Detection of KIT p.D816V is a cornerstone in the diagnosis and classification of mast cell activation syndromes (MCAS) and mastocytosis. However, KIT p.D816V may be undetected due to a low mutated cell burden in blood and bone marrow (BM) of many patients, particularly among those without skin lesions. These findings underscore the need for ultra-sensitive molecular techniques for the detection of KIT p.D816V in these clinical settings. Here, we evaluated for the first time the sensitivity and specificity of a novel Flow-SuperRCA® KIT p.D816V assay, compared to conventional ASOqPCR, through the analysis of 548 blood and BM samples from 337 adults MCAS and mastocytosis patients. Our results demonstrated greater sensitivity of the new technique vs ASOqPCR (limit-of-detection: 0.001% vs 0.01% VAF), with higher rates of positivity for KIT p.D816V in whole-blood and/or BM of 64% of monoclonal-MCAS (MMAS) and 55% of BM mastocytosis patients (p<.0001). Notably, the sensitivity of the new assay went even beyond that of ASOqPCR in purified BM mast cells (p<.0001). Additionally, clonality was newly identified in 18% of patients previously diagnosed with non-clonal-MCAS, who presented with a unique cutaneous MCA-related symptomatic profile. These results confirm the high-specificity and ultra-sensitivity of the Flow-SuperRCA® assay for the detection of KIT p.D816V, emerging as a well-suited whole-blood and whole-BM test for the diagnostic screening and classification of MCAS and mastocytosis patients. These findings further highlight the clonal nature of an unprecedently high fraction of patients who presented with anaphylaxis and MCAS, with important pathogenic, diagnostic and clinical implications.

Recent advances have transformed the treatment landscape for relapsed and refractory follicular lymphoma. While chemotherapy has long served as the backbone of treatment, the availability of novel targeted, immunomodulatory and immunotherapeutic approaches is challenging its relevance. These approaches have focused on targeting epigenetic regulators, components of the B-cell receptor or its downstream intracellular pathways, and the follicular lymphoma tumor microenvironment. The recent development of bispecific antibodies and chimeric antigen receptor T-cell therapies, which target both tumor-associated and host-specific antigens, has enabled a redirection of the immune system, enhancing the innate anti-tumor immune response. Rational combinations of these strategies are actively being evaluated in the relapsed and refractory setting and will inevitably move forward into earlier lines of treatment. The success of these approaches has led to numerous and parallel options for patients and clinicians. The emerging challenge now lies in how best to approach each individual patient with relapsed or refractory follicular lymphoma, addressing complex decision-making that considers a patient's prior treatment history, goals of care, clinical and biological characteristics of recurrence, as well as personal preferences. Understanding the implications of refractory and transformed disease, as well as the timing and biology of relapse will be critical to support a more personalized treatment approach in the modern era.

Blood clotting is triggered in hemostasis and thrombosis when the membrane-bound tissue factor (TF)/factor VIIa (FVIIa) complex activates factor X (FX). There are no structures of TF/FVIIa on membranes, with or without FX. Using cryo-EM to address this gap, we assembled TF/FVIIa complexes on nanoscale membrane bilayers (nanodiscs), bound to XK1 and an antibody fragment. XK1 is a FX mimetic whose protease domain is replaced by the first Kunitz-type (K1) domain of tissue factor pathway inhibitor, while 10H10 is a non-inhibitory, anti-TF antibody. We determined a cryo-EM structure of a TF/FVIIa/XK1/10H10/nanodisc complex with a resolution of 3.7 Å, allowing us to model all the protein backbones. TF/FVIIa extends perpendicularly from the membrane, interacting with a "handle shaped" XK1 at two locations: the K1 domain docks into FVIIa's active site, while the γ-carboxyglutamate-rich (GLA) domain binds to TF's substrate-binding exosite. The FX and FVIIa GLA domains also contact each other and the membrane surface. Except for a minor contact between the first epidermal growth factor (EGF)-like domain of XK1 and TF, the rest of the FX light chain does not interact with TF/FVIIa. The structure reveals a previously unrecognized, membrane-dependent allosteric activation mechanism between FVIIa and TF where a serine-rich loop in TF that partially obscures the TF exosite must undergo a shift to allow access of the FX GLA domain to its final binding location on the membrane-bound TF/FVIIa complex. This mechanism also provides a novel explanation for the otherwise puzzling phenomenon of TF encryption/decryption on cell surfaces.

Venous thromboembolism (VTE) is a frequent (annual incidence of 1-2 per 1,000) and potentially life-threatening (case-fatality rate up to 10%) disease. VTE is associated with serious short-term and long-term complications, including a recurrence rate approaching 20% within five years. Anticoagulant therapy, the mainstay of VTE treatment, drastically reduces the risk of early VTE recurrence, but it exposes patients to a substantial risk of bleeding. We analysed the genomic architecture of VTE recurrence using data from 6,355 patients across eight cohorts (including 1,775 recurrences), enriched by subgroup analyses, according to sex and clinical manifestation of first VTE, which led to the identification of 28 molecular markers. Through genome-wide association studies, we identified one locus associated with VTE recurrence: GPR149/MME. Among all variants known to be associated with first VTE, KNG1 and FGG were associated with recurrence. Additionally, Mendelian Randomization analyses identified seven proteins as risk factors for recurrence: elevated plasma levels of coagulation factor XI, coagulation factor VIII, von Willebrand factor, BGAT and GOLM2; decreased levels of PCSK9 and pro-IL16. Subgroup analyses revealed 18 molecular determinants associated with VTE recurrence, with notable differences between subgroups. For example, the exonic variant SLC4A1 p.Glu40Lys was significantly associated in pulmonary embolism patients (Hazard Ratio (HR)=3.13, P=5.9×10-11) but showed no effect in deep vein thrombosis patients (HR=0.91, P=0.72). These findings emphasize the role of specific genetic loci and protein pathways in influencing VTE recurrence and provide valuable insights into potential therapeutic targets. Further research is needed to clarify the biological mechanisms driving these associations.

Chimeric antigen receptor T (CAR-T) cell therapy has revolutionized treatment for B-cell malignancies, yet over 60% of patients relapse within one year, often due to insufficient CAR-T persistence. While mouse and primary cell models have been instrumental in advancing CAR-T therapy, they frequently fail to predict clinical outcomes, underscoring the need for more translationally relevant models. To address this limitation, we conducted the first systematic evaluation of CAR structure-function relationships in an immunocompetent nonhuman primate (NHP) model. We engineered an array of 20 CD20-targeted CARs with distinct combinations of hinge, transmembrane, and costimulatory domains. Following ex vivo characterization, we administered pooled autologous CAR-T arrays to three NHPs and tracked CAR abundance longitudinally using a novel digital droplet PCR assay. Ex vivo, CAR-T cells incorporating the MyD88-CD40 costimulatory domain exhibited markedly distinct functional profiles, including increased activation, unique cytokine secretion, tonic signaling, and resistance to exhaustion. In vivo, MyD88-CD40 CARs expanded dramatically, comprising up to 100% of peripheral CAR-T cells and significantly outperforming canonical CD28- and 4-1BB-based CARs. This expansion was associated with robust B-cell depletion across all animals. MyD88-CD40 CARs, particularly those with a CD28 hinge and transmembrane domain, demonstrated superior trafficking to secondary lymphoid tissues and persistence through study endpoint, unlike other CARs which waned by day 28. Our findings highlight the value of NHP models for screening CAR designs and identify MyD88-CD40 CARs as candidates with unmatched potency. The unique functional attributes conferred by this domain may provide key insights into features that drive enhanced CAR-T cell activity.

Aberrant activation of BCL11B ("BCL11B-a") defines a subtype of lineage ambiguous leukemias with T-lymphoid and myeloid features, co-occurring activating FLT3 mutations, and a stem/progenitor immunophenotype and gene expression profile. As with other lineage ambiguous leukemias, optimal treatment is unclear and there are limited targeted therapeutic options. Here, we investigated the efficacy of BCL-2 and FLT3 inhibition with venetoclax and gilteritinib, respectively, in preclinical models of BCL11B-a leukemia. Despite variation in response to single agent therapies, the combination of venetoclax plus gilteritinib (VenGilt) was highly effective in all models evaluated. BH3 profiling suggested that resistance to venetoclax monotherapy was due to the tumor-intrinsic dependence on additional BCL-2 family proteins prior to drug treatment. Longitudinal single cell RNA-seq analysis identified mitochondrial pathways and a pro-lymphoid gene expression signature as potential drivers of rare cell survival on VenGilt therapy. These data support clinical evaluation of venetoclax in combination with gilteritinib in BCL11B-a lineage ambiguous leukemias.

Integration of torque teno mini virus (TTMV) generating the TTMV::RARA fusion represents a newly recognized subtype of acute promyelocytic leukemia (APL) that merits detailed investigation. We present the first comprehensive characterization of its epidemiologic profile, clinical presentation, virologic characteristics, and underlying molecular mechanisms. Our findings indicate that TTMV::RARA is more prevalent in pediatric patients and represents the second most common retinoic acid receptor fusion after PML::RARA. Affected patients exhibit a high incidence of extramedullary involvement, particularly myeloid sarcoma. Cytogenetic abnormalities involving i(17)(q10) or 7q22 were identified in 52.0% of cases, largely in a mutually exclusive manner. Co-occurring mutations in epigenetic regulators were present in 76.9% of patients. Although most patients achieved initial remission, relapse was common and associated with rapid acquisition of all-trans retinoic acid (ATRA) resistant mutation and secondary chemoresistance. Venetoclax-containing regimens demonstrated encouraging clinical efficacy. Phylogenetic analysis indicated that patient-derived TTMV strains clustered into a distinct clade. TTMV integration consistently occurred within RARA intron 2, involving a consensus fragment of 510-610 bp encompassing the viral promoter and ORF2 N-terminus, likely mediated by microhomology-driven recombination. Tandem RUNX1-binding motifs within the integrated viral promoter may underlie the myelotropism of these TTMV strains and facilitate transcriptional activation of TTMV::RARA. The chimeric protein retains at least the first 56 N-terminal residues of ORF2 and remains transcriptionally responsive to pharmacological concentrations of ATRA. These findings establish TTMV::RARA-APL as a distinct leukemia entity, laying the foundation for future studies on virus-mediated leukemogenesis and therapeutic strategies.

Asparaginase-associated pancreatitis (AAP) is a significant complication in acute lymphoblastic leukemia (ALL) therapy, often leading to treatment delays or discontinuation. This study aimed to identify AAP risk factors, assess outcomes after first and second episodes, and evaluate the impact of asparaginase rechallenge. We retrospectively analyzed 7,640 patients (aged 1 month-18 years) treated under the Chinese Children Cancer Group ALL 2015 protocol. Patients were stratified as low-risk (LR), intermediate-risk (IR), or high-risk (HR) based on clinical features and measurable residual disease (MRD). AAP was categorized as early- or late-onset depending on treatment phase. Older age and IR/HR status were independent risk factors for AAP. The cumulative AAP incidence was 2.2% in LR and 5.8% in IR/HR groups. Among 298 patients who developed AAP, 92 were rechallenged with asparaginase; second episodes occurred in 20.8% of LR and 33.8% of IR/HR patients, with no increase in severity. Lack of rechallenge (HR, 2.0; 95% CI, 1.1-3.6) and Day 46 MRD ≥0.01% (HR, 3.7; 95% CI, 2.1-6.8) were independently associated with inferior event-free survival (EFS). Among patients with early-onset AAP, rechallenged patients had superior 5-year EFS compared to those not rechallenged (80.1% vs. 60.2%; P = .003). Similarly, among IR/HR group, rechallenged individuals and better 5-year EFS than those not rechallenged (82.4% vs. 60.6%; P = .004). IR/HR patients with early-onset AAP who were not rechallenged had especially poor outcomes (5-year EFS, 53.3%). These findings support considering asparaginase rechallenge in IR/HR patients with early-onset AAP when alternative therapies are limited. Chinese Clinical Trial Registry: ChiCTR2000032211.

The FDA recently licensed 14-day cold-stored platelets for bleeding patients. This policy change represents a reversal from the 1970s when cold-stored platelets were discontinued because of their short circulation time in healthy humans. This change will increase their availability in US hospitals with large trauma populations and in remote and rural settings in the U.S. In some of these hospitals, cold-stored platelets will be the only platelets available. It is currently unclear whether patients with hypoproliferative thrombocytopenia who need platelet transfusion for prophylaxis benefit from cold-stored platelets. However, it is noteworthy that in recent clinical trials using room temperature-stored platelets, the transfusion interval in hematology-oncology patients can be as short as one transfusion per day, very similar to what one would expect to achieve with cold-stored platelets. Furthermore, the emphasis on the post-transfusion count increment and the platelet count as transfusion trigger per se is questionable. In the PLADO trial, there was only a poor correlation between the morning platelet count and bleeding events, implicating other factors, such as red blood cells, coagulation factors, and vascular health, as possible culprits. In this perspective article, we review the history of cold platelets and the reason for their discontinuation, focus on recent clinical trial data using room temperature-stored platelets, and review the platelet count as a transfusion trigger. Overall, using cold platelets for prophylaxis may seem counterintuitive, but a closer look at the available data suggests that the indication expansion may hold more promise.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare but life-threatening condition in which maternal alloantibodies, generated during pregnancy, target human platelet antigens (HPAs) leading to thrombocytopenia and increased risk of bleedings in the fetus or neonate. The most clinically relevant antigen in people of European descent is HPA-1a, located on the integrin β3 subunit. The β3 integrins are conformationally regulated heterodimeric receptors including platelet integrin αIIbβ3, but also αvβ3, expressed strongly on endothelial cells. FNAIT is clinically highly heterogenous, with symptoms ranging from mild thrombocytopenia to intracranial hemorrhage (ICH) which can cause lifelong disabilities or perinatal death. It has been suggested that anti-HPA-1a antibodies that exclusively react with αvβ3 cause ICH, due to induction of endothelial cell damage and/or defects in angiogenesis. Here, we analyzed a large cohort of retrospectively and prospectively collected maternal sera from severe and mild FNAIT cases. Disease severity was associated with the extent of thrombocytopenia, and with high anti-HPA-1a antibody reactivity towards both αIIbβ3 and αvβ3. Exclusive anti-HPA-1a reactivity with αvβ3 or endothelial cells was not found. In contrast, all anti-HPA-1a antibodies reacted with platelets and endothelial cells, and with αvβ3- and αIIbβ3-transduced cells, but reacted generally more with the αIIbβ3 integrin. Furthermore, HPA-1a epitope accessibility and antibody binding is influenced by integrin conformation and activation status. Higher reactivity of anti-HPA-1a antibodies with αIIbβ3 over αvβ3 diminishes upon integrin conformational activation. Together, these data emphasize the need for further investigation into the relation between endothelial properties of anti-HPA-1a antibodies and disease outcome in FNAIT.