Journal of Hematology & Oncology

Background.CAR T-cell therapy is the standard of care for R/R DLBCL, but more than half of patients fail to achieve long-term remission. Identification of cellular biomarkers in CAR T- cell infusion products (IPs) that predict complete remission beyond six months may guide the development of strategies to improve outcomes. Methods.IPs from 13 R/R DLBCL patients were analyzed using a 39-marker mass cytometry panel, comparing cell populations between long-term responders (R) and non-responders (NR). Both unsupervised and supervised analyses were performed. Longitudinal blood samples were analyzed for 30 days to track CAR T-cell subpopulation dynamics. Results.At a median follow-up of 13.5 months, median progression-free survival (PFS) was 13.3 months (95% CI: 9.7-24.3) in R (n=8) versus 3.5 months (95% CI: 0.5-5.4) in NR (n=5). The HR for PFS was 56.67 (95% CI: 7.3-439.3; P=0.0001). A subset of CD3+CXCR3+CD7+ CAR T-cells found in both CD4+ and CD8+ populations was significantly enriched in R and expressed higher levels of perforin, granzyme B, and NKG2D (restricted to CD8+). NR had more CXCR3+CD7+LAG3+ CAR T-cells. CD3, CD7, CXCR3, and NKG2D cell surface levels were higher in R, whereas LAG3, Ki67, and CD71 were elevated in NR. A predictive cut-off ratio of CD3+CXCR3+CD7+LAG3+CAR+ T-cells <0.83 and CD3+CXCR3+CD7+NKG2D+CAR+ T-cells >1.034 yielded a predictive accuracy of 0.92. Serum CXCL9 and CXCL10 levels were not different between groups. Conclusions.Increased frequency of CAR T-cells expressing CD7, CXCR3 and NKG2D in R versus LAG3 and CD71 in NR emerged as strong correlates of therapeutic outcome.

The unique cytoskeletal protein Septin7 is generally considered to be required for cytokinesis in yeast and mammals. Whole body genetic ablation of Septin7 in mice is embryonic lethal. Septin7-deficient fibroblasts and HeLa cells are defective in cytokinesis and undergo obligate multinucleation. Surprisingly, lymphocyte- and myeloid-specific deletion of Septin7 in mice did not result in any detectable abnormalities in blood lineage development, suggesting that Septin7 is dispensable for steady-state hematopoiesis. In contrast, Septin7-deficient hematopoietic stem cells failed to engraft and establish donor chimerism following transplantation, indicating that Septin7 is essential for hematopoietic stem cell function under stress conditions. To reconcile these contradictory findings, we analyzed the effects of Septin7 deletion in hematopoietic cells from mice with either pan-hematopoietic or lymphoid lineage-specific Septin7 deletion. Our results demonstrate that Cre-induced deletion of the floxed Septin7 allele is inefficient during the early stages of hematopoiesis, suggesting strong selection pressure against Septin7 deficiency at this stage. In contrast, deletion of Septin7 at the common lymphoid progenitor stage, as well as in Hoxb8-immortalized hematopoietic progenitors, is efficient and does not result in any noticeable defects in cell division. Taken together, our findings indicate that Septin7 is essential during early hematopoiesis but becomes dispensable at later stages.

BackgroundWhile hypomethylating agents (HMA) are currently used to treat myelodysplastic syndrome (MDS) and patients with cancer, their effects on reactivation and/or upregulation of oncogenes are generally not well elucidated. SALL4 is a known oncogene that plays an important role in MDS. In this study, we examined the impact of HMA on SALL4 methylation and expression. MethodsPaired bone marrow samples from a cohort of MDS patients on the BMT-AZA trial, collected before and after four cycles of azacytidine (AZA) treatment, were used to explore the relationship between changes in SALL4 expression, treatment response and clinical outcome with a follow-up of up to 40 months. No/low-SALL4 expressing leukemic cell lines were used to study the relationship between SALL4 methylation and expression. A novel locus-specific demethylation technology, CRISPR-DNMT1-interacting RNA (CRISPR-DiR), was used to identify the CpG island critical for SALL4 expression. ResultsIn MDS patients, we noted SALL4 upregulation after AZA treatment in 40% of the cases. Significantly, patients with SALL4 upregulation had a worse outcome. Using CRISPR-DiR, we discovered that demethylation of a 500bp CpG island within the 5UTR-Exon1-Intron1 region was critical for SALL4 expression. Importantly, in cell lines and patients, we confirmed that HMA treatment led to demethylation of the same CpG region and upregulation of SALL4 expression. ConclusionsCRISPR-DiR was useful to define the critical region important for gene activation. Along with analysis of patient samples, we demonstrated that demethylation and upregulation of an oncogene after HMA treatment can indeed occur and should be further studied.

Anti-CD38 antibody therapies have transformed multiple myeloma (MM) treatment. However, a large fraction of patients inevitably relapses. To understand this, we investigated 32 relapsed MM patients treated with daratumumab, lenalidomide, and dexamethasone (Dara-Rd; NCT03848676). Whole genome sequencing (WGS) before and after treatment pinpointed genomic drivers associated with early progression, including RPL5 loss and APOBEC mutagenesis. Flow cytometry on 202 blood samples, collected every three months until progression for 31 patients, revealed distinct immune changes significantly impacting clinical outcomes. Progressing patients exhibited significant depletion of CD38+ NK cells, persistence of T cell exhaustion, and reduced depletion of T-reg cells over time. These findings underscore the influence of immune composition and daratumumab-induced immune changes in promoting MM resistance. Integrating genomics and flow cytometry unveiled associations between adverse genomic features and immune patterns. Overall, this study sheds light on the intricate interplay between genomic complexity and the immune microenvironment driving resistance to Dara-Rd.

Hematopoiesis requires the coordinated differentiation of hematopoietic stem cells and progenitor cells (HSPCs) in multiple tissues. Although differentiation of HSPCs in bone marrow (BM) has been well-studied, our knowledge about the migration and differentiation of HSPCs cross tissues is limited. Here, we collected and integrated single-cell RNA-seq data of human CD34+ cells, which represent HSPCs, from BM, peripheral blood (PB), thymus and mobilized PB (mPB), to investigate the hematopoiesis cross tissues. We constructed a cell atlas of HSPCs cross tissues and found most HSPC subsets in BM had counterparts in PB, indicating migration of HSPCs from BM to PB has a much broad spectrum. We found B progenitors highly expressed CXCR4 for anchoring in BM, while cells with low expression of CXCR4 facilitate their migration out of BM. Among the HSPC subsets from thymus, we only found the counterparts of the earliest thymic progenitors (ETPs) in BM and PB, potentially indicating that ETPs were the subsets that migrated from BM to PB and thymus. We found interaction signaling including CD99-CD99, CXCL12-CXCR4 and CCL19-CCR7 played important roles in ETP homing to thymus. Briefly, these data provided a single unified developmental spectrum of hematopoiesis cross different tissues, connected by cell migration.

Chimeric antigen receptor (CAR) natural killer cells (CAR NK) cells, leveraging safety and not requiring HLA match in adoptive infusion, have emerged as promising alternative cells to CAR-T cells for immunotherapies. High and multiple doses of CAR NK cell infusions are essential to maintain therapeutic efficacy in clinical trials. This requires efficient methods for generating large-scale CAR NK cells and significantly reducing CAR engineering costs. In this study, we develop a three-step strategy to generate highly high yields of induced NK (iNK) and CAR iNK cells from human umbilical cord blood CD34+ hematopoietic stem and progenitor cells (CD34+ HSPCs). Starting from a single umbilical cord blood CD34+ HSPC, our reliable method efficiently produces 14-83 million mature iNK cells or 7-32 million CAR iNK cells with high expression levels of CD16 and zero T cell contaminations. Introducing CAR expression elements at the HSPC level reduces the quantities of CAR pseudoviruses to 1 / 140.000 - 1 / 600,000 compared to engineering CARs in mature NK cells. The iNK and CAR iNK cells, including fresh cells and thawed cells from cryopreserved conditions, demonstrate remarkable tumoricidal activities against various human cancer cells and significantly prolong the survival of human tumor-bearing animals. The high yields of CAR NK cells and negligible costs of CAR engineering of our method support the broad applications of CAR NK cells for treating cancer patients.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer worldwide, and exhibits high molecular heterogeneity. Molecular subtypes are characterized by specific chromosomal and molecular alterations, which are critical for guiding risk-adapted therapies. However, the increasing number of recognized prognostic molecular subtypes demands large resources, which are often limited in low/middle-income countries; thereby restricting the molecular characterization. This study aimed to perform an integrated molecular characterization of childhood B-ALL in Argentine patients. We performed RNA-seq on diagnostic bone marrow aspirates from Argentine patients enrolled in the ALLIC-GATLA-2010 protocol. We used different bioinformatic tools to identify and validate single nucleotide variants, fusion transcripts, gene expression profiles and molecular subtypes. We successfully determined transcriptome-based molecular subtype in 93.7% of patients; with high concordance to conventional karyotyping and RT-PCR (17/18 patients with available molecular data). Analysis of chimeric transcripts revealed 82 fusions, both intra- and inter-chromosomal, suggesting that leukemic cells may undergo chromosomal instability. Two of these fusions were novel: SCAF8::FER1L4 and DBF4B::EFTUD2. We also identified 21 different SNVs/InDels in 16 genes, including three novel variants (DUX4 p.I65N, CREBBP p.G1542V, and CSF3R p.G147R) and predicted to alter protein function. Overall, we observed that all patients who relapsed carried high-risk genetic alterations at diagnosis. Whole-transcriptome analysis of leukemic bone marrow enabled molecular subtyping and the identification of both known and novel molecular alterations associated with prognosis.

Targeted immunotherapies against B-cell maturation antigen (BCMA) have transformed the treatment landscape of Multiple Myeloma (MM). Fc receptor-like 5 (FCRL5) has emerged as an alternative target. However, resistance frequently emerges within months, posing a significant clinical challenge. Structural alterations and mutations in BCMA only account for the minority of cases and insights into BCMA antigen escape remain largely unknown. This study investigates novel (epi)genetic mechanisms of antigen escape through comprehensive multi-omic Oxford Nanopore profiling of sequential pre-treatment and relapse samples. We identify acquired DNA-hypermethylation across the entire BCMA gene, and hypermethylation of the FCRL5 promoter, both resulting in epigenetic gene silencing as novel resistance mechanisms through which MM cells evade therapy. These findings underscore the dynamic clonal evolution of MM under therapeutic pressure and highlight the critical role of epigenetic modifications in resistance. Furthermore, we demonstrate the potential of advanced sequencing technologies for capturing epigenetic and complex genomic alterations in clinical settings, paving the way for personalized treatment strategies and predictive biomarkers for early resistance detection. Statement of significanceAcquired DNA hypermethylation of BCMA and FCRL5 regulatory regions, leading to gene expression downregulation, represent novel epigenetic resistance mechanisms to anti-BCMA and anti-FCRL5 immunotherapies. Furthermore, DNA methylation marks serve as a molecular memory of therapeutic pressure, capturing the treatment history of cancer cells.

Lirilumab is a fully human monoclonal antibody designed to block killer inhibitory receptors (KIR), which are major immune checkpoints involved in the regulation of NK cell-mediated killing of HLA-I-expressing tumors. EFFIKIR is a multicenter randomized double-blind 3-arm placebo-controlled phase II trial with lirilumab as single-agent as maintenance therapy of elderly patients with AML in first complete remission (NCT01687387). Two dose schedules led to either continuous or intermittent KIR occupancy. 153 patients were randomized and 152 patients were treated after 3+7 induction therapy. The median follow-up was 36.6 months. Lirilumab was well tolerated, with no significant hematological toxicity. The median LFS were 17.6, 6.7 and 13.9 months in the 0.1mg/kg arm, 1mg/kg arm and placebo arm, respectively. An excess in early relapse led to early termination of treatment in the 1mg/kg arm. Extensive analysis of immune cell fate following KIR blockade evidenced a decrease of KIR+ NK cell absolute counts following KIR blockade, associated with a decrease of Bcl-2. Lirilumab also bound antigen-experienced CD8+ T cells, and induced a transient decrease of CD69 expression. Besides, lirilumab bound v{delta}2+ {gamma}{delta}T cells with a high cytotoxic potential, and induced a decrease of DNAM-1 and Bcl-2, the latter being associated with a decrease of KIR+ {gamma}{delta}T cell, and with a drastic reduction of time to relapse. Overall, the potentially deleterious effects on immune effectors may have resulted in the impairment of immune surveillance associated with an unexpected high rate of early relapse in the group of patients exposed to prolonged full KIR blockade. KEY POINTSO_LIProlonged full KIR blockade leads to potentially deleterious effects on NK cells, CD8+ T cells and v{delta}2+ {gamma}{delta}T cells C_LIO_LICombined inhibitory effects of KIR blockade may have resulted in the impairment of immunosurveillance associated with high rate of relapse C_LI

The mechanisms underlying myeloid malignancies deletions are not well understood, nor is it clear why specific genomic hotspots are predisposed to particular deletions. In the current study we inspected the genomic regions around recurrent deletions in myeloid malignancies, and identified microhomology-mediated end-joining (MMEJ) signatures in recurrent deletions in CALR, ASXL1 and SRSF2 loci. Since MMEJ deletions are the result of DNA double-strand breaks (DSBs), we introduced CRISPR Cas9 DSBs into exon 12 of ASXL1, successfully generating recurrent ASXL1 deletion in human hematopoietic stem and progenitor cells (HSPCs). A systematic search of COSMIC dataset for MMEJ deletions in all cancers revealed that recurrent deletions enrich myeloid malignancies. Despite this myeloid predominance, we provide evidence that MMEJ deletions occur in multipotent HSCs. An analysis of DNA repair pathway gene expression in single human adult bone marrow HSPCs could not identify a subpopulation of multipotent HSPCs with increased MMEJ expression, however exposed differences between myeloid and lymphoid biased progenitors. Our data indicate an association between MMEJ-repaired DSBs and recurrent MMEJ deletions in human HSCs and in myeloid leukemia. A better understanding of the source of these DSBs and the regulation of the HSC MMEJ repair pathway might aid with preventing recurrent deletions in human pre-leukemia.

BackgroundCD19-directed chimeric antigen receptor (CAR) T-cell therapies have transformed the treatment landscape for pediatric B-cell acute lymphoblastic leukemia (B-ALL), yet relapse driven by antigen escape remains a major limitation. Dual-targeting CAR approaches recognizing CD19 and CD22 have shown promising clinical activity. However, sustained remissions are limited by insufficient CAR T-cell persistence. MethodsCAR22.19, a fully human tandem CD19/CD22 CAR, was developed and clinically applied within a named-patient program in nine heavily pretreated pediatric patients with relapsed/refractory B-ALL. Treatment indications were CD19-negative blast population (n=5), relapse after CD19 CAR T (n=3) and/or restricted access to approved CAR T-cell products (n=3). Autologous and donor-derived CAR22.19 T-cells (CART22.19) were manufactured using a GMP-compliant, semi-automated fresh in fresh out process. Safety and efficacy were assessed through standardized clinical monitoring, measurable residual disease analysis, and CAR T-cell kinetics. ResultsPreclinical validation demonstrated antigen-specific cytotoxicity and dual-antigen activity. Clinically, CART22.19 were well tolerated, with no treatment-related deaths and no grade [&ge;]3 neurotoxicity, while grade [&ge;]3 cytokine release syndrome occurred in 38.5% (5/13) of infusions and resolved with standard interventions. An initial complete molecular remission was achieved in 78% (7/9) of patients, with a 12-month overall survival rate of 53.3% (95% CI, 17.7-79.6%). Sustained treatment response in CD19-CD22 cases underscore the functional contribution of the CD22-targeting domain. In contrast, all patients refractory to prior CD19 CAR T-cell therapies relapsed early with retained CD19CD22 expression. Limited in vivo persistence was found to be a key mechanism of treatment failure. Notably, durable remission and sustained functional persistence of CART22.19 was achieved in one patient refractory to autologous CART22.19 following infusion of donor-derived CART22.19 after reduced-intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (alloHSCT) in non-remission. ConclusionsCART22.19 therapy demonstrated a favorable safety profile and promising clinical activity in a high-risk pediatric population, with dual targeting enabling disease control in CD19-negative disease. However, limited CAR T-cell persistence remains a major obstacle to sustained remission. Our findings support further clinical development of CART22.19 and highlight the potential of donor-derived CAR T-cells following RIC alloHSCT as a novel therapeutic strategy to enhance persistence and improve outcomes in heavily pretreated pediatric patients.

Genomic profiling of hematologic malignancies has augmented our understanding of variants that contribute to disease pathogenesis and supported development of prognostic models that inform disease management in the clinic. Tumor only sequencing assays are limited in their ability to identify definitive somatic variants, which can lead to ambiguity in clinical reporting and patient management. Here, we describe the MSK-IMPACT Heme cohort, a comprehensive data set of somatic alterations from paired tumor and normal DNA using a hybridization capture-based next generation sequencing platform. We highlight patterns of mutations, copy number alterations, and mutation signatures in a broad set of myeloid and lymphoid neoplasms. We also demonstrate the power of appropriate matching to make definitive somatic calls, including in patients who have undergone allogeneic stem cell transplant. We expect that this resource will further spur research into the pathobiology and clinical utility of clinical sequencing for patients with hematologic neoplasms.

The loss of chromosome Y (LOY) in leukocytes is the most prevalent form of clonal mosaicism observed in older men. It is associated with all major causes of mortality, including cardiovascular diseases and cancer. However, LOY effect on individual immune-cell lineages remains unclear. Here, we used single-cell RNA sequencing data from Onek1K cohort and showed that LOY has a wide effect on cell fate across immune cell populations with the largest representation of LOY in classical monocytes. These monocytes exhibit a profibrotic signature marked by downregulation of IL1B and MYC-regulated genes, consistent with previous observations of LOY-associated macrophages in cardiac and pulmonary injury. Additionally, we detected an aberrant expression of XIST, the essential X-chromosome inactivation (XCI) lncRNA expressed in females, and not normally expressed in males. Notably, we observed upregulation of genes known to escape X-inactivation, including male-biased cancer-related genes KDM6A, DDX3X, KDM5C, and ZRSR2. Our results demonstrate the effect of LOY on the fate of immune cells, mediated by cell-type specific transcriptional changes and aberrant XCI characteristics.

Reduced intensity conditioning (RIC) is fraught with risk for disease relapse. This may be overcome by donor T cell alloreactivity. Reducing the duration of intense immune suppression in the early term following transplantation may create an immunologic environment favoring rapid T cell reconstitution to influence longer term transplant outcomes. Twenty-six patients were adaptively randomized based on donor-derived T cell recovery, between 2 different dosing schedules of mycophenolate mofetil (MMF): MMF for 30 days post-transplant, with filgrastim for cytokine support (MMF30 arm, 11 patients), or MMF for 15 days post-transplant, with sargramostim (MMF15 arm, 15 patients). All patients were treated with anti-thymocyte globulin at a dose of 1.7 mg/kg/day from day - 9 through day -7 and total body irradiation, 450 cGy given in 3 fractions. Patients were well matched between the study arms and underwent HLA matched related and unrelated donor hematopoietic cell transplantation (HCT). The MMF15 arm demonstrated superior T cell recovery in the first month. This difference persisted through the first year for total T cells, T cell subsets and NK cells. T cell repertoire tended to be more diverse in the MMF15 arm. The long term superior immune recovery in the MMF15 arm is consistent with a disproportionate impact of early interventions in HCT. Clinically, shorter course MMF post-transplant was not associated with increased risk of acute or chronic graft vs. host disease (GVHD), and relapse and there was a trend toward improved overall survival in the MMF15 arm. Reducing the duration of intense immunosuppression in the early term and the use of sargramostim following allogeneic HCT is feasible and leads to superior long-term T cell recovery. This regimen should be studied to improve immune recovery in large cohorts of patients undergoing HCT with RIC.

Chimeric antigen receptor (CAR)-T cell therapies demonstrate potent anti-tumor efficacy in hematologic malignancies, yet clinical outcomes remain unpredictable due to the bespoke nature of the treatment, which is manufactured from each patients own T-cells. While germline variants are known to influence response to immune checkpoint inhibitors1, 2, their role in CAR-T cell therapy is unknown. Here, we pair whole-genome germline sequencing of lymphoma patients from the ZUMA-13 and ZUMA-74 clinical trials of axicabtagene ciloleucel, along with correlative biomarkers and functional assays, to ascertain the impact of germline variants on CAR-T cell behavior. Hypothesizing shared mechanisms of the most common toxicities of CAR-T cells, namely cytokine release syndrome (CRS) with hemophagocytic lymphohistiocytosis (HLH)--a hyperinflammatory syndrome driven by T cell overactivation5, we first looked within 17 canonical HLH-associated genes6, and identified putative deleterious STXBP2 variants in 15% of ZUMA-1 patients with toxicity, which were absent in control subjects who did not experience high grade toxicity. Subjects with these variants had elevated baseline IFN-{gamma} and inflammatory cytokines, findings that were recapitulated in engineered STXBP2-deficient and STXBP2-variant-expressing primary CAR-T cells derived from healthy donors. However, STXBP2 variant enrichment was absent in ZUMA-7 for this toxicity phenotype, possibly reflecting differences in underlying disease burden and evolving clinical management between the trials. A more expansive genome-wide analysis revealed ADAMTSL3 (a negative regulator of TGF{beta}7) as the only gene nominally enriched for putative deleterious variants in both ZUMA-1 and ZUMA-7 among control subjects, suggesting a protective effect. Finally, we focused on associations between germline variants and CAR-T cell expansion after infusion, a more objective and granular continuous variable that is strongly associated with clinical response across most CAR-T products8. We found a strong association between PTPN22, a known negative regulator of T-cell activation 9-12 and an autoimmune risk gene13, 14, variant status and CAR-T cell expansion in both ZUMA-1 and ZUMA-7, with the patients having the highest level of CAR-T expansion across clinical trials harboring variants in the gene. Together, these findings demonstrate the first clear association between germline variants and the clinical behavior of engineered immune cell therapies, which has implications for cellular therapy design, monitoring, testing, clinical trial design, and patient care.

Clinical responses to immune checkpoint blockade (ICB) for metastatic melanoma (MM) are variable, with patients frequently developing immune related adverse events (irAEs). The role played by myeloid populations in modulating responses to ICB remains poorly defined. We explored the effect of MM and the response to ICB across a cohort of patients with MM (n=116) and healthy donors (n=45) using bulk and single cell RNA-seq, and flow cytometry. Monocytes from patients with MM exhibit highly dysregulated baseline transcriptional profiles, whilst ICB treatment elicits induction of interferon signaling, MHC class II antigen presentation and CXCR3 ligand expression. Although both combination (cICB - anti-PD-1 and anti-CTLA) and single-agent (sICB - anti-PD1) ICB therapy modulates a shared set of genes, cICB displays a markedly greater magnitude of transcriptional effect. Notably, we find increased baseline monocyte counts correlate with a monocyte proliferation signature and risk of early death, whilst a gene-signature corresponding to a subset of platelet-binding classical monocytes conversely associates with improved outcome. This work demonstrates a central role for monocytes in the modulation of treatment response to ICB, providing insights into inter-individual variation in immune responses to ICB and further highlighting the multifarious immunological consequences of ICB treatment.

TP53-mutant acute myeloid leukemia (AML) is associated with particularly poor clinical outcomes and resistance to current therapies. While immunotherapy has revolutionized treatment for other hematologic malignancies, its efficacy in TP53-mutant AML remains limited. Although TP53 mutations in leukemic blasts are well characterized, their presence and functional consequences in immune cells have not been fully explored. Here we show that TP53 mutations are also present in T and NK cells from AML patients, where they are associated with increased activation markers but diminished cytotoxic function. T cells engineered to express common TP53 mutations exhibit an exhausted phenotype, characterized by impaired cytokine secretion and reduced tumor-killing capacity. Remarkably, restoring wild-type p53 conformation using a targeted small molecule reactivator reverses these dysfunctions and improves disease control in preclinical AML models. These findings reveal a novel mechanism of immune escape driven by mutant p53 in T cells and highlight the therapeutic potential of p53 reactivation. This work broadens our understanding of immune dysfunction in AML and supports incorporating immune-cell genotyping and correction strategies into future immunotherapy approaches for TP53-mutant disease.

Patients with multiple myeloma (MM), an age-dependent neoplasm of antibody-producing plasma cells, have compromised immune systems and might be at increased risk for severe COVID-19 outcomes. This study characterizes risk factors associated with clinical indicators of COVID-19 severity and all-cause mortality in myeloma patients utilizing NCATS National COVID Cohort Collaborative (N3C) database. The N3C consortium is a large, centralized data resource representing the largest multi-center cohort of COVID-19 cases and controls nationwide (>16 million total patients, and >6 million confirmed COVID-19+ cases to date). Our cohort included myeloma patients (both inpatients and outpatients) within the N3C consortium who have been diagnosed with COVID-19 based on positive PCR or antigen tests or ICD-10-CM diagnosis code. The outcomes of interest include all-cause mortality (including discharge to hospice) during the index encounter and clinical indicators of severity (i.e., hospitalization/emergency department/ED visit, use of mechanical ventilation, or extracorporeal membrane oxygenation (ECMO)). Finally, causal inference analysis was performed using the propensity score matching (PSM) method. As of 05/16/2022, the N3C consortium included 1,061,748 cancer patients, out of which 26,064 were MM patients (8,588 were COVID-19 positive). The mean age at COVID-19 diagnosis was 65.89 years, 46.8% were females, and 20.2% were of black race. 4.47% of patients died within 30 days of COVID-19 hospitalization. Overall, the survival probability was 90.7% across the course of the study. Multivariate logistic regression analysis showed histories of pulmonary and renal disease, dexamethasone, proteasome inhibitor/PI, immunomodulatory/IMiD therapies, and severe Charlson Comorbidity Index/CCI were significantly associated with higher risks of severe COVID-19 outcomes. Protective associations were observed with blood-or-marrow transplant/BMT and COVID-19 vaccination. Further, multivariate cox proportional hazard analysis showed that high and moderate CCI levels, International Staging System (ISS) moderate or severe stage, and PI therapy were associated with worse survival, while BMT and COVID-19 vaccination were associated with lower risk of death. Finally, matched sample average treatment effect on the treated (SATT) confirmed the causal effect of BMT and vaccination status as top protective factors associated with COVID-19 risk among US patients suffering from multiple myeloma. To the best of our knowledge, this is the largest nationwide study on myeloma patients with COVID-19.

XPO1 (Exportin-1) is the nuclear export protein responsible for the normal shuttling of several proteins and RNA species between the nucleocytoplasmic compartment of eukaryotic cells. XPO1 recognizes the nuclear export signal (NES) of its cargo proteins to facilitate its export. Alterations of nuclear export have been shown to play a role in oncogenesis in several types of solid tumor and hematologic cancers. Over more than a decade, there has been substantial progress in targeting nuclear export in cancer using selective XPO1 inhibitors. This has resulted in recent approval for the first-in-class drug selinexor for use in relapsed, refractory multiple myeloma and diffuse large B-cell lymphoma (DLBCL). Despite these successes not all patients respond effectively to XPO1 inhibition and there has been lack of biomarkers for response to XPO1 inhibitors in the clinic. Using hematologic malignancy cell lines and samples from patients with myelodysplastic neoplasms treated with selinexor, we have identified XPO1, NF-{kappa}B(p65), MCL-1 and p53 protein levels as protein markers of response to XPO1 inhibitor therapy. These markers could lead to the identification of response upon XPO1 inhibition for more accurate decision making in the personalized treatment of cancer patients undergoing treatment with selinexor.

Patients with chronic lymphocytic leukemia (CLL) treated with B-cell pathway inhibitors and anti-CD20 antibodies exhibit low humoral response rate (RR) following SARS-CoV-2 vaccination. To investigate the relationship between the initial transcriptional response to vaccination with ensuing B and T cell immune responses, we performed a comprehensive immune transcriptome analysis flanked by antibody and T cell assays in peripheral blood prospectively collected from 15 CLL/SLL patients vaccinated with heterologous BNT162b2/ChAdOx1 with follow up at a single institution. The two-dose antibody RR was 40% increasing to 53% after booster. Patients on BTKi, venetoclax {+/-} anti-CD20 antibody within 12 months of vaccination responded less well than those under BTKi alone. The two-dose T cell RR was 80% increasing to 93% after booster. Transcriptome studies revealed that seven patients showed interferon-mediated signaling activation within 2 days and one at 7 days after vaccination. Increasing counts of COVID-19 specific IGHV genes correlated with B-cell reconstitution and improved humoral RR. T cell responses in CLL patients appeared after vaccination regardless of treatment status. A higher humoral RR was associated with BTKi treatment and B-cell reconstitution. Boosting was particularly effective when intrinsic immune status was improved by CLL-treatment.