Susceptibility to mycobacterial infection in VEXAS syndrome.

OBJECTIVES: VEXAS is a recently described acquired auto-inflammatory and hematologic syndrome caused by somatic mutations in UBA1. To date, VEXAS is not a recognized cause of acquired immunodeficiency. PATIENTS AND METHODS: Two of our 10 VEXAS patients developed a disseminated Mycobacterium avium infection. To shed light on this observation, we retrospectively studied all patients with disseminated non-tuberculous mycobacterial infections (NTMi) seen at our institution over 13 years. Inclusion criteria were a positive blood/bone marrow culture, or 2 positive cultures from distinct sites, or one positive culture with 2 involved sites. RESULTS: patient 1 presented with fever, rash, orbital cellulitis and lung infiltrates. Patient 2 presented with fever and purpura. In both cases, Mycobacterium avium was identified on bone marrow culture. Twenty cases of disseminated NTMi were reviewed. Among 11 HIV-negative patients, three had chronic immune-mediated disease; three had untreated myeloid neoplasm; two had VEXAS; one had undergone kidney transplantation; one had GATA-2 deficiency; and one had no identified aetiology. None had lymphoid neoplasia or had undergone bone marrow transplantation. HIV-negative cases had higher CD4 counts than HIV-positive patients (median CD4: 515/mm3  vs 38/mm3, p< 0.001). Monocytopenia was present in seven cases. At 2 years, six patients had died, including both VEXAS patients. DISCUSSION: VEXAS patients have an intrinsic susceptibility to disseminated NTMi, which may result from monocytic dysfunction. NTMi can mimic VEXAS flare. Clinicians should maintain a high suspicion for opportunistic infections before escalating immunosuppressive therapy. Further studies are needed to confirm and better decipher the herein reported observations.

Role of red cell mass evaluation in myeloproliferative neoplasms with splanchnic vein thrombosis and normal hemoglobin value: a study of the France Intergroupe des Syndromes myeloprolifératifs.

Not available.

Phase I study of zoledronic acid combined with escalated doses of interleukine-2 for early in vivo generation of Vγ9Vδ2 T-cells after haploidentical stem cell transplant with posttransplant cyclophosphamide.

The presence of donor Vγ9Vδ2 T-cells after haploidentical hematopoietic stem cell transplant (h-HSCT) has been associated with improved disease-free survival. These cells kill tumor cells in a non-MHC restricted manner, do not induce graft-versus-host disease (GVHD), and can be generated by stimulation with zoledronic acid (ZA) in combination with interleukin-2 (IL-2). This monocentric phase I, open-label, dose-escalating study (ClinicalTrials.gov: NCT03862833) aimed at evaluating the safety and possibility to generate Vγ9Vδ2 T-cells early after h-HSCT. It applied a standard 3 + 3 protocol to determine the maximum tolerated dose (MTD) of increasing low-doses of IL-2 (5 days [d] per week, 4 weeks) in combination with a single dose of ZA, starting both the first Monday after d + 15 posttransplant. Vγ9Vδ2 T-cell monitoring was performed by multiparameter flow cytometry on blood samples and compared with a control cohort of h-HSCT recipients. Twenty-six patients were included between April 2019 and September 2022, 16 of whom being ultimately treated and seven being controls who received h-HSCT only. At the three dose levels tested, 1, 0, and 1 dose-limiting toxicities were observed. MTD was not reached. A significantly higher number of Vγ9Vδ2 T-cells was observed during IL-2 treatment compared with controls. In conclusion, early in vivo generation of Vγ9Vδ2 T-cells is feasible after h-HSCT by using a combination of ZA and repeated IL-2 infusions. This study paves the way to a future phase 2 study, with the hope to document lesser posttransplant relapse with this particular adaptive immunotherapy.

A p53 score derived from TP53 CRISPR/Cas9 HMCLs predicts survival and reveals a major role of BAX in the response to BH3 mimetics.

ABSTRACT: To establish a strict p53-dependent gene-expression profile, TP53-/- clones were derived from TP53+/+ and TP53-/mut t(4;14) human myeloma cell lines (HMCLs) using CRISPR/Cas9 technology. From the 17 dysregulated genes shared between the TP53-/- clones from TP53+/+ HMCLs, we established a functional p53 score, involving 13 genes specifically downregulated upon p53 silencing. This functional score segregated clones and myeloma cell lines as well as other cancer cell lines according to their TP53 status. The score efficiently identified samples from patients with myeloma with biallelic TP53 inactivation and was predictive of overall survival in Multiple Myeloma Research Foundation-coMMpass and CASSIOPEA cohorts. At the functional level, we showed that among the 13 genes, p53-regulated BAX expression correlated with and directly affected the MCL1 BH3 mimetic S63845 sensitivity of myeloma cells by decreasing MCL1-BAX complexes. However, resistance to S63845 was overcome by combining MCL1 and BCL2 BH3 mimetics, which displayed synergistic efficacy. The combination of BH3 mimetics was effective in 97% of patient samples with or without del17p. Nevertheless, single-cell RNA sequencing analysis showed that myeloma cells surviving the combination had lower p53 score, showing that myeloma cells with higher p53 score were more sensitive to BH3 mimetics. Taken together, we established a functional p53 score that identifies myeloma cells with biallelic TP53 invalidation, demonstrated that p53-regulated BAX is critical for optimal cell response to BH3 mimetics, and showed that MCL1 and BCL2 BH3 mimetics in combination may be of greater effectiveness for patients with biallelic TP53 invalidation, for whom there is still an unmet medical need.

Tocilizumab in combination with a standard induction chemotherapy in acute myeloid leukaemia patients (TOCILAM study): a single-centre, single-arm, phase 1 trial.

Background: In acute myeloid leukaemia (AML), interleukin-6 (IL-6) promotes chemo-resistance and its levels correlate with poor prognosis. IL-6 blockade may represent a promising therapeutic strategy. We aimed to test, tocilizumab, an anti-IL-6 receptor (R) monoclonal antibody in combination with standard intensive AML induction chemotherapy. Methods: This investigator-initiated single-centre phase 1 trial was conducted at Nantes University Hospital in France. According to a continual reassessment method, three escalating doses were tested of intravenous (IV) tocilizumab (4, 6, and 8 mg/kg) administered at day (d) 8 of a standard AML induction chemotherapy (IV idarubicine 8 mg/m2 d1 to d5 + IV cytarabine 100 mg/m2 d1 to d7). All adults (aged ≥ 18 years) with an Eastern Cooperative Oncology Group performance status of 0-2 and with a newly diagnosed (excluding patients with a favourable risk according to ELN-2017 classification if <60 year-old) or a relapsed/refractory AML were eligible. The primary objective was to determine the maximum tolerated dose of tocilizumab to administrate with a standard intensive AML induction. Safety outcomes were continuously monitored for at each participant contact. This trial is registered with ClinicalTrials.gov, NCT04547062. Findings: Between Dec 29, 2020 and Dec 1, 2022, 12 patients were enrolled, of whom 75% had an ELN-2017 high-risk profile, and were treated with tocilizumab- two patients at 4 mg/kg, two at 6 mg/kg and eight at 8 mg/kg of tocilizumab. No dose-limiting toxicity related to tocilizumab was documented. There were nine serious adverse events, none of which were related to tocilizumab, and there was no treatment-related deaths. MTD was thus not reached. Two deaths occurred during induction. In the remaining ten evaluable patients, nine responded to treatment. Interpretation: The combination of tocilizumab with standard AML intensive induction appears to be safe and resulting responses are encouraging. A dose of 8 mg/kg of tocilizumab given at day 8 of induction could be used for further phase 2/3 studies. Funding: The Leucémie Espoir Atlantique Famille (LEAF)-"Tous avec Fabien" association.

CARD11 gain of function upregulates BCL2A1 expression and promotes resistance to targeted therapies combination in B-cell lymphoma.

A strategy combining targeted therapies is effective in B-cell lymphomas (BCL), such as mantle cell lymphoma (MCL), but acquired resistances remain a recurrent issue. In this study, we performed integrative longitudinal genomic and single-cell RNA-sequencing analyses of patients with MCL who were treated with targeted therapies against CD20, BCL2, and Bruton tyrosine kinase (OAsIs trial). We revealed the emergence of subclones with a selective advantage against OAsIs combination in vivo and showed that resistant cells were characterized by B-cell receptor (BCR)-independent overexpression of NF-κB1 target genes, especially owing to CARD11 mutations. Functional studies demonstrated that CARD11 gain of function not only resulted in BCR independence but also directly increased the transcription of the antiapoptotic BCL2A1, leading to resistance against venetoclax and OAsIs combination. Based on the transcriptional profile of OAsIs-resistant subclones, we designed a 16-gene resistance signature that was also predictive for patients with MCL who were treated with conventional chemotherapy, underlying a common escape mechanism. Among druggable strategies to inhibit CARD11-dependent NF-κB1 transduction, we evaluated the selective inhibition of its essential partner MALT1. We demonstrated that MALT1 protease inhibition led to a reduction in the expression of genes involved in OAsIs resistance, including BCL2A1. Consequently, MALT1 inhibition induced synergistic cell death in combination with BCL2 inhibition, irrespective of CARD11 mutational status, both in vitro and in vivo. Taken together, our study identified mechanisms of resistance to targeted therapies and provided a novel strategy to overcome resistance in aggressive BCL. The OAsIs trial was registered at www.clinicaltrials.gov #NCT02558816.

Targeted High-throughput Sequencing for Hematological Malignancies: A GBMHM Survey of Practice and Cost Evaluation in France.

The objective of this study was to assess the clinical impact and financial costs of next-generation sequencing (NGS) in 5 categories of pediatric and adult hematological cancers. NGS prescriptions were prospectively collected from 26 laboratories, with varied technical and reporting practice (all or only significant targets). Impact was defined by the identification of (1) an actionable mutation, (2) a mutation with prognostic and/or theranostic value, and/or (3) a mutation allowing nosological refinement, reported by local investigators. A microcosting study was undertaken in 4 laboratories, identifying the types and volumes of resources required for each procedural step. Individual index prescriptions for 3961 patients were available for impact analysis on the management of myeloid disorders (two thirds) and, mainly mature B, lymphoid disorders (one third). NGS results were considered to impact the management for 73.4% of prescriptions: useful for evaluation of prognostic risk in 34.9% and necessary for treatment adaptation (actionable) in 19.6%, but having no immediate individual therapeutic impact in 18.9%. The average overall cost per sample was 191 € for the restricted mature lymphoid amplicon panel. Capture panel costs varied from 369 € to 513 €. Unit costs varied from 0.5 € to 5.7 € per kb sequenced, from 3.6 € to 11.3 € per target gene/hot-spot sequenced and from 4.3 € to 73.8 € per target gene/hot-spot reported. Comparable costs for the Amplicon panels were 5-8 € per kb and 10.5-14.7 € per target gene/hot-spot sequenced and reported, demonstrating comparable costs with greater informativity/flexibility for capture strategies. Sustainable funding of precision medicine requires a transparent discussion of its impact on care pathways and its financial aspects.

CPX-351 in higher risk myelodysplastic syndrome and chronic myelomonocytic leukaemia: a multicentre, single-arm, phase 2 study.

BACKGROUND: CPX-351, an encapsulated form of cytarabine and daunorubicin, has shown greater efficacy than the classic 3 + 7 treatment administration in secondary acute myeloid leukaemia. Given that higher-risk myelodysplastic syndrome and chronic myelomonocytic leukaemia share similarities with secondary acute myeloid leukaemia, we aimed to investigate the safety and efficacy of CPX-351 in this context. METHODS: This investigator-initiated two-cohort phase 2 trial was conducted by the Groupe Francophone des Myélodysplasies, with 12 participating centres in France. It comprised cohort A (reported here and completed), which included patients in first-line treatment, and cohort B, which was stopped for lack of inclusion (ie, not enough patients met the inclusion criteria), for patients with hypomethylating agent failure that is not reported here. Cohort A enrolled patients with newly diagnosed higher-risk myelodysplastic syndrome or chronic myelomonocytic leukaemia (aged 18-70 years old) with an Eastern Cooperative Oncology Group performance status of 0-1. Intravenous CPX-351 (100 mg/m2 cytarabine and 44 mg/m2 daunorubicin) was given on days 1, 3, and 5, with a second induction cycle given (same daily dose on days 1 and 3) if at least a partial response was not reached. Patients who responded could receive up to four monthly consolidation cycles (same daily dose on day 1) or allogeneic haematopoietic stem-cell transplantation (HSCT). Overall response rate after one or two induction courses according to European LeukemiaNet 2017 acute myeloid leukaemia was the primary endpoint after CPX-351 induction, whether patients received one or two induction cycles. Safety was assessed in all patients enrolled (in cohort A). This trial is registered with ClinicalTrials.gov, NCT04273802. FINDINGS: Between April 29, 2020, and Feb 10, 2021, 21 (68%) male and ten (32%) female patients were enrolled. 27 (87%) of 31 patients responded (95% CI 70-96). 16 (52%) of the 31 patients received at least one consolidation cycle. 30 (97%) of the 31 patients included were initially considered eligible for allogeneic HSCT and 29 (94%) of the 31 patients had the procedure. Median follow-up was 16·1 months (IQR 8·3-18·1). The most common grade 3-4 adverse events were pulmonary (eight [26%] of 31 patients) and cardiovascular (six [19%] of 31 patients). There were 14 serious adverse events (mainly hospitalisation for infection [n=5] and only one was treatment-related) and no treatment-related death. INTERPRETATION: CPX-351 appears to be active and safe in patients with higher-risk myelodysplastic syndrome and chronic myelomonocytic leukaemia, allowing bridging to allogenic HSCT in most patients. FUNDING: Jazz Pharmaceuticals.

Aggressive, early resistant and relapsed mantle cell lymphoma distinct extrinsic microenvironment highlighted by transcriptome analysis.

Immunotherapy strategies relying on innate or adaptive immune components are increasingly used in onco-haematology. However, little is known about the infiltrated lymph nodes (LN) or bone marrow (BM) landscape of mantle cell lymphoma (MCL). The original transcriptomic approach of reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) was applied here to explore the expression of 24 genes of interest in MCL at diagnosis (21 LN and 15 BM) or relapse (18 LN). This allowed us to identify that at baseline, samples from MCL patients with an aggressive morphology (i.e. blastoid or pleomorphic) or a high proliferative profile, displayed significantly higher monocyte/macrophage-associated transcripts (CD14 and CD163) in LN and BM. Regarding T-cells, aggressive MCL forms had significantly lower amounts of LN CD3E transcripts, yet an increased expression of cytotoxic markers in LN (CD8) and BM (CD94). A very high-risk group with early treatment resistance displayed, at diagnosis, high proliferation (KI67) and high macrophages and cytotoxic transcript levels. Post-immunochemotherapy relapsed samples revealed lower levels of T- and natural killer-cells markers, while monocyte/macrophage markers remained similar to diagnosis. This study suggests that rapid analysis of MCL microenvironment transcriptome signatures by RT-MLPA could allow for an early distinction of patient subgroups candidates for adapted treatment strategies.

Impact on outcomes of mixed chimerism of bone marrow CD34+ sorted cells after matched or haploidentical allogeneic stem cell transplantation for myeloid malignancies.

Allogeneic hematopoietic stem cell transplantation (Allo-HSCT), proposed to patients with high-risk myeloid malignancies, may ultimately fail because of disease relapse. Bone marrow (BM) CD34+ cells in Allo-HSCT recipients can be either re-emerging recipient malignant cells or donor cells attesting of hematopoietic reconstitution. In this context, investigating donor/recipient chimerism in the population of BM CD34+ sorted cells (BM-CD34+SC) was performed in 261 Allo-HSCT recipients (matched n = 145, haploidentical n = 65, matched unrelated n = 51) with myeloid malignancies. BM-CD34+SC chimerism was compared to that of whole peripheral blood (PB) cells as well as other Allo-HSCT-related parameters, and impact on relapse and survival was assessed. Thresholds of 98% donor cells for PB and 90% for BM-CD34+SC were found to allow relapse prediction. This was completed by the application of machine learning tools to explore the predictive value of these parameters in multidimensional models with repeated iterations. BM-CD34+SC mixed chimerism stood out with all these methods as the most robust predictor of relapse with a significant impact on disease-free and overall survivals even after haploidentical Allo-HSCT and/or PTCY administration. This marker therefore appears to be of great interest for the decision of preemptive treatment to avoid post-transplant relapse.

Prognostic impact of DDX41 germline mutations in intensively treated acute myeloid leukemia patients: an ALFA-FILO study.

DDX41 germline mutations (DDX41MutGL) are the most common genetic predisposition to myelodysplastic syndrome and acute myeloid leukemia (AML). Recent reports suggest that DDX41MutGL myeloid malignancies could be considered as a distinct entity, even if their specific presentation and outcome remain to be defined. We describe here the clinical and biological features of 191 patients with DDX41MutGL AML. Baseline characteristics and outcome of 86 of these patients, treated with intensive chemotherapy in 5 prospective Acute Leukemia French Association/French Innovative Leukemia Organization trials, were compared with those of 1604 patients with DDX41 wild-type (DDX41WT) AML, representing a prevalence of 5%. Patients with DDX41MutGL AML were mostly male (75%), in their seventh decade, and with low leukocyte count (median, 2 × 109/L), low bone marrow blast infiltration (median, 33%), normal cytogenetics (75%), and few additional somatic mutations (median, 2). A second somatic DDX41 mutation (DDX41MutSom) was found in 82% of patients, and clonal architecture inference suggested that it could be the main driver for AML progression. DDX41MutGL patients displayed higher complete remission rates (94% vs 69%; P < .0001) and longer restricted mean overall survival censored at hematopoietic stem cell transplantation (HSCT) than 2017 European LeukemiaNet intermediate/adverse (Int/Adv) DDX41WT patients (5-year difference in restricted mean survival times, 13.6 months; P < .001). Relapse rates censored at HSCT were lower at 1 year in DDX41MutGL patients (15% vs 44%) but later increased to be similar to Int/Adv DDX41WT patients at 3 years (82% vs 75%). HSCT in first complete remission was associated with prolonged relapse-free survival (hazard ratio, 0.43; 95% confidence interval, 0.21-0.88; P = .02) but not with longer overall survival (hazard ratio, 0.77; 95% confidence interval, 0.35-1.68; P = .5).

The IL32/BAFF axis supports prosurvival dialogs in the lymphoma ecosystem and is disrupted by NIK inhibition.

Aggressive B-cell malignancies, such as mantle cell lymphoma (MCL), are microenvironment-dependent tumors and a better understanding of the dialogs occurring in lymphoma-protective ecosystems will provide new perspectives to increase treatment efficiency. To identify novel molecular regulations, we performed a transcriptomic analysis based on the comparison of circulating MCL cells (n=77) versus MCL lymph nodes (n=107) together with RNA sequencing of malignant (n=8) versus normal B-cell (n=6) samples. This integrated analysis led to the discovery of microenvironment-dependent and tumor-specific secretion of interleukin-32 beta (IL32ß), whose expression was confirmed in situ within MCL lymph nodes by multiplex immunohistochemistry. Using ex vivo models of primary MCL cells (n=23), we demonstrated that, through the secretion of IL32ß, the tumor was able to polarize monocytes into specific MCL-associated macrophages, which in turn favor tumor survival. We highlighted that while IL32ß-stimulated macrophages secreted several protumoral factors, they supported tumor survival through a soluble dialog, mostly driven by BAFF. Finally, we demonstrated the efficacy of selective NIK/alternative-NFkB inhibition to counteract microenvironment-dependent induction of IL32ß and BAFF-dependent survival of MCL cells. These data uncovered the IL32ß/BAFF axis as a previously undescribed pathway involved in lymphoma-associated macrophage polarization and tumor survival, which could be counteracted through selective NIK inhibition.

A phase I/II feasibility vaccine study by autologous leukemic apoptotic corpse-pulsed dendritic cells for elderly AML patients.

This was a phase I/II study testing the feasibility of a vaccine by autologous leukemic apoptotic corpse-pulsed dendritic cells (DC) in elderly acute myeloid leukemia (AML) patients in first or second complete remission. Pulsed DC were administered at doses of 9 × 106 subcutaneously (1 mL) and 1 × 106 intra-dermally (0.1 mL). Five doses of vaccine were planned on days +1 + 7 + 14 + 21 and +35. Five DC-vaccines were produced and injected for all five patients included in the study. No severe adverse event was documented. Larger Phase 2 studies are now required to precise the role of DC-vaccines with leukemic apoptotic bodies in older as well as younger AML populations. (Clinicaltrials.gov NCT01146262).

Genomic analysis of primary and secondary myelofibrosis redefines the prognostic impact of ASXL1 mutations: a FIM study.

We aimed to study the prognostic impact of the mutational landscape in primary and secondary myelofibrosis. The study included 479 patients with myelofibrosis recruited from 24 French Intergroup of Myeloproliferative Neoplasms (FIM) centers. The molecular landscape was studied by high-throughput sequencing of 77 genes. A Bayesian network allowed the identification of genomic groups whose prognostic impact was studied in a multistate model considering transitions from the 3 conditions: myelofibrosis, acute leukemia, and death. Results were validated using an independent, previously published cohort (n = 276). Four genomic groups were identified: patients with TP53 mutation; patients with ≥1 mutation in EZH2, CBL, U2AF1, SRSF2, IDH1, IDH2, NRAS, or KRAS (high-risk group); patients with ASXL1-only mutation (ie, no associated mutation in TP53 or high-risk genes); and other patients. A multistate model found that both TP53 and high-risk groups were associated with leukemic transformation (hazard ratios [HRs] [95% confidence interval], 8.68 [3.32-22.73] and 3.24 [1.58-6.64], respectively) and death from myelofibrosis (HRs, 3.03 [1.66-5.56] and 1.77 [1.18-2.67], respectively). ASXL1-only mutations had no prognostic value that was confirmed in the validation cohort. However, ASXL1 mutations conferred a worse prognosis when associated with a mutation in TP53 or high-risk genes. This study provides a new definition of adverse mutations in myelofibrosis with the addition of TP53, CBL, NRAS, KRAS, and U2AF1 to previously described genes. Furthermore, our results argue that ASXL1 mutations alone cannot be considered detrimental.

Grade 2 acute GVHD is a factor of good prognosis in patients receiving peripheral blood stem cells haplo-transplant with post-transplant cyclophosphamide.

BACKGROUND: The impact of acute graft versus host disease (GVHD) on survivals for patients receiving a haploidentical allogeneic stem-cell transplant (Allo-SCT) with peripheral blood stem-cells (PBSC) complemented by post-transplant cyclophosphamide (PTCY) is ill-known. MATERIAL AND METHODS: This retrospective study included 131 patients who received a PBSC haplograft in order to precise the impact of acute GVHD on outcomes. There were 78 males and 53 females and the median age for the whole cohort was 59 years (range: 20-71). Thirty-five patients were allografted for a lymphoid disease and 96 for a myeloid malignancy, including 67 patients with acute myeloid leukemia (AML). RESULTS: The cumulative incidence (CI) of day 100 grade 2-4 and 3-4 acute GVHD was 43.4 + 4.6% and 16.7 + 3.4%, respectively. The 2-year CI of moderate/severe chronic GVHD was 10.1 + 2.8%. The only factor affecting the occurrence of GVHD was GVHD prophylaxis. Indeed, CI of day 100 grade 2-4 (but not grade 3-4) acute GVHD was significantly reduced when adding anti-thymoglobulin (ATG) to PTCY. However, in multivariate analysis, grade 2 acute GVHD was significantly associated with better disease-free (HR: 0.36; 95%CI: 0.19-0.69, p = .002) and overall (HR: 0.35; 95%CI: 0.1-0.70, p = .003) survivals. The same results were observed when considering only AML patients. CONCLUSION: Acute grade 2 GVHD is a factor of good prognosis after PBSC haplotransplant with PTCY. Further and larger studies are needed to clarify the complex question of GVHD prophylaxis in the setting of haplo-transplant, especially that of combining ATG and PTCY.