Immune effector cell-associated hematotoxicity (ICAHT) was recently introduced as a distinct toxicity category of CAR-T therapy. While a grading system based solely on neutrophil counts was proposed (hereafter termed N-ICAHT), the prevalence and prognostic impact of thrombocytopenia remains poorly defined. In this multicenter observational study, we systematically examined patterns of thrombocytopenia in 744 patients treated with commercial CD19 CAR-T for B-cell Non-Hodgkin Lymphoma (B-NHL). We developed a grading system termed T-ICAHT with thresholds that closely aligned with N-ICAHT - based on depth, duration and timing of thrombocytopenia. In the core NHL dataset, 43% of patients developed any-grade early T-ICAHT (day 0-30), with 23% developing severe (G3+) manifestations. Late T-ICAHT (day 31-100) was observed in 42% (G3+: 13%). While T-ICAHT and N-ICAHT gradings showed some correlation, considerable discordance was noted. On multivariate analysis, bridging therapy, poor performance status, and high Hematotox scores were associated with increased risk of severe early T-ICAHT. Patients with higher T-ICAHT grades showed increased platelet and red blood cell transfusion burden (p<0.001) and more bleeding events (p=0.01). T-ICAHT grades were inversely associated with overall survival (OS), with landmarked 2-year estimates ranging from 67% (G0), to 48% (G1-2) and 35% (G3+). In multivariable Cox regression analysis, the independent prognostic capacity of T-ICAHT for OS was confirmed. Finally, we validated T-ICAHT's clinical and prognostic utility in 3 external cohorts spanning an additional 599 pediatric and adult patients (NHL, MM, B-ALL), confirming its broad applicability. These findings support integrating T-ICAHT into the ICAHT framework to standardize thrombocytopenia grading in CAR-T recipients.

Hematopoietic stem cells (HSCs) are heterogeneous, and the quality of HSCs - that is, 'transplantability' - is a key determinant for posttransplant hematopoietic reconstitution. However, molecular modalities of high-potency HSCs with superior transplantability still remain poorly understood. Here, we conducted large-scale single-clone serial-transplant experiments and tracked descendant cells of 288 HSC clones to quantify their intrinsic capability for hematopoietic reconstitution. Using integrated single-cell transcriptional, immunophenotypical, and Bayesian dynamic analyses, we uncovered three classes of HSC clones - 'Super', 'Flash', and 'Trickle' - that had higher output in the 1st generation but exhibited markedly different behavior in later generations. The 'Super'-class HSC clones comprised 4% of the HSCs and manifested persistent superior transplantability and balanced myeloid/lymphoid lineage outputs across generations in serial transplants. The 'Super'-class HSCs had a unique molecular signature, including low expression of CD27, that was distinct from previously known 'Classical HSC' signatures. Validation experiments indicated that CD27- HSCs had superior transplantability compared to CD27+ HSCs. Our study asserted an operational definition for 'Super' transplantability of HSCs, defined its molecular program, and suggested new directions for enriching high-potency HSCs in grafts.

Chronic myeloid leukemia (CML) has served as a paradigm for the development of effective initial and next-generation targeted therapies. The availability of five effective and generally well-tolerated BCR::ABL1 tyrosine kinase inhibitors for the treatment of newly diagnosed chronic phase CML offers patients and their treating physicians a welcome luxury of choice. The long-term outlook for newly diagnosed chronic phase CML patients is excellent, with expected survival similar to age-matched controls. However, most patients are expected to require lifelong treatment. As a result, important considerations when choosing frontline treatment include not only treatment efficacy, but also response durability, tolerability, maximizing quality of life, avoidance of serious and irreversible toxicities, the ease of treatment administration and increasingly, the cost of treatment to the patient as well as to society.

Chronic granulomatous disease (CGD) is an inborn error of immunity caused by defects in any one of the 6 subunits (gp91phox, p47phox, p22phox, p67phox, p40phox or chaperone EROS) forming the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex 2 (NOX2) and resulting in defective phagocyte-derived reactive oxygen species (ROS) production. Almost 50% of patients with CGD have inflammatory bowel disease (IBD) associated with dysbiosis and age of IBD onset may vary according to CGD genotype. While we previously demonstrated that the intestinal microbiota determines colitis susceptibility in CGD mice, underlying mechanisms remain unknown. We hypothesized that NOX2 defects are associated with distinct intestinal microbiome signatures and immune responses, which impact colitis severity. Chemical colitis susceptibility was evaluated in 2 strains of CGD mice (gp91phox-/- and p47phox-/-) with distinct microbiota, from 2 different animal facilities, while also evaluating the impact of microbiota standardization and colitogenic microbiota transfer on mucosal immune responses at the intestinal barrier. While p47phox-/- and gp91phox-/-mice harbouring colitogenic microbiota had increased colitis severity, the intestinal epithelial cells from p47phox-/- mice produced more ROS which was associated with increased NOX isoform gene expression. In contrast, gp91phox-/- mice had decreased mucin production and a mucosal immune response profile suggestive of increased inflammasome activation at the intestinal barrier compared to control and p47phox-/- mice. Our findings suggest that the microbiota impacts colitis susceptibility in a CGD genotype-specific manner, thereby potentially explaining differences in the timing of IBD onset in patients with different CGD genotypes while identifying potential novel and personalized therapeutic targets.

Richter transformation (RT) is defined as an aggressive lymphoma emerging in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL). Despite novel therapeutics developed in CLL, RT is associated with poor outcomes. In light of recent progress regarding the diagnostic procedures and therapeutic concepts of RT, an international group of experts, under the coordination of the European Research Initiative on CLL (ERIC), has developed consensus recommendations for clinical procedures and future research on this disease. Patients with RT typically present with a rapid clinical decline, worsening B-symptoms, elevated LDH, and/or rapidly enlarging lymphadenopathy. Workup should include a PET-CT for patients with suspected RT. An excisional biopsy should be taken from an accessible lesion, preferably with the highest FDG avidity, and analyzed for the presence of aggressive lymphoma. The molecular relationship to the original CLL clone(s) should be defined. As no effective standard treatment for RT exists, patients should be treated in a clinical trial. Response of both RT and CLL should be assessed at an early time point, and survival endpoints should be prioritized in trial design. We hope that these recommendations can help to harmonize clinical and translational research and improve outcomes for patients with RT.

This study reports outcomes of PLAT-02, a phase 2 trial of SCRI-CAR19, a second-generation CAR T-cell product with FMC63 scFv and 41BB costimulation, in pediatric and young adult patients with B-cell acute lymphoblastic leukemia; and PLAT-03, a companion study evaluating exogenous CD19 antigen stimulation with serial infusions of T cells expressing truncated CD19, T-cell antigen presenting cells (T-APCs). The efficacy cohort of PLAT-02 (n=72 patients, median age 12.5 years) received fludarabine/cyclophosphamide lymphodepletion followed by a dose of 1X106 CAR+ T cells/kg. MRD-negative complete remission rate was 89%. Leukemia free survival (LFS) with 95% CI at 1 and 2 years was 0.71 (0.58, 0.81) and 0.64 (0.51, 0.75). Patients with low disease burden had significantly higher 1-year LFS (0.91 vs. 0.42). Rapid in vivo contraction of CAR T cells after infusion was associated with CAR loss within six months compared to those without rapid contraction (57% vs. 19%, respectively). Most common grade 3/4 adverse events included cytokine release syndrome in 13% and neurotoxicity in 16%. The companion pilot, PLAT-03, enrolled 26 patients, and 19 received T-APCs. Neither cytokine-release syndrome nor neurotoxicity were observed after T-APC infusion. T-APC infusion in patients improved persistence (P=0.03) with rapid CAR T-cell contraction was associated with decreased early CAR loss (20% with T-APC vs. 57% without). Further exploration of serial artificial CD19 antigen exposure is warranted based on these pilot results. PLAT-02 (NCT02028455) and PLAT-03 (NCT03186118).

Breakthrough hemolysis (BTH) is the hemolytic exacerbation occurring in a patient with paroxysmal nocturnal hemoglobinuria (PNH) on treatment with anti-complement therapies. In this review article we analysed the definition, frequency and severity of BTH events across phase 3 clinical trials with terminal (anti-C5 ravulizumab and crovalimab) and complement inhibitors upstream C5 (anti-C3 pegcetacoplan, alternative-pathway inhibitors iptacopan and danicopan), as well as from real-world reports. Furthermore, we reviewed the impact of the various compounds on quality of life and patients reported outcomes. In particular, BTH may occur with all complement inhibitors, with a frequency of 10-15% over 6 months with eculizumab, crovalimab, and pegcetacoplan, and <5% with ravulizumab, iptacopan, and danicopan plus anti-C5. By prolonging the follow-up, the frequency of BTH appeared increased in pegcetacoplan treated patients (nearly 24% at 1 year) as compared to both anti-C5, iptacopan, and double inhibition with danicopan plus anti-C5. BTH risk appears associated with patients' features, particularly suboptimal response/failure of previous complement inhibitor. Transfusions were required in about half of cases and modifications of anti-complement therapy included anticipation of the next anti-C5 dose and addition of eculizumab in patients on proximal inhibitors. Breakthrough thromboses were rare, though anti-coagulant prophylaxis should be considered during severe episodes. Complement amplifying conditions were observed in about half of cases and were more frequently infections. Treatment adherence, optimization of the administration schedule, anticoagulant prophylaxis, as well as education of patients and physicians remain important factors to prevent BTH and its complications.

Patients with acute myeloid leukemia (AML) ineligible for intensive chemotherapy (IC) have limited treatment options. The phase 3 ENHANCE-3 study aimed to determine whether magrolimab (magrolimab arm) was superior to placebo (control arm) when either was combined with venetoclax and azacitidine. Adults with previously untreated AML who were ineligible for IC were randomized to receive magrolimab (1 mg/kg on days 1 and 4, 15 mg/kg on day 8, 30 mg/kg on days 11 and 15, then weekly for 5 weeks, then every 2 weeks) or placebo, venetoclax (100 mg on day 1, 200 mg on day 2, and 400 mg daily thereafter), and azacitidine (75 mg/m2 days 1-7) in 28-day cycles. The primary endpoint was overall survival (OS); key secondary endpoints included complete remission (CR) rate and safety. After randomization of 378 patients, the trial was stopped at a prespecified interim analysis due to futility. At final analysis, with median follow-up of 7.6 months (magrolimab arm) vs 7.4 months (control arm), median OS was 10.7 vs 14.1 months (HR, 1.178 [95% CI, 0.848-1.637]). The CR rate within 6 cycles was 41.3% vs 46.0%. Addition of magrolimab to venetoclax and azacitidine resulted in more fatal adverse events (19.0% vs 11.4%), primarily driven by grade 5 infections (11.1% vs 6.5%) and respiratory events (2.6% vs 0%). There were similar incidences of any-grade infections, febrile neutropenia, and neutropenia between arms. These results highlight the difficulty in improving outcomes for patients with AML ineligible for IC. This trial was registered at www.clinicaltrials.gov as #NCT05079230.

DNMT3A mutations in polycythemia vera (PV) patients were heterogenous and not enriched in interferon-alpha (IFN)-treated patients. DNMT3A mutations had no detectable impact on hematologic response, molecular response or survival outcomes.

The antenatal role of the hepcidin-regulating protease Tmprss6 has never been elucidated as knockout dams are infertile. Utilising an in vivo knockdown approach, we confirm Tmprsss6 is critical for hepcidin suppression in pregnancy, and Tmprss6 inhibition drives deleterious fetal outcomes.

Hereditary stomatocytosis represents a heterogeneous group of inherited erythrocyte membrane defects characterized by hemolytic anemia of variable degree, with alterations in cellular salt and water, ranging from dehydration to overhydration, and the presence of stomatocytes on peripheral blood smear. This condition encompasses various subtypes, each with distinct clinical and genetic features. The pathophysiology underlying these conditions involves altered red blood cell membrane properties, leading to impaired deformability, alterations in cation permeability and volume, causing increased susceptibility to hemolysis. Advancements in genetic testing have enabled the identification of some causative genes in the last years, such as PIEZO1, KCNN4, and ABCB6. These genetic discoveries have facilitated a deeper understanding of the molecular mechanisms underlying the pathogenesis and have paved the way for improved diagnostic accuracy and genetic counseling. This review provides an overview of the clinical presentation, pathophysiology, molecular genetics, diagnosis, and management strategies of hereditary stomatocytosis, highlighting recent advancements in the field of dehydrated hereditary stomatocytosis, or hereditary xerocytosis, and hepatic iron overload. This latter is directly associated with the physiological role of PIEZO1, the causative gene of DHS, at hepatic and macrophagic levels. Particularly, gain-of-function mutations in PIEZO1 account for a pleiotropic syndrome characterized by different phenotypes depending on the expression of PIEZO1 at multiple cells and tissues.

Hematopoietic Stem Cells (HSCs) possess the ability to long-term reconstitute all the blood lineages and generate all blood cell types. As such, the in vitro generation of HSCs remains a central goal in regenerative medicine. Despite many efforts and recent advancements in the field, there is still no robust, reproducible and efficient protocol for generating bona-fide HSCs in vitro. This suggests that certain regulatory elements have yet to be uncovered. Here, we present a novel and unbiased approach to identifying endogenous components to specify HSCs from pluripotent stem cells. We performed a genome-wide CRISPR activator screening during mesodermal differentiation from mouse embryonic stem cells (mESCs). Following in vitro differentiation, mesodermal KDR+ precursors were transplanted into primary and secondary immunodeficient NSG mice. This approach led to the identification of seven genes (Spata2, Aass, Dctd, Eif4enif1, Guca1a, Eya2, Net1) that, when activated during mesoderm specification, induce the generation of hematopoietic stem and progenitor cells (HSPCs). These cells are capable of serial engraftment and multilineage output (erythroid, myeloid and T and B lymphoid) in vivo. Single-cell RNA sequencing further revealed that activating these seven genes biases the embryoid bodies towards intraembryonic development, instead of extraembryonic, increasing the number of mesodermal progenitors that can generate HSCs. Our findings underscore the importance of differentiation during the first germ layer specification to generate definitive blood stem cells.

Targeted therapy with covalent Bruton tyrosine kinase inhibitors (cBTKi) and/or the B-cell lymphoma 2 (BCL-2) inhibitor venetoclax is now well-established in the first-line management of chronic lymphocytic leukemia (CLL). However, patients with 'double refractory' disease due to the acquired resistance to both drug classes represent an increasing clinical challenge for whom few well-tolerated and effective treatment options currently exist. The highly selective, non-covalent BTKi pirtobrutinib and CD19-directed chimeric antigen receptor T-cell therapy lisocabtagene maraleucel have both recently gained FDA approval for use in patients with CLL which has progressed following ≥2 prior lines, including a cBTKi and a BCL-2i. Additionally, novel BTK-directed therapies and T-cell engaging bispecific antibodies have achieved promising responses in pre-treated CLL in early phase clinical trials. Here, we review the mechanisms responsible for resistance to cBTKi and venetoclax in CLL, appraise recent evidence supporting the use of each of the novel and emerging agent classes and then suggest innovative treatment strategies incorporating these in patients with double-refractory disease, remaining cognizant of the variability of access to novel therapies and clinical trials.