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.

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.

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.

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).

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.

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.

Diagnosis and prognosis are supported by integrated assessment of next-generation sequencing in chronic myeloid malignancies. A real-life study.

Next-generation sequencing (NGS) is used to investigate the presence of somatic mutations. The utility of incorporating routine sequencing to guide diagnosis and therapeutic decisions remains unclear. We report the findings of an observational, multicenter study that aimed to assess the impact of somatic mutation testing by NGS in a reallife setting of chronic myeloid malignancies. A total of 177 patients were enrolled, partitioned into two overlapping groups. In group A (n=94), the indication was to search for clonal hematopoiesis, in a context of suspected myelodysplastic syndrome or myeloproliferative neoplasia. In group B (n=95), the theranostic impact of somatic mutations was studied. A panel of 34 genes was used on DNA extracted from blood or bone marrow samples. Within group A, the detection of clonal hematopoiesis supported the diagnosis of chronic myeloid malignancies for 31 patients while the absence of clonal hematopoiesis ruled out the suspected diagnosis in 47 patients. Within group B, NGS identified prognostically relevant somatic mutations in 32 patients, which had a therapeutic impact in 18 cases. By determining the presence or absence of somatic mutations, the application of NGS in daily practice was found to be useful for an integrated final diagnosis in 83% of the patients. Moreover, the search for somatic mutations had a prognostic impact that led to treatment modification in 19% of the cases. This study outlines the fact that adequate implementation of new investigations may have a significant positive medico-economic impact by enabling appropriate management of patients.

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.

Whole genome copy number analysis in search of new prognostic biomarkers in first line treatment of mantle cell lymphoma. A study by the LYSA group.

Mantle cell lymphoma (MCL) is a lymphoproliferative disorder characterized by the t(11;14)(q13;q32) CCND1/IGH translocation. This lymphoma is however extremely heterogeneous in terms of molecular alterations. Moreover, the course of the disease can vary greatly between indolent forms with slow progression and aggressive conditions rapidly pejorative. The identification of early markers allowing to predict individual patients outcome has however been unsuccessful so far. The LyMa trial treated homogeneously a cohort of young MCL patients. This appeared as a good opportunity to search for biomarkers of response to therapy. DNA extracted from diagnostic paraffin-embedded lymph node biopsies from 100 patients with newly diagnosed MCL, homogeneously treated in this prospective clinical trial, were investigated for copy number alterations and copy neutral loss of heterozygosity using the Oncoscan SNP-array scanning the whole genome. An independent confirmatory cohort was used to strengthen the possibly relevant anomalies observed. Here we describe the recurrent anomalies identified with this technique. Deletions of 17p(TP53) and 9p(CDKN2A) were more frequent in refractory or early relapsing patients (10%), but had no significant impact in univariate analysis on progression-free (PFS) or overall survival (OS). Regardless of the presence of TP53 or CDKN2A deletions, gains in 7p22 (8,5%) were associated with better PFS in univariate but not in multivariate analysis including MCL International Prognostic Index and treatment. Gains of 11q(CCDN1), suggesting gains of the CCND1/IGH fusion, were associated with worse OS and PFS in univariate and multivariate analyses. This worse prognosis impact was confirmed by FISH in an independent confirmatory cohort. This work, using a whole genome approach, confirms the broad genomic landscape of MCL and shows that gains of the CCND1/IGH fusion can be considered as a new prognostic structural variant. Genomic abnormalities of prognostic impact could be useful to strengthen or de-escalate treatment schedules or choosing targeted therapies or CART-cells.

Influence of Donor Type (Sibling versus Matched Unrelated Donor versus Haploidentical Donor) on Outcomes after Clofarabine-Based Reduced-Intensity Conditioning Allograft for Myeloid Malignancies.

Clofarabine-based reduced-intensity conditioning (RIC) regimens are well-established schedules for allograft in patients with myeloid malignancies. A retrospective study was conducted including all adults allografted in our department with such a regimen and disease with the aim to assess whether or not the donor type (matched sibling [MSD], matched unrelated [MUD], or haploidentical [haplo]) impacted outcomes. Between October 2009 and February 2018, 118 patients met the inclusion criteria. Thirty-six, 55, and 27 patients received a graft from an MSD, MUD, or haplo donor, respectively. Peripheral blood stem cells (PBSCs) were the source of graft for all patients. The median age of the entire cohort was 62 years (range, 20 to 73), and the median follow-up was 31 months (range, 4.5 to 106). All patients engrafted except 1 haplo recipient. Neutrophils (>.5 × 109/L) and platelets (50 × 109/L) recoveries were significantly delayed in the haplo group (P = .0003 and P < .0001) compared with MSD and MUD. Acute grades II to IV or III to IV graft-versus-host disease (GVHD) incidences were similar between the 3 groups as well as the incidence of moderate or severe chronic GVHD. Also, similar 2-year overall survival (OS; 64.7% versus 73.9% versus 60.2%, P = .39), disease-free survival (DFS; 57.7% versus 70.9% versus and 53.6%, P = .1), and GVHD relapse-free survival (37.9% versus 54.3% versus 38.9%, P = .23) were observed between MSD versus MUD versus haplo groups. The same was true when considering only acute myeloid leukemia (AML) cases. In multivariate analysis the type of donor remained independent of outcomes in this series, whereas myelodysplastic syndrome (versus AML), high disease risk index, and older donor (≥50 years) were associated with lower OS and DFS. These data suggest that haplo donors are an acceptable alternative for patients receiving a clofarabine-based RIC PBSC allograft for myeloid malignancies who lack an MSD or a MUD.

FLT3 ligand plasma levels have no impact on outcomes after allotransplant in acute leukemia.

OBJECTIVE: This study was designed to assess the impact on outcomes of early soluble Fms-like tyrosine kinase 3 ligand concentrations (sFLc) in patients receiving an allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). METHODS: This was a prospective monocentric study including all allo-HSCT patients included in the previous FLAM/FLAL study (Peterlin et al., 2019). Blood samples collected before the start of conditioning then post-transplant were frozen, stored and tested by ELISA. The parameters considered were hematopoietic recoveries, Leukemia Free Survival and Overall Survival, acute and chronic GVHD, grade 3 or 4 acute and/or extensive chronic GVHD-free and relapse-free survival (GRFS). RESULTS: Forty-one patients were included, a total of 179 samples were assayed for sFLc. There was no impact of sFLc levels (<=median vs> median) on acute and chronic GVHD incidences, LFS, OS nor GRFS. CONCLUSION: At variance with induction results for AML (Peterlin et al., 2019) endogenous sFLc do not appear to be a prognostic marker at the time of or after allo-HSCT. Even though the results are negatives, this is, to the best of our knowledge, the only prospective series specifically addressing the question of sFLc impact after allo-HSCT in acute leukemias.

A novel complete blood count-based score to screen for myelodysplastic syndrome in cytopenic patients.

The diagnosis of myelodysplastic syndromes (MDS) is often challenging, time- and resource-consuming. A thorough analysis of complete blood count (CBC) parameters could, however, help to screen for MDS among other causes of cytopenia. To test this hypothesis, 109 newly-diagnosed MDS patients and 399 cytopenic patients older than 50 years with confirmed absence of MDS were enrolled in a prospective study. Multiparametric analysis highlighted three CBC parameters that were significantly different between the two cohorts: mean corpuscular volume, absolute neutrophil count and median neutrophil complexity and width of dispersion of the events measured (Ne-WX), which were used to define an MDS-CBC score. This score enables the prediction of MDS with 86% sensitivity and 88% specificity. The MDS-CBC score excluded MDS in 89% of cytopenic controls. Moreover, high score values at MDS diagnosis significantly correlated with decreased event-free (P = 0·02) and overall survival (P = 0·01). The power of this score was confirmed in an independent validation cohort (MDS n = 34, cytopenic controls n = 28). The MDS-CBC score is an easy and fast tool to exclude or suspect MDS in unselected patients with cytopenia of unknown reasons at the time of analysis, by prompting blood smear examination. It may thus improve allocation of further MDS-specific work-up in patients with cytopenia at the time of CBC assessment.

Major prognostic value of complex karyotype in addition to TP53 and IGHV mutational status in first-line chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder of remarkable heterogeneity as demonstrated by cytogenetics and molecular analyses. Complex karyotype (CK), TP53 deletions and/or mutations (TP53 disruption), IGVH mutational status, and, more recently, recurrent somatic mutations have been identified as prognostic markers in CLL. On a cohort of 110 patients with CLL treated with first-line fludarabin, cyclophosphamide, and rituximab treatment compared with 33 untreated (watch and wait) patients with CLL, we report more frequent complex karyotypes (34 vs 15%; P = .05), unmutated IGHV (70 vs 21%; P < .0001), ATM deletion (25 vs 6%, P = .02), and NOTCH mutation (3 vs 17%, P = .04). Among treated patients, 39 relapsed during the follow-up period. These patients were characterized before treatment by a higher incidence of trisomy 12 (38 vs 11%, P < .001) and TP53 disruption (31 vs 4%, P = .0002). A significantly shorter 5-year overall survival was found for treated patients with CK (72.4 vs 85.8%; P = .007), unmutated IGHV (70 vs 100%; P = .04), or TP53 disruption (55.7 vs 82.7%; P < .0001). Three risk groups were defined based on the status of TP53 disruption or unmutated IGVH, which differed significantly in terms of 5-year overall survival. Moreover, the presence of CK impacted pejoratively 5-year overall survival and progression-free survival in all these 3 groups. Conventional karyotyping therefore appears to be of value, CK being an additional factor, undetectable in classical FISH, in patients with CLL at the stage when therapy becomes required.

Is allogeneic stem cell transplantation for myelofibrosis still indicated at the time of molecular markers and JAK inhibitors era?

OBJECTIVE: The role of allogenic stem cell transplantation (ASCT) is still debated in myelofibrosis (MF). METHODS: A retrospective analyzed was performed to compare the outcome of 71 patients with intermediate-2 or high-risk Dynamic International Prognosis Scoring System+ (DIPSS+) primary (PMF) or secondary (SMF) myelofibrosis with an indication of ASCT as they ultimately underwent the procedure (n=34) or not (n=37). RESULTS: Five-year overall survival (OS) was not statistically different between both groups (allograft: 52% vs no allograft: 34%, P=.12). However, progression to myelodysplastic syndrome or acute myeloid leukemia at 5 years was significantly lower in transplanted patients (14% vs 50%, P=.01). In univariate analysis, 5-year OS was significantly higher for transplanted vs non-transplanted patients with unfavorable karyotype (75% vs 0%, P=.001), SMF (71% vs 20%, P=.001) or high DIPSS+ score (46% vs 15%, P=.03). There was also a trend for better 5-year OS in allografted patients with high JAK2V617F burden (>65%) (75% vs 8%, P=.07). Interestingly, the survival of patients who did not proceed to ASCT was dramatically increased by the use of ruxolitinib. CONCLUSIONS: Not all intermediate-2/high-risk DIPSS+ MF patients benefit from ASCT, especially since the introduction of JAK2 inhibitors.