Higher CD34 Research Articles

Abstract 4123674: The role of CD34+ PI16+ fibroblast progenitor cells in transplant arteriosclerosis

Background: Transplantation arteriosclerosis is one of the major complications for the mid- and long-term survival of recipients with organ transplantation. Although previous studies suggest that CD34 stem/progenitor cells are involved in this process, the heterogeneity and potential adverse effects of CD34 + SPCs have not been fully determined. Methods and Results: A mouse model of transplant arteriosclerosis was established by using Balb/c, C57BL/6J, CD34-CreERT2, R26-tdTomato, Rosa26-iDTR, CD34-Dre, PI16-CreERT2, CAG-LSL-RSR-tdTomato-2A-DTR mice. Using single-cell RNA-seq and the innovative application of genetic lineage tracing with dual recombinases, we identified a subpopulation of fibroblast progenitor cells marked by high CD34 and PI16 expression. Interestingly, this progenitor cell group gave rise to a distinct chemotactic fibroblast subset. Based on chemokine antibody microarray assay and transcriptome analysis, the subgroup with increased CD34 expression and decreased PI16 expression promoted intimal hyperplasia, acting through the paracrine release of a specific chemokine CCL11 (eotaxin-1). Moreover, the binding of CCL11 to its receptor CCR3 triggered the PI3K-AKT signaling pathway in smooth muscle cells, promoting their proliferation and migration to form neointimal lesions. Overexpression of CCL11 by adeno-associated virus can promote neointimal hyperplasia in vivo, while neutralization of CCL11 or inhibition of receptor CCR3 can alleviate neointimal lesions. Conclusion: Our findings identified CD34 + /PI16 + fibroblast progenitors able to differentiate into specific chemotactic fibroblasts that release chemokines pivotal for neointima formation, implicating a therapeutic potential targeting the chemotactic behavior of these cells.

Optimal infused CD34+ cell dose in multiple myeloma patients undergoing upfront autologous hematopoietic stem cell transplantation

Autologous transplantation remains the standard of care for eligible multiple myeloma (MM) patients, yet optimal CD34+ cell dose remains unclear. We conducted a retrospective study on MM patients undergoing upfront transplant between 2005 and 2021 and divided them into low (≤2.5 × 106 cells/kg) and high (>2.5 × 106 cells/kg) CD34+ dose groups. We included 2479 patients, 95 in the low CD34+ group and 2384 in the high CD34+ group. Patients in the low CD34+ group were older (63.2 vs 61.1 years, p = 0.013), more often had R-ISS III (19% vs 9%, p = 0.014), received plerixafor (60% vs 35%, p < 0.001) and transplanted after 2009 (88% vs 80%, p = 0.047). Time to neutrophil and platelet recovery was longer in the low CD34+ group. Median PFS and OS were lower in the low CD34+ group (31.6 vs. 43.6 months, p = 0.011 and 76.4 vs. 108.2 months, p < 0.001, respectively). Evaluation of incrementally higher CD34+ dose did not show significant improvement in survival at thresholds >2.5 × 106 cells/kg. Multivariable analysis affirmed that CD34+ >2.5 × 106 cells/kg was associated with better PFS (HR 0.71, p = 0.008) and OS (0.59, p < 0.001). After propensity score matching, a CD34+ dose >2.5 × 106 cells/kg remained a predictor of better OS (0.42, p < 0.001). In conclusion, CD34+ dose >2.5 × 106 cells/kg was associated with improved survival, without any additional benefit at incrementally higher doses.

Why are Higher CD34+ Cell Doses Associated with Improved Outcomes among Pediatric Patients Undergoing Autologous Hematopoietic Stem Cell Transplant for Central Nervous System Tumors – But Not for High-Risk Neuroblastoma?

Why are Higher CD34+ Cell Doses Associated with Improved Outcomes among Pediatric Patients Undergoing Autologous Hematopoietic Stem Cell Transplant for Central Nervous System Tumors – But Not for High-Risk Neuroblastoma?

Post-transplantation monitoring and quantitation of microparticles in allogeneic hematopoietic cell transplantation

BackgroundAllogeneic hematopoietic stem cell transplantation (allo-HCT) represents a curative treatment for various blood-related disorders, including hematological malignancies and genetic disorders. The success of this procedure hinges on the efficacy of the conditioning regimen and the graft's ability to engraft and function properly. Microparticles (MPs), small vesicles produced from stimulated, apoptotic, or activated cells, are involved in both physiological and pathological processes. However, the impact of MPs on allo-HCT remains poorly understood. ObjectivesThis study aimed to investigate the presence of MPs from different cell types in grafts and patient plasma after allo-HCT, as well as their association with various parameters. We measured MPs from CD34+, CD56+, CD3+, CD19+, and CD33+ cells in grafts and patient plasma from day 0 to day 60 after transplantation. Methods224 blood samples were collected from 19 consecutive allo –HCT recipients at 0, +4, +14,+30 and + 60 day as well as from their grafts. MPs isolated from the plasma and quantified by flow cytometry analysis. ResultsMP levels varied over time. Notably, CD34+ MP levels were linked to both early and late engraftment of neutrophils and platelets. Furthermore, grafts with high CD34+ and CD56+ MP levels in patient plasma on days 0 and + 4 were associated with late engraftment, whereas high CD33+ MP levels in both graft and patient plasma on day +4 were associated with early engraftment. Conditioning regimen affected CD19+ MP levels at day +14, and the number of CD34+, CD56+, and CD19+ MPs 30 days after transplantation was correlated with acute graft-versus-host disease. ConclusionThese findings suggest that MPs derived from hematopoietic cells may play a significant role in the clinical course of patients following allo-HCT.

Changes in hematopoietic stem cell numbers following acute exercise in non-athlete marathon runners

PurposeHematopoietic stem cell (HSC) transplant is one of the curative methods for some patients with hematological malignancies. Granulocyte colony-stimulating factor (G-CSF) is the most common drug used to mobilize CD34+ cells, generally found in small numbers. Recent evidence showed that exercise causes transient mobilization in HSC. However, the type and intensity of exercise have not been fully revealed. We aimed to detect a significant increase in stem cell levels following 60 ​min of running at a personalized running pace. Materials/methodsEighteen runners, 48.2 ​± ​1.9 years with peak oxygen consumption of 46.2 ​± ​1.4 ​ml/kg/min, were enrolled in the study. The cardiopulmonary exercise test was performed to determine the individual running pace, and the participants ran 60-min on a treadmill at an intensity close to their ventilatory threshold (VT). The blood sampling for HSC count was performed before, immediately after, at the 1st, 4th and 24th hour after the 60-min running. ResultsThe CD34+ HSCs were 13.9 ​± ​2.3 ​cells/μl before and significantly increased immediately after to 19.5 ​± ​3.6 ​cells/μl (p ​< ​0.05). The consecutive HSC counts were 15.3 ​± ​2.2, 19.5 ​± ​4.8 and 15.1 ​± ​3.4 ​cells/μl at the 1st, 4th, and 24th hour, respectively. ConclusionThe individual data showed that some runners had higher HSC levels than the transplantation limit before and after the 60-min running trail, which was maintained for 24 ​h. Pre-running high CD34+ HSCs may reflect an adaptive response to regular exercise, with a 60-min run near the VT further elevating HSCs. Individualized exercise may be a valuable tool to mobilize the CD34+ HSCs in peripheral blood for donors.

The role of the primitive marker CD133 in CD34-negative acute myeloid leukemia for the detection of leukemia stem cells.

The most important reason for dismal outcomes in acute myeloid leukemia (AML) is the development of relapse. Leukemia stem cells (LSCs) are hypothesized to initiate relapse, and high CD34+CD38- LSC load is associated with poor prognosis. In 10% of AML patients, CD34 is not or is low expressed on the leukemic cells (<1%), and CD34+CD38- LSCs are absent. These patients are classified as CD34-negative. We aimed to determine whether the primitive marker CD133 can detect LSCs in CD34-negative AML. We retrospectively quantified 148 CD34-negative patients for proportions of CD34-CD133+ and CD133+CD38- cell fractions in the diagnostic samples of CD34-negative patients in the HOVON102 and HOVON132 trials. No prognostic difference was found between patients with high or low proportions of CD34-CD133+, which is found to be aberrantly expressed in AML. A high level of CD133+CD38- cells was not associated with poor overall survival, and expression in AML was similar to normal bone marrow. To conclude, CD133 is useful as an additional primitive marker for the detection of leukemic blast cells in CD34-negative AML. However, CD133+CD38 alone is not suitable for the detection of LSCs at diagnosis.

Mobilization strategies with and without plerixafor for autologous stem cell transplant in patients with multiple myeloma.

Autologous stem cell transplantation is a standard treatment strategy for patients with multiple myeloma that requires effective mobilization and apheresis of peripheral blood progenitor cells; however, in the current era of novel myeloma induction therapies, the optimal mobilization regimen to enhance stem cell yield while limiting toxicity and resource utilization remains unknown. In this multicenter retrospective study, we assessed apheresis and transplant outcomes in myeloma patients mobilized with granulocyte colony stimulating factor (G-CSF) alone (n = 62), G-CSF with chemotherapy (n = 43), or G-CSF with the CXCR4 antagonist plerixafor (n = 417). Compared to patients treated with G-CSF alone, the plerixafor group required significantly fewer median apheresis sessions (1 vs 2, p = 0.0023) with higher CD34+ stem cell yield (9.9 vs 5.8 × 106 cells/kg, p < 0.001) and had significantly faster engraftment of neutrophils (HR 1.54, 95% CI 1.17-2.03) and platelets (HR 2.24, 95% CI 1.69-2.96) after transplant. Additionally, the plerixafor group showed a significantly better toxicity profile and lower adverse event rate than patients treated with G-CSF alone (p = 0.0028) or chemomobilization (p < 0.0001), with a trend toward reduced survival in chemomobilization patients. Taken together, these data support the routine use of plerixafor-based mobilization to increase apheresis efficiency and reduce toxicity in myeloma patients undergoing transplant.

High CD34-positive cell dose in matched unrelated donor allogeneic hematopoietic stem cell transplant is not associated with graft-versus-host disease or mortality.

CD34+ stem cells serve as the primary graft source for allogeneic transplants, with a minimum of 2-4 × 106 cells/kg needed for engraftment. There are conflicting data on outcomes at high stem cell doses, with studies limited by few patients receiving doses far above the minimum target. In this retrospective, single-center study of patients with hematologic malignancies who underwent matched unrelated donor transplants, we assessed outcomes for engraftment, survival, relapse, and graft-versus-host disease (GVHD) for the highest CD34+ dose quintile (>13 × 106 cells/kg, n = 36) compared to the remaining patients (n = 139). Similar analysis was performed correlating T cell dose and outcomes. There was no difference between the groups in neutrophil engraftment, with a trend toward faster platelet engraftment. There was no significant difference in mortality (adjusted risk ratio [aRR] = 1.02, 95% confidence interval [CI] = 0.85-1.22), relapse (aRR = 1.10, 95% CI = 0.85-1.42), or overall survival by Kaplan-Meier analysis (p = .44). High CD34+ dose was not associated with higher incidence of acute GVHD (aRR = 0.99 grades II-IV, aRR = 1.18 grades III-IV) or chronic GVHD (aRR = 0.87 overall, RR = 1.21 severe). There was limited correlation between CD34+ and T cell dose (R2 = .073), and there was no significant difference in survival, relapse, or GVHD in the highest T cell dose quintile (n = 33) compared to the remaining quintiles (n = 132). We found no difference in survival, relapse, or GVHD incidence or severity in patients receiving CD34+ doses above prior cutoffs reported in the literature. These data do not support the routine use of graft CD34+ dose reduction.

Efficacy of prophylactic antibiotics for the prevention of neutropenic fever in patients with multiple myeloma receiving high-dose cyclophosphamide for stem cell mobilization

High-dose cyclophosphamide (HD-Cy) (3 g/m2) plus granulocyte colony-stimulating factor (G-CSF) is a very effective regimen for peripheral blood stem cell (PBSC) mobilization. Unfortunately, it is associated with an increased risk of neutropenic fever (NF). We analyzed the effect of NF on PBSC apheresis results and the efficacy of prophylactic antibiotics for the prevention of NF associated with HD-Cy plus G-CSF for PBSC mobilization in patients with newly diagnosed multiple myeloma (MM). First, patients were divided into NF ( +) and NF ( −) groups according to whether they suffered from NF during mobilization. Second, we divided patients into an antibiotic prophylaxis group and a nonantibiotic prophylaxis group according to whether antibiotic prophylaxis was used during the mobilization period. Our study showed that NF( +) patients (n = 44) had lower CD34 + cell dose collection (median 2.60 versus 5.34 × 106/kg, P < 0.001) and slower neutrophil engraftment and platelet engraftment (median 11 versus 10 days, P = 0.002, and median 13 versus 11 days, P = 0.043, respectively) than NF( −) patients (n = 234). Of note, the nonantibiotic prophylaxis group patients (n = 30) had a 26.7% incidence of NF. In the patients receiving antibiotic prophylaxis (n = 227), the incidence was reduced to 9.3% (P = 0.01). The antibiotic prophylaxis patients had higher CD34 + cell collection (median 5.41 versus 2.27 × 106/kg, P < 0.001) and lower hospitalization cost of mobilization ($ median 3108.02 versus 3702.39, p = 0.012). Thus, our results demonstrate that NF is associated with lower CD34 + cell collection and that antibiotic prophylaxis can reduce the incidence of NF and improve stem cell mobilization and collection outcomes, which reduces the hospitalization cost of mobilization.

Relationship between the cytokine profle of supernatants of invasive breast carcinoma and its molecular and histopathological characteristics

The aim of the study was to analyze the correlation between the cytokine profile of supernatants of invasive breast carcinoma of a nonspecific type (IBC-NST) samples, histopathological and molecular genetic parameters of IBC-NST, expression of the CD34 as a marker of angiogenesis and metastasis to regional lymph nodes (RLN).Material and Methods. The production of 14 cytokines in IBC-NST biopsy samples from 28 patients aged 37–60 years was studied. The concentration of cytokines in the supernatants of biopsies (CCSB) was determined (in pg/ml) using enzyme immunoassay (ELISA). The expression of CD34 and markers of IBC-NST molecular subtypes (HER2/neu, ER, PR, Ki67) in IBC-NST biopsy samples was evaluated by immunohistochemical method. The relative content of tumor cells of different differentiation grade in the IBC- NST samples was evaluated by histopathological analysis.Results. The assessment of CCSB showed statistically significant differences in IFN-γ, G-CSF, IL-2, IL-10 and MCP-1 between patients of group I (with metastases in RLNs) and group II (without metastases in RLNs). In group I, the correlations between histopathological parameters (Her2/neu, CD34 and Ki67 expressions, % of mitoses and poorly-differentiated cancer cells) and CCSB (MCP-1, IL-18) were revealed. In group II, the correlations between CCSB (IL-2, VEGF-A, G-CSF, IL-1Ra) and histopathological parameters, such as expression of Her2/neu, CD34, PR, % of mitoses and well-differentiated cancer cells, were revealed. The ROC analysis showed that the presence or absence of metastases in RLNs can be predicted on the basis of CD34 expression levels and concentrations of IL-10, G-CSF, and MCP-1 in supernatants of IBC-NST biopsy samples. The quality of the model for stratifying patients into groups with and without RLN metastases, based on the assessment of the concentration of MCP-1 in the supernatants of IBC-NST biopsies, reached maximum values (AUC=1.000) with relatively high CD34 expression.Conclusion. The analysis of the data obtained showed that the assessment of CD34 expression and production of cytokines in IBC-NST biopsies is important for predicting the presence or absence of metastases in RLNs.

Development of an umbilical cord blood transplantation–specific nonrelapse mortality risk assessment score

AbstractHigher rate of nonrelapse mortality (NRM) remains yet to be resolved in umbilical cord blood transplantation (UCBT). Considering that UCBT has some unique features compared with allogeneic hematopoietic cell transplantation from other graft sources, a UCBT-specific NRM risk assessment system is required. Thus, in this study, we sought to develop a UCBT-specific NRM Risk Assessment (CoBRA) score. Using a nationwide registry database, we retrospectively analyzed 4437 recipients who had received their first single-unit UCBT. Using the backward elimination method, we constructed the CoBRA score in a training cohort (n = 2687), which consisted of recipients age ≥55 years (score 2), hematopoietic cell transplantation–specific comorbidity index ≥3 (score 2), male recipient, graft-versus-host disease prophylaxis other than tacrolimus in combination with methotrexate, performance status (PS) 2 to 4, HLA allele mismatch ≥ 2, refined Disease Risk Index high risk, myeloablative conditioning, and CD34+ cell doses < 0.82 × 105/kg (score 1 in each). The recipients were categorized into 3 groups: low (0-4 points), intermediate (5-7 points), and high (8-11 points) groups according to the CoBRA score. In the validation cohort (n = 1750), the cumulative incidence of NRM at 2 years was 14.9%, 25.5%, and 47.1% (P < .001), and 2-year overall survival (OS) was 74.2%, 52.7%, and 26.3% (P < .001) in the low, intermediate, and high groups, respectively. In summary, the CoBRA score could predict the NRM risk as well as OS after UCBT. Further external validation will be needed to confirm the significance of the CoBRA score.

The Correlation between VEGF and CD34 Protein Marker and Pyogenic Granuloma.

A typical non-neoplastic connective tissue proliferations called a pyogenic granuloma. A vascular adhesion molecule used to assess angiogenesis is the CD34 marker. The primary memberof a family of growth factors, VEGF helps in generating and maintaining the lymphatic and blood circulation systems. The aim of the study was to know the correlation between VEGF and CD34 protein marker and pyogenic granuloma. Thirty-one formalin fixed paraffin embedded (FFPE) blocks were taken from female pyogenic granuloma patients ranging in age from 29 to 70. The IHC was used to identify VEGF and CD34 expression in the cytoplasm of the cells. Seventeenout of 31 patients had VEGF positive expression. Twenty-sixout of 31 had CD34 positive expression and 5 with no expression (negative expression). Brown-stained cytoplasm showed high VEGF and CD34 expression, whereas blue stained cytoplasm showed no VEGF and CD34 expression in these cells. The results suggest the role of suchbiomarkers in the oral pyogenic granuloma pathogenesis, and it appears that CD34 and VEGF are valuable biomarkers in evaluating vascular and inflammatory diseases like pyogenic granuloma.

Osimertinib Covalently Binds to CD34 and Eliminates Myeloid Leukemia Stem/Progenitor Cells.

Osimertinib binds CD34 and selectively kills CD34+ leukemia cells to induce remission in preclinical models and patients with AML with a high percentage of CD34+ blasts, providing therapeutic options for myeloid leukemia patients.

Higher CD34+ Cell Doses Correlate with Reduced Incidence of Relapse and Better Event-Free Survival after KIR-Ligand Mismatch Cord Blood Transplantation for Childhood Acute Myeloid Leukemia

Introduction In children with acute myeloid leukemia (AML), hematopoietic stem cell transplantation (HSCT) is an important treatment, and cord blood units from unrelated donors provide a well-established source of stem cells for HSCT. The main advantages of using cord blood include rapid availability, allowance of greater HLA disparities due to a relatively low risk of severe graft-versus-host disease (GVHD), and possibly a greater graft-versus-leukemia (GVL) effect. Killer cell immunoglobulin-like receptors (KIRs) are the main receptors on natural killer cells that play an important role in GVL after HSCT. Previous studies on the effects of KIR-ligand (KIR-L) incompatibility in unrelated cord blood transplantation (CBT) for leukemia have resulted in conflicting results, and in some studies, this has led to an increased incidence of acute GVHD. Furthermore, the impact of KIR-L mismatch on outcomes in children with AML remains poorly understood. In this study, we explored the association of KIR-L incompatibility and other clinical factors with patient outcomes in children with AML who received CBT. Methods Data were collected from the Transplant Registry Unified Management Program sponsored by the Japanese Society for Transplantation and Cellular Therapy and the Japanese Data Center for Hematopoietic Cell Transplantation. The patients were selected according to the following criteria: (1) de novo non-M3 AML, (2) age &amp;lt; 16 years and CR1 or CR2 at the time of receiving HSCT, (3) no prior HSCT, and (4) HSCT performed between 2000 and 2021. Patients without information on survival and disease recurrence or those with Down syndrome were excluded. KIR-L mismatch in the graft-versus-host direction was defined as lacking a donor KIR-L group (HLA-C1, C2, Bw4, or A3/A11) in a recipient. A myeloablative conditioning regimen was defined as total-body irradiation (TBI) at &amp;gt; 8 Gy, melphalan at &amp;gt; 140 mg/m 2, or busulfan at ≥ 9 mg/kg. All other regimens were analyzed as reduced-intensity conditioning regimens. Adverse cytogenetics were defined as previously described [Creutzig et al. Blood, 2012]. Results A total of 299 patients were included. The median age at HSCT was 5 years (range, 0-15), and the median follow-up period for survivors was 7.2 years (range, 0.1-22.4). The median total nucleated cell and CD34+ cell doses were 6.7 x 10 7/kg (range 0.01-12.3) and 1.9 x 10 5/kg (range 0.01-59.4), respectively. The study cohort consisted of 240 patients in the KIR-L match group and 59 patients in the KIR-L mismatch group. The background characteristics of these two groups did not differ significantly, except for a trend indicating that the KIR-L mismatch group received CBT more recently ( p = 0.063). The cumulative incidence rates of neutrophil recovery at Day 42 were 93.3% (95% CI, 89.3-95.9%) and 91.5% (95% CI, 80.0-96.5%) ( p = 0.980), and the cumulative incidence rates of platelet engraftment at 6 months were 91.5% (95% CI, 87.1-94.5%) and 89.8% (95% CI, 77.9-95.5%) ( p = 0.314) for the KIR-L match and mismatch groups, respectively. The cumulative incidence rates of grade II-IV acute GVHD at Day 100 were 40.9% (95% CI, 34.6-47.1%) and 40.7% (95% CI, 28.0-52.9%) ( p = 0.709), whereas those of chronic GVHD at one year after transplantation were 15.6% (95% CI, 11.2-20.6%) and 15.9% (95% CI, 7.8-26.6%) ( p = 0.914) for the KIR-L match and mismatch groups, respectively. In univariate analyses, the 5-year event-free survival (EFS) was 70.1% (95% CI, 63.6-75.6%) for the KIR-L match group and 72.9% (95% CI, 59.0-82.8%) for the KIR-L mismatch group ( p = 0.609). Stratification by CD34+ cell doses showed a significant correlation between higher CD34+ cell doses and better EFS in the KIR-L mismatch group (Figure 1), attributed not only to reduced nonrelapse mortality but also to a lower incidence of relapse. In multivariate analysis of EFS, KIR-L mismatch with high CD34+ cell doses (&amp;gt; the median doses) was identified as an independent favorable prognostic factor (Table 1; hazard ratio = 0.21, p = 0.037). Conclusion Our study demonstrates that higher CD34+ cell doses were associated with a lower relapse rate and better EFS in KIR-L mismatch CBT for childhood AML. This finding is noteworthy, as previous literature has primarily emphasized the role of higher CD34+ cell doses in reducing nonrelapse mortality. Our results suggest that higher CD34+ cell doses are also crucial for achieving a high GVL effect in the context of KIR-L mismatch CBT.

Α-Globin Lentiviral Vectors for Hematopoietic Stem Cell Gene Therapy of α-Thalassemia

α-thalassemia is an inherited blood disorder caused by mutations in α-globin genes (HBA1 and HBA2) resulting in the reduction of α-globin chains, the subunit, along with β-globin chain, constituting adult hemoglobin (α 2β 2). While a reduction of α-globin expression of 40% presents asymptomatic phenotypes, expression levels decreasing to &amp;lt;20% or an α/β-globin mRNA ratio of &amp;lt;0.2, causes severe anima. Treatment for severe cases of α-thalassemia entails lifelong, biweekly blood transfusions with daily chelation therapy. While these therapies enable patients to live into mid- to late-adulthood, they continue to engender serious clinical manifestations, such as iron toxicity. To answer this clinical need, our laboratory has developed a stem cell gene therapy in which functional copies of α-globin gene integrate into the genome of patient's hematopoietic and progenitor stem cells (HSPCs) by lentiviral vectors (LVs) to normalize the globin chain imbalance and restore hemoglobin function in derived red blood cells (RBCs). The design of our α-globin LVs (AGLVs) is based on GLOBE β-globin LV utilized in a clinical trial for β-thalassemia (NCT02453477), which has achieved transfusion reduction and independence in patients with transfusion-dependence β-thalassemia major. We have constructed a series of short proviral length AGLVs for optimized titer production, HSPC infectivity, and lineage-specific α-globin gene expression. Twelve AGLVs varying in gene and regulatory element compositions were constructed and assessed for raw titer yields and characterized for gene transfer efficiency, mRNA expression and hemoglobin production in a α-globin knockout (KO) human erythroid cell line. Successfully, all AGLVs confer high raw titers ~ 1e7 TU/mL and enabled the production of adult hemoglobin (HbA). We identified two optimal AGLVs for further characterization in human primary HSPCs: 1) A2-UV for yielding highest raw titers and gene transfer efficiency, and 2) A2-Globe for producing the most α-globin mRNA. Both A2-UV and A2-Globe LVsharbor HBA2, are regulated by the β-globin promoter and enhanced by the core or large β-LCR enhancer region, respectively. To assess candidate AGLVs in human HSPCs, AGLVs were tagged at the transcription level to enable identification and quantification of vector-derived α-globin mRNA proceeding HSPCs transduction anderythroid differentiation. A2-UV demonstrated optimum CD34 + infectivity and gene transfer, and A2-Globe expressed a high ~30% of α-globin mRNA per total β-globin per vector copy number (VCN), achieving levels of one endogenous α-globin gene (~25% per total β-globin). Current studies examined A2-UV and A2-Globe in α-thalassemia major patient HSPCs - lacking all four α-globin genes - harboring a lethal homozygous 20kb deletion of HBA1 and HBA2, known as SEA –/– mutation. Patient HSPCs were transduced and differentiated into RBCs. A high CD34+ infectivity and gene transfer was obtained by both candidate vectors, without utilization of transduction enhancers. Furthermore, a correction of α/β-globin mRNA ratio of ~0.7 was obtained while retaining a low VCN (&amp;lt;3).RBCs were then analyzed at the protein level . Chromatograms of hemoglobin tetramers revealed that HbA represented 98% of all hemoglobin, compared to 0% in non-transduced patient RBCs. Single globin chain chromatograms successfully demonstrated α/β-globin chain ratio of ~0.4 or higher and successfully demonstrated that a low average of copies per cell (~3) restored 60% of adult hemoglobin when normalized to healthy donor RBCs. These data demonstrate α/β-globin correction and hemoglobin restoration in α-thalassemia patient cells with the most severe mutation. Three assays were conducted on patient cells, all with similar and highly encouraging results for disease amelioration in severe cases of α-thalassemia. Overall, this project is paving the way for the first development of a curative gene therapy approach for α-thalassemia.

Platelet Factor 4 Binds to Low-Density Lipoprotein Receptor to Block Leukemic Stem Cell Expansion and Recurrence

Healthy hematopoietic stem cells (HSCs) reside in highly specialized microenvironments within the bone marrow, commonly referred to as niches. HSC activity is tightly regulated by various cellular and molecular niche constituents that ensure healthy hematopoiesis. However, through the accumulation of mutations, hematopoietic stem and progenitor cells can give rise to leukemia-initiating cells or leukemic stem cells (LSCs), which play a vital role in the onset, progression, and treatment outcome of acute myelogenous leukemia (AML). In AML, the expansion of the leukemic clone is associated with impaired healthy hematopoiesis, resulting in severe anemia, thrombocytopenia, and immunodeficiency, which can lead to severe morbidity. Current therapeutic options mainly eliminate the bulk of leukemic cells but do not efficiently target LSCs, leading to the high rates of relapse seen in AML patients. Recent studies suggest that LSCs hijack and alter the healthy bone marrow niche to better support their maintenance and proliferation. Understanding how LSCs interact with the bone marrow microenvironment is crucial for eradicating LSCs and preventing AML relapse. Our previous work showed that platelet factor 4 (PF4), a chemokine stored in platelet alpha-granules and secreted by bone marrow megakaryocytes, can induce quiescence in healthy myeloid-biased HSCs ( Nature Medicine 2014; Developmental Cell 2018). Here, we investigate the regulation of malignant LSCs by PF4 using mouse and human AML models. In an aggressive mouse AML model, MLL-AF9, our results show that recombinant PF4 blocks the proliferation of phenotypic LSCs (Lineage − IL7Rα − Sca1 − MLL-AF9-GFP + c-Kit high CD34 + FcγRII/III high) in vitro (70% reduction; P=0.0279). Strikingly, cell cycle analyses revealed that in vivo, both LSCs and bulk AML proliferation are significantly inhibited 24 hours post-PF4 (80 mg/kg) injection, resulting in a significantly lower leukemic burden. By contrast, Pf4 -/- mice exhibited rapid AML disease progression via increased LSC proliferation. Furthermore, using a genetic mouse model ( Pf4-Cre; iDTR) where PF4-secreting megakaryocyte cells can be depleted in vivo in leukemic mice, our results revealed that megakaryocyte depletion led to poor AML survival due to accelerated disease progression (Log-rank P&amp;lt;0.0017). These results demonstrate that megakaryocyte-derived PF4 can directly regulate the proliferation of leukemia cells. Interestingly, our results indicate that AML drives striking alterations in bone marrow megakaryocytes, including a lower frequency, reduced cell maturation, and reduced secretion of PF4 (~80% reduction, P&amp;lt;0.0001) compared to healthy controls. Accordingly, PF4 levels are reduced in the serum of myelodysplastic syndrome patients where hyperproliferative malignant cells are found alongside dysplastic megakaryocytes. To assess whether recombinant PF4 can alter the course of AML relapse in a preclinical experimental setting, we treated moribund leukemic mice with the chemotherapeutic drug Cytarabine followed by 3 daily I.V. injections of PF4 (80 mg/kg) or saline. Strikingly, this short treatment with PF4 preferentially reduced the frequency of LSCs in vivo during the relapse phase of AML (75% reduction; P=0.05). Furthermore, mice transplanted with MLL-AF9 AML cells that were pre-treated with Cytarabine and PF4 in vitro had prolonged overall survival (Log-rank P&amp;lt;0.0132) compared to Cytarabine alone. Importantly, two of six mice had no detectable AML up to 150 days post-transplantation. Finally, single-cell RNA sequencing experiments revealed that PF4 signals through Low-Density Lipoprotein Receptor (LDLR) on LSCs, whose expression is significantly upregulated upon Cytarabine treatment, during the relapse phase. Functional studies with fluorescently labeled low-density lipoprotein (LDL) confirmed that PF4 limits LSC uptake of LDL via LDLR to block LSC proliferation ( P=0.0044), suggesting an important role for PF4 in AML metabolism. Altogether, our studies reveal an unknown function for PF4 in AML and highlight PF4's potential as an effective adjuvant therapy in preventing AML relapse by inhibiting the recurrence of chemotherapeutic-resistant LSCs.

Comparison of Non-Relapse Mortality after Haploidentical Hematopoietic Stem Cell Transplantation with Post-Transplant Cyclophosphamide Versus Single Umbilical Cord Blood Transplantation for Hematologic Disease

Introduction: Unrelated cord blood transplantation (UCBT) and haploidentical stem cell transplantation with post-transplant cyclophosphamide as graft-versus-host disease (GVHD) prophylaxis (PTCy-haplo) are alternative options for patients with hematologic disease lacking a human leukocyte antigen (HLA)-matched donor. Non-relapse mortality (NRM) after UCBT may vary depending on the cord blood CD34+ cell count, but no study has accounted for CD34+ cell count when comparing the outcomes after UCBT and PTCy-haplo. Therefore, we analyzed transplant outcomes after UCBT and PTCy-haplo in patients with hematologic disease from data of a nationwide registry that included cord blood CD34+ cell count. Methods: Patients with hematologic disease receiving first UCBT or PTCy-haplo between 2014 and 2020 were identified from the nationwide registration data of the Japanese Data for Hematopoietic Cell Transplantation. Acute GVHD-related mortality was defined as death caused primarily by GVHD or occurred secondary to GVHD, and infection-related mortality as death due to graft failure or infection but not acute GVHD. Patients treated by UCBT were stratified by the median CD34+ cell count for comparison (0.84 × 10 5/kg). Results: A total of 7,499 patients were enrolled and classified into 3 groups: PTCy-haplo (n = 1,142), UCBT with high CD34+ cell count (UCB-H; ≥ 0.84 × 10 5/kg, n = 3,185), and UCBT with low CD34+ cell count (UCB-L; &amp;lt; 0.84 × 10 5/kg, n = 3,172). Patients in the PTCy-haplo group were slightly younger than UCB-H and UCB-L patients (median age: 54 vs. 56 and 55 years). The PTCy-haplo group also included a greater proportion of males than the UCB-H group (62.4% vs. 50.0%) but not the UCB-L group (64.2%). A greater proportion of PTCy-haplo patients had a PS score of 0 (53.2% vs. 46.2% and 45.6%), a hematopoietic cell transplantation-specific comorbidity index of 0 (57.6% vs. 52.7% and 54.4%) and donor-specific anti-HLA antibody (2.7% vs. 1.0% and 1.6%), received reduced-intensity conditioning (58.0% vs. 40.5% and 41.6%), calcineurin inhibitor with mycophenolate mofetil (88.5% vs. 38.6% and 38.4%), and underwent transplantation in the later period (82.0% vs. 54.6% and 50.7%). There were no significant differences in disease-risk index between groups. At 2 years post-transplantation, NRM and overall survival (OS) were significantly better in the PTCy-haplo group than the UCB-H and UCB-L groups (NRM: 18.8% vs. 21.6% and 25.3%, P &amp;lt; 0.001; OS: 56.2% vs. 53.3% vs. 49.6%, P &amp;lt; 0.001). Neutrophil engraftment rate at 42 days was also significantly higher in the PTCy-haplo group than the UCB-H and UCB-L groups (93.7% vs. 87.5% and 82.9%, P &amp;lt; 0.001). The PTCy-haplo group less frequently developed grade II-IV (26.4% vs. 37.3% and 34.4%, P &amp;lt; 0.001) and III-IV acute GVHD (7.0% vs. 12.2% and 10.5%, P &amp;lt; 0.001). While neither the differences in NRM nor that in OS remained significant between PTCy-haplo and UCB-H groups in multivariable analysis (NRM: hazard ratio [HR], 1.18, 95% confidence interval [CI], 0.99-1.41, P = 0.073; OS: HR, 1.05, 95% CI, 0.94-1.17, P = 0.42), significant differences were maintained between PTCy-haplo and UCB-L groups (NRM: HR, 1.35, 95% CI, 1.13-1.61, P = 0.001; OS: HR, 1.12, 95% CI, 1.00-1.26, P = 0.042). Regarding the cause-specific mortality, infection-related mortality was also significantly lower in the PTCy-haplo group compared to UCB-H and UCB-L groups (8.1% vs. 11.1% and 13.6%, P &amp;lt; 0.001) while GVHD-related mortality did not differ among groups (5.0% vs. 5.2% and 4.9%, P = 0.69). Conclusion: UCBT-H resulted in similar NRM and OS as patients receiving PTCy-haplo, whereas outcomes were worse in UCBT-L. These findings indicate that high CD34+ cell count in cord blood is a critical criterion for alternative donor selection.

Impact of Allograft T-Cell (CD3) Dose on Outcomes When CD34 Cell Dose Is Capped: Analysis of 811 Consecutive Allogeneic PBSC Transplants from a Single Center

G-CSF mobilized PBSC allografts contain higher T-cell doses than bone marrow grafts and have been associated with a significantly higher risk of chronic GVHD. Higher CD34+ cell content in PBSC grafts has also been shown to predict higher rates of chronic GVHD and associated mortality 1,2 To mitigate this risk our center adopted a policy of capping CD34+ cell dose at 5 x 10e6/kg recipient weight for unmanipulated PBSC grafts. For this study, we assessed the effect of T-cell (CD3) dose administered under this capped CD34 strategy, on GVHD and other transplant outcomes. Consecutive first PBSC allografts for hematologic malignancy performed using the CD34 cap strategy between 2010 and 2022 (n=811) were included in this analysis. GVHD prophylaxis was tacrolimus/methotrexate for MRD and MUD and post-transplant cyclophosphamide based for Haplo. Patients receiving anti-T cell serotherapy or ex-vivo T-depletion were excluded. GVHD was uniformly assessed and graded by a dedicated practitioner for all cases. Patient characteristics were: median age 55 (18-80); 57% male; race -white 74%, black 21%, Asian 5%; diagnosis AML 38%, ALL 18%, MDS 18%, NHL 10%; donor type haplo 42%, MRD 32%, MUD 26%, %; high/v. high 25%, low/intermediate 73%; HCT-CI (0-2) 43%, (&amp;gt;3)57%, regimen - myeloablative 48%, RIC 31%, NMA 21%). Median follow-up for survivors was 64 (16-151) months. Median CD34 and CD3 doses infused were 5.0 x 10e6/kg (min 1.59) and 17.3 x 10e7/kg (range 1.2-83.3) respectively. Median CD3 dose infused were higher with female donors (20.5 vs 15.5 x 10e7/kg, p&amp;lt;0.001) and older donors (18.6 vs 15.9 x 10e7 for donors aged &amp;gt;38 vs &amp;lt;38, p&amp;lt;0.001). The effect of Infused CD3 dose was assessed in terciles (ranges 1.2-13.5, 13.6-22, 22.1-83.3 x 10e7/kg). Median peripheral blood donor CD3 chimerism was 100% for all CD3 dose terciles at d 30, through 180 post-transplant. On univariate analysis - estimated 3-year survival, DFS, NRM, relapse and acute GVHD incidence were not significantly different between the low, middle and high CD3 terciles. In contrast, the cumulative incidence (CI) of moderate-to-severe chronic GVHD at 3 years was significantly higher in the highest vs lowest tercile (36.8% vs 28.6%, p=0.042) (Fig 1), while severe chronic GVHD (14.5% vs 13%) was not significantly different. On stratified Cox multivariable analysis comparing the highest to the lowest CD3 dose terciles - survival, DFS, NRM, relapse and acute GVHD were not significantly different. Moderate-to-severe chronic GVHD was significantly higher in the highest versus lowest tercile (HR 1.53, p=0.005). In order to address specific effects of CD3 dose for particular donor groups, we analyzed incidence of chronic GVHD by CD3 dose terciles separately for the three donor types (MRD n=258, MUD n=210 and Haplo n=343). Whereas the CI of moderate-to-severe chronic GVHD was not significantly different for lowest versus highest CD3 tercile in MRD and Haplo transplant recipients, it was significantly higher for MUD recipients (50% vs 26%, p=0.008, GRFS 29% vs 9%, p=0.005). On multivariable analysis, risk of moderate-to-severe chronic GVHD was significantly higher for the highest versus lowest CD3 dose terciles (HR 2.62, p=0.002) and for the middle vs lowest terciles (HR1.77, p=0.043) in MUD patients (Fig 1). In contrast, CD3 dose had no significant impact on chronic GVHD for MRD and Haplo recipients. This large retrospective analysis of PBSC allografts using a CD34 cap (&amp;lt;5 x 10e6/kg) shows that this approach produced infused CD3 doses ranging between 1.2 and 83.3 x 10e6 /kg. The median and upper limit of infused CD3 dose was lower than previously reported by studies with no CD34 cap 2,3. CD3 dose had a significant impact on the rate of moderate-to-severe chronic GVHD in MUD recipients. A prospective comparison of capped versus uncapped CD34 dosing may help determine the value of this strategy in mitigating GVHD risk of PBSC grafts. These data suggest the need for more aggressive GVHD prophylaxis in patients infused with higher CD3 doses in MUD patients. 1. Mohty M, Bilger K, Jourdan E, et al. Leukemia. 2003;17(5):869-875. 2. Zaucha JM, Gooley T, Bensinger WI, et al. Blood. 2001;98(12):3221-3227. 3. Saad A, Lamb L, Wang T, et al. Biol Blood Marrow Transplant. 2019;25(9):1875-1883.

Indicators of Response to the Wee1 Inhibitor Adavosertib in Acute Myeloid Leukemia

Wee1 is a nuclear kinase that regulates cell cycle progression by inhibiting Cdk1, which is essential for G2 to M phase transition. Inhibition of Wee1 leads to high Cdk1 activity, causing cells to bypass proper DNA repair at the G2/M checkpoint, resulting in mitotic catastrophe and cell death, plus increased Cdk2 activity, causing abnormal DNA replication and DNA double-stranded breaks. Inhibitors of Wee1 are being investigated for the treatment of a variety of cancers including AML. In this study, we aimed to identify biomarkers for Wee1 inhibitor (Wee1i) response in AML using ex vivo drug sensitivity data, bulk RNA sequence, exome sequence, and associated clinical data for a large set of primary AML samples tested against the Wee1i adavosertib. Mononuclear cells from bone marrow aspirates (n=136) or peripheral blood samples (n=11) were collected from AML patients using approved protocols and after informed consent. 101 samples were collected at the time of diagnosis and 35 samples were from relapsed/refractory (R/R) patients. For ex vivo drug-sensitivity testing, freshly isolated cells were tested against a library of up to 634 oncology drugs (215 approved drugs) including adavosertib. The drugs were pre-plated in 5 concentrations in a 10,000-fold concentration range and incubated with cells for 72h. Cell viability was assessed using the CellTiter Glo (CTG) assay, and a drug sensitivity score (DSS) calculated based on the area under the dose response curve. Somatic alterations of the drug tested samples were identified via exome sequencing of DNA from the AML samples and matched skin biopsy controls. Gene expression data were obtained from bulk RNA sequence data of the AML samples. Out of 136 samples analyzed, the median DSS for adavosertib was 8.45. We found positive and significant correlations between sensitivity to adavosertib and two factors: 1) malignant cell percentage (R= 0.31, p-value= 0.0097), and 2) final relative viability percentage (R=0.58, p-value= 4.3e-13). Correlation analysis between the DSS of adavosertib and 633 other drugs revealed several strong positive and significant correlations. These included drugs with specific mechanisms of actions including HSP90 inhibitors (n=5), JAK2 inhibitors (n=4), topoisomerase II inhibitors (n=4), CDK4/6 inhibitors (n=3), mitotic inhibitors, vinca alkaloid microtubule depolymerizers (n=3), and PLK1 inhibitors (n=3). Since adavosertib also targets PLK1 in addition to Wee1, the correlation with PLK1 inhibitor activity confirms the similar target profiles. Based on the specific adavosertib DSS thresholds, the samples were separated in two groups. Samples sensitive to adavosertib (n=35) had DSS between 21.3 (max) and 11.9 (3rd quartile). Samples resistant to adavosertib (n=34) had DSS between 0.0 (min) and 5.65 (1st quartile). Differential gene expression analysis between the sensitive vs resistant samples showed that multiple isoforms of the PCDH and HOXB genes were upregulated in adavosertib sensitive samples. The GLI2, PI15, and SKIDA1 genes were also upregulated in the sensitive samples (Figure A). In contrast, genes such DNTT, PF4V1, TRH, CD1B, S100A16, and CD34 were overexpressed in resistant samples (Figure B). Gene set enrichment analysis revealed positive enrichment of pathways related to MYC targets v2 in the sensitive samples. On the other hand, hallmark pathways associated with cell cycle, apoptosis, RAS signaling, and p53 showed significant negative enrichment scores in the sensitive samples. No statistically significant associations were observed between drug response and frequently occuring somatic mutations in our dataset. However, genetic abberations related MLLT4-MLL/MLL-fusions were only observed in sensitive samples. Collectively, our current findings show that there may be specific gene expression signatures associated with Wee1i sensitivity such high expression of PCDH and HOXB. Also, the positive enrichment of the MYC target v2 pathway in adavosertib sensitive samples suggests that highly proliferating tumor cells might be more sensitive to Wee1i. In addition, high CD34 expression in resistant samples suggests that immature stem and progenitor cells may be less susceptible to Wee1i. In summary, targeting of Wee1 in AML - as studied in NCT05682170 azenosertib study - may be effective for a molecularly defined subset of patients.

Human Adult Long-Term Repopulating Hematopoietic Stem Cells Can be Purified By Surface cMPL Expression

Simple combinations of markers that reliably demarcate human long-term repopulating hematopoietic stem cells (LT-HSCs) would facilitate our understanding of their unique molecular state and the development of stem cell-based therapeutics. The complex CD34+CD38-CD45RA-CD90+CD49f+ phenotype is now routinely accepted to define a subset of cord blood CD34+ cells enriched in LT-HSCs (Notta, Science 2011). The same phenotype with the addition of rhodamine can enrich human LT-HSCs within mobilized peripheral blood (MPB) CD34+ cells, but lower stem cell enrichment is observed (Huntsman, Blood 2015). The hematopoietic cytokine thrombopoietin (TPO) and its receptor cMPL act as primary regulators of HSC function. In recent clinical studies, the synthetic TPO agonist eltrombopag was shown to improve long-term trilineage hematopoiesis in patients with immune aplastic anemia, providing evidence that activation of cMPL receptor stimulates residual multipotent LT-HSCs within the bone marrow (BM). Building on these observations, we hypothesized that cMPL might also be a relevant marker to delineate LT-HSC activity within human adult CD34+ cell populations and provide the basis for a novel LT-HSC purification scheme. To assess the utility of surface cMPL expression for purification of human adult LT-HSCs, we first performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) of human MPB CD34+ cells from a healthy volunteer. CITE-seq was conducted with antibodies designed to react with human cMPL receptor and previously described HSC epitopes (CD34, CD38, CD90, CD45RA, and CD49f). We identified 7 distinct clusters in dimension-reduction (UMAP) analysis. By confirming expression levels of HSPC gene signatures and HSC surface markers, such as CD38, we assigned each CD34+ cell cluster to a distinct HSPC subpopulation, including a single LT-HSC population (cluster 2) (Fig A). Notably, CD34+ cell subsets expressing higher levels of surface cMPL expression were highly enriched in transcriptionally defined HSC population (cluster 2) and consistently co-expressing the known human LT-HSC phenotypic markers, including lower levels of surface CD38 and CD45RA expression and higher levels of surface CD90 and CD49f expression (Fig B). To determine whether surface cMPL expression can enrich functional LT-HSCs, we next performed serial xenotransplantation assays. We used fluorescence-activated cell sorting to partition human MPB CD34+ cells into cMPL high (top 10%) and cMPL low (bottom 10%) populations, and transplanted these cells into immunodeficient NBSGW mice. Serial peripheral blood (PB) sampling of primary xenografted mice revealed a striking difference in patterns of hematopoietic reconstitution over time between the two groups. In mice transplanted with cMPL high cells, human cell chimerism increased gradually throughout the 16-week engraftment period, while a progressive decline in engraftment was observed in the cMPL low group. Notably, mean human cell engraftment within the PB, BM and spleen of mice transplanted with cMPL high cells was 209-fold (p &amp;lt; 0.01), 37-fold (p &amp;lt; 0.0001) and 283-fold (p &amp;lt; 0.001) higher than mean human cell chimerism in the cMPL low group at 16 weeks post-transplantation, respectively. These data suggest that cMPL high and cMPL low populations are distinctly enriched in LT-HSCs and hematopoietic progenitors, respectively. Next, to quantitatively compare the frequency of self-renewing LT-HSCs within the cMPL high and cMPL low HSPC subsets, human CD45+ cells obtained from the BM of primary mice were injected into secondary NBSGW mice at limiting dilution and BM engraftment was measured at 16 weeks post-transplantation (total period of engraftment: 32 weeks). After accounting for engraftment parameters (i.e., cell dose and engraftment levels) in both primary and secondary mice, extreme limiting dilution analysis computed self-renewing LT-HSC frequencies of 1 in 1,267 within the cMPL high CD34+ cell population, and 1 in 149,010 in the cMPL low CD34+ cell fraction, representing a 116-fold enrichment of LT-HSCs using cMPL as a single marker purification strategy. Collectively, these data suggest that cMPL expression can effectively delineate LT-HSCs in human adult CD34+ HSPCs and thus serve as an additional relevant marker for the development of human adult HSC targeting therapeutics.