Cutaneous Manifestations of Myeloid Neoplasms Exhibit Broad and Divergent Morphologic and Immunophenotypic Features but Share Ancestral Clonal Mutations With Bone Marrow.

In this study, we performed a comprehensive molecular analysis of paired skin and peripheral blood/bone marrow (BM) samples from 17 patients with cutaneous myeloid or cutaneous histiocytic-dendritic neoplasms. The cutaneous manifestations included 10 patients with cutaneous acute myeloid leukemia (c-AML), 2 patients with full or partial Langerhans cell differentiation, 2 patients with blastic plasmacytoid dendritic cell neoplasms (BPDCN), 1 patient with both Langerhans cell differentiation and BPDCN, and 2 patients with full or partial indeterminate dendritic cell differentiation. Seven of the 10 c-AML patients (70%) exhibited concurrent or subsequent marrow involvement by acute myeloid leukemia, with all 7 cases (100%) demonstrating shared clonal mutations in both the skin and BM. However, clonal relatedness was documented in one additional case that never had any BM involvement. Nevertheless, NPM1 mutations were identified in 7 of the 10 (70%) of these c-AML cases while one had KMT2A rearrangement and one showed inv(16). All 3 patients (100%) with Langerhans cell neoplasms, 2 patients with BPDCN (100%), and one of the 2 patients (50%) with other cutaneous dendritic cell neoplasms also demonstrated shared mutations between the skin and concurrent or subsequent myeloid neoplasms. Both BM and c-AML shared identical founding drivers, with a predominance of NPM1, DNMT3A, and translocations associated with monocytic differentiation, with common cutaneous-only mutations involving genes in the signal transduction and epigenetic pathways. Cutaneous histiocytic-dendritic neoplasms shared founding drivers in ASXL1, TET2, and/or SRSF2. However, in the Langerhans cell histiocytosis or histiocytic sarcoma cases, there exist recurrent secondary RAS pathway hits, whereas cutaneous BPDCN cases exhibit copy number or structural variants. These results enrich and broaden our understanding of clonally related cutaneous manifestations of myeloid neoplasms and further illuminate the highly diverse spectrum of morphologic and immunophenotypic features they exhibit.

Acute Promyelocytic Leukemia With Torque Teno Mini Virus::RARA Fusion: An Approach to Screening and Diagnosis.

Acute promyelocytic leukemia (APL) with variant RARA translocation is linked to over 15 partner genes. Recent publications encompassing 6 cases have expanded the spectrum of RARA partners to torque teno mini virus (TTMV). This entity is likely underrecognized due to the lack of clinician and pathologist familiarity, inability to detect the fusion using routine testing modalities, and informatic challenges in its recognition within next-generation sequencing (NGS) data. We describe a clinicopathologic approach and provide the necessary tools to screen and diagnose APL with TTMV::RARA using existing clinical DNA- or RNA-based NGS assays, which led to the identification of 4 cases, all without other known cytogenetic/molecular drivers. One was identified prospectively and 3 retrospectively, including 2 from custom automated screening of multiple data sets (50,257 cases of hematopoietic malignancy, including 4809 acute myeloid leukemia/myeloid sarcoma/APL cases). Two cases presented as myeloid sarcoma, including 1 with multiple relapses after acute myeloid leukemia-type chemotherapy and hematopoietic stem cell transplant. Two cases presented as leukemia, had a poor response to induction chemotherapy, but achieved remission upon reinduction (including all-trans retinoic acid in 1 case) and subsequent hematopoietic stem cell transplant. Neoplastic cells demonstrated features of APL including frequent azurophilic granules and dim/absent CD34 and HLA-DR expression. RARA rearrangement was not detected by karyotype or fluorescent in situ hybridization. Custom analysis of NGS fusion panel data identified TTMV::RARA rearrangements and, in the prospectively identified case, facilitated monitoring in sequential bone marrow samples. APL with TTMV::RARA is a rare leukemia with a high rate of treatment failure in described cases. The diagnosis should be considered in leukemias with features of APL that lack detectable RARA fusions and other drivers, and may be confirmed by appropriate NGS tests with custom informatics. Incorporation of all-trans retinoic acid may have a role in treatment but requires accurate recognition of the fusion for appropriate classification as APL.

Primary cutaneous EBV+ extranodal NK/T-cell lymphoma of gamma/delta T-cell lineage in the posttransplantation setting.

Posttransplantation primary cutaneous T-cell lymphomas (PT-CTCL) are a rare complication of sustained immunosuppression in the posttransplant setting. When present, PT-CTCLs are typically EBV- and exhibit features of mycosis fungoides/Sézary syndrome or CD30+ lymphoproliferative disorders. We present a case of a 75-year-old individual who developed skin lesions 30 years after liver transplantation. Pathologic evaluation of the skin biopsy revealed involvement by a clonal, EBV+ T-cell population of gamma/delta lineage with no evidence of systemic disease. Comprehensive genomic profiling was performed, confirming focal one-copy loss of 6q23.3, altogether consistent with the extremely rare and unusual diagnosis of primary cutaneous EBV+ extranodal NK/T-cell lymphoma of gamma/delta T-cell lineage in the posttransplantation setting.

T Cell Transcriptional Profiling and Immunophenotyping Uncover LAG3 as a Potential Significant Target of Immune Modulation in Multiple Myeloma.

Autologous stem cell transplant (ASCT) is the standard of care for patients with multiple myeloma (MM). The clinical significance of peripheral blood T lymphocyte (PBTL) immunologic changes associated with ASCT is poorly understood. Here we evaluated T cell transcriptional messenger RNA profiles and immunophenotypes to correlate immunologic senescence, exhaustion, and anergy with clinical endpoints in a cohort of patients with MM undergoing ASCT. ASCT induced global transcriptional T cell changes and altered molecular levels of markers of T cell subtypes, T cell activation, and exhaustion. These included reduced CD4/CD8 ratio, skewing toward the Th1 subset, reduced expression of costimulatory receptors CD27 and CD28, heightened T cell activation, and increased expression of immune modulatory molecules LAG3 and PD1. Multicolor flow cytometry experiments confirmed altered circulating CD4 and CD8 subsets and skewing toward differentiated effector cells. Moreover, ASCT promoted an exhausted immunophenotype in CD3+CD4+ subsets and a senescent immunophenotype in CD3+CD8+ subsets. Subset-specific altered expression was also seen for surface molecules with immunomodulatory function. ASCT affected soluble levels of molecules with immunomodulatory function by increasing plasma HVEM and TIM3. High molecular LAG3 level was associated with inferior event-free survival post-ASCT (hazard ratio = 5.44; confidence interval, 1.92 to 15.46; P = .001; adjusted P [controlling for false discovery rate] = .038). Using a comprehensive evaluation of PBTLs on a molecular and phenotypic level, we have identified that ASCT induces global T cell alterations with CD4 and CD8 subset-specific changes. Moreover, LAG3 emerged as an early biomarker of adverse events post-ASCT. These findings will support the development of treatment strategies targeting immune defects in MM to augment or restore T cell responses.

MiSet RFC Standards: Defining a Universal Minimum Set of Standards Required for Reproducibility and Rigor in Research Flow Cytometry Experiments.

Poor adherence to best practices, insufficient training, and pressure to produce data quickly may lead to publications of suboptimal biomedical research flow cytometry data, which contributes to the body of irreproducible research findings. In addition, documentation of compliance with best flow cytometry practices for submission, visualization, and publication of flow cytometry data is currently endorsed by very few scientific journals, which is particularly concerning as numerous peer-reviewed flow cytometry publications emphasize instrumentation, experimental design, and data analysis as important sources of variability. Guidelines and resources for adequate reporting, annotation and deposition of flow cytometry experiments are provided by MIFlowCyt and the FlowRepository database, and comprehensive expert recommendations covering principles and techniques of field-specific flow cytometry applications have been published. To facilitate the integration of quality-defining parameters into manuscript and grant submission and publication requirements across biomedical fields that rely on the use of flow-cytometry-based techniques, a single comprehensive yet easily and universally applicable document is needed. To produce such a list of gold-standard parameters that assess whether a research flow cytometry experiment has been planned, conducted, interpreted, and reported at the highest standard, a new initiative defining the minimum set of standards a robust and rigorous research flow experiment must fulfill (MiSet RFC Standards) was proposed at CYTO 2019. MiSet RFC Standards will integrate and simplify existing resources to provide a universal benchmark a flow cytometry experiment can easily be measured against. The goal of MiSET RFC Standards is its integration into peer-review and publication procedures through partnership with stakeholders, journals and publishers in biomedical and translational research. This article introduces the aims and anticipated timeline and discusses strategies for interdisciplinary consensus and implementation. A single-resource broadly applicable guideline will harmonize standards across different fields of biomedical research and lead to publication of more robust research findings. © 2019 International Society for Advancement of Cytometry.

Tumor necrosis factor receptor signaling is a driver of chronic lymphocytic leukemia that can be therapeutically targeted by the flavonoid wogonin.

Chronic lymphocytic leukemia is a malignancy of mature B cells that strongly depend on microenvironmental factors, and their deprivation has been identified as a promising treatment approach for this incurable disease. Cytokine array screening of 247 chronic lymphocytic leukemia serum samples revealed elevated levels of tumor necrosis factor (TNF) receptor-1 which were associated with poor clinical outcome. We detected a microenvironment-induced expression of TNF receptor-1 in chronic lymphocytic leukemia cells in vitro, and an aberrantly high expression of this receptor in the proliferation centers of patients' lymph nodes. Stimulation of TNF receptor-1 with TNF-α enhanced nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) activity and viability of chronic lymphocytic leukemia cells, which was inhibited by wogonin. The therapeutic effects of wogonin were analyzed in mice after adoptive transfer of Eµ-T-cell leukemia 1 (TCL1) leukemic cells. Wogonin treatment prevented leukemia development when given early after transplantation. The treatment of full-blown leukemia resulted in the loss of the TNF receptor-1 on chronic lymphocytic leukemia cells and their mobilization to blood. Targeting TNF receptor-1 signaling is therefore proposed for the treatment of chronic lymphocytic leukemia.

Biphasic Effects of Vitamin D and FGF23 on Human Osteoclast Biology.

Vitamin D and FGF23 play a major role in calcium/phosphate balance. Vitamin D may control bone resorption but the potential role of FGF23 has never been evaluated. The objective of this study was therefore to compare the effects of vitamin D and FGF23 on osteoclast differentiation and activity in human monocyte-derived osteoclasts. Human monocytes, purified from blood of healthy donors, were incubated with M-CSF and RANKL to obtain mature multinucleated osteoclasts (MNC). Experiments were carried out to assess the effects of FGF23 as compared to native vitamin D (25-D) and active vitamin D (1,25-D) on osteoclast differentiation and on bone-resorbing osteoclast activity. Additional experiments with the pan fibroblast growth factor receptor inhibitor (FGFR-i) were performed. Phosphorylation Akt and Erk pathways were analyzed by Western blot analyses. Both 1,25-D and FGF23, to a lesser extent, significantly inhibited osteoclastogenesis at early stages; when adding FGFR-i, osteoclast formation was restored. Biochemical experiments showed an activation of the Akt and Erk pathways under FGF23 treatment. In contrast, in terms of activity, 1,25-D had no effect on resorption, whereas FGF23 slightly but significantly increased bone resorption; 25-D had no effects on either differentiation or on activity. These data show that 1,25-D inhibits osteoclastogenesis without regulating osteoclast-mediated bone resorption activity; FGF23 has biphasic effects on osteoclast physiology, inhibiting osteoclast formation while stimulating slightly osteoclast activity. These results may be of importance and taken into account in chronic kidney disease when therapies modulating FGF23 are available.

Translating the regulatory landscape of medical devices to create fit-for-purpose artificial intelligence (AI) cytometry solutions.

The implementation of medical software and artificial intelligence (AI) algorithms into routine clinical cytometry diagnostic practice requires a thorough understanding of regulatory requirements and challenges throughout the cytometry software product lifecycle. To provide cytometry software developers, computational scientists, researchers, industry professionals, and diagnostic physicians/pathologists with an introduction to European Union (EU) and United States (US) regulatory frameworks. Informed by community feedback and needs assessment established during two international cytometry workshops, this article provides an overview of regulatory landscapes as they pertain to the application of AI, AI-enabled medical devices, and Software as a Medical Device in diagnostic flow cytometry. Evolving regulatory frameworks are discussed, and specific examples regarding cytometry instruments, analysis software and clinical flow cytometry in-vitro diagnostic assays are provided. An important consideration for cytometry software development is the modular approach. As such, modules can be segregated and treated as independent components based on the medical purpose and risk and become subjected to a range of context-dependent compliance and regulatory requirements throughout their life cycle. Knowledge of regulatory and compliance requirements enhances the communication and collaboration between developers, researchers, end-users and regulators. This connection is essential to translate scientific innovation into diagnostic practice and to continue to shape the development and revision of new policies, standards, and approaches.

Case Report: Post-transplant lymphoproliferative disorder as a serious complication of vascularized composite allotransplantation.

Vascularized composite allotransplantation (VCA) is an emerging field in transplant surgery. Despite overall positive outcomes, VCA confers risk for multiple complications related to the procedure and subsequent immunosuppression. Post-transplant lymphoproliferative disorder (PTLD) is a heterogeneous group of lymphoproliferative disorders occurring after solid organ and hematopoietic stem cell transplant. A patient with PTLD after bilateral upper extremity transplantation is presented as well as a review of all known cases of PTLD after VCA, with a focus on the unique epidemiology, presentation, and treatment in this population.

Recommendations for using artificial intelligence in clinical flow cytometry.

Flow cytometry is a key clinical tool in the diagnosis of many hematologic malignancies and traditionally requires close inspection of digital data by hematopathologists with expert domain knowledge. Advances in artificial intelligence (AI) are transferable to flow cytometry and have the potential to improve efficiency and prioritization of cases, reduce errors, and highlight fundamental, previously unrecognized associations with underlying biological processes. As a multidisciplinary group of stakeholders, we review a range of critical considerations for appropriately applying AI to clinical flow cytometry, including use case identification, low and high risk use cases, validation, revalidation, computational considerations, and the present regulatory frameworks surrounding AI in clinical medicine. In particular, we provide practical guidance for the development, implementation, and suggestions for potential regulation of AI-based methods in the clinical flow cytometry laboratory. We expect these recommendations to be a helpful initial framework of reference, which will also require additional updates as the field matures.

Severe enterovirus infections in patients with immune-mediated inflammatory diseases receiving anti-CD20 monoclonal antibodies.

OBJECTIVE: Patients with X linked agammaglobulinemia are susceptible to enterovirus (EV) infections. Similarly, severe EV infections have been described in patients with impaired B-cell response following treatment with anti-CD20 monoclonal antibodies (mAbs), mostly in those treated for haematological malignancies. We aimed to describe severe EV infections in patients receiving anti-CD20 mAbs for immune-mediated inflammatory diseases (IMIDs). METHODS: Patients were included following a screening of data collected through the routine surveillance of EV infections coordinated by the National Reference Center and a review of the literature. Additionally, neutralising antibodies were assessed in a patient with chronic EV-A71 meningoencephalitis. RESULTS: Nine original and 17 previously published cases were retrieved. Meningoencephalitis (n=21/26, 81%) associated with EV-positive cerebrospinal fluid (n=20/22, 91%) was the most common manifestation. The mortality rate was high (27%). EV was the only causal agents in all reported cases. Patients received multiple anti-CD20 mAbs infusions (median 8 (5-10)), resulting in complete B-cell depletion and moderate hypogammaglobulinemia (median 4.9 g/L (4.3-6.7)), and had limited concomitant immunosuppressive treatments. Finally, in a patient with EV-A71 meningoencephalitis, a lack of B-cell response to EV was shown. CONCLUSION: EV infection should be evoked in patients with IMIDs presenting with atypical organ involvement, especially meningoencephalitis. Anti-CD20 mAbs may lead to impaired B-cell response against EV, although an underlying primary immunodeficiency should systematically be discussed.

Hematopathology of Severe Acute Respiratory Syndrome Coronavirus 2 Infection and Coronavirus Disease-19.

Coronavirus disease 2019 is caused by severe acute respiratory syndrome coronavirus 2 and is associated with pronounced hematopathologic findings. Peripheral blood features are heterogeneous and very often include neutrophilia, lymphopenia, myeloid left shift, abnormally segmented neutrophils, atypical lymphocytes/plasmacytoid lymphocytes, and atypical monocytes. Bone marrow biopsies and aspirates are often notable for histiocytosis and hemophagocytosis, whereas secondary lymphoid organs may exhibit lymphocyte depletion, pronounced plasmacytoid infiltrates, and hemophagocytosis. These changes are reflective of profound innate and adaptive immune dysregulation, and ongoing research efforts continue to identify clinically applicable biomarkers of disease severity and outcome.

Advances in Acute Myeloid Leukemia Classification, Prognostication and Monitoring by Flow Cytometry.

Although final classification of acute myeloid leukemia (AML) integrates morphologic, cytogenetic, and molecular data, flow cytometry remains an essential component of modern AML diagnostics. Here, we review the current role of flow cytometry in the classification, prognostication, and monitoring of AML. We cover immunophenotypic features of key genetically defined AML subtypes and their effects on biological and clinical behaviors, review clinically tractable strategies to differentiate leukemias with ambiguous immunophenotypes more accurately and discuss key principles of standardization for measurable residual disease monitoring. These advances underscore flow cytometry's continued growth as a powerful diagnostic, management, and discovery tool.

Correctly Establishing and Interpreting Oxygenation Status in Sickle Cell Disease.

BACKGROUND: As hypoxemia and hypoxia are central elements of disease pathophysiology and disease-related morbidity and mortality in individuals affected by sickle cell disease (SCD), clinical management aims to optimize oxygenation. CONTENT: Hypoxemia is primarily screened for with pulse oximetry. However, in SCD pulse oximetry can inaccurately reflect arterial saturation, posing the risk of undetected (occult) hypoxemia. Solely relying on pulse oximetry might therefore lead to misdiagnosis or mismanagement, with devastating effects on tissue oxygenation. The interpretation of oxygenation status is multifaceted, and "oxygen saturation" is often used as an umbrella term to refer to distinctly different measured quantities-estimated oxygen saturation (O2Sat), hemoglobin oxygen saturation (SO2) by either pulse oximetry or co-oximetry, and fractional oxyhemoglobin (FO2Hb). While in many clinical situations this ambiguous use is of little consequence, O2Sat, SO2, and FO2Hb cannot be used interchangeably in the setting of SCD, as dyshemoglobins, anemia, cardiopulmonary comorbidities, concomitant medications, and frequent transfusions need to be accounted for. This article describes the parameters that determine blood and tissue oxygen concentration, discusses laboratory method performance characteristics and the correct interpretation of currently available clinical laboratory testing, and reviews the literature on noninvasive vs invasive oxygenation measurements in SCD. SUMMARY: By correctly establishing and interpreting oxygenation parameters, clinical and laboratory teams can ensure high-quality, equitable healthcare, counteracting systemic exacerbations of health disparities frequently experienced by individuals with SCD.

One-year retrospective analysis of anti-FXa apixaban and rivaroxaban levels demonstrates utility for management decisions in various urgent and nonurgent clinical situations.

OBJECTIVES: Quantification of direct oral anticoagulant (DOAC) plasma levels can guide clinical management, but insight into clinical scenarios surrounding DOAC-calibrated anti-FXa assays is limited. METHODS: Apixaban- and rivaroxaban-calibrated chromogenic anti-Xa assays performed over a 1-year period were retrospectively analyzed. Patient demographics, DOAC history, concomitant medications, and renal/liver comorbidities were obtained. Indications for testing and associated clinical actions were reviewed. Machine learning (ML) models predicting clinical actions were evaluated. RESULTS: In total, 371 anti-FXa apixaban and 89 anti-FXa rivaroxaban tests were performed for 259 and 67 patients in recurring urgent (acute bleeding, unplanned procedures) and nonurgent situations, including several scenarios not captured by existing testing recommendations (eg, drug monitoring, recurrent thromboembolic events, bleeding tendency). In urgent settings, andexanet reversal was guided by radiologic and clinical findings over DOAC levels in 14 of 32 instances, while 51% of apixaban patients qualified for nonreversal strategies through the availability of levels. Levels also informed procedure/intervention timing and supported management decisions when DOAC clearance or DOAC target levels were in question. The importance of clinical context was emphasized by exploratory ML models predicting particular clinical actions. CONCLUSIONS: Although clinical situations are complex, DOAC testing facilitates clinical decision-making, including reversal, justifying more widespread implementation of these assays.

DeepHeme: A generalizable, bone marrow classifier with hematopathologist-level performance.

Morphology-based classification of cells in the bone marrow aspirate (BMA) is a key step in the diagnosis and management of hematologic malignancies. However, it is time-intensive and must be performed by expert hematopathologists and laboratory professionals. We curated a large, high-quality dataset of 41,595 hematopathologist consensus-annotated single-cell images extracted from BMA whole slide images (WSIs) containing 23 morphologic classes from the clinical archives of the University of California, San Francisco. We trained a convolutional neural network, DeepHeme, to classify images in this dataset, achieving a mean area under the curve (AUC) of 0.99. DeepHeme was then externally validated on WSIs from Memorial Sloan Kettering Cancer Center, with a similar AUC of 0.98, demonstrating robust generalization. When compared to individual hematopathologists from three different top academic medical centers, the algorithm outperformed all three. Finally, DeepHeme reliably identified cell states such as mitosis, paving the way for image-based quantification of mitotic index in a cell-specific manner, which may have important clinical applications.

In Vivo Modeling of CLL Transformation to Richter Syndrome Reveals Convergent Evolutionary Paths and Therapeutic Vulnerabilities.

Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy. SIGNIFICANCE: Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease. This article is highlighted in the In This Issue feature, p. 101.