: a localized indolent posttransplant lymphoproliferative disorder.

Genetics play an increasingly important role in the risk stratification and management of acute myeloid leukemia (AML) patients. Traditionally, AML classification and risk stratification relied on cytogenetic studies; however, molecular detection of gene mutations is playing an increasingly important role in classification, risk stratification, and management of AML. Molecular testing does not take the place of cytogenetic testing results, but plays a complementary role to help refine prognosis, especially within specific AML subgroups. With the exception of acute promyelocytic leukemia, AML therapy is not targeted but the intensity of therapy is driven by the prognostic subgroup. Many prognostic scoring systems classify patients into favorable, poor, or intermediate prognostic subgroups based on clinical and genetic features. Current standard of care combines cytogenetic results with targeted testing for mutations in FLT3, NPM1, CEBPA, and KIT to determine the prognostic subgroup. Other gene mutations have also been demonstrated to predict prognosis and may play a role in future risk stratification, although some of these have not been confirmed in multiple studies or established as standard of care. This paper will review the contribution of cytogenetic results to prognosis in AML and then will focus on molecular mutations that have a prognostic or possible therapeutic impact.

https://www.mdpi.com/2077-0383/4/3/460/pdf

Molecular Genetic Markers in Acute Myeloid Leukemia

Context.—Soft tissue tumors are composed of numerous and complex diagnostic entities. Because of this complexity and the recognition of an intermediate malignancy category including some tumors with a deceptively bland histologic appearance, soft tissue tumors may represent a major diagnostic challenge to the general practicing pathologist. Objective.—To correctly diagnose soft tissue tumors with the ancillary use of immunohistochemistry. Data Sources.—Review of the current literature with emphasis on those tumors for which immunohistochemistry has proven to be particularly useful. Conclusions.—Immunohistochemistry plays an important role in the diagnosis of soft tissue tumors. One of its major utilities is to correctly identify a tumor as being of mesenchymal or nonmesenchymal origin. Once mesenchymal origin has been established, histologic subtyping according to specific cell lineage may be achieved with the use of lineage-specific markers. Tumors of uncertain cell lineage and tumors with primi...

Application of immunohistochemistry to soft tissue neoplasms.

The global COVID-19 pandemic has led to an urgent need for scalable methods for clinical diagnostics and viral tracking. Next generation sequencing technologies have enabled large-scale genomic surveillance of SARS-CoV-2 as thousands of isolates are being sequenced around the world and deposited in public data repositories. A number of methods using both short- and long-read technologies are currently being applied for SARS-CoV-2 sequencing, including amplicon approaches, metagenomic methods, and sequence capture or enrichment methods. Given the small genome size, the ability to sequence SARS-CoV-2 at scale is limited by the cost and labor associated with making sequencing libraries. Here we describe a low-cost, streamlined, all amplicon-based method for sequencing SARS-CoV-2, which bypasses costly and time-consuming library preparation steps. We benchmark this tailed amplicon method against both the ARTIC amplicon protocol and sequence capture approaches and show that an optimized tailed amplicon approach achieves comparable amplicon balance, coverage metrics, and variant calls to the ARTIC v3 approach. The tailed amplicon method we describe represents a cost-effective and highly scalable method for SARS-CoV-2 sequencing.

/pdf/a-rapid-cost-effective-tailed-amplicon-method-for-sequencing-4t0pcem1pb.pdf

Sophia Yohe

Papers

Review of Clinical Next-Generation Sequencing.

EBV-positive mucocutaneous ulcer in organ transplant recipients: a localized indolent posttransplant lymphoproliferative disorder.

Molecular Genetic Markers in Acute Myeloid Leukemia

Application of immunohistochemistry to soft tissue neoplasms.

A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2.