Comparison of fludarabine/melphalan (FluMel) with fludarabine/melphalan/BCNU or thiotepa (FBM/FTM) in patients with AML in first complete remission undergoing allogeneic hematopoietic stem cell transplantation - a registry study on behalf of the EBMT Acute Leukemia Working Party.

Conditioning protocols for patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT) are being developed continuously to improve their anti-leukemic efficacy and reduce their toxicity. In this study, we compared the conditioning protocol of fludarabine with melphalan 140 mg/m2 (FluMel) with conditioning protocols based on this same backbone but with an additional alkylating agent i.e., either fludarabine/BCNU (also known as carmustine)/melphalan (FBM), or fludarabine/thiotepa/melphalan (FTM) 110 mg/m2. We included 1272 adult patients (FluMel, n = 1002; FBM/FTM, n = 270) with acute myeloid leukemia (AML) with intermediate/poor cytogenetic risk in first complete remission (CR) from the registry of the EBMT Acute Leukemia Working Party. Despite patients in the FBM/FTM group were older (64.1 years vs. 59.8 years, p < 0.001) and had a worse Karnofsky performance score (KPS < 90, 33% vs. 24%, p = 0.003), they showed a better overall survival (OS) (2 y OS: 68.3% vs. 58.1%, p = 0.02) and less non-relapse mortality (NRM) (2 y NRM: 15.8% vs. 22.2%, p = 0.009) compared to patients treated with FluMel. No significant differences were observed in relapse incidence (RI) (2 y RI: 24.9% vs. 23.7%, p = 0.62). In conclusion, the addition of a second alkylating agent (BCNU/carmustine or thiotepa) to FluMel as FBM/FTM conditioning, improves OS in AML patients in first CR with intermediate/poor risk cytogenetics after allo-HCT.

Posttransplant MRD and T-cell chimerism status predict outcomes in patients who received allografts for AML/MDS.

Allogeneic stem-cell transplant allows for the delivery of curative graft-versus-leukemia (GVL) in patients with acute myeloid leukemia/myelodysplasia (AML/MDS). Surveillance of T-cell chimerism, measurable residual disease (MRD) and blast HLA-DR expression may inform whether GVL effectiveness is reduced. We report here the prognostic impact of these biomarkers in patients allografted for AML/MDS. One hundred eighty-seven patients from FIGARO, a randomized trial of reduced-intensity conditioning regimens in AML/MDS, were alive and relapse-free at the first MRD time-point and provided monitoring samples for flow cytometric MRD and T-cell chimerism, requested to month+12. Twenty-nine (15.5%) patients had at least 1 MRD-positive result posttransplant. MRD-positivity was associated with reduced overall survival (OS) (hazard ratio [HR], 2.18; P = .0028) as a time-varying Cox variable and remained significant irrespective of pretransplant MRD status in multivariate analyses (P < .001). Ninety-four patients had sequential MRD with T-cell chimerism results at months+3/+6. Patients with full donor T-cell chimerism (FDTC) had an improved OS as compared with patients with mixed donor T-cell chimerism (MDTC) (adjusted HR=0.4; P = .0019). In patients with MDTC (month+3 or +6), MRD-positivity was associated with a decreased 2-year OS (34.3%) vs MRD-negativity (71.4%) (P = .001). In contrast, in the group with FDTC, MRD was infrequent and did not affect the outcome. Among patients with posttransplant MRD-positivity, decreased HLA-DR expression on blasts significantly reduced OS, supporting this as a mechanism for GVL escape. In conclusion, posttransplant MRD is an important predictor of the outcome in patients allografted for AML/MDS and is most informative when combined with T-cell chimerism results, underlining the importance of a GVL effect in AML/MDS.

Use of eculizumab in children with allogeneic haematopoietic stem cell transplantation associated thrombotic microangiopathy - a multicentre retrospective PDWP and IEWP EBMT study.

Terminal complement blockade by humanised monoclonal antibody eculizumab has been used to treat transplantation-associated thrombotic microangiopathy (TA-TMA) in recent years. This retrospective international study conducted by the Paediatric Diseases (PDWP) and Inborn Error Working Party (IEWP) of the European Society for Blood and Marrow Transplantation (EBMT) describes outcome and response of 82 paediatric patients from 29 centres who developed TA-TMA and were treated with eculizumab between January 2014 and May 2019. The median time from hematopoietic stem cell transplantation (HSCT) to TA-TMA manifestation was 92 days (range: 7-606) and from TA-TMA diagnosis to the start of eculizumab treatment 6 days (range: 0-135). Most patients received eculizumab weekly (72%, n = 55) with a standard weight (kg)-based dose (78%, n = 64). Six months from beginning of eculizumab therapy, the cumulative incidence of TA-TMA resolution was 36.6% (95% CI: 26.2-47) and the overall survival (OS) was 47.1% (95% CI: 35.9-57.5). All 43 patients with unresolved TA-TMA died. The cause of death was HSCT-related in 41 patients. This study also documents poor outcome of patients without aGvHD and their frequent concomitant viral infections. Considering recent publications, intensified eculizumab dosing and complement monitoring could potentially improve upon outcomes observed in this study.

Comparison of fludarabine-melphalan and fludarabine-treosulfan as conditioning prior to allogeneic hematopoietic cell transplantation-a registry study on behalf of the EBMT Acute Leukemia Working Party.

In recent years considerable variations in conditioning protocols for allogeneic hematopoietic cell transplantation (allo-HCT) protocols have been introduced for higher efficacy, lower toxicity, and better outcomes. To overcome the limitations of the classical definition of reduced intensity and myeloablative conditioning, a transplantation conditioning intensity (TCI) score had been developed. In this study, we compared outcome after two frequently used single alkylator-based conditioning protocols from the intermediate TCI score category, fludarabine/melphalan 140 mg/m2 (FluMel) and fludarabine/treosulfan 42 g/m2 (FluTreo) for patients with acute myeloid leukemia (AML) in complete remission (CR). This retrospective analysis from the registry of the Acute Leukemia Working Party (ALWP) of the European Society of Bone Marrow Transplantation (EBMT) database included 1427 adult patients (median age 58.2 years) receiving either Flu/Mel (n = 1005) or Flu/Treo (n = 422). Both groups showed similar 3-year overall survival (OS) (54% vs 51.2%, p value 0.49) for patients conditioned with FluMel and FluTreo, respectively. However, patients treated with FluMel showed a reduced 3-year relapse incidence (32.4% vs. 40.4%, p value < 0.001) and slightly increased non-relapse mortality (NRM) (25.7% vs. 20.2%, p value = 0.06) compared to patients treated with FluTreo. Our data may serve as a basis for further studies examining the role of additional agents/ intensifications in conditioning prior to allo-HCT.

Azacitidine for the treatment of steroid-refractory chronic graft-versus-host disease: the results of the phase II AZTEC clinical trial.

Chronic graft-versus-host disease (cGvHD) is a major cause of non-relapse morbidity and mortality following allogeneic stem cell transplant. Over half of patients with moderate or severe cGvHD fail to respond adequately to first-line treatment with systemic steroids, and although a range of second-line options have been employed, a lack of prospective evidence means there is no standard of care. The AZTEC trial is a prospective, single-arm, phase II study investigating the safety and activity of azacitidine for the treatment of cGvHD in patients who are resistant to, or intolerant of, systemic steroid therapy. The co-primary outcomes were treatment tolerability, and activity measured as objective response according to modified National Institutes of Health criteria. Fourteen patients were recruited to the first stage of the trial, of whom seven completed the planned six cycles of azacitidine 36 mg/m2 days 1-5 per 28-day cycle. Azacitidine was tolerated by 13/14 patients, and 7/14 showed an objective response. Clinical responses were mirrored by improvements in patient-reported cGvHD symptoms and quality of life. AZTEC demonstrates that azacitidine is a safe and promising option for the treatment of cGvHD, and continued evaluation in the second stage of this phase II efficacy study is supported.

Augmented Reduced-Intensity Regimen Does Not Improve Postallogeneic Transplant Outcomes in Acute Myeloid Leukemia.

PURPOSE: Reduced-intensity conditioning (RIC) regimens have extended the curative potential of allogeneic stem-cell transplantation to older adults with high-risk acute myeloid leukemia (AML) and myelodysplasia (MDS) but are associated with a high risk of disease relapse. Strategies to reduce recurrence are urgently required. Registry data have demonstrated improved outcomes using a sequential transplant regimen, fludarabine/amsacrine/cytarabine-busulphan (FLAMSA-Bu), but the impact of this intensified conditioning regimen has not been studied in randomized trials. PATIENTS AND METHODS: Two hundred forty-four patients (median age, 59 years) with high-risk AML (n = 164) or MDS (n = 80) were randomly assigned 1:1 to a fludarabine-based RIC regimen or FLAMSA-Bu. Pretransplant measurable residual disease (MRD) was monitored by flow cytometry (MFC-MRD) and correlated with outcome. RESULTS: There was no difference in 2-year overall survival (hazard ratio 1.05 [85% CI, 0.80 to 1.38] P = .81) or cumulative incidence of relapse (CIR) (hazard ratio 0.94 [95%CI, 0.60 to 1.46] P = .81) between the control and FLAMSA-Bu arms. Detectable pretransplant MFC-MRD was associated with an increased CIR (2-year CIR 41.0% v 20.0%, P = .01) in the overall trial cohort with a comparable prognostic impact when measured by an unsupervised analysis approach. There was no evidence of interaction between MRD status and conditioning regimen intensity for relapse or survival. Acquisition of full donor T-cell chimerism at 3 months abrogated the adverse impact of pretransplant MRD on CIR and overall survival. CONCLUSION: The intensified RIC conditioning regimen, FLAMSA-Bu, did not improve outcomes in adults transplanted for high-risk AML or MDS regardless of pretransplant MRD status. Our data instead support the exploration of interventions with the ability to accelerate acquisition of full donor T-cell chimerism as a tractable strategy to improve outcomes in patients allografted for AML.

BEAM-Campath Allogeneic Stem Cell Transplant for Patients with Relapsed/Refractory Lymphoma: High Incidence of Long-Term Mixed Donor-Recipient Chimerism and the Response to Donor Lymphocyte Infusions.

BiCNU (carmustine), etoposide, Ara-C, melphalan (BEAM) and Campath conditioning was developed to reduce the high transplant-related mortality in patients with lymphoma while delivering intensive antilymphoma immunotherapy, as well as to some extent a platform for allogeneic stem cell engraftment. Significant numbers of patients appeared to have persistent recipient-derived hematopoiesis, and therefore we retrospectively analyzed patients with lymphoma undergoing BEAM-Campath conditioned allogeneic stem cell transplantation at our center (2003 to 2017) to characterize the patterns of chimerism and patient outcomes. Chimerism was analyzed with short tandem repeat PCR. Mixed donor-recipient chimerism (MDRC) was defined as 5% to 94.9% donor. Fifty-two patients (n = 30 male), with a median age of 45 years, were identified with histologic diagnoses of Hodgkin lymphoma (n = 13), diffuse large B cell lymphoma (n = 7), low-grade non-Hodgkin lymphoma (n = 16), mantle cell lymphoma (n = 10), and T cell lymphoma (n = 6). Pretransplant, 93% achieved complete response (52%) or partial response (41%) with a median of 3 prior therapies (n = 3 prior autologous stem cell transplantation). Donors were Matched sibling donors (MSD) (n = 21), matched unrelated donors (MUD) (n = 24), miss-matched unrelated donors (MMUD) (n = 6), and syngeneic (n = 1). Acute graft-versus host disease (GVHD) developed in 52% (81% grade I to II) and chronic GVHD (83% extensive) in 12%. MDRC of T cells (MDRCt) developed in 62% (n = 32), and 29% (n = 15) developed MDRC of myeloid cells (MDRCm) at a median onset of 100 days. Donor lymphocyte infusion (DLI) was given to 17 patients, with a median starting dose of 1 × 106/kg. The first DLI was given at a median of 225 days post-transplant (range, 99 days to 5.3 years). Of these, 9 developed acute post-DLI GVHD and 2 limited chronic GVHD. Conversion to full donor occurred in 47% MDRCt and 50% MDRCm. Multivariate analysis identified sibling donor type as associated with increased MDRCt (P = .035; hazard ratio [HR], 0.17) and reduced total nucleated cell dose with increased MDRCm (P = .021; HR, 0.76). The median follow-up was 6 years, and 2-year NRM cumulative incidence was 16% (95% confidence interval [CI], 7% to 27%). Ten-year progression and extensive GVHD-free survival was 45% (95% CI, 28% to 61%), and overall survival was 66% (95% CI, 50% to 78%). One-year landmark analysis identified no increased GVHD or relapse risk with MDRCt or MDRCm but reduced nonrelapse mortality (NRM) risk with MDRCt (P = .001). BEAM-Campath allografts for high-risk lymphoma achieve long-term disease-free survival with low rates of GVHD and transplant-related mortality. The frequent development of myeloid MDRC demonstrates that BEAM-Campath is a nonmyeloablative conditioning regimen in almost a third of patients. MDRCt is associated with reduced NRM, but neither MDRCt or MDRCm is associated with increased GVHD or relapse.

The complex genetic landscape of familial MDS and AML reveals pathogenic germline variants.

The inclusion of familial myeloid malignancies as a separate disease entity in the revised WHO classification has renewed efforts to improve the recognition and management of this group of at risk individuals. Here we report a cohort of 86 acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) families with 49 harboring germline variants in 16 previously defined loci (57%). Whole exome sequencing in a further 37 uncharacterized families (43%) allowed us to rationalize 65 new candidate loci, including genes mutated in rare hematological syndromes (ADA, GP6, IL17RA, PRF1 and SEC23B), reported in prior MDS/AML or inherited bone marrow failure series (DNAH9, NAPRT1 and SH2B3) or variants at novel loci (DHX34) that appear specific to inherited forms of myeloid malignancies. Altogether, our series of MDS/AML families offer novel insights into the etiology of myeloid malignancies and provide a framework to prioritize variants for inclusion into routine diagnostics and patient management.

Intracellular cytarabine triphosphate in circulating blasts post-treatment predicts remission status in patients with acute myeloid leukemia.

Cytarabine remains the backbone of therapy in acute myeloid leukemia (AML). The ability to assess intracellular cytarabine triphosphate (ara-CTP) levels in patients receiving cytarabine represents a major goal in the prediction of treatment response. This study, conducted within a clinical setting, aimed to assess ara-CTP levels in circulating peripheral blasts from non-M3 AML patients receiving cytarabine at one of three dosing levels, using a novel biosensor assay. Results from the initial 72 hours post-commencement were correlated with day 28 remission status, with feasibility parameters concurrently assessed. Intracellular ara-CTP was detectable in ex vivo blasts post-treatment for standard-dose (SD) and high-dose (HD) patients (p < 0.05), and quantification revealed a 27-fold increase in intracellular steady-state concentration between the two dosing levels. For low-dose cytarabine, high rates of patient discharge and low intracellular concentrations limited analysis; however, assessment of intracellular ara-CTP concentration was achievable in a dwindling population of blasts for SD and HD treatment cohorts, with 4 hours post-treatment commencement potentially being most predictive of clinical response (r = -0.912, p = 0.0113). Concurrent assessment of peripheral leukemia-associated immunophenotype (LAIP)-positive cells revealed a decline in burden (0-72 hours), which correlated with remission status (p < 0.05). Unexpectedly high rates of night sampling led to challenges associated with sampling rates, but did not have an impact on patient compliance. Additional training of night staff improved feasibility substantially. Multiple peripheral sampling during the initial 72 hours of treatment is feasible in newly diagnosed patients, and ara-CTP is detectable over the initial 24 hours, facilitating prediction of chemosensitivity of leukemic blasts to cytarabine.

Are reduced-intensity transplants safe in older patients with hematologic malignancies?

Non-myeloablative allogeneic hematopoietic cell transplantation (HCT) offers a potentially curative therapy for older patients with hematologic malignancies. However, the treatment modality is underutilized and, to date, there are only limited data on outcomes in people older than 60 years. This study describes the toxicities and outcomes of non-myeloablative allogeneic HCT, conditioned with low-dose total body irradiation with or without fludarabine, in patients aged 60-75 years enrolled in prospective multicenter clinical trials. Increasing age was not associated with adverse outcome. Relapse risk and comorbidity burden predicted survival; fit patients with low risk of disease relapse did well after transplant regardless of age. Despite demonstrating the feasibility of non-myeloablative allogeneic HCT in patients aged 60-75 years, the study raises important questions regarding how best to manage older patients with high-risk hematologic malignancies and how to optimize our approach to allogeneic HCT in this age group.

Isolation and characterization of human interleukin-10-secreting T cells from peripheral blood.

Recent studies have expanded our understanding of the role of the anti-inflammatory cytokine interleukin (IL)-10, produced by multiple lineages of both human and murine T cells, in regulating the immune response. Here, we demonstrate that the small percentage of circulating CD4(+) T cells that secrete IL-10 can be isolated from human peripheral blood and, importantly, we have optimized a protocol to expand these cells in both antigen-specific and polyclonal manners. Expanded CD4(+)IL-10(+) T cells abrogate proliferation and T helper (Th) 1-like cytokine production in an antigen-specific manner, and to a lesser extent exhibit bystander suppressive capacity. CD4(+)IL-10(+) T cells are suppressive in a cell contact-dependent way, though they do not require secretion of IL-10 for their suppressive role in vitro. CD4(+)IL-10(+) T cells have an activated phenotype, with high expression of CD25, CD69, and cytotoxic T-lymphocyte antigen-4, and are largely FoxP3 negative. This novel method for the isolation and expansion of suppressive IL-10-secreting T cells has important implications both for further research and clinical therapeutic development.