CAR T-cell therapy induces a high rate of prolonged remission in relapsed primary CNS lymphoma: Real-life results of the LOC network.

The prognosis of relapsed primary central nervous system lymphoma (PCNSL) remains dismal. CAR T-cells are a major contributor to systemic lymphomas, but their use in PCNSL is limited. From the LOC network database, we retrospectively selected PCNSL who had leukapheresis for CAR-T cells from the third line of treatment, and, as controls, PCNSL treated with any treatment, at least in the third line and considered not eligible for ASCT. Twenty-seven patients (median age: 68, median of three previous lines, including ASCT in 14/27) had leukapheresis, of whom 25 received CAR T-cells (tisa-cel: N = 16, axi-cel: N = 9) between 2020 and 2023. All but one received a bridging therapy. The median follow-up after leukapheresis was 20.8 months. The best response after CAR-T cells was complete response in 16 patients (64%). One-year progression-free survival from leukapheresis was 43% with a plateau afterward. One-year relapse-free survival was 79% for patients in complete or partial response at CAR T-cell infusion. The median overall survival was 21.2 months. Twenty-three patients experienced a cytokine release syndrome and 17/25 patients (68%) a neurotoxicity (five grade ≥3). The efficacy endpoints were significantly better in the CAR T-cell group than in the control group (N = 247) (median PFS: 3 months; median OS: 4.7 months; p < 0.001). This series represents the largest cohort of PCNSL treated with CAR T-cells reported worldwide. CAR T-cells are effective in relapsed PCNSL, with a high rate of long-term remission and a reassuring tolerance profile. The results seem clearly superior to those usually observed in this setting.

Antileukemic activity of the VPS34-IN1 inhibitor in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive disease with a poor prognosis. Vacuolar protein sorting 34 (VPS34) is a member of the phosphatidylinositol-3-kinase lipid kinase family that controls the canonical autophagy pathway and vesicular trafficking. Using a recently developed specific inhibitor (VPS34-IN1), we found that VPS34 inhibition induces apoptosis in AML cells but not in normal CD34+ hematopoietic cells. Complete and acute inhibition of VPS34 was required for the antileukemic activity of VPS34-IN1. This inhibitor also has pleiotropic effects against various cellular functions related to class III PI3K in AML cells that may explain their survival impairment. VPS34-IN1 inhibits basal and L-asparaginase-induced autophagy in AML cells. A synergistic cell death activity of this drug was also demonstrated. VPS34-IN1 was additionally found to impair vesicular trafficking and mTORC1 signaling. From an unbiased approach based on phosphoproteomic analysis, we identified that VPS34-IN1 specifically inhibits STAT5 phosphorylation downstream of FLT3-ITD signaling in AML. The identification of the mechanisms controlling FLT3-ITD signaling by VPS34 represents an important insight into the oncogenesis of AML and could lead to new therapeutic strategies.

Plasmodium falciparum sexual parasites develop in human erythroblasts and affect erythropoiesis.

Plasmodium falciparum gametocytes, the sexual stage responsible for malaria parasite transmission from humans to mosquitoes, are key targets for malaria elimination. Immature gametocytes develop in the human bone marrow parenchyma, where they accumulate around erythroblastic islands. Notably though, the interactions between gametocytes and this hematopoietic niche have not been investigated. Here, we identify late erythroblasts as a new host cell for P falciparum sexual stages and show that gametocytes can fully develop inside these nucleated cells in vitro and in vivo, leading to infectious mature gametocytes within reticulocytes. Strikingly, we found that infection of erythroblasts by gametocytes and parasite-derived extracellular vesicles delay erythroid differentiation, thereby allowing gametocyte maturation to coincide with the release of their host cell from the bone marrow. Taken together, our findings highlight new mechanisms that are pivotal for the maintenance of immature gametocytes in the bone marrow and provide further insights on how Plasmodium parasites interfere with erythropoiesis and contribute to anemia in malaria patients.

Cost and efficacy of peripheral stem cell mobilization strategies in multiple myeloma.

Mobilization of peripheral blood stem cells (PBSC) can be performed using plerixafor, which is expensive, or high-dose cyclophosphamide (HDCy). We hypothesized that the overall cost of mobilization with plerixafor might not be greater if the cost of complication management was considered. We performed a cost analysis of these two strategies. This multicentric observational study recruited patients with myeloma who underwent a first PBSC mobilization. We considered direct medical costs, including hospitalization, mobilization agents, apheresis, and supportive treatments. We included 111 patients, 54 and 57 in the HDCy and plerixafor groups, respectively. Cost of mobilization with HDCy was 5097 ± 2982€ vs. 10958 ± 1789€ for plerixafor (p < 0.0001). Cost of agents used was 1287 ± 779€ vs. 6552 ± 509€, respectively (p = 0.0009). The mean number of days of hospitalization was 2 and 2.1 days, respectively (p = 0.035). All patients achieved the minimum PBSC collection target (p = 1.0); however, ASCT was performed with HDCy in 67% patients and with plerixafor in 86% (p = 0.02). Plerixafor mobilization incurred a greater cost, mostly due to the greater cost of the drug. Hospitalization length in the two groups was similar in our series. Interestingly, plerixafor appeared to be a very effective and safe mobilizing approach translating into a greater ASCT success.

A Recurrent Activating Missense Mutation in Waldenström Macroglobulinemia Affects the DNA Binding of the ETS Transcription Factor SPI1 and Enhances Proliferation.

The ETS-domain transcription factors divide into subfamilies based on protein similarities, DNA-binding sequences, and interaction with cofactors. They are regulated by extracellular clues and contribute to cellular processes, including proliferation and transformation. ETS genes are targeted through genomic rearrangements in oncogenesis. The PU.1/SPI1 gene is inactivated by point mutations in human myeloid malignancies. We identified a recurrent somatic mutation (Q226E) in PU.1/SPI1 in Waldenström macroglobulinemia, a B-cell lymphoproliferative disorder. It affects the DNA-binding affinity of the protein and allows the mutant protein to more frequently bind and activate promoter regions with respect to wild-type protein. Mutant SPI1 binding at promoters activates gene sets typically promoted by other ETS factors, resulting in enhanced proliferation and decreased terminal B-cell differentiation in model cell lines and primary samples. In summary, we describe oncogenic subversion of transcription factor function through subtle alteration of DNA binding leading to cellular proliferation and differentiation arrest. SIGNIFICANCE: The demonstration that a somatic point mutation tips the balance of genome-binding pattern provides a mechanistic paradigm for how missense mutations in transcription factor genes may be oncogenic in human tumors.This article is highlighted in the In This Issue feature, p. 681.

Finely-tuned regulation of AMP-activated protein kinase is crucial for human adult erythropoiesis.

AMP-activated protein kinase (AMPK) is a heterotrimeric complex containing α, ß, and γ subunits involved in maintaining integrity and survival of murine red blood cells. Indeed, Ampk α1-/- , Ampk ß1-/- and Ampk γ1-/- mice develop hemolytic anemia and the plasma membrane of their red blood cells shows elasticity defects. The membrane composition evolves continuously along erythropoiesis and during red blood cell maturation; defects due to the absence of Ampk could be initiated during erythropoiesis. We, therefore, studied the role of AMPK during human erythropoiesis. Our data show that AMPK activation had two distinct phases in primary erythroblasts. The phosphorylation of AMPK (Thr172) and its target acetyl CoA carboxylase (Ser79) was elevated in immature erythroblasts (glycophorin Alow), then decreased conjointly with erythroid differentiation. In erythroblasts, knockdown of the α1 catalytic subunit by short hairpin RNA led to a decrease in cell proliferation and alterations in the expression of membrane proteins (band 3 and glycophorin A) associated with an increase in phosphorylation of adducin (Ser726). AMPK activation in mature erythroblasts (glycophorin Ahigh), achieved through the use of direct activators (GSK621 and compound 991), induced cell cycle arrest in the S phase, the induction of autophagy and caspase-dependent apoptosis, whereas no such effects were observed in similarly treated immature erythroblasts. Thus, our work suggests that AMPK activation during the final stages of erythropoiesis is deleterious. As the use of direct AMPK activators is being considered as a treatment in several pathologies (diabetes, acute myeloid leukemia), this observation is pivotal. Our data highlighted the importance of the finely-tuned regulation of AMPK during human erythropoiesis.

Plerixafor in poor mobilizers with non-Hodgkin's lymphoma: a multi-center time-motion analysis.

High-dose chemotherapy alongside peripheral blood stem cell (PBSC) infusion has become the standard of care in different hematologic malignancies. The goal of PBSC mobilization is to allow collection of sufficient CD34+ cells to proceed to transplantation. The current mobilization regimen with granulocyte colony-stimulating factor (G-CSF), alone or in combination with chemotherapy, still fails in 10-25% of patients. Plerixafor is able to rescue most of these patients from mobilization failure. In this study, we investigated the impact of plerixafor on the cost and time spent on apheresis in patients who were considered poor mobilizers, with <20 × 106/µl peripheral CD34+ cells after mobilization but prior to apheresis. Patient hospital records from ten centers in three European countries were reviewed and compared during two time periods, namely prior and after plerixafor introduction to the market. During the plerixafor period, patients spent less time on apheresis (350 vs. 461 min). Poor mobilizers given plerixafor collected more CD34+ cells during the first apheresis session, leading to a decrease in the average number of apheresis sessions needed. The total apheresis yield was unaffected. This analysis shows that the use of plerixafor lessens the time-effort associated with the management of poor mobilizers and reduces apheresis costs.

Impact of plerixafor (mozobil) on hospital efficiency: A single center experience.

Plerixafor (Mozobil) in combination with granulocyte colony-stimulating factor (G-CSF) has shown to increase mobilization of peripheral blood stem cells (PBSC) as compared to G-CSF alone in patients undergoing autologous stem cell transplantation (ASCT). However, up to 25% of patients treated with G-CSF alone still fail mobilization. Adding plerixafor to poor mobilizers allows to rescue these patients from mobilization failure and to reduce the number of apheresis sessions. The goal of this retrospective study was to capture the impact of plerixafor on treatment outcome and on apheresis department efficiency. The latter was measured in terms of time-slots lost, that is, the number of apheresis sessions scheduled but not carried out due to poor mobilization, and the number of elective apheresis sessions performed for patients undergoing extracorporeal photopheresis (ECP). Hospital records of patients treated before and after introduction of plerixafor were collected and analyzed. With plerixafor, the mobilization failure rate dropped from 12% to 4% and the mean number of time-slots lost per patient dropped from 1.39 to 0.89. Additional drug costs due to plerixafor were partially balanced by a reduction in apheresis sessions, resulting in an additional cost of 759€ per ASCT candidate. More importantly, with the use of plerixafor, the availability of time-slots turned from erratic to predictable such that freed capacity could be dedicated to other apheresis procedures. As a result, the number of ECP sessions increased from 0 in 2005 to 685 sessions in 2014.

Does bendamustine impact the mobilization of peripheral blood stem cells? A multicenter retrospective study of 23 cases.

Bendamustine is used in the treatment of different relapsing or refractory subtypes of lymphoma. Its impact on the yield of peripheral blood stem cells is not well known. Twenty three patients who received bendamustine followed immediately or after another chemotherapy by stem cell mobilization (SCM) were included. The patients were divided into two groups: group 1 (n=17), in whom SCM was performed immediately after bendamustine chemotherapy, and group 2 (n=6), in whom SCM was performed after another cycle of chemotherapy. The success rate of mobilization after Bendamustine+/-plerixafor was 36% (eight cytapheresis succeeded for a total number of 22 cytapheresis); and 75% after other approaches (chemotherapy based or steady state) used for patients who received bendamustine previously. Although bendamustine used alone was not an effective drug to mobilize stem cells, this agent does not seem to have detrimental effects on subsequent SCM.

Cell penetrating peptides as a therapeutic strategy in chronic lymphocytic leukemia.

PP2A is a serine/threonine phosphatase critical to a number of physiological and developmental processes. In this manuscript, we show that a peptide, specifically blocking the caspase- 9/PP2A interaction, DPT-C9h, induces apoptosis in primary tumour B cells isolated from peripheral blood mononuclear cells or bone marrow of chronic lymphocytic leukemia (CLL) patients, but not on B cells obtained from healthy donors (HD). Moreover, in both CLL patients and HD, DPT-C9h does not induce apoptosis on T- and NKcells and monocytes. Our results strongly suggest that DPT-C9h peptide has tumour specificity and that caspase-9/PP2Ac interaction constitutes a novel therapeutic approach for the treatment in CLL patients.

Current status of haematopoietic autologous stem cell transplantation in lymphoid malignancies: a European perspective.

The use of autologous haematopoietic stem cell transplantation (ASCT) has increased considerably in lymphoid malignancies in the last decade, and it is now considered as the standard of care in particular circumstances. This review aims to present an overview of the current situation with ASCT in lymphoid malignancies in Europe, in terms of both current use and issues. It will also look briefly at ASCT in rarer haematological malignancies and at the future. It is intended as a reflection of opinion from selected centres in Europe and as an aid to understanding for those who are new to the area. The review is based on a series of four preceptorship meetings held in Europe in 2013.

Infusion of allogeneic natural killer cells in a patient with acute myeloid leukemia in relapse after haploidentical hematopoietic stem cell transplantation.

BACKGROUND: Allogeneic donor natural killer (NK)-cell infusion (NK-DLI) is a promising immunotherapy for patients with hematologic disorders. CASE REPORT: This report describes the case of a patient who received a single haploidentical NK-DLI for a relapse of acute myeloid leukemia (AML) after haploidentical hematopoietic stem cell transplantation. He underwent a cytoreductive, immunosuppressive regimen before NK-DLI and received high-dose interleukin-2 in vivo for 8 weeks afterward. RESULTS: No major adverse effect was observed. Prospective phenotypic and functional studies of the NK cells showed major expansion of infused NK cells and, more importantly, of the alloreactive KIR2DL1+KIR2DL2/DL3-NKG2A- subset, which reached 117×10(6) cells/L on Day +14 after NK-DLI, the greatest expansion of infused alloreactive NK cells reported so far. Infused NK cells conserved their lytic capacities against K562 target cells and primary AML-mismatched blasts. CONCLUSION: We review the literature to clarify these data and to detail the indications for allogeneic NK-DLI, the criteria for determining the most suitable donor, the types of conditioning regimens, and the procedures for selecting and activating NK cells.

CD4+CD25+ regulatory T cell depletion improves the graft-versus-tumor effect of donor lymphocytes after allogeneic hematopoietic stem cell transplantation.

Donor T cells play a pivotal role in the graft-versus-tumor effect after allogeneic hematopoietic stem cell transplantation. Regulatory T cells (T(reg)s) may reduce alloreactivity, the major component of the graft-versus-tumor effect. In the setting of donor lymphocyte infusion after hematopoietic stem cell transplantation, we postulated that T(reg) depletion could improve alloreactivity and likewise the graft-versus-tumor effect of donor T cells. The safety and efficacy of T(reg)-depleted donor lymphocyte infusion was studied in 17 adult patients with malignancy relapse after hematopoietic stem cell transplantation. All but one had previously failed to respond to at least one standard donor lymphocyte infusion, and none had experienced graft-versus-host disease. Two of the 17 patients developed graft-versus-host disease after their first T(reg)-depleted donor lymphocyte infusion and experienced a long-term remission of their malignancy. Four of the 15 patients who did not respond after a first T(reg)-depleted donor lymphocyte infusion received a second T(reg)-depleted donor lymphocyte infusion combined with lymphodepleting chemotherapy aimed to also eliminate recipient T(reg)s. All four developed acute-like graft-versus-host disease that was associated with a partial or complete and durable remission. In the whole cohort, graft-versus-host disease induction through T(reg) depletion was associated with improved survival. These results suggest that T(reg)-depleted donor lymphocyte infusion is a safe, feasible approach that induces graft-versus-host or graft-versus-tumor effects in alloreactivity-resistant patients. In patients not responding to this approach, the combination of chemotherapy-induced lymphodepletion of the recipient synergizes with the effect of T(reg)-depleted donor lymphocyte infusion. These findings offer a rational therapeutic approach for cancer cellular immunotherapy.

Delayed recovery after autologous peripheral hematopoietic cell transplantation: potential effect of a high number of total nucleated cells in the graft.

BACKGROUND: Some patients demonstrate delayed recoveries after autologous hematopoietic stem cell transplantation despite infusion of an adequate number of CD34+ cells/kg and clinically stable status. Factors considered being possible predictors of this outcome in this context were explored. STUDY DESIGN AND METHODS: A total of 246 patients were evaluated in terms of engraftment. Delayed recovery was defined by white blood cell recovery time exceeding mean+1 SEM. Clinical factors and graft characteristics were examined. Comparisons between patients with normal or delayed engraftment were made. Proinflammatory cytokines and proteolytic enzyme quantification and CXCR4+ and CD44+ cell enumeration were performed on peripheral hematopoietic stem cells (PHSC) product samples of patients with delayed engraftment and patients with usual engraftment time. RESULTS: Sixteen patients, who received at least 3 × 10(6) CD34+ cells/kg without known clinical factors likely to affect engraftment, demonstrated a delayed recovery time of over 20 days. Some graft variables were found to be significantly increased in these patients by univariate analysis. One variable was the total number of nucleated cells cryopreserved and infused. Among the nucleated cells, the absolute number of granulocytes before and after cryopreservation also differed significantly between the two groups. A multivariate analysis showed that the main predictive factor for delayed recovery was the number of nucleated cells in the graft (p=0.0044). The influence of contaminating cells might be related to the release of elastase, matrix metalloproteinase-9, interleukin (IL)-1ß, and IL-6 involved in stem cell homing. CONCLUSION: Therefore, the numeration of total nucleated cells and granulocytes should be considered as a possible quality control variable of PHSCs submitted for cryopreservation.

Massive expansion of regulatory T-cells following interleukin 2 treatment during a phase I-II dendritic cell-based immunotherapy of metastatic renal cancer.

Cytotoxic chemotherapy is ineffective in metastatic renal cancer. However, systemic administration of interleukin 2 (IL-2) or infusion of dendritic cells (DCs) loaded with tumor extracts can lead to some response rates with concomitant survival improvements. We report the results of a phase I-II pilot study combining DCs and IL-2 where six patients were included. DCs were derived from bone marrow CD34+ cells and loaded with autologous tumor extracts. CD34-DC vaccines were infused subcutaneously at day 45, 52, 59, 90 and 120 following surgery in combination with IL-2, that was subsequently administrated after the 3rd and 4th DC vaccinations. Preparation of tumor extracts and CD34-DCs were satisfactory in all patients but one. Due to rapid tumor progression, one patient was excluded before vaccination. In the 4 remaining patients, two received 3 vaccinations, while the 2 others received 5 vaccinations and the full IL-2 treatment. No adverse effect due to the vaccinations was observed. A specific immune response against autologous tumor cells was observed in the 2 patients who completed the treatment. Interestingly, these 2 patients had a more prolonged survival than the patients receiving 3 vaccinations. Importantly, a transient and massive increase of circulating natural regulatory T-cells (nTregs) was evidenced in 3 patients following IL-2 administration. Overall, the use of CD34-DC vaccines is feasible, safe and non-toxic. A specific anti-tumor immune response can be detected. However, our data highlights that IL-2 is a potent inducer of nTregs in vivo and as such may have a negative impact on cancer immunotherapy.