Epigenetic regulator genes direct lineage switching in MLL/AF4 leukemia.

The fusion gene MLL/AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukemia. Relapse can be associated with a lineage switch from acute lymphoblastic to acute myeloid leukemia, resulting in poor clinical outcomes caused by resistance to chemotherapies and immunotherapies. In this study, the myeloid relapses shared oncogene fusion breakpoints with their matched lymphoid presentations and originated from various differentiation stages from immature progenitors through to committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programs, including alternative splicing. These findings indicate that the execution and maintenance of lymphoid lineage differentiation is impaired. The relapsed myeloid phenotype is recurrently associated with the altered expression, splicing, or mutation of chromatin modifiers, including CHD4 coding for the ATPase/helicase of the nucleosome remodelling and deacetylation complex. Perturbation of CHD4 alone or in combination with other mutated epigenetic modifiers induces myeloid gene expression in MLL/AF4+ cell models, indicating that lineage switching in MLL/AF4 leukemia is driven and maintained by disrupted epigenetic regulation.

Comparison of minimal residual disease measurement by multicolour flow cytometry and PCR for fusion gene transcripts in infants with acute lymphoblastic leukaemia with KMT2A gene rearrangements.

This study aimed to evaluate the concordance between minimal residual disease (MRD) results obtained by multicolour flow cytometry (MFC) and polymerase chain reaction for fusion gene transcripts (FGTs) in infants with acute lymphoblastic leukaemia (ALL) associated with rearrangement of the KMT2A gene (KMT2A-r). A total of 942 bone marrow (BM) samples from 123 infants were studied for MFC-MRD and FGT-MRD. In total, 383 samples (40.7%) were concordantly MRD-negative. MRD was detected by the two methods in 441 cases (46.8%); 99 samples (10.5%) were only FGT-MRD-positive and 19 (2.0%) were only MFC-MRD-positive. A final concordance rate of 87.4% was established. Most discordance occurred if residual leukaemia was present at levels close to the sensitivity limits. Neither the type of KMT2A fusion nor a new type of treatment hampering MFC methodology had an influence on the concordance rate. The prognostic value of MFC-MRD and FGT-MRD differed. MFC-MRD was able to identify a rapid response at early time-points, whereas FGT-MRD was a reliable relapse predictor at later treatment stages. Additionally, the most precise risk definition was obtained when combining the two methods. Because of the high comparability in results, these two rather simple and inexpensive approaches could be good options of high clinical value.

A simple procedure to identify children with B-lineage acute lymphoblastic leukemia who can be successfully treated with low or moderate intensity: Sequential versus single-point minimal residual disease measurement.

Sequential monitoring of minimal residual disease (MRD) by molecular techniques or multicolor flow cytometry (MFC) has emerged over the past two decades as the primary tool to optimize treatment in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The aim of our study was to compare the prognostic power of repeated MFC-MRD measurement with single-point MRD assessment in children with BCP-ALL treated with the reduced-intensity protocol ALL-MB 2008. Data from consecutive MFC-MRD at day 15 and day 36 (end of induction, EOI) were available for 507 children with Philadelphia-negative BCP-ALL. They were stratified into standard risk (SR, n = 265), intermediate risk (ImR, n = 211), and high risk (HR, n = 31) according to the initial clinical characteristics defined in the ALL-MB 2008 protocol. Quantitative (relative to quantitative thresholds) and kinetic (logarithmic reduction) assessments of MFC-MRD at both time points effectively separated patients into three groups with different risk of recurrence. On the other hand, starting with low (for the SR group) and moderate (for the ImR group) induction therapy, a single MFC-MRD measurement at EOI proved sufficient to unequivocally identify patients in whom this therapy is highly effective and distinguish them from those who cannot be successfully treated with such therapy. Therefore, initiating treatment with low or moderate treatment from the start, together with careful consideration of initial clinical risk factors and just one EOI-MFC-MRD measurement is simple, inexpensive, and entirely sufficient for treatment optimization. Furthermore, for a large proportion of patients, this approach allows better adjustment, in particular also reduction of therapy intensity than sequential MRD measurements.

Genetic characteristics and treatment outcome in infants with KMT2A germline B-cell precursor acute lymphoblastic leukemia: Results of MLL-Baby protocol.

The aim of this study was to present the diagnostic and outcome characteristics of infants with germline status of KMT2A gene (KMT2A-g) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treated consistently according to the MLL-Baby protocol, a moderate-intensity protocol. Of the 139 patients enrolled in the MLL-Baby study, 100 (71.9%) carried different types of rearranged KMT2A (KMT2A-r), while the remaining 39 infants (28.1%) had KMT2A-g. KMT2A-g patients were generally older (77% older than 6 months), less likely to have a very high white blood cell count (greater than 100 × 109 /L), less likely to be central nervous system (CNS)-positive, and more likely to be CD10-positive. The 6-year event-free survival and overall survival rates for all 39 patients were 0.74 (standard error [SE] 0.07) and 0.80 (SE 0.07), respectively. Relapse was the most common adverse event (n = 5), with a cumulative incidence of relapse (CIR) of 0.13 (SE 0.06), while the incidence of a second malignancy (n = 1) and death in remission (n = 3) was 0.03 (SE 0.04) and 0.08 (SE 0.04), respectively. None of the initial parameters, including genetics and the presence of recently described fusions of NUTM1 and PAX5 genes, was able to distinguish patients with different outcomes. Only rapidity of response, measured as minimal residual disease (MRD) by flow cytometry, showed a statistically significant impact. Moderate-intensity therapy, as used in the MLL-Baby protocol in infants with KMT2A-g BCP-ALL, yields results comparable to other infant studies. Patients with a slow multicolor flow cytometry (MFC)-MRD response should be subjected to advanced therapies, such as targeted or immunotherapies.

Incidence and prognostic value of central nervous system involvement in infants with B-cell precursor acute lymphoblastic leukemia treated according to the MLL-Baby protocol.

AIM: The aim of the study was to evaluate the incidence and prognostic impact of central nervous system (CNS) involvement in infants with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), as well as its relation with minimal residual disease (MRD) data. METHODS: A total of 139 consecutive infants with BCP-ALL from the MLL-Baby trial were studied. Cerebrospinal fluid (CSF) samples were investigated by microscopy of cytospin slides. MRD was evaluated according to the protocol schedule by flow cytometry and PCR for fusion gene transcripts (FGT). RESULTS: Involvement of the CNS at any level was found in 50 infants (36.0%). The incidence of CNS involvement was higher in patients with KMT2A gene rearrangements (44.0% for KMT2A-r vs. 15.4% for KMT2A-g, p = .003). The outcome of CNS-positive infants was significantly worse than that of CNS-negative infants, although this prognostic impact was limited to the KMT2A-r group (event-free survival 0.21 for CNS-positive vs. 0.48 for CNS-negative infants, p = .044). CNS-positive infants could not be treated successfully by conventional chemotherapy alone, irrespective of the rapidity of MRD response. In contrast, the combination of initial CNS negativity and FGT-MRD negativity identified a group comprising up to one-third of infants with KMT2A-r ALL who can be treated with chemotherapy and achieve very good outcomes (disease-free survival above 95%), and remaining patients should be allocated to receive other types of treatment. CONCLUSION: We can conclude that this combination of initial CNS involvement and MRD data can significantly improve risk-group allocation in future clinical trials enrolling infants with ALL.

Blinatumomab following haematopoietic stem cell transplantation - a novel approach for the treatment of acute lymphoblastic leukaemia in infants.

Blinatumomab with subsequent haematopoietic stem cell transplantation was applied in 13 infants with acute lymphoblastic leukaemia (ALL). Eight patients were treated in first remission due to slow clearance of minimal residual disease (MRD); one for MRD-reappearance after long MRD negativity, one for primary refractory disease and three during relapse treatment. In slow MRD responders, complete MRD response was achieved prior to transplantation, with an 18-month event-free survival of 75%. In contrast, only one of five patients with relapsed/refractory ALL is still in complete remission. These data provide a basis for future studies of immunotherapy in very high-risk infant ALL.

Primary post-transplant lymphoproliferative disorder of the central nervous system: characteristics, management and outcome in 25 paediatric patients.

Primary central nervous system (CNS) post-transplant lymphoproliferative disorder (PTLD) in childhood is rare. Twenty-five patients were retrieved from nine European Intergroup for Childhood Non-Hodgkin's Lymphoma and/or international Berlin-Frankfurt-Münster Study Group members. Types of allografts included kidney (n = 11), liver (n = 4), heart (n = 5), bowel (n = 1) and haematopoietic stem cells (n = 4). Eighteen were male, 16 ≥ 10 years old, 21 had monomorphic disease and 24 solid intracranial tumour masses. Four-year event-free and overall survival rates were 50% ± 10% and 74% ± 9% respectively. This report represents the largest paediatric series of CNS PTLD reported to date, showing favourable survival odds following systemic and intrathecal chemotherapy and rituximab administration.

Relative expansion of CD19-negative very-early normal B-cell precursors in children with acute lymphoblastic leukaemia after CD19 targeting by blinatumomab and CAR-T cell therapy: implications for flow cytometric detection of minimal residual disease.

CD19-directed treatment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) frequently leads to the downmodulation of targeted antigens. As multicolour flow cytometry (MFC) application for minimal/measurable residual disease (MRD) assessment in BCP-ALL is based on B-cell compartment study, CD19 loss could hamper MFC-MRD monitoring after blinatumomab or chimeric antigen receptor T-cell (CAR-T) therapy. The use of other antigens (CD22, CD10, CD79a, etc.) as B-lineage gating markers allows the identification of CD19-negative leukaemia, but it could also lead to misidentification of normal very-early CD19-negative BCPs as tumour blasts. In the current study, we summarized the results of the investigation of CD19-negative normal BCPs in 106 children with BCP-ALL who underwent CD19 targeting (blinatumomab, n = 64; CAR-T, n = 25; or both, n = 17). It was found that normal CD19-negative BCPs could be found in bone marrow after CD19-directed treatment more frequently than in healthy donors and children with BCP-ALL during chemotherapy or after stem cell transplantation. Analysis of the antigen expression profile revealed that normal CD19-negative BCPs could be mixed up with residual leukaemic blasts, even in bioinformatic analyses of MFC data. The results of our study should help to investigate MFC-MRD more accurately in patients who have undergone CD19-targeted therapy, even in cases with normal CD19-negative BCP expansion.

Prognostic value of minimal residual disease measured by fusion-gene transcript in infants with KMT2A-rearranged acute lymphoblastic leukaemia treated according to the MLL-Baby protocol.

The prognostic value of minimal residual disease (MRD) measured by fusion-gene transcript (FGT) detection was investigated in 76 infants (aged ≤1 year) with acute lymphoblastic leukaemia (ALL) with lysine methyltransferase 2A (KMT2A) rearrangements. Either at the end of induction or at later time-points, FGT-MRD-positivity was associated with poor outcome. FGT-MRD-positivity after first consolidation or first high-risk block detected 46·5% of infants with extremely poor outcome [disease-free survival (SE) 0·06 (0·06), cumulative incidence of relapse (SE) 0·91 (0·05)], which was also confirmed in multivariable analysis. Thus, FGT-MRD measurement at a single time-point clearly identifies infants with ALL who are curable with conventional chemotherapy and those who would benefit only from other treatment approaches.

Immunophenotypic changes of leukemic blasts in children with relapsed/refractory B-cell precursor acute lymphoblastic leukemia, who have been treated with Blinatumomab.

Not available.

Non-Hodgkin lymphoma and pre-existing conditions: spectrum, clinical characteristics and outcome in 213 children and adolescents.

Children and adolescents with pre-existing conditions such as DNA repair defects or other primary immunodeficiencies have an increased risk of non-Hodgkin lymphoma. However, large-scale data on patients with non-Hodgkin lymphoma and their entire spectrum of pre-existing conditions are scarce. A retrospective multinational study was conducted by means of questionnaires sent out to the national study groups or centers, by the two largest consortia in childhood non-Hodgkin lymphoma, the European Intergroup for Childhood non-Hodgkin Lymphoma, and the international Berlin-Frankfurt-Münster Study Group. The study identified 213 patients with non-Hodgkin lymphoma and a pre-existing condition. Four subcategories were established: a) cancer predisposition syndromes (n=124, 58%); b) primary immunodeficiencies not further specified (n=27, 13%); c) genetic diseases with no increased cancer risk (n=40, 19%); and d) non-classifiable conditions (n=22, 10%). Seventy-nine of 124 (64%) cancer predispositions were reported in groups with more than 20 patients: ataxia telangiectasia (n=32), Nijmegen breakage syndrome (n=26), constitutional mismatch repair deficiency (n=21). For the 151 patients with a known cancer risk, 5-year event-free survival and overall survival rates were 40%±4% and 51%±4%, respectively. Five-year cumulative incidences of progression/relapse and treatment-related death as a first event were 22%±4% and 24%±4%, respectively. Ten-year incidence of second malignancy was 24%±5% and 7-year overall survival of the 21 patients with a second malignancy was 41%±11%. Patients with non-Hodgkin lymphoma and pre-existing conditions have an inferior survival rate with a large proportion of therapy-related deaths compared to patients with non-Hodgkin lymphoma and no pre-existing conditions. They may require special vigilance when receiving standard or modified/reduced-intensity chemotherapy or when undergoing allogeneic stem cell transplantation.

Very long survival in complete cytogenetic remission in an adolescent with lymphoid blast crisis of chronic myeloid leukemia after treatment with intensive ALL-directed chemotherapy combined with continuous imatinib.

An 11-year-old male was diagnosed with chronic-phase chronic myeloid leukemia (CML) in 1998 and received therapy with interferon-α2b and low-dose cytarabine. In 6 years, he progressed to lymphoid blast crisis and received induction chemotherapy with prednisolone, vincristine, daunorubicin, and l-asparaginase concomitantly with imatinib 400 mg/day, and continuation with vincristine + prednisolone, cytarabine + etoposide, vincristine + l-asparaginase, cyclophosphamide + etoposide, and 6-mercaptopurine + methotrexate. Complete molecular response (MR) was achieved and therapy was continued with imatinib 800 mg/day. He relapsed to chronic-phase CML after interruption of imatinib and regained MR after its restart. The patient is alive 17.5 years after CML diagnosis and 11.5 years after lymphoid blast crisis.

Blinatumomab as postremission therapy replaces consolidation and substantial parts of maintenance chemotherapy and results in stable MRD negativity in children with newly diagnosed B-lineage ALL.

The bispecific T cell-binding antibody blinatumomab (CD19/CD3) is widely and successfully used for the treatment of children with relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here, we report the efficacy of a single course of blinatumomab instead of consolidation chemotherapy to eliminate minimal residual disease (MRD) and maintain stable MRD-negativity in children with primary BCP-ALL.Between February 2020 and November 2022, 177 children with non-high-risk BCP-ALL were enrolled in the ALL-MB 2019 pilot study (NCT04723342). Patients received the usual risk-adapted induction therapy according to the ALL-MB 2015 protocol. Those who achieved a complete remission at the end of induction (EOI) received treatment with blinatumomab immediately after induction at a dose of 5 µg/m2/day for 7 days and 21 days at a dose of 15 µg/m2/day, followed by 12 months of maintenance therapy. MRD was measured using multicolor flow cytometry (MFC) at the EOI, then immediately after blinatumomab treatment, and then four times during maintenance therapy at 3-month intervals.All 177 patients successfully completed induction therapy and achieved a complete hematological remission. In 174 of these, MFC-MRD was measured at the EOI. 143 patients (82.2%) were MFC-MRD negative and the remaining 31 patients had varying degrees of MFC-MRD positivity.MFC-MRD was assessed in all 176 patients who completed the blinatumomab course. With one exception, all patients achieved MFC-MRD negativity after blinatumomab, regardless of the MFC-MRD score at EOI. One adolescent girl with high MFC-MRD positivity at EOI remained MFC-MRD positive. Of 175 patients who had completed 6 months of maintenance therapy, MFC-MRD data were available for 156 children. Of these, 155 (99.4%) were MFC-MRD negative. Only one boy with t(12;21) (p13;q22)/ETV6::RUNX1 became MFC-MRD positive again. The remaining 174 children had completed the entire therapy. MFC-MRD was examined in 154 of them, and 153 were MFC-MRD negative. A girl with hypodiploid BCP-ALL showed a reappearance of MFC-MRD with subsequent relapse.In summary, a single 28-day course of blinatumomab immediately after induction, followed by 12 months of maintenance therapy, is highly effective in achieving MRD-negativity in children with newly diagnosed non-high risk BCP-ALL and maintaining MRD-negative remission at least during the treatment period.

Laboratory characterization of the pediatric B/T subtype of mixed-phenotype acute leukemia: Report of a case series.

OBJECTIVES: Mixed-phenotype acute leukemia (MPAL) is a rare disease associated with difficulties in the correct lineage assignment of leukemic cells. One of the least common subtypes within this category is characterized by the simultaneous presence of B- and T-lineage-defining antigens. Each case of suspected B/T MPAL should be considered in light of all available laboratory and clinical data to avoid misdiagnosis. METHODS: In this study, we describe 6 pediatric patients who presented with leukemic blasts bearing B- and T-lineage antigens at diagnosis, including their clinical, immunophenotypic, morphologic, and cytogenetic characteristics. RESULTS: In 3 patients, more or less distinct populations of B- and T-lymphoid origin were found; the other 3 patients had a single mixed-phenotype blast population. All cases fulfilled the World Health Organization criteria, but not all of them turned out to be bona fide cases of B/T MPAL according to the available clinical and laboratory data. Found genetic lesions were helpful for the confirmation of MPAL instead of 2 concomitant tumors, but for a general B/T MPAL diagnosis, genetic studies provided the only descriptive data. CONCLUSIONS: The accurate diagnosis of B/T MPAL requires a multidisciplinary approach combining high-tech laboratory methods and close cooperation between treating physicians and pathologists.

Flow cytometric minimal residual disease measurement accounting for cytogenetics in children with non-high-risk acute lymphoblastic leukemia treated according to the ALL-MB 2008 protocol.

BACKGROUND: Quantitative measurement of minimal residual disease (MRD) is the "gold standard" for estimating the response to therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Nevertheless, the speed of the MRD response differs for different cytogenetic subgroups. Here we present results of MRD measurement in children with BCP-ALL, in terms of genetic subgroups with relation to clinically defined risk groups. METHODS: A total of 485 children with non-high-risk BCP-ALL with available cytogenetic data and MRD studied at the end-of-induction (EOI) by multicolor flow cytometry (MFC) were included. All patients were treated with standard-risk (SR) of intermediate-risk (ImR) regimens of "ALL-MB 2008" reduced-intensity protocol. RESULTS AND DISCUSSION: Among all study group patients, 203 were found to have low-risk cytogenetics (ETV6::RUNX1 or high hyperdiploidy), while remaining 282 children were classified in intermediate cytogenetic risk group. For the patients with favorable and intermediate risk cytogenetics, the most significant thresholds for MFC-MRD values were different: 0.03% and 0.04% respectively. Nevertheless, the most meaningful thresholds were different for clinically defined SR and ImR groups. For the SR group, irrespective to presence/absence of favorable genetic lesions, MFC-MRD threshold of 0.1% was the most clinically valuable, although for ImR group the most informative thresholds were different in patients from low-(0.03%) and intermediate (0.01%) cytogenetic risk groups. CONCLUSION: Our data show that combining clinical risk factors with MFC-MRD measurement is the most useful tool for risk group stratification of children with BCP-ALL in the reduced-intensity protocols. However, this algorithm can be supplemented with cytogenetic data for part of the ImR group.

One-point flow cytometric MRD measurement to identify children with excellent outcome after intermediate-risk BCP-ALL: results of the ALL-MB 2008 study.

BACKGROUND:  Measurement of minimal residual disease (MRD) with multicolor flow cytometry (MFC) has become an important tool in childhood acute lymphoblastic leukemia (ALL), mainly to identify rapid responders and reduce their therapy intensity. Protocols of the Moscow-Berlin (MB) group use a comparatively low (for standard risk; SR) or moderate (for intermediate risk; ImR) treatment intensity from the onset, based on initial patient characteristics. Recently, we reported that 90% of SR patients-50% B cell precursor (BCP-ALL)-MFC-MRD negative at end of induction (EOI)-had 95% event-free survival (EFS).  METHODS: In the present study, we applied this method to children with initial ImR features. RESULTS:  In study MB 2008, 1105 children-32% of BCP-ALL patients-were assigned to the ImR group. Of these, 227 were treated in clinics affiliated with MFC laboratories of the MB group network, and included in this MFC-MRD pilot study. A single-point MFC-MRD measurement at the EOI with the threshold of 0.01% identified 65% of patients-20% of all BCP-ALL patients-with EFS of 93.5%. CONCLUSION:  Taking both studies together, the combination of clinical parameters and a one-point MRD measurement identifies 70% of BCP-ALL patients with an excellent outcome after low- or moderate-intensity therapy and avoids overtreatment of a significant proportion of patients.

Reliable Flow-Cytometric Approach for Minimal Residual Disease Monitoring in Patients with B-Cell Precursor Acute Lymphoblastic Leukemia after CD19-Targeted Therapy.

We aimed to develop an antibody panel and data analysis algorithm for multicolor flow cytometry (MFC), which is a reliable method for minimal residual disease (MRD) detection in patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treated with CD19-directed therapy. The development of the approach, which was adapted for the case of possible CD19 loss, was based on the additional B-lineage marker expression data obtained from a study of primary BCP-ALL patients, an analysis of the immunophenotypic changes that occur during blinatumomab or CAR-T therapy, and an analysis of very early CD19-negative normal BCPs. We have developed a single-tube 11-color panel for MFC-MRD detection. CD22- and iCD79a-based primary B-lineage gating (preferably consecutive) was recommended. Based on patterns of antigen expression changes and the relative expansion of normal CD19-negative BCPs, guidelines for MFC data analysis and interpretation were established. The suggested approach was tested in comparison with the molecular techniques: IG/TR gene rearrangement detection by next-generation sequencing (NGS) and RQ-PCR for fusion-gene transcripts (FGTs). Qualitative concordance rates of 82.8% and 89.8% were obtained for NGS-MRD and FGT-MRD results, respectively. We have developed a sensitive and reliable approach that allows MFC-MRD monitoring after CD19-directed treatment, even in the case of possible CD19 loss.

Additional flow cytometric studies for differential diagnosis between Burkitt lymphoma/leukemia and B-cell precursor acute lymphoblastic leukemia.

The differentiation between Burkitt lymphoma/leukemia (BL) and B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is sometimes complicated. Laboratory findings that favor BL (e.g., surface expression of µ heavy chain and/or one of the light chains of immunoglobulin, FAB L3 morphology of blasts, MYC gene rearrangements) are not always present simultaneously. Our previous work demonstrated that BL differed from Ig(+) BCP-ALL by expression of Ig and other surface markers. In the current study, we have evaluated additional flow cytometric markers for reliable differentiation between BL and BCP-ALL. Among three studied surface antigens (CD44, CD38, CD58), only CD58 demonstrated significantly higher expression in BL as compared to BCP-ALL. Moreover, BL cases were associated with an increased level of Ki-67 and a higher percentage of cells in the S-phase of cell cycle. These two features reflect an aggressive proliferative potential of BL. Thus, when BL is suspected and results of surface Ig evaluation are controversial, the flow cytometric analysis of CD58, Ki-67 and cell cycle could assist in the differential diagnosis.

Safety and efficacy of the low-dose memory (CD45RA-depleted) donor lymphocyte infusion in recipients of αß T cell-depleted haploidentical grafts: results of a prospective randomized trial in high-risk childhood leukemia.

Depletion of αß T cells from the graft prevents graft-vs.-host disease (GVHD) and improves outcome of HSCT from haploidentical donors. In a randomized trial, we aimed to evaluate the safety and efficacy of low-dose memory (CD45RA-depleted) donor lymphocytes (mDLI) after HSCT with αß T-cell depletion. A cohort of 149 children was enrolled, 76 were randomized to receive scheduled mDLI and 73 received standard care. Conditioning was based on either 12 Gy total body irradiation or treosulfan. Rabbit antithymocyte globulin was replaced by tocilizumab and abatacept. Primary end points were the incidence of acute GVHD grades II-IV and the incidence of cytomegalovirus (CMV) viremia. The incidence of grades II-IV aGVHD was 14% in the experimental arm and 12% in the control arm, p-0.8. The incidence of CMV viremia was 45% in the experimental arm and 55% in the control arm, p-0.4. Overall, in the total cohort 2-year NRM was 2%, cumulative incidence of relapse was 25%, event-free survival 71%, and overall survival 80%, without difference between the study arms. Memory DLI was associated with improved recovery of CMV-specific T-cell responses in a subcohort of CMV IgG seropositive recipients.