Fifth Edition of the World Health Organization Classification of Tumors of the Hematopoietic and Lymphoid Tissues: Acute Lymphoblastic Leukemias, Mixed-Phenotype Acute Leukemias, Myeloid/Lymphoid Neoplasms With Eosinophilia, Dendritic/Histiocytic Neoplasms, and Genetic Tumor Syndromes.

This manuscript represents a review of lymphoblastic leukemia/lymphoma (acute lymphoblastic leukemia/lymphoblastic lymphoma), acute leukemias of ambiguous lineage, mixed-phenotype acute leukemias, myeloid/lymphoid neoplasms with eosinophilia and defining gene rearrangements, histiocytic and dendritic neoplasms, and genetic tumor syndromes of the 5th edition of the World Health Organization Classification of Tumors of the Hematopoietic and Lymphoid Tissues. The diagnostic, clinicopathologic, cytogenetic, and molecular genetic features are discussed. The differences in comparison to the 4th revised edition of the World Health Organization classification of hematolymphoid neoplasms are highlighted.

Structural determinants of mitochondrial STAT3 targeting and function.

Signal transducer and activator of transcription (STAT) 3 has been found within mitochondria in addition to its canonical role of shuttling between cytoplasm and nucleus during cytokine signaling. Mitochondrial STAT3 has been implicated in modulation of cellular metabolism, largely through effects on the respiratory electron transport chain. However, the structural requirements underlying mitochondrial targeting and function have remained unclear. Here, we show that mitochondrial STAT3 partitions between mitochondrial compartments defined by differential detergent solubility, suggesting that mitochondrial STAT3 is membrane associated. The majority of STAT3 was found in an SDS soluble fraction copurifying with respiratory chain proteins, including numerous components of the complex I NADH dehydrogenase, while a minor component was found with proteins of the mitochondrial translation machinery. Mitochondrial targeting of STAT3 required the amino-terminal domain, and an internal linker domain motif also directed mitochondrial translocation. However, neither the phosphorylation of serine 727 nor the presence of mitochondrial DNA was required for the mitochondrial localization of STAT3. Two cysteine residues in the STAT3 SH2 domain, which have been previously suggested to be targets for protein palmitoylation, were also not required for mitochondrial translocation, but were required for its function as an enhancer of complex I activity. These structural determinants of STAT3 mitochondrial targeting and function provide potential therapeutic targets for disrupting the activity of mitochondrial STAT3 in diseases such as cancer.

High-resolution melting assay for rapid, simultaneous detection of JAK2, MPL and CALR variants.

AIMS: Identification of recurrent genetic alterations in JAK2, MPL and CALR remains crucial in the diagnosis of Philadelphia-negative myeloproliferative neoplasms (MPNs). Current laboratory testing algorithms may entail batching and/or sequential testing, involving multiple testing modalities and sometimes send-out testing that increase the technical and economic demands on laboratories while delaying patient diagnoses. To address this gap, an assay based on PCR and high-resolution melting (HRM) analysis was developed for simultaneous evaluation of JAK2 exons 12-14, MPL exon 10 and CALR exon 9, embodied in the HemeScreen® (hereafter 'HemeScreen') MPN assay. METHODS: The HemeScreen MPN assay was validated with blood and bone marrow samples from 982 patients with clinical suspicion for MPN. The HRM assay and Sanger sequencing were performed in independent Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories with Sanger sequencing (supported by droplet digital PCR) serving as the gold standard. RESULTS: HRM and Sanger sequencing had an overall concordance of 99.4% with HRM detecting 133/139 (96%) variants confirmed by sequencing (9/10 MPL, 25/25 CALR, 99/104 JAK2), including 114 single nucleotide variants and 25 indels (3-52 bp). Variants consisted of disease-associated (DA) variants (89%), variants of unclear significance (2%) and non-DA variants (9%) with a positive predictive value of 92.3% and negative predictive value of 99.5%. CONCLUSIONS: These studies demonstrate the exquisite accuracy, sensitivity and specificity of the HRM-based HemeScreen MPN assay, which serves as a powerful, clinically applicable platform for rapid, simultaneous detection of clinically relevant, somatic disease variants.

Proteogenomic Profiling of High-Grade B-Cell Lymphoma With 11q Aberrations and Burkitt Lymphoma Reveals Lymphoid Enhancer Binding Factor 1 as a Novel Biomarker.

High-grade B-cell lymphomas with 11q aberrations (HGBL-11q) represent a World Health Organization-defined group of lymphomas that harbor recurrent chromosome 11q aberrations involving proximal gains and telomeric losses. Although a limited number of HGBL-11q cases evaluated thus far appear to show a similar course and prognosis as Burkitt lymphoma (BL), many molecular differences have been appreciated, most notably the absence of MYC rearrangement. Despite biological differences between BL and HGBL-11q, histomorphologic and immunophenotypic distinction remains challenging. Here, we provide a comparative whole proteomic profile of BL- and HGBL-11q-derived cell lines, identifying numerous shared and differentially expressed proteins. Transcriptome profiling performed on paraffin-embedded tissue samples from primary BL and HGBL-11q lymphomas was additionally performed to provide further molecular characterization. Overlap of proteomic and transcriptomic data sets identified several potential novel biomarkers of HGBL-11q, including diminished lymphoid enhancer-binding factor 1 expression, which was validated by immunohistochemistry staining in a cohort of 23 cases. Altogether, these findings provide a comprehensive multimodal and comparative molecular profiling of BL and HGBL-11q and suggest the use of enhancer-binding factor 1 as an immunohistochemistry target to distinguish between these aggressive lymphomas.

Rapid and Automated Semiconductor-Based Next-Generation Sequencing for Simultaneous Detection of Somatic DNA and RNA Aberrations in Myeloid Neoplasms.

Evaluation of suspected myeloid neoplasms involves testing for recurrent, diagnostically and therapeutically relevant genetic alterations. Current molecular testing requires multiple technologies, different domains of expertise, and unconnected workflows, resulting in variable, lengthy turnaround times that can delay treatment. To address this unmet clinical need, we evaluated the Oncomine Myeloid Assay GX panel on the Ion Torrent Genexus platform, a rapid, integrated nucleic acid to report next-generation sequencing platform for detecting clinically relevant genetic aberrations in myeloid disorders. Specimens included synthetic DNA (101 targets) and RNA (9 targets) controls and real-world nucleic acid material derived from bone marrow or peripheral blood samples (40 patients). Ion Torrent Genexus results and performance indices were compared with those obtained from clinically validated genomic testing workflows in 2 separate clinical laboratories. The Ion Torrent Genexus identified 100% of DNA and RNA control variants. For primary patient specimens, the Ion Torrent Genexus reported 82 of 107 DNA variants and 19 of 19 RNA gene fusions identified on clinically validated assays, yielding an 80% overall detection rate. Reanalysis of exported, unfiltered Ion Torrent Genexus data revealed 15 DNA variants not called by the filtered on-board bioinformatics pipeline, yielding a 92% potential detection rate. These results hold promise for the implementation of an integrated next-generation sequencing system to rapidly detect genetic aberrations, facilitating accurate, genomics-based diagnoses and accelerated time to precision therapies in myeloid neoplasms.

FBXW7ß isoform drives transcriptional activation of the proinflammatory TNF cluster in human pro-B cells.

Noncanonical exon usage plays many important roles in cellular phenotypes, but its contribution to human B-cell development remains sketchily understood. To fill this gap, we collected various B-cell fractions from bone marrow (BM) and tonsil donors, performed RNA sequencing, and examined transcript variants. We identified 150 genes that harbor local splicing variations in all pairwise comparisons. One of them encodes FBXW7, an E3 ubiquitin ligase implicated as a driver in several blood cancers. Surprisingly, we discovered that in normal human pro-B cells, the predominant transcript used an alternative first exon to produce the poorly characterized FBXW7ß isoform, previously thought to be restricted to neural tissues. The FBXW7ß transcript was also abundant in cell lines and primary samples of pediatric B-cell acute lymphoblastic leukemia (B-ALL), which originates in the BM. When overexpressed in a heterologous cell system, this transcript yielded the expected protein product, as judged by anti-FLAG immunoblotting and mass spectrometry. Furthermore, in REH B-ALL cells, FBXW7ß mRNA was the only FBXW7 isoform enriched in the polyribosome fraction. To shed light on possible functions of FBXW7ß, we used gain- and loss-of-function approaches and identified an FBXW7-dependent inflammatory gene signature, apparent in a subset of B-ALL with high FBXW7ß expression. This signature contained several members of the tumor necrosis factor superfamily, including those comprising the HLA Class III cluster (LTB, LST1, NCR3, LTA, and NFKBIL1). Our findings suggest that FBXW7ß expression drives proinflammatory responses, which could contribute to normal B-cell development, leukemogenesis, and responses to anticancer therapies.

BCL10 Mutations Define Distinct Dependencies Guiding Precision Therapy for DLBCL.

Activated B cell-like diffuse large B-cell lymphomas (ABC-DLBCL) have unfavorable outcomes and chronic activation of CARD11-BCL10-MALT1 (CBM) signal amplification complexes that form due to polymerization of BCL10 subunits, which is affected by recurrent somatic mutations in ABC-DLBCLs. Herein, we show that BCL10 mutants fall into at least two functionally distinct classes: missense mutations of the BCL10 CARD domain and truncation of its C-terminal tail. Truncating mutations abrogated a motif through which MALT1 inhibits BCL10 polymerization, trapping MALT1 in its activated filament-bound state. CARD missense mutations enhanced BCL10 filament formation, forming glutamine network structures that stabilize BCL10 filaments. Mutant forms of BCL10 were less dependent on upstream CARD11 activation and thus manifested resistance to BTK inhibitors, whereas BCL10 truncating but not CARD mutants were hypersensitive to MALT1 inhibitors. Therefore, BCL10 mutations are potential biomarkers for BTK inhibitor resistance in ABC-DLBCL, and further precision can be achieved by selecting therapy based on specific biochemical effects of distinct mutation classes. SIGNIFICANCE: ABC-DLBCLs feature frequent mutations of signaling mediators that converge on the CBM complex. We use structure-function approaches to reveal that BCL10 mutations fall into two distinct biochemical classes. Both classes confer resistance to BTK inhibitors, whereas BCL10 truncations confer hyperresponsiveness to MALT1 inhibitors, providing a road map for precision therapies in ABC-DLBCLs. See related commentary by Phelan and Oellerich, p. 1844. This article is highlighted in the In This Issue feature, p. 1825.

The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms.

We herein present an overview of the upcoming 5th edition of the World Health Organization Classification of Haematolymphoid Tumours focussing on lymphoid neoplasms. Myeloid and histiocytic neoplasms will be presented in a separate accompanying article. Besides listing the entities of the classification, we highlight and explain changes from the revised 4th edition. These include reorganization of entities by a hierarchical system as is adopted throughout the 5th edition of the WHO classification of tumours of all organ systems, modification of nomenclature for some entities, revision of diagnostic criteria or subtypes, deletion of certain entities, and introduction of new entities, as well as inclusion of tumour-like lesions, mesenchymal lesions specific to lymph node and spleen, and germline predisposition syndromes associated with the lymphoid neoplasms.

Functional characterization of NPM1-TYK2 fusion oncogene.

Gene fusions are known to drive many human cancers. Therefore, the functional characterization of newly discovered fusions is critical to understanding the oncobiology of these tumors and to enable therapeutic development. NPM1-TYK2 is a novel fusion identified in CD30 + lymphoproliferative disorders, and here we present the functional evaluation of this fusion gene as an oncogene. The chimeric protein consists of the amino-terminus of nucleophosmin 1 (NPM1) and the carboxyl-terminus of tyrosine kinase 2 (TYK2), including the kinase domain. Using in vitro lymphoid cell transformation assays and in vivo tumorigenic xenograft models we present direct evidence that the fusion gene is an oncogene. NPM1 fusion partner provides the critical homodimerization needed for the fusion kinase constitutive activation and downstream signaling that are responsible for cell transformation. As a result, our studies identify NPM1-TYK2 as a novel fusion oncogene and suggest that inhibition of fusion homodimerization could be a precision therapeutic approach in cutaneous T-cell lymphoma patients expressing this chimera.

Isolation and characterization of new phenolic siderophores with antimicrobial properties from Pseudomonas sp. UIAU-6B.

Five new phenolic siderophores 1-5 were isolated from the organic extract of a culture broth in a modified SGG medium of Pseudomonas sp. UIAU-6B, obtained from sediments collected from the Oyun river in North Central Nigeria. The structure of the new compounds, pseudomonin A-C (1-3) and pseudomobactin A and B (4 and 5) isolated alongside two known compounds, pseudomonine (6) and salicylic acid (7), were elucidated based on high-resolution mass spectrometry, 1D and 2D NMR analyses. The absolute configuration of the threonine residue in compounds 1-5 was determined by Marfey analysis. The antimicrobial evaluation of compound 4 exhibited the most potent activity against vancomycin-sensitive Enterococcus faecium VS144754, followed by 3 and 5, with MIC values ranging from 8 to 32 µg/mL. Compounds 2 and 3 exhibited moderate activity against Mycobacterium tuberculosis H37Rv, with MIC values of 7.8 and 15.6 µg/mL, respectively. Plausible biosynthetic hypotheses toward the new compounds 1-5 were proposed.

Optimization of Sources of Circulating Cell-Free DNA Variability for Downstream Molecular Analysis.

Circulating cell-free DNA (ccfDNA) is used increasingly as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream molecular analysis. Determining optimal blood processing and ccfDNA quantification are crucial for ccfDNA to serve as an accurate biomarker as it moves into the clinical realm. Whole blood was collected from 50 subjects, processed to plasma, and used immediately or frozen at -80°C. Plasma ccfDNA was extracted and concentration was assessed by real-time quantitative PCR (qPCR), fluorimetry, and droplet digital PCR (ddPCR). For the 24 plasma samples from metastatic pancreatic cancer patients, the variant allele fractions (VAF) of KRAS G12/13 pathogenic variants in circulating tumor DNA (ctDNA) were measured by ddPCR. Using a high-speed (16,000 × g) or slower-speed (4100 × g) second centrifugation step showed no difference in ccfDNA yield or ctDNA VAF. A two- versus three-spin centrifugation protocol also showed no difference in ccfDNA yield or ctDNA VAF. A higher yield was observed from fresh versus frozen plasma by qPCR and fluorimetry, whereas a higher yield was observed for frozen versus fresh plasma by ddPCR, however, no difference was observed in ctDNA VAF. Overall, our findings suggest factors to consider when implementing a ccfDNA extraction and quantification workflow in a research or clinical setting.

International evidence-based consensus diagnostic and treatment guidelines for unicentric Castleman disease.

Castleman disease (CD) includes a group of rare and heterogeneous disorders with characteristic lymph node histopathological abnormalities. CD can occur in a single lymph node station, which is referred to as unicentric CD (UCD). CD can also involve multicentric lymphadenopathy and inflammatory symptoms (multicentric CD [MCD]). MCD includes human herpesvirus-8 (HHV-8)-associated MCD, POEMS-associated MCD, and HHV-8-/idiopathic MCD (iMCD). The first-ever diagnostic and treatment guidelines were recently developed for iMCD by an international expert consortium convened by the Castleman Disease Collaborative Network (CDCN). The focus of this report is to establish similar guidelines for the management of UCD. To this purpose, an international working group of 42 experts from 10 countries was convened to establish consensus recommendations based on review of treatment in published cases of UCD, the CDCN ACCELERATE registry, and expert opinion. Complete surgical resection is often curative and is therefore the preferred first-line therapy, if possible. The management of unresectable UCD is more challenging. Existing evidence supports that asymptomatic unresectable UCD may be observed. The anti-interleukin-6 monoclonal antibody siltuximab should be considered for unresectable UCD patients with an inflammatory syndrome. Unresectable UCD that is symptomatic as a result of compression of vital neighboring structures may be rendered amenable to resection by medical therapy (eg, rituximab, steroids), radiotherapy, or embolization. Further research is needed in UCD patients with persisting constitutional symptoms despite complete excision and normal laboratory markers. We hope that these guidelines will improve outcomes in UCD and help treating physicians decide the best therapeutic approach for their patients.

Mastermind: A Comprehensive Genomic Association Search Engine for Empirical Evidence Curation and Genetic Variant Interpretation.

Design and interpretation of genome sequencing assays in clinical diagnostics and research labs is complicated by an inability to identify information from the medical literature and related databases quickly, comprehensively and reproducibly. This challenge is compounded by the complexity and heterogeneity of nomenclatures used to describe diseases, genes and genetic variants. Mastermind is a widely-used bioinformatic platform of genomic associations that has indexed more than 7.5 M full-text articles and 2.5 M supplemental datasets. It has automatically identified, disambiguated and annotated >6.1 M genetic variants and identified >50 K disease-gene associations. Here, we describe how Mastermind improves the sensitivity and reproducibility of clinical variant interpretation and produces comprehensive genomic landscapes of genetic variants driving pharmaceutical research. We demonstrate an alarmingly high degree of heterogeneity across commercially available panels for hereditary cancer that is resolved by evidence from Mastermind. We further examined the sensitivity of Mastermind for variant interpretation by examining 108 clinically-encountered variants and comparing the results to alternate methods. Mastermind demonstrated a sensitivity of 98.4% compared to 4.4, 45.6, and 37.4% for alternatives PubMed, Google Scholar, and ClinVar, respectively, and a specificity of 98.5% compared to 45.1, 57.6, and 68.8% as well as an increase in content yield of 22.6-, 2.2-, and 2.6-fold. When curated for clinical significance, Mastermind identified more than 4.9-fold more pathogenic variants than ClinVar for representative genes. For structural variants, we compared Mastermind's ability to sensitively identify evidence for 10 representative disease-causing CNVs versus results identified in PubMed, as well as its ability to identify evidence for fusion events compared to COSMIC. Mastermind demonstrated a 4.0- to 43.9-fold increase in references for specific CNVs compared to PubMed, as well as 5.4-fold more fusion genes when compared with COSMIC's curated database. Additionally, Mastermind produced an 8.0-fold increase in reference citations for fusion events common to Mastermind and outside databases. Taken together, these results demonstrate the utility and superiority of Mastermind in terms of both sensitivity and specificity of automated results for clinical diagnostic variant interpretation for multiple genetic variant types and highlight the potential benefit in informing pharmaceutical research.

FBXW7 Triggers Degradation of KMT2D to Favor Growth of Diffuse Large B-cell Lymphoma Cells.

Mature B-cell neoplasms are the fifth most common neoplasm. Due to significant heterogeneity at the clinical and genetic levels, current therapies for these cancers fail to provide long-term cures. The clinical success of proteasome inhibition for the treatment of multiple myeloma and B-cell lymphomas has made the ubiquitin pathway an important emerging therapeutic target. In this study, we assessed the role of the E3 ligase FBXW7 in mature B-cell neoplasms. FBXW7 targeted the frequently inactivated tumor suppressor KMT2D for protein degradation, subsequently regulating gene expression signatures related to oxidative phosphorylation (OxPhos). Loss of FBXW7 inhibited diffuse large B-cell lymphoma cell growth and further sensitized cells to OxPhos inhibition. These data elucidate a novel mechanism of regulation of KMT2D levels by the ubiquitin pathway and uncover a role of FBXW7 in regulating oxidative phosphorylation in B-cell malignancies. SIGNIFICANCE: These findings characterize FBXW7 as a prosurvival factor in B-cell lymphoma via degradation of the chromatin modifier KMT2D.

Enabling Precision Oncology Through Precision Diagnostics.

Genomic testing enables clinical management to be tailored to individual cancer patients based on the molecular alterations present within cancer cells. Genomic sequencing results can be applied to detect and classify cancer, predict prognosis, and target therapies. Next-generation sequencing has revolutionized the field of cancer genomics by enabling rapid and cost-effective sequencing of large portions of the genome. With this technology, precision oncology is quickly becoming a realized paradigm for managing the treatment of cancer patients. However, many challenges must be overcome to efficiently implement the transition of next-generation sequencing from research applications to routine clinical practice, including using specimens commonly available in the clinical setting; determining how to process, store, and manage large amounts of sequencing data; determining how to interpret and prioritize molecular findings; and coordinating health professionals from multiple disciplines.

Impact of Genetics on Mature Lymphoid Leukemias and Lymphomas.

Recurrent genetic aberrations have long been recognized in mature lymphoid leukemias and lymphomas. As conventional karyotypic and molecular cloning techniques evolved in the 1970s and 1980s, multiple cytogenetic aberrations were identified in lymphomas, often balanced translocations that juxtaposed oncogenes to the immunoglobulin (IG) or T-cell receptor (TR) loci, leading to dysregulation. However, genetic characterization and classification of lymphoma by conventional cytogenetic methods is limited by the infrequent occurrence of recurrent karyotypic abnormalities in many lymphoma subtypes and by the frequent difficulty in growing clinical lymphoma specimens in culture to obtain informative karyotypes. As higher-resolution genomic techniques developed, such as array comparative genomic hybridization and fluorescence in situ hybridization, many recurrent copy number changes were identified in lymphomas, and copy number assessment of interphase cells became part of routine clinical practice for a subset of diseases. Platforms to globally examine mRNA expression led to major insights into the biology of several lymphomas, although these techniques have not gained widespread application in routine clinical settings. With the advent of next-generation sequencing (NGS) techniques in the early 2000s, numerous insights into the genetic landscape of lymphomas were obtained. In contrast to the myeloid malignancies, most common lymphomas exhibit an at least somewhat mutationally complex genome, with few single driver mutations in the majority of patients. However, many recurrently mutated pathways have been identified across lymphoma subtypes, informing targeted therapeutic approaches that are beginning to make meaningful changes in the treatment of lymphoma. In addition to the ability to identify possible therapeutic targets, NGS techniques are highly amenable to the tracking of residual lymphoma following therapy, because of the presence of unique genetic "fingerprints" in lymphoma cells due to V(D)-J recombination at the antigen receptor loci. This review will provide an overview of the impact of novel genetic technologies on lymphoma classification, biology, and therapy.

What is new in mature T-cell leukemias?

Mature T-cell and NK-cell leukemias represent a clinically heterogeneous group of diseases, ranging from indolent expansions of large granular lymphocytes, to aggressive diseases that are associated with a fulminant clinical course. Recent advances in genomic methodologies have massively increased the understanding of the pathogenesis of this group of diseases. While the entities are genetically heterogeneous, JAK-STAT pathway activation appears to be important across these disorders. The identification of constitutively activated pathways and the emergence of novel targeted pharmaceutical agents raise the expectation that more effective therapies will be identified for these disorders in the coming years.