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Pulmonary arterial hypertension is characterized by endothelial dysfunction and microthrombi formation. The role of anticoagulation remains controversial, with studies demonstrating inconsistent effects on pulmonary arterial hypertension mortality. Clinical anticoagulation practices are currently heterogeneous, reflecting physician preference. This study uses thrombelastography and hematology markers to evaluate whether clot formation and fibrinolysis are abnormal in pulmonary arterial hypertension patients. Venous blood was collected from healthy volunteers (n = 20) and patients with pulmonary arterial hypertension (n = 20) on stable medical therapy for thrombelastography analysis. Individual thrombelastography parameters and a calculated coagulation index were used for comparison. In addition, hematologic markers, including fibrinogen, factor VIII activity, von Willebrand factor activity, von Willebrand factor antigen, and alpha2-antiplasmin, were measured in pulmonary arterial hypertension patients and compared to healthy volunteers. Between group differences were analyzed using t tests and linear mixed models, accounting for repeated measures when applicable. Although the degree of fibrinolysis (LY30) was significantly lower in pulmonary arterial hypertension patients compared to healthy volunteers (0.3% ± 0.6 versus 1.3% ± 1.1, p = 0.04), all values were within the normal reference range (0–8%). All other thrombelastography parameters were not significantly different between pulmonary arterial hypertension patients and healthy volunteers (p ≥ 0.15 for all). Similarly, alpha2-antiplasmin activity levels were higher in pulmonary arterial hypertension patients compared to healthy volunteers (103.7% ± 13.6 versus 82.6% ± 9.5, p < 0.0001), but all individual values were within the normal range (75–132%). There were no other significant differences in hematologic markers between pulmonary arterial hypertension patients and healthy volunteers (p ≥ 0.07 for all). Sub-group analysis comparing thrombelastography results in patients treated with or without prostacyclin pathway targeted therapies were also non-significant. In conclusion, treated pulmonary arterial hypertension patients do not demonstrate abnormal clotting kinetics or fibrinolysis by thrombelastography.

Predictors, genetic characteristics, and long-term outcomes of patients with SAA who clonally evolved after immunosuppressive therapy (IST) were assessed. SAA patients were treated with IST from 1989-2020. Clonal evolution was categorized as “high-risk” (overt myeloid neoplasm [meeting WHO criteria for dysplasia, MPN or acute leukemia] or isolated chromosome-7 abnormality/complex karyotype without dysplasia or overt myeloid neoplasia) or “low-risk” (non-7 or non-complex chromosome abnormalities without morphological evidence of dysplasia or myeloid neoplasia). Univariate and multivariate analysis using Fine-Gray competing risk regression model determined predictors. Long-term outcomes included relapse, overall survival (OS) and hematopoietic stem cell transplant (HSCT). Somatic mutations in myeloid cancer genes were assessed in evolvers and in 407 patients 6 months after IST. Of 663 SAA patients, 95 developed clonal evolution. Pre-treatment age >48 years and ANC > 0.87 × 109/L were strong predictors of high-risk evolution. OS was 37% in high-risk clonal evolution by 5 years compared to 94% in low-risk. High-risk patients who underwent HSCT had improved OS. Eltrombopag did not increase high-risk evolution. Splicing factors and RUNX1 somatic variants were detected exclusively at high-risk evolution; DNMT3A, BCOR/L1 and ASXL1 were present in both. RUNX1, splicing factors and ASXL1 somatic mutations detected at 6 months after IST predicted high-risk evolution.

BackgroundHematologic toxicities, including coagulopathy, endothelial activation, and cytopenias, with CD19-targeted chimeric antigen receptor (CAR) T-cell therapies correlate with cytokine release syndrome (CRS) and neurotoxicity severity, but little is known about the extended toxicity profiles of CAR T-cells targeting alternative antigens. This report characterizes hematologic toxicities seen following CD22 CAR T-cells and their relationship to CRS and neurotoxicity.MethodsWe retrospectively characterized hematologic toxicities associated with CRS seen on a phase 1 study of anti-CD22 CAR T-cells for children and young adults with relapsed/refractory CD22+ hematologic malignancies. Additional analyses included correlation of hematologic toxicities with neurotoxicity and exploring effects of hemophagocytic lymphohistiocytosis-like toxicities (HLH) on bone marrow recovery and cytopenias. Coagulopathy was defined as evidence of bleeding or abnormal coagulation parameters. Hematologic toxicities were graded by Common Terminology Criteria for Adverse Events V.4.0.ResultsAcross 53 patients receiving CD22 CAR T-cells who experienced CRS, 43 (81.1%) patients achieved complete remission. Eighteen (34.0%) patients experienced coagulopathy, of whom 16 had clinical manifestations of mild bleeding (typically mucosal bleeding) which generally subsided following CRS resolution. Three had manifestations of thrombotic microangiopathy. Patients with coagulopathy had higher peak ferritin, D-dimer, prothrombin time, international normalized ratio (INR), lactate dehydrogenase (LDH), tissue factor, prothrombin fragment F1+2 and soluble vascular cell adhesion molecule-1 (s-VCAM-1). Despite a relatively higher incidence of HLH-like toxicities and endothelial activation, overall neurotoxicity was generally less severe than reported with CD19 CAR T-cells, prompting additional analysis to explore CD22 expression in the central nervous system (CNS). Single-cell analysis revealed that in contrast to CD19 expression, CD22 is not on oligodendrocyte precursor cells or on neurovascular cells but is seen on mature oligodendrocytes. Lastly, among those attaining CR, grade 3–4 neutropenia and thrombocytopenia were seen in 65% of patients at D28.ConclusionWith rising incidence of CD19 negative relapse, CD22 CAR T-cells are increasingly important for the treatment of B-cell malignancies. In characterizing hematologic toxicities on CD22 CAR T-cells, we demonstrate that despite endothelial activation, coagulopathy, and cytopenias, neurotoxicity was relatively mild and that CD22 and CD19 expression in the CNS differed, providing one potential hypothesis for divergent neurotoxicity profiles. Systematic characterization of on-target off-tumor toxicities of novel CAR T-cell constructs will be vital as new antigens are targeted.Trial registration numberNCT02315612.

Double minutes (dmins), a form of extrachromosomal DNA (ecDNA), represent a rare cytogenomic event in myeloid neoplasms and are most commonly associated with amplification of oncogenes such as MYC or KMT2A . Dmins derived from the 11q24 region that exclude KMT2A are exceedingly uncommon, and their pathogenic significance remains poorly understood. We report a 74‐year‐old female initially diagnosed with myelodysplastic syndrome (MDS) with isolated del(5) (q13q33) and mutations in TP53 and SF3B1 . After eight years and treatment with lenalidomide with excellent clinical response, she developed progressive cytopenias and transformation to acute myeloid leukemia, myelodysplasia‐related (AML‐MR). Cytogenomic analysis at the time of leukemic transformation revealed del(5) (q13q33) in all 20 metaphase cells analyzed and loss of the EGR1 (5q31.2) gene in 94% of interphase nuclei by fluorescence in situ hybridization (FISH). Notably, 18 of 20 metaphase cells also harbored dmins, ranging from 2 to 22 copies per cell. Array‐based comparative genomic hybridization and single nucleotide polymorphism array (array‐CGH + SNP) identified a 5.57 Mb amplification of chromosome 11q24.2–q25 encompassing at least 40 genes, including FLI1 and ETS1 but excluding KMT2A . Metaphase FISH confirmed localization of the amplified 11q24 segment within the dmins, and immunohistochemistry demonstrated nuclear FLI1 expression in myeloblasts. The patient was treated with combination azacitidine and venetoclax and an investigational immunotherapy within a clinical trial. This case represents the third reported instance of dmins derived from the 11q24 region involving FLI1 and ETS1 and the first identified in the context of AML evolved from del(5q) MDS. Dmins in myeloid neoplasms have been linked to genomic instability, clonal evolution, and therapeutic resistance. Amplification and expression of FLI1 in blasts, a hematopoietic transcription factor implicated in leukemogenesis and poor prognosis in AML, suggest a potential pathogenic role for 11q24‐derived dmins in disease progression. Our findings expand the spectrum of dmin‐associated oncogenic amplifications in myeloid neoplasms and highlight FLI1 and ETS1 as recurrent targets of 11q24‐derived ecDNA amplification. Recognition of such rare events underscores the importance of integrative cytogenomic profiling for uncovering novel mechanisms of leukemic transformation and potential therapeutic targets.

Abstract Objectives To determine fluorescently labeled aerolysin (FLAER) binding and glycophosphatidylinositol–anchored protein expression in bone marrow (BM) cells of healthy volunteers and patients with paroxysmal nocturnal hemoglobinuria (PNH) detected in peripheral blood (PB); compare PNH clone size in BM and PB; and detect PNH in BM by commonly used antibodies. Methods Flow cytometry analysis of FLAER binding to leukocytes and expression of CD55/CD59 in erythrocytes. Analysis of CD16 in neutrophils and CD14 in monocytes in BM. Results FLAER binds to all normal BM leukocytes, and binding increases with cell maturation. In PNH, lymphocytic clones are consistently smaller than clones of other BM cells. PNH clones are detectable in mature BM leukocytes with high specificity and sensitivity using common antibodies. Conclusions PNH clone sizes measured in mature BM leukocytes and in PB are comparable, making BM suitable for PNH assessment. We further demonstrate that commonly used reagents (not FLAER or CD55/CD59) can reliably identify abnormalities of BM neutrophils and monocytes consistent with PNH cells.

Late-onset inflammatory toxicities resembling hemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS) occur after chimeric antigen receptor T cell (CAR T cell) infusion and represent a therapeutic challenge. Given the established link between perforin deficiency and primary HLH, we investigated the role of perforin in anti-CD19 CAR T cell efficacy and HLH-like toxicities in a syngeneic murine model. Perforin contributed to both CD8+ and CD4+ CAR T cell cytotoxicity but was not required for in vitro or in vivo leukemia clearance. Upon CAR-mediated in vitro activation, perforin-deficient CAR T cells produced higher amounts of proinflammatory cytokines compared with WT CAR T cells. Following in vivo clearance of leukemia, perforin-deficient CAR T cells reexpanded, resulting in splenomegaly with disruption of normal splenic architecture and the presence of hemophagocytes, which are findings reminiscent of HLH. Notably, a substantial fraction of patients who received anti-CD22 CAR T cells also experienced biphasic inflammation, with the second phase occurring after the resolution of cytokine release syndrome, resembling clinical manifestations of HLH. Elevated inflammatory cytokines such as IL-1β and IL-18 and concurrent late CAR T cell expansion characterized the HLH-like syndromes occurring in the murine model and in humans. Thus, a murine model of perforin-deficient CAR T cells recapitulated late-onset inflammatory toxicities occurring in human CAR T cell recipients, providing therapeutically relevant mechanistic insights.

Double minutes (dmins), a form of extrachromosomal DNA (ecDNA), represent a rare cytogenomic event in myeloid neoplasms and are most commonly associated with amplification of oncogenes such as MYC or KMT2A. Dmins derived from the 11q24 region that exclude KMT2A are exceedingly uncommon, and their pathogenic significance remains poorly understood. We report a 74-year-old female initially diagnosed with myelodysplastic syndrome (MDS) with isolated del(5) (q13q33) and mutations in TP53 and SF3B1. After eight years and treatment with lenalidomide with excellent clinical response, she developed progressive cytopenias and transformation to acute myeloid leukemia, myelodysplasia-related (AML-MR). Cytogenomic analysis at the time of leukemic transformation revealed del(5) (q13q33) in all 20 metaphase cells analyzed and loss of the EGR1 (5q31.2) gene in 94% of interphase nuclei by fluorescence in situ hybridization (FISH). Notably, 18 of 20 metaphase cells also harbored dmins, ranging from 2 to 22 copies per cell. Array-based comparative genomic hybridization and single nucleotide polymorphism array (array-CGH+SNP) identified a 5.57Mb amplification of chromosome 11q24.2-q25 encompassing at least 40 genes, including FLI1 and ETS1 but excluding KMT2A. Metaphase FISH confirmed localization of the amplified 11q24 segment within the dmins, and immunohistochemistry demonstrated nuclear FLI1 expression in myeloblasts. The patient was treated with combination azacitidine and venetoclax and an investigational immunotherapy within a clinical trial. This case represents the third reported instance of dmins derived from the 11q24 region involving FLI1 and ETS1 and the first identified in the context of AML evolved from del(5q) MDS. Dmins in myeloid neoplasms have been linked to genomic instability, clonal evolution, and therapeutic resistance. Amplification and expression of FLI1 in blasts, a hematopoietic transcription factor implicated in leukemogenesis and poor prognosis in AML, suggest a potential pathogenic role for 11q24-derived dmins in disease progression. Our findings expand the spectrum of dmin-associated oncogenic amplifications in myeloid neoplasms and highlight FLI1 and ETS1 as recurrent targets of 11q24-derived ecDNA amplification. Recognition of such rare events underscores the importance of integrative cytogenomic profiling for uncovering novel mechanisms of leukemic transformation and potential therapeutic targets.

We reviewed CD56 expression on bone marrow (BM) granulocytes and monocytes by flow cytometry in 101 cases of myeloproliferative neoplasm (MPN) and compared them with those from 42 cases of myelodysplastic syndrome (MDS), 47 cases of negative BM after stem cell transplantation, and 24 cases of negative BM after chemotherapy. Forty cases of negative staging BM for lymphoma were used as negative controls. CD56 expression on granulocytes was also compared with corresponding morphologic and molecular genetic findings in five patients who underwent serial BM sampling. Using 10 % as positive threshold, aberrant CD56 expression was detected on granulocytes and monocytes in 20 and 34 % cases, respectively, in MPN, and in 18 and 36 % cases, respectively, in high-grade MDS. Aberrant CD56 expression was present in all subtypes of MPN with the highest frequency seen in primary myelofibrosis (37 % on granulocytes, 40 % on monocytes). Compared to monocytes, granulocytes had lower frequencies of CD56 expression but higher specificity for abnormal cells. Low levels of CD56 expression were detected on the granulocytes in normal BM after chemotherapy and stem cell transplantation, but the expression levels were generally below 10 %. In the patients with serial samples, CD56 expression paralleled with disease status by BM morphology, BCR/ABL1 transcripts, or percentage of BM recipient cells. We conclude that aberrant CD56 expression on granulocytes is present in a subset of patients in all subtypes of MPN. Identification of abnormal CD56-positive granulocytes is helpful in both initial diagnosis and follow-up of patients in MPN.

Abstract Background Microcytic erythrocytosis is an underrecognized and underevaluated complete blood count (CBC) finding. The literature pertaining to the determination of its etiology specifically by hemoglobin variant analysis is limited. Methods We performed hemoglobin variant analysis by high performance liquid chromatography on 137 patients who revealed microcytic erythrocytosis on CBC, and reviewed the results for the diagnosis of hemoglobin-associated disorders. Results A diagnosis of thalassemia trait and/or a hemoglobinopathy was established in 93 of 137 (67.9%) patients. Amongst these, ß-thalassemia trait topped the list with 69 cases (74.1%), followed by hereditary persistence of fetal hemoglobin with 5 cases (5.5%), Hemoglobin E disease with 4 cases (4.3%), and ∂/ß-thalassemia with 2 cases (2.1%). Compound heterozygous conditions with 1 or more hemoglobinopathies and/or thalassemias were diagnosed in 13 cases (14.0%). Abnormal hemoglobins in the compound heterozygosity group included C, S, HPFH, and 2 unknowns. Conclusion Hemoglobin variant analysis provided a very high positive yield in determining the etiology of microcytic erythrocytosis.