Background: Severe steroid-refractory acute graft-versus-host disease (SR-aGvHD) following allogeneic stem cell transplantation (HSCT) is associated with a high mortality rate. Mesenchymal stromal cell (MSC) therapy has emerged as a promising treatment option for SR-aGvHD, yet its clinical efficacy has never been conclusively demonstrated. Aims: The randomized double-blind placebo-controlled HOVON-113 trial was designed as an international effort to evaluate whether addition of MSC to standardized second line treatment of SR-aGVHD improves clinical outcome. Patients developing aGvHD grade II to IV after alloSCT or donor lymphocyte infusions (DLI) with gut and/or liver involvement and stable or progressive disease or mixed response after 5 to 10 days of 2 mg/kg steroids or equivalent, were eligible for the study. Methods: Patients were randomized to receive 2 intravenous infusions at a 7-day interval of either MSC 2 x 106/kg body weight or placebo. In addition, all patients received mycophenolate mofetil (MMF) as standardized second line treatment. Patients who developed aGvHD while on prophylactic MMF were ineligible for inclusion. The trial’s primary endpoint was complete or partial response at 28 days following the 1st MSC or placebo infusion, reported as overall response (OR). Secondary endpoints included overall survival (OS), cumulative incidence of chronic GvHD, and adverse events. A sample size of 150 patients was calculated to detect an increase in the aGvHD response rate from 40% to 65% with a power of 0.80 and a 2-sided significance level of 0.05. Results: In the course of 5 years, 41 eligible patients were included in the Netherlands, Germany, Spain, Italy, and Belgium. 7 of these were < 18 years (age range 1-16). 20 patients were randomized to the MSC arm and 21 to the placebo arm. At diagnosis, 12% of patients had grade II aGvHD, 68% grade III, and 20% grade IV. In 63% of cases, aGvHD had been elicited by alloSCT and in 37% of cases by DLI. These variables were distributed evenly between the treatment arms. Of the 41 patients, 36 (85%) received both infusions. 3 did not receive any infusions and 2 received only 1 infusion due to refusal (1) or swift clinical deterioration and death (4). No infusion toxicity was observed. OR at day 28 was reached by 8 (38%) of patients in the placebo arm and by 12 (60%) in the MSC arm (odds ratio = 2.48, 95% confidence interval (CI) 0.65-9.51, p = 0.19). 7 patients died before day 28 and could not be evaluated, 6 (29%) in the placebo arm and 1 (5%) in the MSC arm, and were considered as non-responders in the primary analysis. OS at 1 year after randomization was 33% (95% CI 15-53) in the placebo arm and 45% (95% CI 23-65) in the MSC arm (HR = 0.81, 95% CI 0.37-1.77, p = 0.60). Infectious complications were comparable between treatments arms; placebo arm: 25% grade 3, 45% grade 4; MSC arm: 22% grade 3, 39% grade 4. The main causes of death were GvHD and infections. The median follow-up of the 15 patients still alive was 24.0 months (range 9.4 – 73.8 months). Summary/Conclusion: Due to regulatory issues and insufficient accrual, the trial could only include 41 patients and did not have sufficient power to detect the anticipated difference between the two treatment arms. No statistically significant effect of MSC infusions on aGvHD response on day 28, overall survival, or safety could be demonstrated. An extensive biomarker analysis, immunological monitoring, quality of life analysis, health technology assessment, and ethical examination are still ongoing.

The therapeutic landscape of chronic myeloid leukemia (CML) has profoundly changed over the past 7 years. Most patients with chronic phase (CP) now have a normal life expectancy. Another goal is achieving a stable deep molecular response (DMR) and discontinuing medication for treatment-free remission (TFR). The European LeukemiaNet convened an expert panel to critically evaluate and update the evidence to achieve these goals since its previous recommendations. First-line treatment is a tyrosine kinase inhibitor (TKI; imatinib brand or generic, dasatinib, nilotinib, and bosutinib are available first-line). Generic imatinib is the cost-effective initial treatment in CP. Various contraindications and side-effects of all TKIs should be considered. Patient risk status at diagnosis should be assessed with the new EUTOS long-term survival (ELTS)-score. Monitoring of response should be done by quantitative polymerase chain reaction whenever possible. A change of treatment is recommended when intolerance cannot be ameliorated or when molecular milestones are not reached. Greater than 10% BCR-ABL1 at 3 months indicates treatment failure when confirmed. Allogeneic transplantation continues to be a therapeutic option particularly for advanced phase CML. TKI treatment should be withheld during pregnancy. Treatment discontinuation may be considered in patients with durable DMR with the goal of achieving TFR.

Background: Patients (pts) with AML harboring FLT3 internal tandem duplications (ITD) have poor outcomes, in particular pts with a high ITD mutant to wild-type (wt) allelic ratio (AR; ≥0.5). The 2017 update of the European LeukemiaNet (ELN) recommendations addressed the importance of the ITD AR in the genetic risk stratification and defined distinct NPM1/FLT3-ITD genotypes. Aims: In this exploratory post-hoc analysis from randomized pts treated within the RATIFY trial evaluating the addition of midostaurin to standard chemotherapy, we investigated the prognostic impact of the NPM1/FLT3-ITD genotypes according to the 2017 ELN risk categories. Methods: In total, 319 of 717 pts with FLT3-ITD positive AML were included who gave informed consent for biomarker analyses and who could be categorized according to 2017 ELN genetic risk stratification. Median age was 47.9 years (range, 18–60); median follow-up time was 4.8 years. Results: Pts were categorized as follows: (i) favorable-risk (n = 85), NPM1mut/FLT3-ITDlow, irrespective of secondary chromosome abnormalities and concurrent gene mutations; (ii) intermediate-risk (n = 111), NPM1mut/FLT3-ITDhigh and NPM1wt/FLT3-ITDlow, both subgroups without adverse cytogenetics and without concurrent RUNX1, ASXL1, TP53 mutations; (iii) adverse-risk (n = 123), NPM1wt/FLT3-ITDhigh; as well as NPM1mut/FLT3-ITDhigh and NPM1wt/FLT3-ITDlow with adverse-risk cytogenetic and/or molecular markers; frequencies of RUNX1, ASXL1, and TP53 mutations in adverse-risk AML were 29.2%, 19.8%, and 1.9%, respectively. Rates of allogeneic transplantation (alloSCT) in first complete remission (CR) were not different among the groups, with 29.4%, 35.1%, and 35.0% in pts. with favorable-, intermediate-, and adverse-risk AML, respectively. Rates of CR (including responses after induction 1 and 2) were negatively associated with adverse-risk genetics (51.2% vs 69.4% and 64.0% in favorable- and intermediate-risk, respectively; p = 0.02); multivariable logistic regression analysis revealed adverse-risk as significantly different compared to favorable-risk (odds ratio [OR] 0.534, 95% confidence interval [CI] 0.287–0.979; p = 0.044), whereas no difference was seen between the two other risk groups; treatment with midostaurin had no impact (p = 0.32). Overall survival (OS) differed among the 3 ELN-risk groups; 5-year survival rates were 63%, 43%, 33% for favorable, intermediate- and adverse-risk groups, respectively. The OS curves did not show differential effects of midostaurin among the 3 risk groups (Figure 1), with a benefical effect seen for midostaurin vs placebo in all 3 groups with hazard ratios (HR) estimated as HR = 0.52, HR = 0.51, and HR = 0.55 for favorable-, intermediate-, and adverse-risk groups, respectively. A multivariable Cox model adjusted for age, WBC, sex, and bone marrow blasts was used to test the effects of treatment and risk classification. The resulting model revealed an increasing HR with increasing risk category (p < 0.001; intermediate vs. favorable, HR = 1.70, 95% CI 1.08–2.68; adverse vs. favorable risk, HR = 2.48, 95% CI 1.59–3.86), while treatment with midostaurin significantly reduced risk compared to placebo (p < 0.001; HR = 0.53, 95% CI 0.38–0.72).Summary/Conclusion: Data from this post-hoc exploratory analysis confirm the prognostic value of the 2017 ELN risk categories also among AML pts with the distinct NPM1/FLT3-ITD genotypes. A Cox model revealed a significant effect for midostaurin compared to placebo independent of the ELN risk groups. Analysis with regard to potential confounding effects of allogenic transplantation are ongoing.

Background: Myelodysplastic syndrome (MDS) and myelodysplatic/myeloproliferative neoplasm (MDS/MPN) belong to a heterogeneous group of bone marrow (BM) diseases. Immunophenotyping is part of the basic diagnostic workup, but the biological implications and clinical consequences of distinct surface antigen expression patterns in MDS have not been reported. Aims: To assess pre‐treatment immunophenotypic features in MDS patients (pts) and to identify associated disease characteristics and outcome. Methods: We analyzed 175 pts with MDS (n = 155) or MDS/MPN (n = 20) who received an allogeneic cell transplantation (HCT, median age 59.2 [range 18.5–74.4] years) using peripheral blood (n = 174) or cord blood (n = 1) stem cells after myeloablative (n = 45), reduced‐intensity (n = 58) or non‐myeloablative (n = 72) conditioning. Donors were matched related (n = 31) or unrelated (n = 96) or had at least one allelic mismatch (n = 48). Prior to HCT, 53% of pts received cytoreductive therapy (24% azacitidine, 21% intensive chemotherapy, 8% both). Pts were grouped according to IPSS‐R (0% very good, 8% good, 31% intermediate, 25% poor, 37% very poor). Flow cytometric analysis was performed centrally on pre‐treatment bone marrow mononuclear cells. Median follow up after HCT was 3.9 years. Results: We identified four pts subgroups with distinct pre‐treatment immunophenotypic features by unsupervised hierarchical clustering (Figure 1A). For pts in the first cluster (n = 12) a particularly adverse outcome after HCT was observed. Compared to all other clusters, this cluster was characterized by a higher expression of immature surface antigens (CD34, P < .001; CD117, P < .001) and a higher expression of the leukemic stem cell population CD34+/CD38‐ ( P = .001), of myeloid antigens (CD11b, P = .05; CD13 P < .01; CD33, P < .01; CD15, P < .01; CD65, P = .05 and CD64, P < .01), of pan‐leukocyte antigens (CD45, P = .03) and a lower expression of T‐cell antigens (CD2, P < .01 & CD7, P < .01). Pts in cluster 1 were more likely to present with an excess of blasts ( P = .03) and to harbor a complex karyotype (KT, P = .02). Although pts in cluster 1 also more often received cytoreductive treatment prior to HCT ( P = .04) they had a higher cumulative incidence of relapse/progression (CIR, P < .01, Figure 1B) including a higher incidence of secondary acute myeloid leukemia (CIsAML, P < .01, Figure 1C). This adverse prognostic impact was also seen in multivariate analyses for CIR after adjustment for age at HCT and IPSS‐R (Hazard ratio [HR] 3.2, 95% Confidence interval [CI] 1.1–9.6, P = .04) and for CIsAML after adjustment for excess of blasts and blast count prior to HCT (HR 3.2, 95% CI 1.0–9.6, P = .04). Cluster 1 immunophenotypic features also remained predictive for higher CIR and CIsAML in bivariate analyses including the presence of a monosomal (HR 2.9, P = .04 & HR 3.4, P = .02, respectively) or complex KT (HR 2.6, P = .08 & HR 3.0, P = .04, respectively). Summary/Conclusion: Assessment of pre‐treatment surface antigen expression patterns allows for identifying a subgroup of MDS pts with adverse clinical outcome after HCT irrespective of clinical prognosticators or sensitivity to pre‐HCT treatment approaches. This subgroup of pts requires post‐HCT strategies to maintain disease control. image

Introduction: Acute myeloid leukemia (AML) developing secondary after other hematologic diseases, or therapy related after cytotoxic treatment for solid tumors or rheumatologic diseases (s/tAML) is clinically, genetically & prognostically distinct from de novo diseases. Data indicate that s/tAML patients (pts) have inferior outcome compared to de novo cases after chemotherapy & therefore often require consolidation therapy using allogeneic stem cell transplantation (HSCT). Leukemic stem cells (LSC) initiate & maintain AML. They are also believed to exist within the CD34+/CD38- &/or high GPR56 expressing bone marrow (BM) population, which have been shown to impact adversely on outcome. The prognostic impact of LSC markers in de novovs s/tAML after HSCT with non-myeloablative conditioning intensity - where the therapeutic approach also relies on immunological graft-versus-leukemia effects - is unknown. Methods: We analyzed 379 AML pts who received an allogeneic peripheral blood HSCT in complete remission (CR, 82%) or CR with incomplete peripheral recovery (CRi, 18%) between 1999 & 2018 after non-myeloablative (3x30 mg/m2 Fludarabine & 2 Gy total body irradiation) conditioning. At diagnosis, cytogenetic & flow cytometric analyses were performed centrally. For pts with pre-treatment BM available the mutation status of CEBPA, NPM1 & presence of FLT3-ITD by fragment analyses as well as expression levels of GPR56 by qPCR were assessed. Using a next-generation targeted amplicon sequencing approach we analyzed a panel comprising 54 recurrently mutated (mut) genes in myeloid malignancies on the MiSeq platform (Illumina). Median follow up after HSCT was 3.7 years. Results: 229 pts (60%) had de novo & 150 pts (40%) had AML secondary to myelodysplastic syndrome (MDS, n=82), myeloproliferative neoplasm (MPN, n=22) or MDS/MPN (n=10), or therapy related after Non-Hodgkin lymphoma (n=9), solid tumors (n=25) or rheumatologic diseases (n=2). At diagnosis, s/tAML pts had lower white blood counts (P=.03), lower blasts in BM (P<.001) or blood (P=.007) & a higher BM CD34+/CD38- cell burden (P=.01) & GPR56 expression (P=.04). They also had worse European LeukemiaNet risk (P=.007), were less likely to have a normal karyotype by trend (P=.06), to have a core binding factor AML (P=.02), to be NPM1mut (P=.003), DNMT3Amut (P=.03) & to harbor a FLT3-ITD (P=.002) but more likely to be JAK2mut (P<.001). Comparing pts with s/tAML vsde novo AML, there was no significant different cumulative incidence of relapse (CIR, P=.85) or overall survival (OS, P=.29). Next, we evaluated the prognostic impact of the LSC-associated populations in pts with de novo or s/tAML separately. In pts with de novo AML, we observed a significantly higher CIR & shorter OS for pts harboring a high CD34+/CD38- cell burden (high vs low, 6% cut, P=.006 [Fig. 1A] & P=.003) & a higher CIR but not significantly different OS for pts with a low GPR56 expression (high vs low, median cut, P=.03 [Fig. 1B] & P=.95). Combining both parameters, we observed a stepwise higher CIR & shorter OS for pts with low expression of both variables vs pts with a low CD34+/CD38- cell burden but high GPR56 expression vs pts with a high CD34+/CD38-cell burden (P=.003 [Fig. 1C] & P=.05). In contrast, in pts with s/tAML, there was no prognostic significance of the CD34+/CD38- cell burden (CIR P=.38 [Fig. 1D] & OS P=.95), the GPR56 expression (CIR P=.64 [Fig. 1E] & OS P=.82) & both markers combined (CIR P=.57 [Fig. 1F] & OS P=.98). Also in multivariate analyses, the combination of both markers significantly impacted CIR (Hazard ratio 2.49, P<.001 after adjustment for donor type) & was the only significant factor for OS (Odds Ratio 0.68, P=.04) in de novo AML but not in s/tAML. Conclusion: While there was no significantly different CIR or OS in s/tAML compared to de novo AML pts undergoing non-myeloablative HSCT we observed a significant impact on outcome for the known LSC-associated prognosticators CD34+/CD38- cell burden & GPR56 expression levels at diagnosis only in de novo AML pts. Different underlying disease biology & possibly different LSC-associated populations may be relevant for disease reoccurrence in s/tAML. Figure Jentzsch: Novartis: Honoraria; Jazz Pharmaceuticals: Honoraria. Niederwieser:Daichii: Speakers Bureau; Cellectis: Consultancy. Platzbecker:Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Schwind:Daiichi Sankyo: Honoraria; Novartis: Honoraria, Research Funding.