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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A Potassium-Based Quality-of-Service Metric Reduces Phlebotomy Errors, Resulting in Improved Patient Safety and Decreased Cost.

OBJECTIVES: Poor phlebotomy technique can introduce pseudohyperkalemia without hemolysis, requiring additional workup and placing a significant burden on patients, clinical teams, and laboratories. Such preanalytical biases can be detected through systematic evaluation of potassium concentrations on a per-phlebotomist basis. We report our long-term experience with a potassium-based quality-of-service phlebotomy metric and its effects on resource utilization. METHODS: Potassium monitoring and retraining of 26 full-time phlebotomists were piloted as a quality-of-service intervention. Changes in potassium concentrations and impact on resource utilization were assessed. An algorithm for data monitoring and phlebotomist feedback was developed, followed by institution-wide implementation. RESULTS: Systematic intervention and retraining normalized K+ concentrations and lowered the percentage of venipunctures with K+ above 5.2 mmol/L, leading to a marked increase in phlebotomist compliance. This change resulted in resources savings of 13% to 100% for individual phlebotomists, reducing the total extra laboratory time required for repeat phlebotomies to determine hyperkalemia, mostly in the high-volume phlebotomist group. CONCLUSIONS: A quality-of-service algorithm that involved monitoring potassium concentrations on a per-phlebotomist basis with feedback and retraining contributed to a concrete, data-based quality improvement plan. The institution-wide implementation of this metric allowed for significant cost savings and a reduction in critical value alerts, directly affecting the quality of patient care.

Adding venetoclax to fludarabine/busulfan RIC transplant for high-risk MDS and AML is feasible, safe, and active.

Adding the selective BCL-2 inhibitor venetoclax to reduced-intensity conditioning chemotherapy (fludarabine and busulfan [FluBu2]) may enhance antileukemic cytotoxicity and thereby reduce the risk of posttransplant relapse. This phase 1 study investigated the recommended phase 2 dose (RP2D) of venetoclax, a BCL-2 selective inhibitor, when added to FluBu2 in adult patients with high-risk acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and MDS/myeloproliferative neoplasms (MPN) undergoing transplant. Patients received dose-escalated venetoclax (200-400 mg daily starting day -8 for 6-7 doses) in combination with fludarabine 30 mg/m2 per day for 4 doses and busulfan 0.8 mg/kg twice daily for 8 doses on day -5 to day -2 (FluBu2). Transplant related-toxicity was evaluated from the first venetoclax dose on day -8 to day 28. Twenty-two patients were treated. At study entry, 5 patients with MDS and MDS/MPN had 5% to 10% marrow blasts, and 18 (82%) of 22 had a persistent detectable mutation. Grade 3 adverse events included mucositis, diarrhea, and liver transaminitis (n = 3 each). Neutrophil/platelet recovery and acute/chronic graft-versus-host-disease rates were similar to those of standard FluBu2. No dose-limiting toxicities were observed. The RP2D of venetoclax was 400 mg daily for 7 doses. With a median follow-up of 14.7 months (range, 8.6-24.8 months), median overall survival was not reached, and progression-free survival was 12.2 months (95% confidence interval, 6.0-not estimable). In patients with high-risk AML, MDS, and MDS/MPN, adding venetoclax to FluBu2 was feasible and safe. To further address relapse risk, assessment of maintenance therapy after venetoclax plus FluBu2 transplant is ongoing. This study was registered at clinicaltrials.gov as #NCT03613532.

Rivaroxaban-calibrated chromogenic anti-Xa assay in cirrhosis: Use to rule out disseminated intravascular coagulation.

Peritoneovenous shunts (PVSs) are used to relieve ascites in cirrhosis. Disseminated intervascular coagulation (DIC) is a complication of PVSs requiring immediate PVS removal. We report a patient who developed new elevations of prothrombin time (PT) and activated partial thromboplastin time (aPTT) 6 hours after PVS placement, concerning for new-onset DIC. We address the key clinical question of distinguishing DIC from rivaroxaban effect on labs. The patient took rivaroxaban 3 hours after PVS placement, suggesting rivaroxaban effect. Rivaroxaban-calibrated anti-Xa level was in the expected treatment range. Over 12 hours, coagulation labs and rivaroxaban levels declined, with no evidence of DIC. The sudden PT/aPTT increase was attributed to rivaroxaban, however, the distinction between DIC and rivaroxaban effect was possible only with the rapid availability of rivaroxaban levels. While there are no US Food and Drug Administration-approved tests for rivaroxaban levels in the United States, this case demonstrates they can have significant clinical impact, encouraging more widespread adaptation of these assays.

Activation of Notch and Myc Signaling via B-cell-Restricted Depletion of Dnmt3a Generates a Consistent Murine Model of Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by disordered DNA methylation, suggesting these epigenetic changes might play a critical role in disease onset and progression. The methyltransferase DNMT3A is a key regulator of DNA methylation. Although DNMT3A somatic mutations in CLL are rare, we found that low DNMT3A expression is associated with more aggressive disease. A conditional knockout mouse model showed that homozygous depletion of Dnmt3a from B cells results in the development of CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3a depletion yields a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haploinsufficient tumor suppressor. B1a cells were confirmed as the cell of origin of disease in this model, and Dnmt3a depletion resulted in focal hypomethylation and activation of Notch and Myc signaling. Amplification of chromosome 15 containing the Myc gene was detected in all CLL mice tested, and infiltration of high-Myc-expressing CLL cells in the spleen was observed. Notably, hyperactivation of Notch and Myc signaling was exclusively observed in the Dnmt3a CLL mice, but not in three other CLL mouse models tested (Sf3b1-Atm, Ikzf3, and MDR), and Dnmt3a-depleted CLL were sensitive to pharmacologic inhibition of Notch signaling in vitro and in vivo. Consistent with these findings, human CLL samples with lower DNMT3A expression were more sensitive to Notch inhibition than those with higher DNMT3A expression. Altogether, these results suggest that Dnmt3a depletion induces CLL that is highly dependent on activation of Notch and Myc signaling. SIGNIFICANCE: Loss of DNMT3A expression is a driving event in CLL and is associated with aggressive disease, activation of Notch and Myc signaling, and enhanced sensitivity to Notch inhibition.

Inhibition of androgen/AR signaling inhibits diethylnitrosamine (DEN) induced tumour initiation and remodels liver immune cell networks.

A promotional role for androgen receptor (AR) signaling in hepatocellular carcinogenesis is emerging. In pre-clinical models, including diethylnitrosamine- (DEN-) induced hepatocellular carcinoma (HCC), anti-androgen therapies delay hepatocarcinogenesis. However, pharmacologic anti-androgen therapy in advanced HCC patients fails, suggesting that AR plays a role in HCC onset. This study aims to characterize AR expression and function throughout DEN-induced liver inflammation and carcinogenesis and evaluate the efficacy of prophylactic AR antagonism to prevent hepatocarcinogenesis. We demonstrate that pharmacologic AR antagonism with enzalutamide inhibits hepatocellular carcinogenesis. With enzalutamide treatment, we observe decreased CYP2E1 expression, reducing DEN-induced hepatocyte death and DNA ethyl-adducts. AR protein expression analyses show that DEN causes an initial upregulation of AR in portal fibroblasts and leukocytes, but not hepatocytes, suggesting that hepatocyte-autonomous AR signaling is not essential for DEN-induced carcinogenesis. Ablating androgen signaling by surgical castration reduced pre-carcinogen Kupffer cell populations but did not alter DEN-mediated immune cell recruitment nor AR expression. In this study, we identified that anti-androgen interventions modulate mutagenic DNA adducts, tumour initiation, and immune cell composition. Additionally, we find that AR expression in hepatocytes is not present during nor required for early DEN-mediated carcinogenesis.