Bone Marrow CD34 Research Articles

Correlation between PAFAH1B3 Expression Level and Risk of Disease Progression After Autologous Hematopoietic Stem Cell Transplantation in Multiple Myeloma Patients

To investigate the association between the expression level of platelet-activating factor acetylhydrolase 1B3 (PAFAH1B3 ) gene in bone marrow CD138+ cells of patients with multiple myeloma (MM) treated with autologous hematopoietic stem cell transplantation (AHSCT) and the prognosis within 2 years. 147 MM patients treated with AHSCT in The First and The Second Affiliated Hospital of Nantong University from May 2014 to May 2019 were included in the study. Expression level of PAFAH1B3 mRNA in bone marrow CD138+ cells of the patients was detected. Patients with disease progression or death during 2 years of follow-up were included in progression group, and the rest were included in good prognosis group. After comparing the clinical data and PAFAH1B3 mRNA expression levels of the two groups, the patients were divided into high PAFAH1B3 expression group and low PAFAH1B3 expression group based on the median PAFAH1B3 mRNA expression level of the enrolled patients. Progression-free survival rate (PFSR) between the two groups was compared by the Kaplan-Meier method. The related factors of prognosis within 2 years were analyzed by univariate analysis and multivariate COX regression analysis. At the end of follow-up, there were 13 patients lost to follow-up. Finally, 44 patients were included in the progression group and 90 patients were included in the good prognosis group. Age in the progression group was higher than that in the good prognosis group, the proportion of patients with CR+VGPR after transplantation in the progression group was lower than that in the good prognosis group, and there was a statistical difference between two groups in the cases distribution of ISS stage (all P<0.05). PAFAH1B3 mRNA expression level and the proportion of patients with LDH>250U/L in the progression group were higher than those in the good prognosis group, and platelet count in the progression group was lower than that in the good prognosis group (all P<0.05). Compared with the low PAFAH1B3 expression group, the 2-year PFSR of the high PAFAH1B3 expression group was significantly lower (log-rank χ2=8.167, P=0.004). LDH>250U/L (HR=3.389, P=0.010), PAFAH1B3 mRNA expression (HR=50.561, P=0.001) and ISS stage Ⅲ(HR=1.000, P=0.003) were independent risk factors for prognosis in MM patients, and ISS stage Ⅰ (HR=0.133, P=0.001) was independent protective factor. The expression level of PAFAH1B3 mRNA in bone marrow CD138+ cells is related to the prognosis of MM patients treated with AHSCT, and detecting PAFAH1B3 mRNA expression can bring some information for predicting PFSR and prognostic stratification of patients.

5612617 EFFICACY AND SAFETY OF A SINGLE DOSE OF EXAGAMGLOGENE AUTOTEMCEL FOR TRANSFUSION-DEPENDENT-THALASSEMIA AND SEVERE SICKLE CELL DISEASE

Background: Elevated fetal hemoglobin(HbF) is associated with improved outcomes in patients(pts) with transfusion-dependent β-thalassemia(TDT) and sickle cell disease(SCD). Exagamglogene autotemcel(exa-cel; formerly known as CTX001) is a cell therapy designed to reactivate HbF via non-viral, ex vivo CRISPR/Cas9 gene-editing at the erythroid enhancer region of BCL11A in autologous CD34+ hematopoietic stem and progenitor cells(HSPCs). Early data from the pivotal trials CLIMB THAL-111(NCT03655678) and CLIMB SCD-121(NCT03745287) showed that a single dose of exa-cel increased HbF and total Hb sufficiently to eliminate red blood cell(RBC) transfusions(TDT) and vaso-occlusive crises(VOCs; SCD). Aims: To report efficacy and safety data from the first 75 pts dosed with exa-cel in the ongoing CLIMB THAL-111 and CLIMB SCD-121 trials. Methods: Following pharmacokinetic-adjusted busulfan myeloablation and exa-cel infusion, pts(12-35y) are monitored for engraftment, total Hb, HbF, BCL11A edited alleles, transfusions, VOCs(SCD only), and adverse events(AEs). Data presented as mean(min-max) unless noted. Results: 44 pts with TDT(age 21.3 [12-35] y) and 31 pts with SCD(age 22.5 [12-34} y) had been infused with exa-cel at data cut(follow-up 12.3 [1.2-37.2] mo and 9.6 [2.0-32.3] mo, respectively). 26/44 pts with TDT(59.1%) had β0/β0 or a β0/β0-like genotype(β0/IVS-I-110, IVS-I-110/IVS-I-110). In the 2-yr period before screening, pts with TDT received 36.0(15.0-71.0) units RBC/yr and pts with SCD had 3.9(2.0-9.5) severe VOCs/yr. After exa-cel infusion, all pts engrafted neutrophils and platelets. Median time to neutrophil and platelet engraftment was 29 and 43 days in pts with TDT and 27 and 32 days in those with SCD, respectively. Overall, 42 of 44 pts with TDT stopped RBC transfusions(duration 0.8 to 36.2 mo); 2 pts had not yet stopped transfusions but had 75% and 89% reductions in transfusion volume. By Month 3, increases in HbF and mean total Hb levels(>9 g/dL) were achieved, with mean total Hb levels increasing to >11 g/dL thereafter and were maintained. All pts with SCD(n=31) no longer had severe VOCs after exa-cel infusion(duration 2.0 to 32.3 mo). The mean proportion of HbF was >20% by Month 3, increasing to ~40% at Month 4 and was stable thereafter, with mean total Hb levels >11 g/dL after Month 3. Pts with TDT and SCD with ≥1 yr follow-up had stable proportions of edited BCL11A alleles in bone marrow CD34+ HSPCs and peripheral blood mononuclear cells. Two pts with TDT had serious AEs(SAEs) considered related to exa-cel. The first pt was previously reported. The second pt had delayed neutrophil engraftment and thrombocytopenia, which were considered related to both exa-cel and busulfan. All SAEs resolved. No pts with SCD had SAEs considered related to exa-cel. There were no deaths, discontinuations, or malignancies. Conclusions: Exa-cel infusion led to elimination of transfusions in almost all patients with TDT and elimination of VOCs in all patients with SCD, with associated clinically meaningful increases in HbF and total Hb that were maintained over time. Proportions of CRISPR/Cas9-edited BCL11A alleles remained stable after more than 1 year, indicating that long-term HSCs were successfully edited. The safety profile was generally consistent with busulfan myeloablation and autologous transplant. These results indicate exa-cel has the potential to be the first CRISPR/Cas9-based therapy to provide a one-time functional cure for TDT and severe SCD. Reference 1. Frangoul et al, N Engl J Med 2021; 384:252-260.

Bone marrow CD34+ molecular chimerism as an early predictor of relapse after allogeneic stem cell transplantation in patients with acute myeloid leukemia.

Minimal residual disease (MRD) monitoring is an important tool to optimally address post-transplant management of acute myeloid leukemia (AML) patients. We retrospectively analyzed the impact of bone marrow CD34+ molecular chimerism and WT1 on the outcome of a consecutive series of 168 AML patients submitted to allogeneic stem cell transplantation. The cumulative incidence of relapse (CIR) was significantly lower in patients with donor chimerism on CD34+ cells ≥ 97.5% and WT1 < 213 copies/ABL x 10^4 both at 1st month (p=0.008 and p<0.001) and at 3rd month (p<0.001 for both). By combining chimerism and WT1 at 3rd month, 13 patients with chimerism < 97.5% or WT1 > 213 showed intermediate prognosis. 12 of these patients fell in this category because of molecular chimerism < 97.5% at a time-point in which WT1 was < 213. Our results confirm that lineage-specific molecular chimerism and WT1 after allo-SCT (1st and 3rd month) are useful MRD markers. When considered together at 3rd month, CD34+ molecular chimerism could represent an earlier predictor of relapse compared to WT1. Further studies are necessary to confirm this preliminary observation.

TIGIT inhibition and lenalidomide synergistically promote antimyeloma immune responses after stem cell transplantation in mice.

Autologous stem cell transplantation (ASCT) with subsequent lenalidomide maintenance is standard consolidation therapy for multiple myeloma, and a subset of patients achieve durable progression-free survival that is suggestive of long-term immune control. Nonetheless, most patients ultimately relapse, suggesting immune escape. TIGIT appears to be a potent inhibitor of myeloma-specific immunity and represents a promising new checkpoint target. Here we demonstrate high expression of TIGIT on activated CD8+ T cells in mobilized peripheral blood stem cell grafts from patients with myeloma. To guide clinical application of TIGIT inhibition, we evaluated identical anti-TIGIT antibodies that do or do not engage FcγR and demonstrated that anti-TIGIT activity is dependent on FcγR binding. We subsequently used CRBN mice to investigate the efficacy of anti-TIGIT in combination with lenalidomide maintenance after transplantation. Notably, the combination of anti-TIGIT with lenalidomide provided synergistic, CD8+ T cell-dependent, antimyeloma efficacy. Analysis of bone marrow (BM) CD8+ T cells demonstrated that combination therapy suppressed T cell exhaustion, enhanced effector function, and expanded central memory subsets. Importantly, these immune phenotypes were specific to the BM tumor microenvironment. Collectively, these data provide a logical rationale for combining TIGIT inhibition with immunomodulatory drugs to prevent myeloma progression after ASCT.

The density of bone marrow mononuclear cells and CD34+ cells in patients with three neurologic conditions

BackgroundThis study aimed to identify the density of mononuclear cells (MNCs) and CD34+ cells in the bone marrow of patients with three neurologic conditions.MethodsThe study included 88 patients with three neurologic conditions: 40 with cerebral palsy (CP) due to oxygen deprivation (OD), 23 with CP related to neonatal icterus (NI), and 25 with neurological sequelae after traumatic brain injury. Bone marrow aspiration was conducted from the patients’ bilateral anterior iliac crest under general anesthesia in an operating theater. MNCs were isolated by Ficoll gradient centrifugation and then infused intrathecally.ResultsThere was a significant difference in the average MNC per ml and percentage of CD34+ cells by the type of disease, age group, and infusion time (p value < 0.05). The multivariable regression model showed the percentage of CD34+ association with the outcome (gross motor function 88 items- GMFM-88) in patients with CP.ConclusionsThe density of MNCs was 5.22 million cells per mL and 5.03% CD34+ cells in patients with three neurologic conditions. The highest density of MNCs in each ml of bone marrow was found in patients with CP due to OD, whereas the percentage of CD34+ cells was the highest among patients with CP related to NI.

TET1-mediated microRNA-188-5p promoter hydroxymethylation regulates PTEN/PI3K/AKT signaling pathway in acute myeloid leukemia cells

ObjectiveAcute myeloid leukemia (AML) remains a common hematopoietic malignancy, and drug resistance greatly blunts the efficacy of chemotherapy in AML treatment. Adriamycin (ADM, also called doxorubicin), is one of the most widely used chemotherapeutics for treating cancers. Herein, we studied the molecular mechanisms underlying microRNA-188-5p (miR-188-5p)-mediated ADM resistance in AML. MethodsDifferentially expressed miRNAs were screened in normal and malignant hematopoietic cells by bioinformatics tools. MiR-188-5p expression in primary bone marrow CD34+ cells and AML cells was evaluated. AML/ADM cells were established using THP-1 and Kasumi-1 cells. The effect of miR-188-5p on the drug resistance in AML/ADM cells was examined by delivery of miR-188-5p-inhibitor. The binding relationship between TET1 and miR-188-5p was analyzed by ChIP, and the downstream target of miR-188-5p was predicted by bioinformatics analysis and validated by dual-luciferase assay. Finally, rescue experiments were carried out in vitro and in vivo. ResultsmiR-188-5p was highly expressed in AML cells, and miR-188-5p-inhibitor sensitized the AML/ADM cells to ADM. Inhibition of TET1 reduced miR-188-5p promoter hydroxymethylation and downregulated miR-188-5p. miR-188-5p bound to the 3′UTR of PTEN to inhibit PTEN expression, and the PI3K/AKT signaling was activated upon inhibition of PTEN. Suppression of PTEN conferred resistance again to AML/ADM cells in the presence of miR-188-5p inhibitor. ConclusionTET1 elevates miR-188-5p expression by promoting miR-188-5p promoter hydroxymethylation, and miR-188-5p inhibits PTEN expression to induce PI3K/AKT signaling pathway activation, leading to ADM resistance in AML.

Therapeutic Angiogenesis Using Autologous CD34-Positive Cells for Vascular Diseases.

CD34 is a cell surface marker, which is expressed in various somatic stem/progenitor cells such as bone marrow (BM)-derived hematopoietic stem cells and endothelial progenitor cells (EPCs), skeletal muscle satellite cells, epithelial hair follicle stem cells, and adipose tissue mesenchymal stem cells. CD34+ cells in BM and peripheral blood are known as a rich source of EPCs. Thus, vascular regeneration therapy using granulocyte colony stimulating factor (G-CSF) mobilized- or BM CD34+ cells has been carried out in patients with various vascular diseases such as chronic severe lower limb ischemia, acute myocardial infarction, refractory angina, ischemic cardiomyopathy, and dilated cardiomyopathy as well as ischemic stroke. Pilot and randomized clinical trials demonstrated the safety, feasibility, and effectiveness of the CD34+ cell therapy in peripheral arterial, cardiovascular, and cerebrovascular diseases. This review provides an overview of the preclinical and clinical reports of CD34+ cell therapy for vascular regeneration.

Efficacy and Safety of a Single Dose of Exagamglogene Autotemcel for Severe Sickle Cell Disease

Background: Elevated fetal hemoglobin (HbF) is associated with improved outcomes in patients with sickle cell disease (SCD). Exagamglogene autotemcel (exa-cel; formerly known as CTX001) is a cell therapy designed to reactivate HbF via non-viral, ex vivo CRISPR/Cas9 gene-editing at the erythroid enhancer region of BCL11A in autologous CD34+ hematopoietic stem and progenitor cells (HSPCs). Recent data from the pivotal CLIMB SCD-121 (NCT03745287) trial showed that a single dose of exa-cel increased HbF and total hemoglobin (Hb) sufficiently to eliminate vaso-occlusive crises (VOCs) in patients with severe SCD. Here, we report efficacy and safety data from the first 31 patients dosed with exa-cel in the ongoing CLIMB SCD-121 trial. Methods: Patients aged 12 to 35 years with severe SCD and a history of ≥2 VOCs per year in the previous 2 years before screening were eligible. Following pharmacokinetic-adjusted, busulfan myeloablation and infusion of exa-cel, patients are monitored for engraftment, total Hb, HbF, BCL11A-edited alleles, transfusions, VOCs, and adverse events (AEs). The primary endpoint is the proportion of patients who have not experienced a severe VOC for at least 12 months after the infusion of exa-cel, starting 60 days after their last RBC transfusion. Updated efficacy and safety results on all dosed patients will be included in the presentation. Data are reported as mean (min-max), unless noted. Results: At the most recent data cut in February 2022, 31 patients with SCD (age 22.5 [12-34] years) had been infused with exa-cel (follow-up 9.6 [2.0-32.3] months), of whom 6 (19.4%) were between the ages of 12 and <18 years and 29 (93.5%) had the βs/βs genotype. In the 2-year period before screening, patients experienced 3.9 (2.0-9.5) severe VOCs per year. After exa-cel infusion, all patients engrafted neutrophils and platelets with a median time of 27 and 32 days, respectively. All patients were VOC-free at the time of the data cut (duration of follow-up 2.0-32.3 months after exa-cel infusion; Figure). Median time from exa-cel infusion to last RBC transfusion was 19 (11-52) days. The mean proportion of HbF was >20% by Month 3, with mean total Hb levels >11 g/dL on and after Month 3. All 11 patients who have at least 12 months of follow-up after exa-cel infusion have maintained HbF levels >20% while experiencing no VOCs. At Month 6, the mean proportion of edited BCL11A alleles in bone marrow CD34+ HSPCs and peripheral blood mononuclear cells was 86.6% and 76.0%, respectively. These proportions remained stable in all patients who had ≥1 year of follow-up (Figure). There were no patients who had serious AEs considered related to exa-cel. There were no deaths, discontinuations, or malignancies. Conclusions: Exa-cel infusion led to the elimination of VOCs in all patients with SCD, with associated clinically meaningful increases in HbF and total Hb that were maintained over time. Proportions of CRISPR/Cas9-edited BCL11A alleles have remained stable after ≥1 year, indicating that long-term HSCs were successfully edited. The safety profile was generally consistent with that of busulfan myeloablation and autologous transplant. Exa-cel has the potential to be the first CRISPR/Cas9-based therapy to provide a one-time functional cure for SCD. Figure. Clinical data for patients with SCD (N=31) infused with exa-cel. Patients were VOC-free after exa-cel infusion. Time (months) since exa-cel infusion is indicated by the dark blue bar. Inset depicts the percentage of BCL11A-edited alleles in bone marrow CD34+ HSPCs and peripheral blood mononuclear cells of patients with SCD infused with exa-cel over time. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Uncovering Transcriptome Characteristics in Bone Marrow Hematopoiesis in Immune Thrombocytopenia at Single-Cell Resolution

Immune thrombocytopenia (ITP) is an autoimmune disorder, in which megakaryocyte dysfunction caused by an autoimmune reaction can lead to thrombocytopenia, although the underlying mechanisms remain unclear. Here, we hypothesized that bone marrow (BM) CD34+ hematopoietic stem and progenitor cells (HSPCs) participated in the defective megakaryogenesis in ITP patients, and we performed single-cell RNA-seq (scRNA-seq) of BM CD34+ HSPCs to reveal the overall transcriptome alterations in HSPCs in ITP. Dimensional reduction using uniform manifold approximation and projection (UMAP) showed the effective integration of the datasets from different samples. We manually annotated the cell clusters into 17 different cell types with distinct gene expression patterns. By performing differential expression analysis, enrichment analysis, trajectory analysis, transcriptional regulatory network analysis and cell-cell interaction analysis, we found that gene expression, transcriptional regulatory networks and cell-cell interactions varied in HSPCs of ITP, particularly in a pre-B cell population and natural killer/T cell progenitors, highlighting the selective immune aberration associated with defective megakaryopoiesis in ITP. Differentially expressed gene (DEG) analysis indicated that the top upregulated genes in a megakaryocytic progenitors (MkP) population of ITP were associated with erythropoiesis, suggesting a bias toward erythrocytes in megakaryopoiesis in ITP. Moreover, Flow cytometry confirmed that the number of CD9+ cells from CD34+ HSPCs decreased in ITP. Liquid culture assays demonstrated that CD9+ CD34+ HSPCs from healthy controls tended to differentiate into megakaryocytes; however, this tendency was not observed in ITP patients. The percentage of megakaryocytes differentiated from CD9+CD34+ HSPCs was higher than that of the CD9- counterparts from healthy controls, whereas in ITP patients, the percentage decreased to only 1/4th of that in the healthy controls and was comparable to that from the CD9- HSPCs, indicating that differentiation of CD9+ HSPCs toward the megakaryopoietic lineage was impaired in ITP. Further analysis revealed the universality of their heterogeneity and provided an analytical basis for the study of megakaryocyte-related diseases, including ITP. Enrichment analysis confirmed the definition of MkP in our ITP integrated dataset. To further analyze the transcriptional difference between ITP and HC, we pooled MkP from all samples. The expression of several molecular features of MkP was visualized, and seven transcriptionally heterogeneous subpopulations of MkP were identified. We then assessed the expression of marker genes in each subpopulation and characterized the gene sets enriched in different clusters, which showed pronounced heterogeneity within MkP, consistent with the results of previous analyses of MkP/Mk from multi-hematopoietic tissues. We evaluated ITP-associated DEGs in each MkP subcluster, and we found that most of these DEGs were subtype-specific, indicative of the subtype-specific effects of the disorder. In summary, this study dissects the significant heterogeneity and disease characteristics of BM CD34+ HSPCs from ITP patients at single-cell resolution, revealing new insights regarding the pathophysiology of ITP.

DNA Replication Stress Due to Loss of R-Loops in Myelodysplastic Syndromes with SF3B1 Mutation

Introduction: Myelodysplastic syndromes (MDS) with SF3B1 mutation represent a subtype of hematopoietic stem cell disorders with bone marrow (BM) erythroid dysplasia and ring sideroblasts. This mutation in a splicing factor gene causes multiple alterations of mRNA maturation. Other mutated splicing factors SRSF2 or U2AF1, produce splicing alterations and formation of unscheduled R-loops, which trigger DNA damage response and genetic instability. Mechanistically, mutant SRSF2 impairs the transcription pause release downstream of TSS allowing invasion of relaxed DNA double helix by nascent RNA. Although previous studies suggested that R-loops inappropriately form in SF3B1 mutant cells, investigations are required to elucidate why MDS with SF3B1 mutation are less prone to genetic instability. Methods: A total of 96 patients including 53 MDS with SF3B1 mutation and 43 other low risk MDS was enrolled in this study. RNA-seq of BM mononuclear cells (MNC) was performed in 21 MDS with and 6 MDS w/o SF3B1 mutation. BM CD34+-derived erythroblasts were expanded from 69 MDS and 22 healthy controls to analyze transcriptome, proteome, R-loop landscape, and DNA replication stress. For global proteome analysis, after protein digestion, peptides were fractionated on SCX column and analyzed by mass spectrometry. R-loops were detected by DNA-RNA immunoprecipitation (DRIP-seq) using S9.6 antibody. Immunofluorescence labeling of pRPA32, 53BP1 and γH2AX, DNA combing, BrdU assay and metabolomics were used to investigate DNA replication stress and DNA biosynthesis in human primary erythroblasts or in murine proerythroblastic (proE) G1E-ER4 Crispr-cas9 Sf3b1K700E/+ cell line. expressing GATA1 under the control of estrogen receptor. Results: In SF3B1-mutated BM MNC samples, DeSeq2 analysis of RNA-seq data revealed 1,058 upregulated genes (log2(FC)>0.6, BH-adj P-value<0.05) involved in DNA replication, cell cycle, nucleotide biosynthesis, DNA repair, and erythroid differentiation. Analysis of splicing identified 3,937 differential splicing events (ΔPSI>0.10, BH-adj P-value<0.05) including 1,256 events of intron retention (IR). Among IR events, 384 loss of IR events correlated with altered expression of 293 genes. In SF3B1-mutated erythroblasts, loss of IR events were more frequent in polychromatophilic erythroblasts (polyE: 383) than in proE/early basophilic erythroblasts (eBasoE: 171) suggesting that gene extinction mediated by IR during normal differentiation was altered. Quantitative proteomics revealed 924 significantly deregulated proteins with little overlap with IR transcripts, consistently involved in DNA damage, DNA repair, and nucleotide biosynthesis pathways. DRIP-seq was performed in proE/eBasoE of 8 MDS w/o or with SF3B1 mutation to investigate R-loop formation. Stringent peak calling identified 2,208 shared peaks in wild-type samples compared to 159 shared peaks in SF3B1-mutated samples, suggesting a loss of R-loops. Integrative analysis and DRIP-qPCR demonstrated that R-loop losses overlapped IR loss events, consistent with the notion that intron excision prevents R-loop formation. Compared to SRSF2 or U2AF1-mutated erythroblasts, SF3B1-mutated cells exhibited a significant increase of DNA synthesis by BrdU labeling and of replication fork speed by DNA combing. Immunofluorescence analysis detected pRPA32 foci marking single strand DNA but no evidence of double strand breaks in contrast to SRSF2 mutant cells in which 53BP1 or γH2AX foci were detected. Compared to isogenic cell lines, Sf3b1K700E/+ proE G1E-ER4 cells demonstrated higher proliferation rate and fork speed in proliferation and in estradiol-induced differentiation conditions, and significantly lower amounts of deoxynucleotides (dATP, dCTP, TTP) suggesting persistence of DNA synthesis during differentiation. Targeting of DNA polymerase by aphidicolin or ribonucleotide reductase by low doses of hydroxyurea partially rescued erythroid differentiation. Conclusion: Our data demonstrated a highly specific signature of DNA replication stress at transcriptomic and proteomic level in SF3B1-mutant cells. High replication fork speed reflects the loss of R-loops in these cells, which may reduce the risk of genetic instability at the expense of normal differentiation. Pharmacological targeting of the DNA replication stress could improve erythropoiesis.

KarMMa-2 Cohort 2a: Efficacy and Safety of Idecabtagene Vicleucel in Clinical High-Risk Multiple Myeloma Patients with Early Relapse after Frontline Autologous Stem Cell Transplantation

**Co-lead author Introduction Patients with multiple myeloma (MM) who relapse early after frontline therapy with autologous stem cell transplant (ASCT) have a poor prognosis. Novel therapies are needed to improve outcomes (Paiva Blood 2012; Bygrave Br J Haematol 2020). In the pivotal phase 2 KarMMa study (NCT03361748), treatment with the B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T cell therapy idecabtagene vicleucel (ide-cel) resulted in frequent, deep, and durable responses in pts with relapsed and refractory MM (RRMM) who were triple-class exposed and refractory to last treatment (Munshi N Engl J Med 2021). KarMMa-2 is a multicohort, phase 2, multicenter trial (NCT03601078) of ide-cel in RRMM and in clinical high-risk MM, defined as early relapse after frontline therapy (cohorts 2a, 2b) or inadequate response after frontline ASCT (2c). The efficacy and safety of ide-cel in cohort 2a are presented. Methods Eligible pts in cohort 2a had early relapse after frontline therapy, defined as progressive disease (PD) <18 mo from starting treatment containing induction, ASCT (single or tandem), and lenalidomide-containing maintenance. After lymphodepletion (cyclophosphamide 300 mg/m2 + fludarabine 30 mg/m2 × 3), pts received a single infusion of ide-cel at 150-450 × 106 CAR+ T cells. The primary efficacy endpoint was complete response (CR) rate (CRR; CR and stringent CR) by investigator per IMWG criteria. Secondary endpoints included overall response rate (ORR; ≥ partial response), time to response (TTR), duration of response (DOR), PFS, OS, safety, and pharmacokinetics (PK). Exploratory endpoints included assessment of soluble BCMA (sBCMA) level and minimal residual disease negativity (MRD-) by next-generation sequencing (<10-5 nucleated cells). Efficacy and safety were analyzed in all pts who received ide-cel; PK and sBCMA were analyzed in evaluable pts. MRD- is reported for evaluable pts with ≥CR. Results Ide-cel was successfully manufactured and infused in 37/39 pts. Median age was 57 y; median time since diagnosis was 1.6 y. A total of 62.2% pts had ECOG PS 0, and 70.3% received bridging therapies (corticosteroids, alkylating agents, immunomodulatory agents, proteasome inhibitors [PI], and/or anti-CD38 antibodies) for MM. At study entry, 13.5%/51.4%/5.4% pts had R-ISS stage I/II/III disease, 32.4% had high-risk cytogenetics, 18.9% had bone marrow biopsy-determined high tumor burden (≥50% bone marrow CD138+ plasma cells), and 8.1% had extramedullary disease. Most pts had disease refractory to an immunomodulatory agent (86.5%) or PI (89.2%); 86.5% had double refractory disease. At data cut-off (14 Mar 2022), median follow-up was 21.5 mo (range 2-31). CRR was 45.9% (95% CI 29.5-63.1), and ORR was 83.8% (95% CI 68.0-93.8) (Table 1). At 6 mo post-ide-cel, MRD− was observed in 11/13 (85%; 95% CI 57.8-95.7) pts. At 12 mo, MRD− was observed in 7/10 (70%; 95% CI 39.7-89.2) pts; of the 7 pts, 1 had PD at 20.8 mo, 5 sustained MRD− at ≥18 mo, and >12 mo data was unavailable for 1 pt. Median TTR was 1 mo (range 0.9-2.9). Median DOR was 15.7 mo (95% CI 7.62-19.81). Median PFS was 11.4 mo (95% CI 5.55-19.58); median OS was not reached. Time to event endpoint results for DOR, PFS, and OS are shown in Table 1. Grade (Gr) 3-4 AEs on or after ide-cel infusion occurred in all pts, most commonly neutropenia in 35 (94.6%) pts, anemia in 17 (45.9%), and thrombocytopenia in 14 (37.8%). Two pts died due to pneumonia and pseudomonal sepsis. Gr 1/2 cytokine release syndrome (CRS) occurred in 30 (81.1%) pts; 1 (2.7%) pt had a Gr 3 event (Table 1). Gr 1/2 investigator-identified neurotoxicity (NT) occurred in 8 (21.6%) pts; no pts had ≥Gr 3 NT. Robust cell expansion was seen in 36 evaluable pts (Table 2). Ide-cel cellular expansion levels were higher in pts who had ≥CR vs those who had <CR with ide-cel (Table 2). sBCMA (range 15.0-737.0 ng/mL at infusion, n = 36) was cleared within 2 mo post-ide-cel infusion in 25/37 (67.6%) pts, including all pts who had ≥CR. Conclusions Ide-cel treatment demonstrated frequent, deep responses in pts who experienced an early relapse after frontline therapy with ASCT. MRD− was sustained in a subset of pts >18 mo. The incidence of CRS and NT were similar in these pts vs those treated with ide-cel in later lines. These results support a favorable clinical benefit-risk profile of ide-cel and its potential use in earlier lines of treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

KarMMa-2 Cohort 2c: Efficacy and Safety of Idecabtagene Vicleucel in Patients with Clinical High-Risk Multiple Myeloma Due to Inadequate Response to Frontline Autologous Stem Cell Transplantation

**Co-lead authors Introduction Patients (pts) with multiple myeloma (MM) who do not achieve a deep response post-autologous stem cell transplantation (ASCT) have an increased risk of progressive disease (PD) death (van de Velde Eur J Haematol 2017) and a high unmet medical need. In the pivotal phase 2 KarMMa study (NCT03361748), treatment with the B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T cell therapy idecabtagene vicleucel (ide-cel) led to frequent, deep, and durable responses in pts with relapsed and refractory MM (RRMM) who were triple-class exposed and refractory to last treatment (Munshi N Engl J Med 2021). KarMMa-2 is a multicohort, phase 2, multicenter trial (NCT03601078) of ide-cel in RRMM and in clinical high-risk MM. The efficacy and safety of ide-cel in cohort 2c pts with MM who had an inadequate response to frontline therapy with ASCT are presented. Methods Eligible pts in cohort 2c had newly diagnosed MM (NDMM), received ≥3 cycles of induction therapy (including a proteasome inhibitor, immunomodulatory agent, and dexamethasone), and had an inadequate response to ASCT (single or tandem), defined as less than very good partial response (VGPR) at 70-110 days after last ASCT, without use of consolidation or maintenance therapy. Pts had ECOG performance status (PS) ≤1. After lymphodepletion (cyclophosphamide 300 mg/m2 + fludarabine 30 mg/m2 × 3), pts received a single infusion of ide-cel at dose range 150-450 × 106 CAR+ T cells. At investigator discretion, pts could receive maintenance therapy post-infusion. The primary endpoint was complete response (CR) rate (CRR; CR and stringent CR) per investigator and IMWG Uniform Response Criteria. Secondary endpoints included overall response rate (ORR; ≥ partial response), VGPR rate (VGPRR), time to response (TTR), duration of response, PFS, overall survival, safety, and pharmacokinetics (PK). Exploratory endpoints included assessment of soluble BCMA (sBCMA) level, and minimal residual disease negativity (MRD-) by aggregation of next-generation sequencing and EuroFlow (<10-5 nucleated cells) data with interpolation to reduce missing/indeterminate data points. Efficacy and safety were analyzed in all pts who received ide-cel; PK, sBCMA, and MRD- were analyzed in evaluable pts. Results Ide-cel was successfully manufactured and infused in 31/32 enrolled pts. Median age was 64 y; 67.7% of pts had ECOG PS 0. At study entry, 41.9% pts had R-ISS stage I disease, 16.1% had stage II, and none had stage III; 9.7% pts had high-risk cytogenetics, 3.2% had bone marrow biopsy-determined high tumor burden (≥50% bone marrow CD138+ plasma cells), and 6.5% had extramedullary disease. At data cut-off (14 Mar 2022), median follow-up was 27.5 mo (range 22-31). CRR was 74.2% (95% CI 55.4-88.1), and ORR was 87.1% (95% CI 70.2-96.4) (Table 1). Median TTR was 1 mo (range 0.9-2.8). Time to event endpoint results are in Table 1. Grade (Gr) 3-4 adverse events (AEs) on or after ide-cel infusion were reported in 29 (93.5%) pts, most commonly, neutropenia in 25 (80.6%) pts, leukopenia in 9 (29.0%), and anemia in 7 (22.6%). No Gr 5 AEs were reported. Grade 1 CRS occurred in 14 (45.2%) pts, Gr 2 in 4 (12.9%), and no pts had ≥Gr 3 (Table 1). Investigator-identified neurotoxicity (NT) occurred in 2 (6.5%) pts, one had Gr 1 and one had Gr 3. Robust cell expansion was seen in 31 evaluable pts (Table 2) despite low tumor burden, as assessed by sBCMA level (range 6.5-154.0 ng/mL at infusion, n = 30). Ide-cel cellular expansion was higher in pts who achieved ≥CR vs <CR with ide-cel (Table 2). sBCMA was cleared within 2 mo post-ide-cel infusion in 24/31 (77.4%) evaluable pts, including in 22 who had ≥CR. Among 23 pts with ≥CR, 16 (69.6%; 95% CI, 49.1-84.4) pts had MRD− at 6 mo post-ide-cel infusion. At 12 mo, MRD− was seen in 17/27 (62.9%; 95% CI 44.2-78.5) evaluable pts, regardless of CR status and in 16/23 (69.6%; 95% CI 49.1-84.4) evaluable pts who had ≥CR. Of note, of the 16 pts, 11 sustained MRD− at 24 mo, 2 pts did not have 24 mo data available, and data for 3 pts were indeterminate. Conclusions Ide-cel treatment resulted in frequent, deep, and durable responses in pts with an inadequate response to frontline ASCT. Early deep clearance of tumor was seen in pts with ≥CR after ide-cel treatment and was sustained at 2 y. Lower incidence of CRS and NT were seen in these pts vs those treated with ide-cel in later lines. These results support a favorable clinical benefit-risk profile of ide-cel in NDMM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Jak2V617F Reversible Activation Shows an Essential Requirement for Jak2V617F in Myeloproliferative Neoplasms (MPNs)

JAK inhibitors (JAKi) improve symptoms and outcomes in MPNs; however, mutant allele burden does not substantively change with chronic therapy. Alternative MAPK signaling and/or cooperating mutations are thought to contribute to MPN cell persistence; however, we hypothesized current JAKi fail to effectively abrogate mutant JAK2 signaling. To that end, we developed a conditionally inducible mouse model allowing for sequential activation, then deletion, of Jak2VF from its endogenous locus using a Dre-rox/Cre-lox (Jak2RL) dual recombinase system (Figure 1A). Dre mRNA electroporation of lineage-negative UbcCreER-Jak2RL bone marrow (BM) was used to induce Jak2VFex vivo, followed by transplant into lethally-irradiated recipient mice in competition with Cd45.1 BM. Transplanted mice developed a highly penetrant MPN characterized by leukocytosis, polycythemia, hepatosplenomegaly, and BM megakaryocytic hyperplasia. Tamoxifen administration 12 weeks post-transplant to delete Jak2VF resulted in abrupt normalization of blood cell parameters, spleen weights, and serum pro-inflammatory cytokines, with a marked prolongation in overall survival (OS) compared to MPN control mice. Reduced peripheral blood (PB) and BM Cd45.2 mutant fraction was also observed, including within hematopoietic stem/progenitor cell (HSPC) compartments and to a greater degree than ruxolitinib (RUX) therapy. Notably, treatment with CHZ868, a type II JAKi, showed improvements in regard to hematocrit and spleen weight reduction on par with deletion, as well as significant reductions in BM HSPC mutant fractions, albeit to a lesser degree than with Jak2VF reversion (Figure 1B). Transplant of Jak2VF deleted BM failed to form phenotypic disease in secondary recipient mice consistent with loss of disease-propagating MPN stem cells. RNA-Seq on sorted LSKs following Jak2VF deletion showed loss of STAT5, MAPK, and MTORC1 target gene expression, as well as negative enrichment in TGFb and TNFa/NFkB pathways consistent with reduced pro-inflammatory and proliferative output. The alterations in MAPK signaling were supported by Ybx1 RT-PCR of BM cKit+ cells and BM pERK IHC, both of which were increased with RUX but potently suppressed with deletion suggesting improved JAK2 targeting can overcome MAPK-mediated persistence. The initial proximity of Jak2RLlox sites grants Cre-mediated recombination prior to Dre-mediated Jak2VF knock-in allowing for assessment of Jak2VF oncogenic dependency alone or in the context of co-occurring mutation. Given the known effects of Tet2 loss on enhanced MPN HSC fitness, we evaluated Tet2 loss on sustaining MPN with Jak2VF reversion. UbcCreER-Jak2RL/Tet2f/f mice were pulsed with TAM to knock-out Tet2 followed by sacrifice and Dre Jak2RL knock-in 6 weeks later. Mice transplanted with UbcCreER-Jak2VF/Tet2-/- cells exhibited enhanced leukocytosis, spleen weights and PB/BM chimerism compared to Jak2VF mice; however, Jak2VF reversion resulted in similar reductions in MPN disease parameters, including Cd45.2 fraction, suggesting Tet2loss does not dramatically alter dependence on mutant JAK/STAT signaling. Notably, while Jak2VF/Tet2-/- mutant cells displayed increased serial replating capacity, this was abrogated with Jak2VF reversion suggesting despite the enhanced fitness of Tet2-/- HSCs, Jak2VF creates a unique dependency that renders double-mutant cells susceptible to eradication with Jak2VF reversion. In sum, Jak2VF deletion abrogates MPN features, extends OS, and reduces mutant cell fraction with depletion of disease-sustaining MPN HSCs, either alone or in the setting of cooperating Tet2 loss. These data suggest JAK2VF mutant-selective inhibition offers greater therapeutic potential than current JAKi and a potential curative option for the treatment of MPN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Regulatory T Cell Depletion during Stem Cell Mobilization Enhances Anti-Myeloma Immunity after Experimental Autologous Stem Cell Transplantation

Autologous stem cell transplantation (ASCT) with G-CSF/plerixafor mobilized peripheral blood stem cells (PBSC) remains the standard of care for newly diagnosed eligible multiple myeloma (MM) patients and prolongs progression-free survival. However, most patients eventually progress and require additional therapy. We have previously demonstrated that experimental ASCT can enhance myeloma-specific immunity and limits disease progression characterized by immunological escape. Here we explored the ability of stem cell mobilization (SCM) to mobilize putative myeloma-specific T cells from the bone marrow (BM) and concurrent effects of regulatory T cell depletion (Treg), hypothesizing that this may represent an opportunity to optimize myeloma-specific immunity prior to ASCT. In myeloma patients we noted increased proportions of subsets of effector memory CD8 T cells, including a CX3CR1/granzyme B (GzmB)+ subset in PBSC grafts relative to BM (P<0.01). TIGIT+ Treg were also significantly expanded in PBSC (P <0.01). To study the relevance of these effects, we transferred congenically marked BM T cells into syngeneic mice. We noted that BM-derived effector (CD44+) CD8 cells preferentially homed back to BM (vs. spleen/blood), consistent with T cell residency at this site. SCM subsequently promoted the migration of these resident effector CD8 T cells from BM (4-fold reduction, P <0.001) to blood (5-fold expansion, P <0.001), confirming that PBSC contain BM resident T cells. BM Foxp3+ Treg were also reduced in BM and increased in blood after SCM. Mobilized Treg were characterized by an immunosuppressive phenotype with high levels of CD73, Nrp-1, CTLA4 and TIGIT, and this represents a possible suppressive pathway invoked by SCM. We thus examined the role of BM Treg in limiting myeloma-specific immunity during SCM with Foxp3-GFP-DTR mice. Depletion of Treg with diphtheria toxin (DT) during SCM promoted the expansion of antigen experienced CD8 T cells that secreted IFNg, TNF and GzmB. We next hypothesized that expansion of these polyfunctional cells would enhance myeloma-specific immunity after ASCT. To test this, we treated Vk*MYC bearing Foxp3-GFP-DTR donor mice with DT (vs. saline) 1 week after tumor injection followed by SCM. CD8 T cells from mobilized grafts were transplanted with BM and CD4 T cells from naïve B6 into lethally irradiated Vk*MYC bearing recipients (Figure 1A). Recipients transplanted with CD8 T cells from Treg depleted donors had dramatically improved tumor control (Figure 1B) after ASCT compared to recipients of cells from Treg intact donors (survival 100 days post-ASCT; 100% for DT group vs 13% for saline group, P <0.0001). Rechallenge of mice surviving over 100 days after ASCT confirmed MM clone-specific immunity with complete elimination of the MM seen at initial ASCT, but not an unrelated clone. We next studied clinically relevant approaches to mimic this scenario. Administration of TIGIT, CTLA4, CD25 or LAG3 antibodies resulted in inadequate Treg depletion in BM to recapitulate this effect. In contrast, a synthetic IL-2/IL-15 mimetic NL-201 that selectively binds the beta and gamma chains of IL-2 receptor but not the high affinity IL-2Ra (CD25) reduced BM Treg (45.9% vs 18.6%, P <0.05) and expanded polyfunctional effector cells expressing IFNg+TNF+GzmB+ in SCM grafts. MM-bearing mice transplanted with NL-201 mobilized CD8 T cells were also protected against relapse after ASCT (survival 100 days post-ASCT; 87.5% for NL-201 group vs 25% for saline group, P <0.05). This protection was characterized by expansion of antigen experienced (CD38+) and cytokine secreting (TNF, IFNg) memory (CD44+) CD8 T cells in the BM 3 weeks after ASCT. BM CD8 T cells and Treg are efficiently mobilized after G-CSF/plerixafor. Depletion of Treg in donors during SCM dramatically increases myeloma specific T cells in stem cell grafts and enhances myeloma-specific immunity after ASCT. Stem cell mobilization in the presence of the synthetic IL-2/15 mimetic NL-201 is a promising clinically tractable approach to recapitulate these effects. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD69+ Bone Marrow-Resident CD8+ T Cells Represent a Functionally Impaired Tumor-Reactive Population and Can be Restored By Blocking PD-1 and Tigit in Multiple Myeloma

Recently, novel anti-myeloma therapies utilizing T cell-mediated immune responses, including chimeric antigen receptor (CAR) T cells or bispecific antibodies engaging T cells to target tumor cells, have been actively studied in clinical trials and shown significant clinical efficacy. To enhance the clinical activity of T cell-exploiting immunotherapies in MM further, a better understanding of immune evasion mechanisms by characterizing myeloma-specific T cells and the strategic reinvigoration of the impaired T cell functions are essential prerequisites. In the present study, we demonstrated that bone marrow (BM)-resident CD69-expressing CD8+ T cells showed more prominent pathologic features including tumor-antigen associated exhaustion and impaired function in MM patients. In addition, using direct ex vivo experimental techniques, we examined the strategy how to restore anti-myeloma responses of the tumor-specific T cells, based on the characteristics of BM-resident CD69+ CD8+ T cell population. We first tried to evaluate various immunophenotypes of CD8+ T cells in BM and peripheral blood (PB) compartments of MM patients and found that the frequency of CD69+ cells among CD8+ T cells was significantly higher in BM than in peripheral circulation. These CD69+CD8+ T cells in BM had an effector memory phenotype negative for CCR7. Also, CD8+ T cells obtained from BM of normal controls contained similar frequency of CD69+ cells to the CD8+ T cells from BM of MM patients, suggesting that the CD69+ population could represent a BM-resident memory population even in a physiologic condition. To this end, we isolated CD69+ and CD69- memory CD8+ T cells from BM of MM patients and analyzed their transcriptomic difference using RNA-sequencing analysis. CD69, EPAS1, EOMES, PDCD1, TIGIT, and TOX were significantly upregulated in CD69+ cells when compared to CD69- cells among memory CD8+ T cells. On the contrary, S1PR1, KLF2, KLF4, SELL, GZMB, and GNLY were downregulated in CD69+ cells when compared to CD69- cells. These results indicate that transcriptomic signatures of CD69+ cells were similar to previously known features of tissue-resident memory T cells and functionally exhausted CD8+ tumor-infiltrating lymphocytes. We then compared the surface expression level of immune checkpoint receptors between CD69+ and CD69- cells among BM CD8+ T cells. CD69+ cells showed significantly higher positive frequencies of PD-1, Lag-3 and TIGIT than CD69- cells, but expression of Tim-3 did not differ between two populations. Notably, TOX, the key transcription factor regulating T cell exhaustion, was higher in CD69+ cells than in CD69- cells. When we examined the expression of other transcription factors as indicators of T cell exhaustion, the EomeshiT-betlo cells were significantly enriched in CD69+ population, indicating that CD69+ cells have features of terminally exhausted CD8+ T cells. More importantly, the myeloma antigen (NY-ESO-1157-165 and HM1.2422-30)-specific CD8+ T cells in BM of MM patients, identified with the MHC-multimer technique, also enriched in CD69+ population. When we compared the expression of immune checkpoint receptors among myeloma antigen-specific CD8+ T cells, the frequencies of PD-1+, TIGIT+ and PD-1+TIGIT+ cells were higher in CD69+ cells than CD69- cells. In fact, CD69+CD8+ T cells from BM of MM patients showed reduced production of effector cytokines in response to direct ex vivo TCR stimulation, compared to CD69- counterpart. Next, we investigated whether blocking PD-1 and/or TIGIT could restore the function of BM-resident CD8+ T cells of MM patients. Although a single blockade using either anti-PD-1 or anti-TIGIT antibody was not effective, their combination resulted in significant increase of IFN-γ+ cells in total CD8+ T cells and CD69+CD8+ T cells but not in CD69-CD8+ T cells Taken together, we demonstrated that BM-resident CD69+CD8+ T cells represent a population of exhausted T cells which are functionally impaired but potentially restorable. Indeed, we provide an ex vivo evidence of dual blockade of immune checkpoint receptors to enhance anti-tumor T cell responses in MM. Detailed understanding of BM-resident CD8+ T cells could provide more fundamental information for improving the current T cell-based immunotherapeutic approaches against MM.

Hematopoietic and Immunological Assessment in Fanconi Anemia after Ex Vivo Lentiviral FANCA Gene Therapy with RP-L102

Introduction: Fanconi anemia (FA) is the most frequent cause of genetic bone marrow failure (BMF) resulting from inherited defects in the FA pathway, one of several essential deoxyribonucleic acid (DNA) repair mechanisms. Although allogeneic hematopoietic stem cell transplantation (alloHSCT) offers hematolymphoid cure, it confers significant morbidity and mortality and is thereby indicated only after development of severe BMF or dysplasia. Ex vivo hematopoietic stem and progenitor cell (HSPC)-based gene therapy (GT) offers an alternative strategy for earlier and potentially safer intervention with the goal of preventing BMF and the need for alloHSCT. Core assessments of safety and efficacy are conducted as part of ongoing GT clinical trials for FA-type A patients who have received ex vivo lentiviral FANCA GT. In these additional exploratory research studies we seek to better understand how GT may affect HSPC behavior, mutational acquisition and immune function. Methods: To deepen our understanding of the effects of ex vivo lentiviral FANCA GT on FA biology, we collected serial bone marrow (BM) and peripheral blood (PB) samples from FA-Type A patients who were enrolled at Stanford University on the Phase I and II RP-L102-0418 and RP-L102-0319 clinical trials. We initiated the assessment of Phase I subjects at 18 months post treatment and the assessment of Phase II subjects at study enrollment. BM was assessed to characterize HSPC distribution by flow cytometry, proliferative capacity by colony-forming count (CFC) assays, transcriptomics by single-cell RNA sequencing (scRNASeq) and mutation acquisition by targeted next generation sequencing (NGS). Immune system composition and function were assessed using PB flow cytometry, immunoglobulin levels and T cell proliferative assays. Results: Of the 8 enrolled patients, 5 subjects met eligibility criteria and were treated with RP-L102 GT. As of April 2022, 4 remain on study, now 18 to 36 months post-treatment. Of these 4 subjects, 3 have demonstrated increasing vector copy number and MMC-resistance. At time of enrollment, HSCs were markedly reduced in these patients compared to healthy controls, even in patients with no or mild cytopenia, and to-date have remained decreased post-GT. Functional HSPC proliferation capacity, as assessed by CFCs, was reduced in all patients at screening but showed evidence of improvement or stabilization in 3 of the 4 post-GT patients, as did BM total cellularity and CD34+ counts. scRNASeq showed reduced proportions of HSCs pre- and post-GT relative to healthy controls. Expression of ribosome biogenesis genes, an indicator of cancer risk, was downregulated post-GT. Targeted bulk-NGS revealed that none of the patients evaluated on these studies had mutations known to be associated with myeloid malignancies pre- or post-GT treatment. Detailed immune monitoring revealed that 3 patients had B-cell lymphopenia at time of enrollment. Additionally, 1 patient showed an increase in B-cell numbers post-GT. IgG levels were normal in all 8 screened patients pre-GT and remained stable post-GT. Interestingly, among 7 vaccinated patients evaluated at enrollment, 3 did not have protective IgG levels to Hepatitis B, 1 did not have protective IgG levels to Varicella Zoster Virus (VZV), and 3 showed equivocal levels of anti-VZV IgG. Conclusions: Although our results are based on a small cohort and are interim within ongoing clinical trials, they indicate the need for more thorough assessment of BM and immune characterization in FA patients beyond reliance on blood counts. Importantly, the paucity of HSCs in FA patients even prior to the development of cytopenias supports the need for early intervention for HSC preservation which may be possible with GT upon diagnosis. Additionally, these studies show that RP-L102 GT appears to be a safe approach for hematopoietic preservation in FA without acquisition of detectable mutations in HSPCs to-date. Moreover, downregulation of ribosome biogenesis genes can potentially lead to decreased leukemogenesis which could decrease cancer risk post-GT. Finally, we show laboratory evidence of baseline immune dysfunction in FA patients mainly consisting of B-cell lymphopenia and poor response to certain vaccines. Given this, post-GT re-vaccination of patients may be clinically indicated and would enable assessment of functional immune recovery potentially associated with the GT treatment.

A Retrospective Study of Antibody Titers in Patients with Plasma Cell Disorders

Background: Recent studies have estimated that infections occur with increased risk in patients with plasma cell disorders as compared to the general population. Our group has previously shown that patients who show response to vaccination following autologous hematopoietic stem cell transplantation (AHCT) have better overall survival and progression free survival than those who do not. In clinical practice, antibody titers against pathogens that are targeted by vaccines are measured infrequently in patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM) and multiple myeloma (MM) at the time of diagnosis. Moreover, studies have previously shown that AHCT and maintenance therapy can cause seroconversion of titers in MM patients but the effect of induction chemotherapy on seroconversion of antibody titers is unknown. Methods: The study population comprised of controls and patients with plasma cell disorders who were seen at Roswell Park Comprehensive Cancer Center from 2018-2022. Controls were defined as patients consulted to our service to rule out gammopathies but found to be negative on initial testing. Immunoparesis was defined as deficiency of uninvolved immunoglobulins. All individuals had antibody titers tested at the time of first diagnosis. Patients with MM who underwent induction chemotherapy followed by AHCT had antibody titers tested prior to AHCT. Serological titers were measured according to Roswell Park Central Clinical Laboratory standards. Interpretation of individual titer results was in accordance with the manufacturers’ recommendations and published literature. Differences of the titers were compared by non-parametric Kruskal-Wallis Test /General Linear Regression Model (for continuous variables) and Fisher's Exact Test. SAS version 9.4 (SAS Institute, Cary, NC) was used for statistical analyses. All tests were two-sided and performed at a nominal significance level of 0.05. Results: A total of 101 patients including 24 (23.8%) controls, 21 (20.8%) MGUS patients, 16 (15.8%) SMM and 40 (39.6%) MM patients were included in the study. At the time of first diagnosis, controls had the greatest number of positive titers (mean 14.21, SD 3.48) followed by patients with MGUS (mean 13.86, SD 3.18), SMM (mean 11.06, SD 3.89) and MM (mean 9.33, SD 4) (p<0.0001), respectively (Figure 1). A total of 66 (65%) patients had a bone marrow biopsy performed at the time of diagnosis, including 12 (11.9%) patients with MGUS, 16 (15.8%) patients with SMM and 38 (37.6%) patients with MM. On linear regression analysis, there was a negative correlation between the number of positive titers and bone marrow plasma cell percentage (p=0.0009). Next we stratified patients with plasma cell disorders into those with immunoparesis (n=54) and those without immunoparesis (n=23) at the time of measurement of antibody titers. The number of positive titers were significantly higher in patients without (Mean 13.57, SD 3.53) than those with immunoparesis (Mean 9.80, SD 3.97) (p=0.0002). In total 48 patients including 12 (11.9%) MGUS patients, 10 (9.9%) SMM patients and 26 (25.7%) MM patients had peripheral blood flow cytometry for CD19+ B cell percentage at the time of measurement of antibody titers. On linear regression analysis, there was a positive correlation between the number of positive titers and the CD19+ B cell percentage (p=0.018). We further studied the seroconversion of antibody titers with induction chemotherapy and 22 out of 40 patients had both pre-and post induction treatment titers. Among them, 8 (36.4%) had seroconversion of titers from negative to positive for at least 3 pathogens without revaccination. Conclusion: Antibody titers for vaccine preventable diseases are lower in patients with MGUS, SMM and MM as compared to controls at the time of diagnosis. Current guidelines for revaccination are not extended to patients with MGUS and SMM and have to be investigated in prospective clinical trials before they can be generally recommended. Factors such as bone marrow plasma cell percentage, immunoparesis and CD19+ B cell percentage correlate with the number of positive titers. In MM patients undergoing AHCT, more than a third showed seroconversion of antibody titers from negative to positive prior to AHCT without revaccination, suggesting a role of induction chemotherapy in augmenting the immune response by reducing tumor burden. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Correlation of Clonal Hematopoiesis in Vexas Syndrome with Immunophenotype Alterations in CD34+ HSPCs (Hematopoietic Stem Progenitors Cells): Multiparametric Flow Cytometry Heterogeneity in CD34+CD38- Stem Cells Fraction and Upregulation of CD90 Thy-1 By Unsupervised Strategy Using PCA, Tsnee and Flowsom

Introduction: VEXAS (vacuoles,E1enzyme,X-linked, autoinflammatory,somatic) syndrome is caused by somatic UBA1mutations affecting myeloid compartment of hematopoietic stem and progenitor cells (HSPC).VEXAS syndrome is a unique systemic autoinflammatory disease associated with cytopenias with marrow dysplasia(1).There is currently no published data concerning the immunophenotypic characteristics pattern of CD34+ HSC in UBA1 mutated patients w/wo MDS.The identification of an easily-accessible profile in CD34+CD38- most immature HSC for UBA1 mutations might help to identify UBA1 patients beside molecular testing and potential target of therapeutic interest. Methods: Here we report flow cytometry of bone marrow CD34+ HSPCs in a cohort of 11 UBA1 mutated patients from the Lyon University Hospital. Our routine 2-tubes panel used in the follow-up of AML MRD, were applied with minimal mandatory 8 markers per tube, according to ELN recommendations (to identify the pattern of LAIP/DfN in bulk and LSC in CD34+CD38- fraction). A «backbone» of CD34/CD38/CD45/CD117 , and CD7,CD56, CD13, CD33, CD19 for the 1st tube and CD90,CLL1+TIM3+CD97, CD123, CD45RA for the 2nd one. Acquired data in BD Canto and Lyric cytometers were analysed using DIVA/FlowJo and KALUZA softwares for classical strategy completed with Tsnee and FlowSom software for unsupervised analyse. Control group was healthy donor or regenerating marrow and MDS patients UBA1 negative. A total of minimal 500000 to one million of cells was acquired in each tube. Results: The clinical and biological characteristics of the 11 UBA1 mutated patients are summarized in Fig1A. Parameters as MFI in cell lineage and LSC markers, percentage of targeted populations (lympho-myeloid primed progenitor (LMPP), Multipotential Progenitor (MPP), Common myeloid progenitor (CMP), granulocyte/monocyte progenitor (GMP) and megakaryocyte/erythroid progenitor (MEP), were integrated in principal component analysis (PCA) (Fig1B). PCA analysed compared our VEXAS cohort with normal marrow (n=10) and MDS patients without UBA1 mutation (n=23), and clusterized properly UBA1 mutated patients from normal marrowand MDS patients. The most contributive variables for appropriate clusterizing were LSC/progenitors markers CD90 Thy1/ CD45RA and CLL1/TIM3 /CD97, CD123 and GMP, MEP, MPP, LMPP-like populations. Interestingly, all normal marrow clusterized together, highly separated from UBA1 mutated cases. However, MDS UBA1 negative patients were heterogeneously distributed, suggesting a strong variability in CD34+ fraction. No lineage marker (i.e. CD13) or aberrant markers (i.e. CD7) was involved in PCA clustering, suggesting that alteration in LSC:HSCP fractions are mostly relevant for distinction between clonal haematopoiesis in UBA1 mutated patients compared to MDS UBA1 negative and normal marrow. A correlation matrix showed a close correlation between LSC/progenitors markers and CD34+CD38- frequency, mostly based on CD90/Thy-1 overexpressed in clonal hematopoiesis observed in UBA1 mutated patients (Fig 1C). We merged data from 2 UBA1 mutated, 2 MDS UBA1 negative and 4 healthy donors to perform unsupervised analysis Tsnee and FlowSom (Fig1D). Interestingly, both UBA1 mutated patients showed different pattern in CD34+ compared to MDS and normal healthy donors suggesting most primitive alteration in CD90 Thy-1 expression in CD34+CD38-cells. Flowsom map allowed to identify nodes of stem cell with abnormal phenotype clearly different in UBA1 mutated patients, backgating of this node showed the overexpression of CD90 in CD34+CD38- cells in UBA1 pos. Conclusions: This is the first large immunophenotypic characterisation by flow of clonal haematopoiesis in UBA1 mutated patients. Our results showed that VEXAS syndrome is characterized by overexpression of CD90 Thy-1 primitive Stem cell markers in CD34+ HSPCs and in most immature CD34+CD38- fraction. Altogether, we can hypothesize that such phenotype might represent specific disease hallmark involved in physiopathology of niche deregulation, which should be confirmed in a larger cohort of patients in a future multicentric clinical trials to improve biological diagnosis and comprehension of kinetics of clonal haematopoiesis in VEXAS patients. 1) Beck DB, Ferrada MA, Sikora KA, et al. Somatic mutations in UBA1 and severe adult-onset autoinflammatory disease. N Engl J Med. 2020;383:2628-2638. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment with Oral Azacitidine in Elderly Patients with Higher Risk Myelodysplastic Syndromes after Response to Subcutaneous Azacitidine

Background Hypomethylating agents (HMAs) are the standard of care for (elderly) IPSS-R higher risk MDS (HR-MDS) patients, but HMA treatment for those patients who are not eligible for hematopoietic stem cell transplant has to be continued for very long time to maintain efficacy. This prolonged continuous treatment renders patients dependent on hospital and on caregivers, and as a result, decreases QoL and too often provokes premature interruption of therapy. Oral azacitidine is approved for maintenance therapy of acute myeloid leukemia patients in complete remission after intensive chemotherapy, and it has shown activity in lower risk MDS. Oral decitabine has been recently approved by FDA for treatment of MDS. There are no data regarding maintenance of response achieved by sc azacitidine in HR MDS by switching to the oral formulation. Aim The primary objective of the study is to explore the feasibility of replacing subcutaneous (sc) azacitidine by the oral formulation, in patients with HR-MDS and responding to sc azacitidine as well as the maintenance or improvement of the achieved hematological response (time to relapse/progression).Methods A monocentric, pilot phase 2 study (ClinicalTrials.gov: NCT04806906) was planned to enroll 10 subjects with confirmed diagnosis of MDS or dysplastic non proliferative CMML, IPSS-R higher risks, aged ≥ 65 years, in CR/CRi, PR or SD with HI stable for additional 2 cycles after response at 4-6 cycles of azacitidine therapy, hence a total of at minimum 6 to 8 cycles of sc azacitidine. At screening, morphological MDS diagnosis, cytogenetic analysis, IWG response assessment, bone marrow and peripheral blood sampling for flow cytometric analysis, NGS evaluation of somatic mutations, DNA methylation analysis and HRQoL are performed. During treatment, subjects are assessed for safety, tolerability and efficacy of oral azacitidine. Assessments during treatment include monitoring for AEs, monitoring for loss of response and SPMs, physical examination, vital signs and weight measurement, ECOG performance status, hematology and serum chemistry, serum ferritin level, concomitant medications, therapies and procedures, review of bone marrow aspirate or biopsy. Patients receive 300 mg oral azacitidine for the first 14 days of each 28-day treatment cycle until no longer receiving the benefit or until progression. Dose of oral azacitidine can be de-escalated based on toxicity. Besides feasibility of treatment with oral azacitidine, we evaluate safety/tolerability, time to treatment discontinuation, and patient-reported outcomes, utilizing the EQ-5D, and measures of healthcare resource utilization. DNA methylation pattern of bone marrow CD34 positive cells at diagnosis, after response to sc azacitidine, and after 2 and 4 cycles of oral azacitidine is also determined by ERRBS for all patients. Results As per August 1, 2022, with a follow up ranging from 1-10 months, 5 HR MDS patients have been enrolled in the study. Median age is 81 yrs (79-86). Male/female ratio: 4/1. At start of therapy with sc azacitidine IPSS-R risk was high 3/5, very high 2/5. Median number of cycles of sc azacitidine is 25 (7-56), median number of cycles of oral azacitidine is 4 (1-9). The dose of 300mg/day was maintained for all patients. Pattern of DNA methylation determined by ERRBS is available for all treated patients and confirms the activity of oral azacitidine. Two patients evolved to AML after 9 and 4 cycles of oral azacitidine, with progression free survival (PFS) from onset of therapy of 17 and 12 months respectively. None of those 2 patients had adverse events during therapy. Treatment was interrupted after 2 cycles in 1 patient following a GI event grade 3 and following patient decision; 2 patients are still ongoing without report of hematological or non-hematological toxicity. At present 1/5 patients died with AML. Conclusions. The patients included in this study were HR MDS, particularly frail, not eligible to investigational studies because of poor compliance and very old median age. The administration of oral azacitidine in patients who responded to sc azacitidine is feasible, the treatment is very well tolerated with no AEs different from those observed in sc azacitidine treatment. Two of five treated patients experienced progressive disease after >12 months of HMA therapy from diagnosis. Oral azacitidine may allow prolonged and continuous treatment in frail HR MDS, optimizing outcome.

Glutamate Metabolism Shapes Osteoclast Differentiation: New Potential Therapeutic Target to Block Osteoclastic Bone Resorption in Multiple Myeloma Patients

Multiple Myeloma (MM) cells are characterized by an impaired Glutamine (Gln) metabolism and Gln-addiction feature. As a result of this, MM patients have lower levels of Gln and higher levels of its main metabolite Glutamate (Glu) in the bone marrow (BM) microenvironment as compared with Smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS) patients. Moreover, we have recently shown that MM-dependent Gln-exhausted microenvironment impairs osteoblast differentiation. Based on this evidence, we have hypothesized that alterations of Gln/Glu axis may have a significant impact on the activation of osteoclasts (OCLs) and in the development of osteolytic lesions. We purified BM CD14+ monocytes from a cohort of 30 patients with monoclonal gammopathies (5 MGUS, 12 SMM and 13 newly diagnosed MM (ND-MM)). Cells have been incubated in osteoclastogenic medium in the presence or absence of Glu. The samples were subsequently analyzed by real-time PCR, Western blot and TRAP staining after 3 or 14 days. Amino acid uptake and amino acid content have been assessed under the same conditions. Glu uptake inhibitors have been tested throughout osteoclastogenesis. Our data showed that the presence of Glu significantly increases the number of OCLs generated from MGUS/SMM CD14+ compared with cells differentiated in the absence of the amino acid (p<0.01). In these patients, Glu increases the expression of RANK as well as NF-κB and NFATc1 protein level pointing to a stimulatory effect on osteoclastogenic signaling pathway. On the contrary, CD14+ obtained from MM patients have a lower response to Glu probably as a consequence of the high Glu microenvironment imposed by MM cells. Consistently, MM CD14+ cells are prone to generate more OCLs also in the absence of Glu compared with CD14+ cells obtained from premalignant samples (median number of OCLs: MGUS/SMM 33, MM 123, p=0.02). The analysis of intracellular amino acids (Gln, Glu, and α-ketoglutarate (2-OG)) indicated that Glu content significantly increases after 8 days of differentiation while decreasing after 14 days pointing to a fast accumulation of Glu upon differentiation. The content of both Gln and 2-OG do not vary appreciably throughout the differentiation. These data suggest that OCLs differentiation is associated with an increased uptake of extracellular Glu. Consistently, the activity of the sodium-dependent Glu transporters EAATs, increases already after 3 days of incubation in differentiating medium without Glu, with a further increase in the presence of the amino acid. Expression analysis revealed that, among EAATs, EAAT1 (encoded by SLC1A3 gene) is responsible for the uptake of extracellular Glu upon differentiation both in the presence and in the absence of the amino acid. The expression of other Glu transporters do not change during differentiation. Selective inhibitors of EAAT1, UCPH101 and TFB-TBOA completely block the formation of multinucleated OCLs in the presence of Glu (p<0.01). In parallel to EAAT1 induction, Glu treatment increases the expression of Calcineurin, the serine/threonine phosphatase activated by increased intracellular calcium levels indicating the possible modulation of calcium signaling by Glu. In conclusion, our results indicate that high BM Glu level sustains OCLs formation by stimulating of NF-κB/NFATc1/calcineurin pathway. EAAT1 transporter is responsible for the uptake of extracellular Glu during osteoclastogenesis while its inhibition markedly blocks OCL formation. Therefore, targeting glutamate metabolism could be a therapeutic strategy to block OCL formation in MM patients.