Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell ...transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD
leukemia cells. This synergized with the allogeneic CD8
T cell response, leading to long-term survival in six mouse models of FLT3-ITD
AML. Sorafenib-related IL-15 production caused an increase in CD8
CD107a
IFN-γ
T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD
AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8
T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT.
The impact of early human cytomegalovirus (HCMV) replication on leukemic recurrence was evaluated in 266 consecutive adult (median age, 47 years; range, 18-73 years) acute myeloid leukemia patients, ...who underwent allogeneic stem cell transplantation (alloSCT) from 10 of 10 high-resolution human leukocyte Ag-identical unrelated (n = 148) or sibling (n = 118) donors. A total of 63% of patients (n = 167) were at risk for HCMV reactivation by patient and donor pretransplantation HCMV serostatus. In 77 patients, first HCMV replication as detected by pp65-antigenemia assay developed at a median of 46 days (range, 25-108 days) after alloSCT. Taking all relevant competing risk factors into account, the cumulative incidence of hematologic relapse at 10 years after alloSCT was 42% (95% confidence interval CI, 35%-51%) in patients without opposed to 9% (95% CI, 4%-19%) in patients with early pp65-antigenemia (P < .0001). A substantial and independent reduction of the relapse risk associated with early HCMV replication was confirmed by multivariate analysis using time-dependent covariate functions for grades II to IV acute and chronic graft-versus-host disease, and pp65-antigenemia (hazard ratio = 0.2; 95% CI, 0.1-0.4, P < .0001). This is the first report that demonstrates an independent and substantial reduction of the leukemic relapse risk after early replicative HCMV infection in a homogeneous population of adult acute myeloid leukemia patients.
Introduction:
Both post-transplant cyclophosphamide (PT-Cy) and anti-T-lymphocyte globulin (ATLG) eliminate proliferating allo-reactive T cells after allogeneic hematopoietic cell transplantation ...(HCT) and therefore contribute to reduce the incidence of graft-versus-host disease (GVHD). Exposure to ATLG has been previously associated with delayed T cell reconstitution (Gooptu et al. BBMT 2018). Yet, no study has compared PT-Cy to ATLG for its effect on cellular immune reconstitution and only one small study compared it to anti-thymocyte globulin (Retiere et al. Oncotarget 2018). Hence, we analyzed the dynamics of immune reconstitution after HCT in patients that received either PT-Cy or ATLG as additional GVHD prophylaxis.
Methods:
We retrospectively analyzed 247 patients (138 male, 109 female) from a single-center, who received HCT from HLA-identical siblings (n=29), haploidentical family donors (n=21), or matched unrelated donors (n=197) between January 2017 and December 2018. All patients were transplanted for hematologic malignancies (49% acute myeloid leukemia). Median age was 56 (range, 18-76) years. Myeloablative conditioning regimen was performed in 119 patients and reduced intensity conditioning in 128 patients. PT-Cy (n=59) was dosed 50 mg/kg/day intravenously (i.v.) on days HCT +3 and +4, followed by tacrolimus in combination with mycophenolate mofetil from day +5. In 188 patients, ATLG was administered at 10 mg/kg bodyweight i.v. on days -3, -2 and -1 in combination with cyclosporine 3 mg/kg i.v. from day -1 and methotrexate (15 mg/m2 on day +1 and 10mg/m2 on days +3, +6, and +11 i.v.). All patients received HCT using peripheral blood stem cells with amedian dose of 6.3x106CD34+ cells/kg (range, 1.3 to 25). Blood samples were collected on days +30, +90, +180, +270 and +365 and analyzed by multiparametric flow cytometry for the following cell subsets: T lymphocytes (CD3+), T helper cells (CD3+/CD4+); cytotoxic T cells (CD3+/CD8+), regulatory T cells (CD3+/CD4+/CD25+/CD127+), T cell receptor αβ(CD3+/TCRαβ), T cell receptor γδ(CD3+/TCRγδ), NK T-cells (CD3+/CD16+/CD56+), NK-cells (CD3-/CD16+/CD56+), naïve helper T cell (CD4+/CD45RA), memory helper T cells (CD4+/CD45RO) and B cells (CD19+).
Results:
Immune cell reconstitution differed significantly between the PT-Cy and the ATLG cohorts. The use of PT-Cy associated with significantly higher median counts of helper T cells during the first 6 months after HCT (p<0.0001, Fig. 1A). In particular, naïve helper T cells (Fig. 1B; median absolute (abs.) cell counts of PT-Cy versus (vs) ATLG cohort: month 1, 15 cells/µL vs 12 cells/µL , p<0.0001; month 3, 13 vs 3 cells/µL, p<0.0001; month 6, 25 vs 4 cells/µL, p<0.0001) and memory helper T cells (median abs. counts month 1, 94 vs 3 cells/µL, p<0.0001; month 3, 116 vs 64 cells/µL, p<0.0001; month 6, 189 vs 89 cells/µL, p =0.004) were significantly higher in the PT-Cy cohort. Cytotoxic T cells (Fig. 1C) and NK cells did not differ between PT-Cy and ATLG cohorts. Interestingly, γδ T cells were significantly higher in the ATLG cohort (Fig. 1D; median abs. counts month 1, 14 cells/µL vs 3 cells/µL; p =0.019). For B cells or NKT cells the use of PT-Cy associated with earlier immune reconstitution with significant differences only at month 1 after HCT (median abs. cell counts 10 cells/µL vs 1 cell/µL, p=0.007 and 11 vs 2 cells/µL, p=0.03, respectively), regulatory T cells differed significantly in months 3 and 6 (median abs. count 9 vs 2 cells/µL, p<0.0001; 10 vs 4 cells/µL, p<0.0001).
The incidence of grade II to IV acute GVHD was significantly lower in the PT-Cy cohort as compared to the ATLG cohort (Hazard ratio 0.48, 95% Confidence interval, 0.30-0.78, p=0.003). Within a median follow up of 11 months, no significant differences in overall survival, relapse incidence and non-relapse mortality were observed between the PT-Cy and ATLG cohorts.
Conclusions:
Our data suggest that the choice of the additional T cell depleting regimen using either ATLG or PT-Cy significantly affects immune reconstitution after HCT. Knowledge of the distinct immune reconstitution profiles should assist clinical decision-making and help optimizing GVHD prophylaxis.
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Bogdanov:Jazz Pharmaceuticals, MSD.: Other: Travel subsidies. Beelen:Medac GmbH Wedel Germany: Consultancy, Honoraria. Turki:Jazz Pharmaceuticals, CSL Behring, MSD.: Consultancy; Neovii Biotech, all outside the submitted work: Other: Travel subsidies.
•An IFN-γ value >10 IU/ml is associated with protection from high-level CMV viremia.•Re-establishment of the CMV-specific immunity is lower in D−/R+ than in D+/R+ patients.•CMV replication has to be ...considered as primary infection in D−/R+ patients.•Only transfer of CMV immunity, not of virus was seen in the D+/R− patients.
CMV reactivation is a major cause of severe complications in allogeneic hematopoietic stem cell transplant (HSCT) recipients. The risk of CMV reactivation depends on the serostatus (+/−) of the donor (D) and recipient (R). The reconstitution of CMV-specific T-cell responses after transplantation is crucial for the control of CMV reactivation.
The study aimed to determine the cellular immune status correlating with protection from high-level CMV viremia (>5000 copies/ml) and disease.
We monitored CMV-specific cellular immune responses in 9 high-risk (D−/R+), 14 intermediate risk (D+/R+) and 3 low risk individuals (D+/R−), and 8 CMV negative controls (D−/R−). Interferon- γ (IFN-γ) levels as a marker for the CD8+ T-cell response were determined by the QuantiFERON-CMV-assay and compared to viral loads determined by PCR.
Early CMV reactivation was detected in all high-risk and 13/14 intermediate risk individuals. High-level viremia was detected in 5/7 high and 7/14 intermediate risk patients. Reconstitution of the CMV-specific cellular immune response started from 3 months after transplantation and resulted in protection against CMV reactivation. Re-establishing of CMV-specific T-cell immune responses with IFN- γ levels >8.9 IU/ml is crucial for protection from high-level CMV viremia.
Monitoring of HSCT-recipients with the QuantiFERON-CMV-assay might be of great benefit to optimize antiviral treatment.
Introduction
Primary and post‐ET/PV myelofibrosis are myeloproliferative neoplasms harboring in most cases driving mutations in JAK2, CALR or MPL, and a variable number of additional mutations in ...other genes. Molecular analysis represents a powerful tool to guide prognosis and clinical management. Only about 10% of patients with myelofibrosis harbor alterations in MPL gene. No data are available about the transplantation outcome in the specific MPL‐mutated group.
Patients
We collected the data of 18 myelofibrosis patients(primary: 14; post‐ET: 4) transplanted in 4 EBMT centers (Hamburg, Paris, Essen, and Hannover) between 2005 and 2016.
Results
Before the transplant, we explored the molecular profile by NGS and reported the frequency of mutations occurring in a panel of genes including JAK2, MPL, CALR, U2AF1, SRSF2, SF3B1, ASXL1, IDH1, IDH2, CBL, DNMT3A, TET2, EZH2, TP53, IKZF1, NRAS, KRAS, FLT3, SH2B3, and RUNX1. The 1‐year transplant‐related mortality was 16.5%, 5‐years overall survival and 5‐y relapse‐free survival 83.5%. The only relapse occurred in a patient who harbored mutations in both ASXL1 and EZH2 genes.
Conclusion
These retrospective data suggest that MPL‐mutated myelofibrosis patients have a favorable outcome after allogeneic transplantation with very low rate of disease relapse (5.5%) in comparison with the available historical controls regarding myelofibrosis in all.
Background
The dynamic International Prognostic Scoring System (DIPSS) is commonly applied to predict survival among patients with primary myelofibrosis (PMF) but has been shown to perform less ...precisely in secondary myelofibrosis (SMF) and after transplantation. Furthermore, the prognostic relevance of mutation profile resulted in the mutation-enhanced IPSS (MIPSS) in PMF, as well as in a model specific to SMF (MYSEC-PM) after essential thrombocythemia (ET) or polycythemia vera (PV). The aim of the current study was to develop a comprehensive prognostic system including clinical and molecular information, specifically in myelofibrosis undergoing transplantation.
Methods
Previously published methods were used to sequence myelofibrosis-associated genes (i.a. CALR1/2, JAK2, MPL,ASXL1, SRSF2, EZH2, IDH1/2, DNMT3A, TET2, TP53). Outcome was calculated from date of transplant (95% confidence interval). Variables associated with overall survival (OS) constructed a Cox regression with a stepwise selection procedure. Hazard ratios (HR) were used as weights for model development. Validation was done using repeated random subsampling. Performance of the model was verified via Harrel's concordance index C and was also tested in predefined cohorts: disease (PMF, SMF), conditioning, and ruxolitinib pretreatment.
Results
Population. The total cohort consisted of 361 patients from four different centers in Germany and France (260 PMF, 101 SMF). Median age at transplant was 57 years (range, 22-75), 58% were male and 42% had a Karnofsky performance score (KPS) <90. The median follow-up was 62 months and was similar between PMF and SMF (p=0.50). Overall 5-year OS was 60% (54-67) being similar in PMF (63%) and SMF after ET (59%) and slightly lower after PV (45%). Most frequent mutations were: JAK2 V617F (57%), CALR (20%; types 1/2/other 66%/23%/11%), MPL (5%), ASXL1 (31%), TET2 (19%), SRSF2 (9%), DNMT3A (6%), TP53 (6%). Two or more mutations were present in 60%. Most transplants were received from matched unrelated (46%), mismatched unrelated donors (MMUD, 27%), identical siblings (27%), and mismatched siblings (1%). Reduced intensity was given more frequently (64%) than myeloablative conditioning (36%). Frequencies at transplant were 9% (low), 29% (intermediate-1), 48% (intermediate-2), 14% (high) according to DIPSS and 3% (low), 40% (intermediate), and 57% (high) for MIPSS.
Factors on outcome. In univariate analysis, mutations in CALR and MPL showed better OS (79% and 76%) vs. JAK2 (53%) and triple negative (50%; p=0.001). Outcome was similar according to CALR type (p=0.99). ASXL1 and DNMT3A mutations also entered the multivariate model. The following eight clinical, molecular and transplant-related variables were identified (corresponding HR): leukocytes >25x109/l (1.71), platelets <150x109/l (1.53), KPS <90 (1.63), age >57 years (1.69), recipient/donor CMV serostatus (+/- vs. other, 1.68), ASXL1 (1.74), JAK2/triple negative (2.10), and MMUD (2.11).
Myelofibrosis Transplant Scoring System (MTSS). A weighted score of 1 was assigned to leukocytosis, thrombocytopenia, KPS <90, age >57, recipient/donor CMV serostatus (+/-), and ASXL1 mutation, whereas 2 points were assigned to JAK2/triple negative and MMUD. Four risk groups constructed the MTSS: low (score 0-2), intermediate (score 3-4), high (score 5-6), and very high (score 7-9). The 5-year OS according to risk groups was 88%, 71%, 50%, and 20% (Figure 1). The hazard for death (with low-risk as reference) was 2.36 for intermediate-risk, 4.65 for high-risk, and 9.72 for very high-risk. The score was predictive of OS overall as well as for PMF and SMF (p<0.001, respectively). The MTSS showed overall C statistics of 0.718 (0.707-0.730) after cross-validation yielding a median of 0.727 in PMF and 0.708 in SMF indicating improved performance and replicability vs. DIPSS (0.572), MIPSS (0.577), and MYSEC-PM (0.601). The system was also predictive of OS in different conditioning settings (reduced intensity and myeloablative) and in patients with ruxolitinib pretreatment (p<0.001, respectively).
Conclusions
The new MTSS includes modern disease- and transplant-associated risk variables pertinent to both PMF and SMF. This proposed system consistently predicts outcome facilitating posttransplant decision-making and can be applied to different conditioning settings and to patients receiving ruxolitinib pretreatment.
Beelen:Medac: Consultancy, Other: Travel Support. Kroeger:Novartis: Honoraria, Research Funding; Riemser: Honoraria, Research Funding; Neovii: Honoraria, Research Funding; Sanofi: Honoraria; JAZZ: Honoraria; Celgene: Honoraria, Research Funding.
Summary
Considering the unsatisfactory results of salvage therapies for patients with relapsed/refractory acute myeloid leukaemia (R/R‐AML), their value before allogeneic haematopoietic stem cell ...transplantation (HSCT) remains questionable. However, direct allogeneic HSCT following established conditioning regimens applied in patients with R/R‐AML during active disease has been equally disappointing. In this retrospective observational study, high‐dose melphalan, as part of a sequential preparative regimen, followed by a total body irradiation (4 × 2 Gy)‐based or a treosulfan‐based dose‐adapted conditioning therapy for allogeneic HSCT was administered to 292 adult patients (median age 56 years, range 17–74) with primary refractory (144 patients), secondary refractory (97 patients) or relapsed AML (51 patients). Overall survival rates at 3 years were 34%, 29% and 41%, respectively. Risk factors associated with an inferior survival were higher age, transplantation from a human leucocyte antigen‐mismatched donor and high disease burden. Patients transplanted with blast infiltration <20% showed a notable survival rate of 51% at 3 years. In particular, patients with primary refractory AML showed a more favourable outcome when transplanted early during their disease course. Thus, high‐dose melphalan‐based sequential conditioning chemotherapy followed by an allogeneic HSCT is feasible and enables long‐term remission to be achieved in a substantial proportion of patients with active R/R‐AML.
Prophylaxis of graft‐versus‐host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HCT) remains challenging. Because prospective randomized trials of in‐vivo T cell depletion ...using anti‐T‐lymphocyte globulin (ATLG) in addition to a calcineurin inhibitor and methotrexate (MTX) led to conflicting outcome results, we evaluated the impact of ATLG on clinical outcome, lymphocyte‐ and immune reconstitution survival models. In total, 1500 consecutive patients with hematologic malignancies received matched unrelated donor (MUD) HCT with cyclosporin and MTX (N = 723, 48%) or with additional ATLG (N = 777, 52%). In the ATLG cohort, grades III‐IV acute (12% vs 23%) and extensive chronic GVHD (18% vs 34%) incidences were significantly reduced (P < .0001). Nonrelapse mortality (27% vs 45%) and relapse (30% vs 22%) differed also significantly. Event‐free and overall survival estimates at 10 years were 44% and 51% with ATLG and 33% and 35% without ATLG (P < .002 and <.0001). A dose‐dependent ATLG effect on lymphocyte‐ and neutrophil reconstitution was observed. At ATLG exposure, lymphocyte counts and survival associated through a logarithmically increasing function. In this survival model, the lymphocyte count optimum range at exposure was between 0.4 and 1.45/nL (P = .001). This study supports additional ATLG immune prophylaxis and is the first study to associate optimal lymphocyte counts with survival after MUD‐HCT.
While the role of anti‐T‐lymphocyte globulin as graft‐versus‐host disease prophylaxis in allogeneic cell transplantation recipients is controversial, Turki et al show a long‐term outcome benefit in a large patient cohort and present a novel model associating optimum lymphocyte counts at anti‐T‐lymphocyte globulin exposure with improved survival.
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