Benefits and risks of clofarabine in adult acute lymphoblastic leukemia investigated in depth by multi-state modeling.

BACKGROUND: We recently reported results of the prospective, open-label HOVON-100 trial in 334 adult patients with acute lymphoblastic leukemia (ALL) randomized to first-line treatment with or without clofarabine (CLO). No improvement of event-free survival (EFS) was observed, while a higher proportion of patients receiving CLO obtained minimal residual disease (MRD) negativity. AIM: In order to investigate the effects of CLO in more depth, two multi-state models were developed to identify why CLO did not show a long-term survival benefit despite more MRD-negativity. METHODS: The first model evaluated the effect of CLO on going off-protocol (not due to refractory disease/relapse, completion or death) as a proxy of severe treatment-related toxicity, while the second model evaluated the effect of CLO on obtaining MRD negativity. The subsequent impact of these intermediate events on death or relapsed/refractory disease was assessed in both models. RESULTS: Overall, patients receiving CLO went off-protocol more frequently than control patients (35/168 [21%] vs. 18/166 [11%], p = 0.019; HR 2.00 [1.13-3.52], p = 0.02), especially during maintenance (13/44 [30%] vs. 6/56 [11%]; HR 2.85 [95%CI 1.08-7.50], p = 0.035). Going off-protocol was, however, not associated with more relapse or death. Patients in the CLO arm showed a trend towards an increased rate of MRD-negativity compared with control patients (HR MRD-negativity: 1.35 [0.95-1.91], p = 0.10), which did not translate into a significant survival benefit. CONCLUSION: We conclude that the intermediate states, i.e., going off-protocol and MRD-negativity, were affected by adding CLO, but these transitions were not associated with subsequent survival estimates, suggesting relatively modest antileukemic activity in ALL.

Molecular and clinical presentation of UBA1-mutated myelodysplastic syndromes.

Mutations in UBA1, which are disease-defining for VEXAS syndrome, have been reported in patients diagnosed with myelodysplastic syndromes (MDS). Here, we define the prevalence and clinical associations of UBA1 mutations in a representative cohort of patients with MDS. Digital droplet PCR profiling of a selected cohort of 375 male patients lacking MDS disease-defining mutations or established WHO disease classification identified 28 patients (7%) with UBA1 p.M41T/V/L mutations. Using targeted sequencing of UBA1 in a representative MDS cohort (n=2,027), we identified an additional 27 variants in 26 patients (1%), which we classified as likely/pathogenic (n=12) and unknown significance (n=15). Among the total 40 patients with likely/pathogenic variants (2%), all were male and 63% were classified by WHO2016 as MDS-MLD/SLD. Patients had a median of one additional myeloid gene mutation, often in TET2 (n=12), DNMT3A (n=10), ASXL1 (n=3), or SF3B1 (n=3). Retrospective clinical review where possible showed that 83% (28/34) UBA1-mutant cases had VEXAS-associated diagnoses or inflammatory clinical presentation. The prevalence of UBA1-mutations in MDS patients argues for systematic screening for UBA1 in the management of MDS.

Myelodysplastic neoplasms dissected into indolent, leukaemic and unfavourable subtypes by computational clustering of haematopoietic stem and progenitor cells.

Myelodysplastic neoplasms (MDS) encompass haematological malignancies, which are characterised by dysplasia, ineffective haematopoiesis and the risk of progression towards acute myeloid leukaemia (AML). Myelodysplastic neoplasms are notorious for their heterogeneity: clinical outcomes range from a near-normal life expectancy to leukaemic transformation or premature death due to cytopenia. The Molecular International Prognostic Scoring System made progress in the dissection of MDS by clinical outcomes. To contribute to the risk stratification of MDS by immunophenotypic profiles, this study performed computational clustering of flow cytometry data of CD34+ cells in 67 MDS, 67 AML patients and 49 controls. Our data revealed heterogeneity also within the MDS-derived CD34+ compartment. In MDS, maintenance of lymphoid progenitors and megakaryocytic-erythroid progenitors predicted favourable outcomes, whereas expansion of granulocyte-monocyte progenitors increased the risk of leukaemic transformation. The proliferation of haematopoietic stem cells and common myeloid progenitors with downregulated CD44 expression, suggestive of impaired haematopoietic differentiation, characterised a distinct MDS subtype with a poor overall survival. This exploratory study demonstrates the prognostic value of known and previously unexplored CD34+ populations and suggests the feasibility of dissecting MDS into a more indolent, a leukaemic and another unfavourable subtype.

Impact of hemodilution on flow cytometry based measurable residual disease assessment in acute myeloid leukemia.

Measurable residual disease (MRD) measured in the bone marrow (BM) of acute myeloid leukemia (AML) patients after induction chemotherapy is an established prognostic factor. Hemodilution, stemming from peripheral blood (PB) mixing within BM during aspiration, can yield false-negative MRD results. We prospectively examined hemodilution by measuring MRD in BM aspirates obtained from three consecutive 2 mL pulls, along with PB samples. Our results demonstrated a significant decrease in MRD percentages between the first and second pulls (P = 0.025) and between the second and third pulls (P = 0.025), highlighting the impact of hemodilution. Initially, 39% of MRD levels (18/46 leukemia-associated immunophenotypes) exceeded the 0.1% cut-off, decreasing to 30% (14/46) in the third pull. Additionally, we assessed the performance of six published methods and parameters for distinguishing BM from PB samples, addressing or compensating for hemodilution. The most promising results relied on the percentages of CD16dim granulocytic population (scarce in BM) and CD117high mast cells (exclusive to BM). Our findings highlight the importance of estimating hemodilution in MRD assessment to qualify MRD results, particularly near the common 0.1% cut-off. To avoid false-negative results by hemodilution, it is essential to collect high-quality BM aspirations and preferably utilizing the initial pull for MRD testing.

ATG versus PTCy in matched unrelated donor haematopoietic stem cell transplantations with non-myeloablative conditioning.

Graft-versus-host disease (GvHD) is a serious complication of allogeneic haematopoietic stem cell transplantation (HSCT). Both anti-thymocyte globulin (ATG) and post-transplant cyclophosphamide (PTCy) are used as lymphocyte-depleting strategies, yet a systematic comparison of transplantation outcomes between these two methods in matched unrelated donors (MUD) transplantations with non-myeloablative conditioning (NMC) is lacking. Adult patients with haematological malignancies who had undergone MUD HSCT with NMC regimens between 2014 and 2021 at 2 centres in Amsterdam (ATG: n = 95, PTCy: n = 90), were included in this retrospective study. Patient characteristics were comparable between the groups. The cumulative incidence of acute GvHD grade II-IV was 48% in the ATG group compared to 21% in the PTCy group (p < 0.001). The 3-year moderate/severe chronic GvHD was similar in both groups (p = 0.69). While the relapse rate was comparable between the groups (ATG 31% vs. PTCy 34%, p = 0.94), non-relapse mortality tended to be higher in the ATG group (17% vs. 9%, p = 0.069). Overall survival was similar in both groups (p = 0.12). In conclusion, PTCy-based regimens resulted in a significantly lower rate of acute GvHD than ATG-containing regimens in MUD transplantations with NMC. Whether PTCy results in improved overall survival as compared to ATG needs to be elucidated in larger prospective studies.

Age and sex associate with outcome in older AML and high risk MDS patients treated with 10-day decitabine.

Treatment choice according to the individual conditions remains challenging, particularly in older patients with acute myeloid leukemia (AML) and high risk myelodysplastic syndrome (MDS). The impact of performance status, comorbidities, and physical functioning on survival is not well defined for patients treated with hypomethylating agents. Here we describe the impact of performance status (14% ECOG performance status 2), comorbidity (40% HCT-comorbidity index ≥ 2), and physical functioning (41% short physical performance battery < 9 and 17% ADL index < 6) on overall survival (OS) in 115 older patients (age ≥ 66 years) treated on a clinical trial with a 10-day decitabine schedule. None of the patient-related variables showed a significant association with OS. Multivariable analysis revealed that age > 76 years was significantly associated with reduced OS (HR 1.58; p = 0.043) and female sex was associated with superior OS (HR 0.62; p = 0.06). We further compared the genetic profiles of these subgroups. This revealed comparable mutational profiles in patients younger and older than 76 years, but, interestingly, revealed significantly more prevalent mutated ASXL1, STAG2, and U2AF1 in male compared to female patients. In this cohort of older patients treated with decitabine age and sex, but not comorbidities, physical functioning or cytogenetic risk were associated with overall survival.

Prevalence and treatment of anemia and secondary iron overload in patients with a myelodysplastic syndrome: Real-world data from a multicenter cohort study.

BACKGROUND: anemia is the most common finding in patients with a myelodysplastic syndrome (MDS). Repetitive red blood cell (RBC) transfusions and disease-related low hepcidin levels induce secondary iron overload. Real-world data on the prevalence and treatment strategies of anemia and secondary iron overload in MDS patients, is limited. METHODS: three years of data on MDS diagnosis, anemia and ferritin management was collected in 230 MDS patients from seven non-academic hospitals in the Netherlands. Descriptive statistics and linear mixed models were used to analyze the data. RESULTS: transfusion dependent (TD) patients (n = 49) needed 1-3 RBC transfusions per month. Serum hemoglobin remained stable in both TD and transfusion-independent (TI) patients over 3 years. In the TD patients, serum ferritin increased 63 pmol/L/month. Overall, 19 (39%) were diagnosed with secondary hemochromatosis, of which 13 (68%) received chelation therapy with a heterogeneous response. CONCLUSIONS: mean hemoglobin remains stable over time in both TD and TI MDS patients. Approximately 40% of TD patients develop secondary hemochromatosis. Treatment and monitoring of secondary hemochromatosis as well as the response on chelation therapy vary substantially.

Immunophenotypic aberrant hematopoietic stem cells in myelodysplastic syndromes: a biomarker for leukemic progression.

Myelodysplastic syndromes (MDS) comprise hematological disorders that originate from the neoplastic transformation of hematopoietic stem cells (HSCs). However, discrimination between HSCs and their neoplastic counterparts in MDS-derived bone marrows (MDS-BMs) remains challenging. We hypothesized that in MDS patients immature CD34+CD38- cells with aberrant expression of immunophenotypic markers reflect neoplastic stem cells and that their frequency predicts leukemic progression. We analyzed samples from 68 MDS patients and 53 controls and discriminated HSCs from immunophenotypic aberrant HSCs (IA-HSCs) expressing membrane aberrancies (CD7, CD11b, CD22, CD33, CD44, CD45RA, CD56, CD123, CD366 or CD371). One-third of the MDS-BMs (23/68) contained IA-HSCs. The presence of IA-HSCs correlated with perturbed hematopoiesis (disproportionally expanded CD34+ subsets beside cytopenias) and an increased hazard of leukemic progression (HR = 25, 95% CI: 2.9-218) that was independent of conventional risk factors. At 2 years follow-up, the sensitivity and specificity of presence of IA-HSCs for predicting leukemic progression was 83% (95% CI: 36-99%) and 71% (95% CI: 58-81%), respectively. In a selected cohort (n = 10), most MDS-BMs with IA-HSCs showed genomic complexity and high human blast counts following xenotransplantation into immunodeficient mice, contrasting MDS-BMs without IA-HSCs. This study demonstrates that the presence of IA-HSCs within MDS-BMs predicts leukemic progression, indicating the clinical potential of IA-HSCs as a prognostic biomarker.

Consensus proposal for revised International Working Group 2023 response criteria for higher-risk myelodysplastic syndromes.

Myelodysplastic syndromes/myelodysplastic neoplasms (MDS) are associated with variable clinical presentations and outcomes. The initial response criteria developed by the International Working Group (IWG) in 2000 have been used in clinical practice, clinical trials, regulatory reviews, and drug labels. Although the IWG criteria were revised in 2006 and 2018 (the latter focusing on lower-risk disease), limitations persist in their application to higher-risk MDS (HR-MDS) and their ability to fully capture the clinical benefits of novel investigational drugs or serve as valid surrogates for longer-term clinical end points (eg, overall survival). Further, issues related to the ambiguity and practicality of some criteria lead to variability in interpretation and interobserver inconsistency in reporting results from the same sets of data. Thus, we convened an international panel of 36 MDS experts and used an established modified Delphi process to develop consensus recommendations for updated response criteria that would be more reflective of patient-centered and clinically relevant outcomes in HR-MDS. Among others, the IWG 2023 criteria include changes in the hemoglobin threshold for complete remission (CR), the introduction of CR with limited count recovery and CR with partial hematologic recovery as provisional response criteria, the elimination of marrow CR, and specific recommendations for the standardization of time-to-event end points and the derivation and reporting of responses. The updated criteria should lead to a better correlation between patient-centered outcomes and clinical trial results in an era of multiple emerging new agents with novel mechanisms of action.

Multiparameter flow cytometry in the evaluation of myelodysplasia: Analytical issues: Recommendations from the European LeukemiaNet/International Myelodysplastic Syndrome Flow Cytometry Working Group.

Multiparameter flow cytometry (MFC) is one of the essential ancillary methods in bone marrow (BM) investigation of patients with cytopenia and suspected myelodysplastic syndrome (MDS). MFC can also be applied in the follow-up of MDS patients undergoing treatment. This document summarizes recommendations from the International/European Leukemia Net Working Group for Flow Cytometry in Myelodysplastic Syndromes (ELN iMDS Flow) on the analytical issues in MFC for the diagnostic work-up of MDS. Recommendations for the analysis of several BM cell subsets such as myeloid precursors, maturing granulocytic and monocytic components and erythropoiesis are given. A core set of 17 markers identified as independently related to a cytomorphologic diagnosis of myelodysplasia is suggested as mandatory for MFC evaluation of BM in a patient with cytopenia. A myeloid precursor cell (CD34+ CD19- ) count >3% should be considered immunophenotypically indicative of myelodysplasia. However, MFC results should always be evaluated as part of an integrated hematopathology work-up. Looking forward, several machine-learning-based analytical tools of interest should be applied in parallel to conventional analytical methods to investigate their usefulness in integrated diagnostics, risk stratification, and potentially even in the evaluation of response to therapy, based on MFC data. In addition, compiling large uniform datasets is desirable, as most of the machine-learning-based methods tend to perform better with larger numbers of investigated samples, especially in such a heterogeneous disease as MDS.

Characterization of myelodysplastic syndromes hematopoietic stem and progenitor cells using mass cytometry.

BACKGROUND: Myelodysplastic syndromes (MDS) at risk of transformation to acute myeloid leukemia (AML) are difficult to identify. The bone marrows of MDS patients harbor specific hematopoietic stem and progenitor cell (HSPC) abnormalities that may be associated with sub-types and risk-groups. Leukemia-associated characteristics of such cells may identify MDS patients at risk of progression to AML and provide insight in the pathobiology of MDS. METHODS: Bone marrow samples from healthy donors (n = 10), low risk (n = 12) and high risk (n = 13) MDS patients were collected, in addition, AML samples for 5 out of 6 MDS patients that progressed. Mass cytometry was applied to assess expression of stem cell subset and leukemia-associated immunophenotype markers. RESULTS: We analyzed the data using FlowSOM to cluster cells with similar expression of 10 commonly used stem cell markers. Metaclusters (n = 20) of these clusters represented populations of cells with a related phenotype, largely resembling known stem cell subsets. Within specific subsets, intra-cellular expression levels of pCREB, IkBα, or pS6 differed significantly between healthy bone marrow (HBM) and MDS or consecutive secondary AML samples. CD34, CD44, and CD49f expression was significantly increased in high risk MDS and AML-associated metaclusters. We identified MDS/sAML cells with aberrant phenotypes when compared to HBM. Such cells were observed in clusters of both primary MDS and secondary AML samples. CONCLUSIONS: High-dimensional mass cytometry and computational data analyses enabled characterization of HSPC subsets in MDS and identification of leukemia stem cell populations based on their immunophenotype. Stem cells in MDS that display leukemia-associated features may predict the risk of developing AML.

Dysplasia and PNH-type cells in bone marrow aspirates of myelodysplastic syndromes.

BACKGROUND: Flow cytometry is increasingly applied in cytopenic patients suspected for myelodysplastic syndromes (MDS). Analysis includes evaluation of antigen expression patterns in granulocytes of which, for example, partial lack of CD16 may indicate dysplasia, but presence of paroxysmal nocturnal hemoglobinuria (PNH)-type cells should be considered. However, diagnostic bone marrow (BM) samples hamper PNH analysis because immature stages in the granulo-/monocytic compartment lack expression of certain glycophosphatidyl-inositol-anchored proteins. In this prospective study, we evaluated the presence of PNH-type cells in BM next to aberrancies from routine MDS immunophenotyping. METHODS: We combined antibodies defining maturation trajectories with FLAER. Validation of the designed method against routine PNH analysis and parallel analysis of BM and blood samples revealed similar results (granulocytes: Wilcoxon p = 0.25 and p = 0.82, respectively). We analyzed BM samples from 134 MDS, 17 chronic myelomonocytic leukemia, 15 aplastic anemia (AA), 1 PNH, 51 non-clonal cytopenic controls, and 12 normal controls. RESULTS: Most AA/PNH-BM samples showed clear PNH clones: median 1.1% (0%-35%); CD16 loss on mature neutrophils paralleled PNH-clone sizes. In MDS-BM, only 3.7% of cases showed ≥0.1% PNH-type cells, whereas partial CD16 loss was more frequent and abundant. CONCLUSIONS: Our findings confirm that dysplastic features in MDS-BM may point to presence of PNH-type cells, though only few cases displayed FLAER-negative cells. We showed that identification of these cells in the granulocyte compartment of BM specimen is feasible, but-according to international guidelines-results need to be confirmed in peripheral blood.

Clinical application of flow cytometry in patients with unexplained cytopenia and suspected myelodysplastic syndrome: A report of the European LeukemiaNet International MDS-Flow Cytometry Working Group.

This article discusses the rationale for inclusion of flow cytometry (FCM) in the diagnostic investigation and evaluation of cytopenias of uncertain origin and suspected myelodysplastic syndromes (MDS) by the European LeukemiaNet international MDS Flow Working Group (ELN iMDS Flow WG). The WHO 2016 classification recognizes that FCM contributes to the diagnosis of MDS and may be useful for prognostication, prediction, and evaluation of response to therapy and follow-up of MDS patients.

A series of case studies illustrating the role of flow cytometry in the diagnostic work-up of myelodysplastic syndromes.

Current guidelines recommend flow cytometric analysis as part of the diagnostic assessment of patients with cytopenia suspected for myelodysplastic syndrome. Herein we describe the complete work-up of six cases using multimodal integrated diagnostics. Flow cytometry assessments are illustrated by plots from conventional and more recent analysis tools. The cases demonstrate the added value of flow cytometry in case of hypocellular, poor quality, or ambiguous bone marrow cytomorphology. Moreover, they demonstrate how immunophenotyping results support clinical decision-making in inconclusive and clinically 'difficult' cases.

ELN iMDS flow working group validation of the monocyte assay for chronic myelomonocytic leukemia diagnosis by flow cytometry.

BACKGROUND: It was proposed that peripheral blood (PB) monocyte profiles evaluated by flow cytometry, called "monocyte assay," could rapidly and efficiently distinguish chronic myelomonocytic leukemia (CMML) from other causes of monocytosis by highlighting an increase in the classical monocyte (cMo) fraction above 94%. However, the robustness of this assay requires a large multicenter validation and the assessment of its feasibility on bone marrow (BM) samples, as some centers may not have access to PB. METHODS: PB and/or BM samples from patients displaying monocytosis were assessed with the "monocyte assay" by 10 ELN iMDS Flow working group centers with harmonized protocols. The corresponding files were reanalyzed in a blind fashion and the cMo percentages obtained by both analyses were compared. Confirmed diagnoses were collected when available. RESULTS: The comparison between cMo percentages from 267 PB files showed a good global significant correlation (r = 0.88) with no bias. Confirmed diagnoses, available for 212 patients, achieved a 94% sensitivity and an 84% specificity. Hence, 95/101 CMML patients displayed cMo ≥94% while cMo <94% was observed in 83/99 patients with reactive monocytosis and in 10/12 patients with myeloproliferative neoplasms (MPN) with monocytosis. The established Receiver Operator Curve again provided a 94% cut-off value of cMo. The 117 BM files reanalysis led to an 87% sensitivity and an 80% specificity, with excellent correlation between the 43 paired samples to PB. CONCLUSIONS: This ELN multicenter study demonstrates the robustness of the monocyte assay with only limited variability of cMo percentages, validates the 94% cutoff value, confirms its high sensitivity and specificity in PB and finally, also confirms the possibility of its use in BM samples.