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.

Digital pathology operations at a tertiary cancer center: Infrastructure requirements and operational cost.

Whole slide imaging is revolutionizing the field of pathology and is currently being used for clinical, educational, and research initiatives by an increasing number of institutions. Pathology departments have distinct needs for digital pathology systems, yet the cost of digital workflows is cited as a major barrier for widespread adoption by many organizations. Memorial Sloan Kettering Cancer Center (MSK) is an early adopter of whole slide imaging with incremental investments in resources that started more than 15 years ago. This experience and the large-scale scan operations led to the identification of required framework components of digital pathology operations. The cost of these components for the 2021 digital pathology operations at MSK were studied and calculated to enable an understanding of the operation and benchmark the accompanying costs. This paper describes the unique infrastructure cost and the costs associated with the digital pathology clinical operation use cases in a large, tertiary cancer center. These calculations can serve as a blueprint for other institutions to provide the necessary concepts and offer insights towards the financial requirements for digital pathology adoption by other institutions.

Quality Management System in Clinical Digital Pathology Operations at a Tertiary Cancer Center.

Digital pathology workflows can improve pathology operations by allowing reliable and fast retrieval of digital images, digitally reviewing pathology slides, enabling remote work and telepathology, use of computer-aided tools, and sharing of digital images for research and educational purposes. The need for quality systems is a prerequisite for successful clinical-grade digital pathology adoption and patient safety. In this article, we describe the development of a structured digital pathology laboratory quality management system (QMS) for clinical digital pathology operations at Memorial Sloan Kettering Cancer Center (MSK). This digital pathology-specific QMS development stemmed from the gaps that were identified when MSK integrated digital pathology into its clinical practice. The digital scan team in conjunction with the Department of Pathology and Laboratory Medicine quality team developed a QMS tailored to the scanning operation to support departmental and institutional needs. As a first step, systemic mapping of the digital pathology operations identified the prescan, scan, and postscan processes; instrumentation; and staffing involved in the digital pathology operation. Next, gaps identified in quality control and quality assurance measures led to the development of standard operating procedures and training material for the different roles and workflows in the process. All digital pathology-related documents were subject to regulatory review and approval by departmental leadership. The quality essentials were developed into an extensive Digital Pathology Quality Essentials framework to specifically address the needs of the growing clinical use of digital pathology technologies. Using the unique digital experience gained at MSK, we present our recommendations for QMS for large-scale digital pathology operations in clinical settings.

MLL-AF4 cooperates with PAF1 and FACT to drive high-density enhancer interactions in leukemia.

Aberrant enhancer activation is a key mechanism driving oncogene expression in many cancers. While much is known about the regulation of larger chromosome domains in eukaryotes, the details of enhancer-promoter interactions remain poorly understood. Recent work suggests co-activators like BRD4 and Mediator have little impact on enhancer-promoter interactions. In leukemias controlled by the MLL-AF4 fusion protein, we use the ultra-high resolution technique Micro-Capture-C (MCC) to show that MLL-AF4 binding promotes broad, high-density regions of enhancer-promoter interactions at a subset of key targets. These enhancers are enriched for transcription elongation factors like PAF1C and FACT, and the loss of these factors abolishes enhancer-promoter contact. This work not only provides an additional model for how MLL-AF4 is able to drive high levels of transcription at key genes in leukemia but also suggests a more general model linking enhancer-promoter crosstalk and transcription elongation.

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.

Oncogenic role and target properties of the lysine-specific demethylase KDM1A in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), epigenetic alterations are considered to centrally shape the transcriptional signatures that drive disease evolution and underlie its biological and clinical subsets. Characterizations of epigenetic regulators, particularly histone-modifying enzymes, are very rudimentary in CLL. In efforts to establish effectors of the CLL-associated oncogene T-cell leukemia 1A (TCL1A), we identified here the lysine-specific histone demethylase KDM1A to interact with the TCL1A protein in B cells in conjunction with an increased catalytic activity of KDM1A. We demonstrate that KDM1A is upregulated in malignant B cells. Elevated KDM1A and associated gene expression signatures correlated with aggressive disease features and adverse clinical outcomes in a large prospective CLL trial cohort. Genetic Kdm1a knockdown in Eµ-TCL1A mice reduced leukemic burden and prolonged animal survival, accompanied by upregulated p53 and proapoptotic pathways. Genetic KDM1A depletion also affected milieu components (T, stromal, and monocytic cells), resulting in significant reductions in their capacity to support CLL-cell survival and proliferation. Integrated analyses of differential global transcriptomes (RNA sequencing) and H3K4me3 marks (chromatin immunoprecipitation sequencing) in Eµ-TCL1A vs iKdm1aKD;Eµ-TCL1A mice (confirmed in human CLL) implicate KDM1A as an oncogenic transcriptional repressor in CLL which alters histone methylation patterns with pronounced effects on defined cell death and motility pathways. Finally, pharmacologic KDM1A inhibition altered H3K4/9 target methylation and revealed marked anti-B-cell leukemic synergisms. Overall, we established the pathogenic role and effector networks of KDM1A in CLL via tumor-cell intrinsic mechanisms and its impacts in cells of the microenvironment. Our data also provide rationales to further investigate therapeutic KDM1A targeting in CLL.

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.

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.

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.

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.

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.

ACCELERATE: A Patient-Powered Natural History Study Design Enabling Clinical and Therapeutic Discoveries in a Rare Disorder.

Geographically dispersed patients, inconsistent treatment tracking, and limited infrastructure slow research for many orphan diseases. We assess the feasibility of a patient-powered study design to overcome these challenges for Castleman disease, a rare hematologic disorder. Here, we report initial results from the ACCELERATE natural history registry. ACCELERATE includes a traditional physician-reported arm and a patient-powered arm, which enables patients to directly contribute medical data and biospecimens. This study design enables successful enrollment, with the 5-year minimum enrollment goal being met in 2 years. A median of 683 clinical, laboratory, and imaging data elements are captured per patient in the patient-powered arm compared with 37 in the physician-reported arm. These data reveal subgrouping characteristics, identify off-label treatments, support treatment guidelines, and are used in 17 clinical and translational studies. This feasibility study demonstrates that the direct-to-patient design is effective for collecting natural history data and biospecimens, tracking therapies, and providing critical research infrastructure.

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.