Association study of candidate DNA-repair gene variants and acute graft versus host disease in pediatric patients receiving allogeneic hematopoietic stem-cell transplantation.

Acute Graft versus Host Disease (aGvHD) grades 2-4 occurs in 15-60% of pediatric patients undergoing allogeneic haematopoietic stem-cell transplantation (allo-HSCT). The collateral damage to normal tissue by conditioning regimens administered prior to allo-HSCT serve as an initial trigger for aGvHD. DNA-repair mechanisms may play an important role in mitigating this initial damage, and so the variants in corresponding DNA-repair protein-coding genes via affecting their quantity and/or function. We explored 51 variants within 17 DNA-repair genes for their association with aGvHD grades 2-4 in 60 pediatric patients. The cumulative incidence of aGvHD 2-4 was 12% (n = 7) in the exploratory cohort. MGMT rs10764881 (G>A) and EXO rs9350 (c.2270C>T) variants were associated with aGvHD 2-4 [Odds ratios = 14.8 (0 events out of 40 in rs10764881 GG group) and 11.5 (95% CI: 2.3-191.8), respectively, multiple testing corrected p ≤ 0.001]. Upon evaluation in an extended cohort (n = 182) with an incidence of aGvHD 2-4 of 22% (n = 40), only MGMT rs10764881 (G>A) remained significant (adjusted HR = 2.05 [95% CI: 1.06-3.94]; p = 0.03) in the presence of other clinical risk factors. Higher MGMT expression was seen in GG carriers for rs10764881 and was associated with higher IC50 of Busulfan in lymphoblastoid cells. MGMT rs10764881 carrier status could predict aGvHD occurrence in pediatric patients undergoing allo-HSCT.

Pharmacotherapy in Pediatric Hematopoietic Cell Transplantation.

Hematopoietic cell transplantation (HCT) is a curative treatment option for both malignant and nonmalignant diseases. Success of the procedure mainly depends on disease control and treatment-related complications. Pharmacotherapy plays a major role in HCT and significantly impacts the outcomes. Main drug use within HCT includes conditioning, GvHD prophylaxis, and prevention/treatment of infections.Increasing evidence suggests individualized dosing in (pediatric) HCT may improve outcome. Dose individualization may result in a better predictable drug treatment in terms of safety and efficacy, including timely immune reconstitution after HCT and optimal tumor or disease control, which may result in improved survival chances.

Cognate CD4 T-cell licensing of dendritic cells heralds anti-cytomegalovirus CD8 T-cell immunity after human allogeneic umbilical cord blood transplantation.

UNLABELLED: Reactivation of human cytomegalovirus (CMV) is hazardous to patients undergoing allogeneic cord blood transplantation (CBT), lowering survival rates by approximately 25%. While antiviral treatment ameliorates viremia, complete viral control requires CD8+ T-cell-driven immunity. Mouse studies suggest that cognate antigen-specific CD4+ T-cell licensing of dendritic cells (DCs) is required to generate effective CD8+ T-cell responses. For humans, this was not fully understood. We here show that CD4+ T cells are essential for licensing of human DCs to generate effector and memory CD8+ T-cell immunity against CMV in CBT patients. First, we show in CBT recipients that clonal expansion of CMV-pp65-specific CD4+ T cells precedes the rise in CMV-pp65-specific CD8+ T cells. Second, the elicitation of CMV-pp65-specific CD8+ T cells from rare naive precursors in cord blood requires DC licensing by cognate CMV-pp65-specific CD4+ T cells. Finally, also CD8+ T-cell memory responses require CD4+ T-cell-mediated licensing of DCs in our system, by secretion of gamma interferon (IFN-γ) by pp65-specific CD4+ T cells. Together, these data show that human DCs require licensing by cognate antigen-specific CD4+ T cells to elicit effective CD8+ T-cell-mediated immunity and fight off viral reactivation in CBT patients. IMPORTANCE: Survival rates after stem cell transplantation are lowered by 25% when patients undergo reactivation of cytomegalovirus (CMV) that they harbor. Immune protection against CMV is mostly executed by white blood cells called killer T cells. We here show that for generation of optimally protective killer T-cell responses that respond to CMV, the early elicitation of help from a second branch of CMV-directed T cells, called helper T cells, is required.

Increased proportion of perforin-expressing CD8+T-cells indicates control of herpesvirus reactivation in children after stem cell transplantation.

Hematopoietic stem cell transplantation (HSCT) is frequently complicated by viral reactivations. Early diagnosis of viral reactivations and preemptive therapy relies on frequent viralload monitoring. An easy marker of effective cytotoxicity in lymphopenia is lacking and therefore we studied perforin-expression in CD8+T-cells in children following HSCT. Prospectively, we weekly monitored viral loads and perforin-expression of CD8+T-cells in whole blood by FACS, until 4months after HSCT in children. 27 patients were included (median age 4,3, range 0.3-20,1years) of whom 19 developed viral reactivations. These patients showed higher percentages of perforin-expressing CD8+T-cells (17,2%, range 0-63%) than those without (6,8%; range 0-16%) (p=0.001). The increased percentage of perforin-expressing CD8+T-cells coincided with a decrease in viral load with a median interval between maximum viral load and maximum level of perforin-expression of 0,4weeks (range 0.1-7.1). We conclude that perforin-expression in CD8+T-cells may be a marker for effective antiviral T-cell reconstitution early after HSCT in children.

First analysis of human herpesvirus 6T-cell responses: specific boosting after HHV6 reactivation in stem cell transplantation recipients.

Early human herpesvirus 6 (HHV6) reactivation after hematopoietic stem cell transplantation (HSCT) is associated with poor survival. We characterized HHV6 immuneresponses in HSCT patients during lymphopenia. Prospectively, HHV6 DNA-load was measured weekly by realtime-PCR. Numbers of IFNγ-producing HHV6-T-cells were retrospectively determined by enzyme-linked immunospot assay 2 months after HSCT. HHV6-specific T-cell proliferative capacity was analyzed with a newly developed assay using antigen-presenting autologous HHV6-infected PBMC. Fifty-six patients were included (median age 4.6 years; range 0.2-21.2 years). HHV6-reactivation occurred in 29/56 (52%) patients with a median time of 14 (range 1-41) days after HSCT. The median number of IFN-γ producing HHV6-specific T-cells at 2 months and the HHV6-specific CD8+ T-cell proliferative capacity at 6 months after HSCT was increased after HHV6-reactivation compared to non-reactivating patients (P=0.006 and p=0.019). In conclusion, HHV6-specific immuneresponses can be initiated during lymphopenia early after HSCT, which implicates a potential window for development of HHV6-specific (immuno)therapy.

Therapeutic Drug Monitoring of Anti-Thymocyte Globulin in Allogeneic Stem Cell Transplantation: Proof of Concept.

Anti-thymocyte globulin (ATG), a polyclonal antibody, is used in allogeneic hematopoietic cell transplantation (HCT) to prevent graft-vs.-host-disease (GvHD) and graft failure (GF). Overexposure to ATG leads to poor early T-cell recovery, which is associated with viral infections and poor survival. Patients with severe inflammation are at high risk for GF and GvHD, and may have active infections warranting swift T-cell recovery. As ATG exposure may be critical in these patients, individualized dosing combined with therapeutic drug monitoring (TDM) may improve outcomes. We describe the individualized dosing approach, an optimal sampling scheme, the assay to measure the active fraction of ATG, and the workflow to perform TDM. Using a previously published population pharmacokinetic (PK) model, we determine the dose to reach optimal exposures associated with low GvHD and rejection, and at the same time promote T-cell recovery. Based on an optimal sampling scheme, peak and trough samples are taken during the first 3 days of once-daily dosing. The fraction of ATG able to bind to T-cells (active ATG) is analyzed using a bio-assay in which Jurkat cells are co-cultured with patient's plasma and the binding is quantified using flow cytometry. TDM is performed based on these ATG concentrations on the third day of dosing; subsequent doses can be adjusted based on the expected area under the curve. We show that individualized ATG dosing with TDM is feasible. This approach is unique in the setting of antibody treatment and may result in better immune reconstitution post-HCT and subsequently better survival chances.

B-cell depletion abrogates immune mediated cytopenia and rejection of cord blood transplantation in Hurler syndrome.

Umbilical cord blood is the preferred donor cell source for children with Inherited Metabolic disorders undergoing Hematopoietic Cell Transplant (HCT), and its use has been associated with improved "engrafted survival" and higher donor chimerism compared to other cell sources. However, as in other pediatric cord blood transplants for non-malignant disease, immune-mediated cytopenia and primary graft failure limit its use, and the latter remains the commonest cause of death following cord blood transplant for non-malignant disease. We have previously shown an association between immune-mediated cytopenia and graft failure in inherited metabolic diseases suggesting that both immune-mediated cytopenia and graft failure could be mediated by antibodies from the residual recipient B cells. Since rituximab is effective in depletion of B cells and management of refractory immune-mediated cytopenia following HCT, we have added rituximab to the conditioning regimen. We studied 57 patients in 2 centers who received myeloablative conditioning for cord blood transplant in Hurler syndrome, and report a significant improvement in event-free survival with reduced incidence of graft failure and without any evidence of immune-mediated cytopenia in those patients that had received rituximab.

Antigen cross-presentation: extending recent laboratory findings to therapeutic intervention.

The initiation of adaptive immune responses requires antigen presentation to lymphocytes. In particular, dendritic cells (DCs) are equipped with specialized machinery that promote effective display of peptide/major histocompatibility complexes (MHC), rendering them the most potent stimulators of naive T lymphocytes. Antigen cross-presentation to CD8(+) T cells is an important mechanism for the development of specific cytotoxic T lymphocyte (CTL) responses against tumours and viruses that do not infect antigen-presenting cells. Here, we review recent findings concerning antigen cross-presentation to CD8(+) T lymphocytes. Specific subtypes of DCs in the mouse have been defined as being especially endowed for antigen cross-presentation, and a human homologue of these DCs has recently been described. DC vaccination strategies for the prevention and treatment of human diseases have been under investigation in recent years, but have not generally reached satisfying results. We here provide an overview of new findings in antigen cross-presentation research and how they can be used for development of the next generation of human DC vaccines.

Familial hemophagocytic lymphohistiocytosis in a pediatric patient diagnosed by brain magnetic resonance imaging.

Familial hemophagocytic lymphohistiocytosis (fHLH) is an autosomal recessive disorder characterized by proliferation and infiltration of several organs by activated lymphocytes and macrophages. Without allogeneic stem cell transplantation, fHLH is fatal. We describe a previously healthy 11-month-old boy with a rapidly progressive encephalopathy. An older brother died at 8 months following a subacute encephalopathy diagnosed as meningoencephalitis. The family history led to the suspicion of a metabolic disease, but metabolic studies were unrevealing. MRI showed multiple inhomogeneous signal abnormalities in the cortex and white matter, most prominent in the cerebral hemispheres and around the dentate nucleus. Gadolinium-enhanced T1-weighted images showed a multitude of enhancing foci, suggestive of perivascular enhancement. Based on MRI pattern with multiple lesions, perivascular enhancement and family history, fHLH was suspected. DNA analysis showed that the patient was compound-heterozygous for the c.445 G>A (p.Gly149Ser) mutation in exon 1 and the c.757 G>A (p.Glu253Lys) mutation in exon 2 of the perforin 1 gene. The patient was treated according to the international HLH-2004 protocol (dexamethasone, etoposide, cyclosporine, intrathecal methotrexate and prednisolone) followed by allogeneic cord blood transplantation. He showed a significant neurological and radiological improvement. The reported case demonstrates that MRI pattern recognition can lead to early diagnosis of fHLH, with subsequent adequate treatment.

Hematopoietic cell transplant in pediatric acute myeloid leukemia after similar upfront therapy; a comparison of conditioning regimens.

The impact of conditioning regimen prior to hematopoietic cell transplant (HCT) in pediatric AML-patients is not well studied. We retrospectively analyzed the impact of Busulfan-Cyclophosphamide (BuCy), Busulfan-Cyclophosphamide-Melphalan (BuCyMel) and Clofarabine-Fludarabine-Busulfan (CloFluBu) in pediatric AML-patients, with similar upfront leukemia treatment (NOPHO-DBHconsortium), receiving an HCT between 2010 and 2015. Outcomes of interest were LFS, relapse, TRM and GvHD. 103 patients were included; 30 received BuCy, 37 BuCyMel, and 36 CloFluBu. The 5-years LFS was 43.3% (SE ± 9.0) in the BuCy group, 59.2 % (SE ± 8.1) after BuCyMel, and 66.7 % (SE ± 7.9) after CloFluBu. Multivariable Cox regression analysis showed a trend to lower LFS after BuCy compared to CloFluBu (p = 0.07). BuCy was associated with a higher relapse incidence compared to the other regimens (p = 0.06). Younger age was a predictor for relapse (p = 0.02). A strong correlation between Busulfan Therapeutic Drug Monitoring (TDM) and lower incidence of aGvHD (p < 0.001) was found. In conclusion, LFS after BuCyMel and CloFluBu was comparable, lower LFS was found after BuCy, due to higher relapse incidence. CloFluBu was associated with lower incidence of aGvHD, suggesting lower toxicity with this type of conditioning. This finding is also explained by the impact of Busulfan monitoring.

Fludarabine exposure in the conditioning prior to allogeneic hematopoietic cell transplantation predicts outcomes.

Fludarabine is the most frequently used agent in conditioning regimens for allogeneic hematopoietic cell transplantation (HCT). Body surface area-based dosing leads to highly variable fludarabine exposure. We studied the relation between fludarabine exposure and clinical outcomes. A retrospective, pharmacokinetic-pharmacodynamic analysis was conducted with data from patients undergoing HCT with fludarabine (160 mg/m2) as part of a myeloablative conditioning (busulfan targeted to an area under the plasma-concentration-time curve [AUC] of 90 mg*h/L) and rabbit antithymocyte globulin (6-10 mg/kg; from day -9/-12) between 2010 and 2016. Fludarabine exposure as AUC was calculated for each patient using a previously published population pharmacokinetic model and related to 2-year event-free survival (EFS) by means of (parametric) time-to-event models. Relapse, nonrelapse mortality (NRM), and graft failure were considered events. One hundred ninety-two patients were included (68 benign and 124 malignant disorders). The optimal fludarabine exposure was determined as an AUC of 20 mg*h/L. In the overexposed group, EFS was lower (hazard ratio [HR], 2.0; 95% confidence interval [CI], 1.1-3.5; P = .02), due to higher NRM (HR, 3.4; 95% CI, 1.6-6.9; P <001) associated with impaired immune reconstitution (HR, 0.43; 95% CI, 0.26-0.70; P <001). The risks of NRM and graft failure were increased in the underexposed group (HR, 3.3; 95% CI, 1.2-9.4; P = .02; HR, 4.8; 95% CI, 1.2-19; P = .02, respectively). No relationship with relapse was found. Fludarabine exposure is a strong predictor of survival after HCT, stressing the importance of optimum fludarabine dosing. Individualized dosing, based on weight and "renal function" or "therapeutic drug monitoring," to achieve optimal fludarabine exposure might improve survival.

Human herpesvirus type 6 reactivation after haematopoietic stem cell transplantation.

Human herpesvirus type 6 (HHV6) is known to reactivate after hematopoetic stem cell transplantation (HSCT) and has been suggested to be associated with increased mortality and severe clinical manifestations, including graft versus host disease (GvHD). The exact etiological role of HHV6 reactivation in increased morbidity and mortality after HSCT remains unclear. This review will focus on the current available evidence of HHV6 reactivation after HSCT and its immuno-modulatory capacities, with particular emphasis on the severe complication GvHD. At present, no effective specific antiviral treatment for HHV6 reactivation has been identified. The currently available antiviral agents are outlined, as well as possible future strategies for the treatment of HHV6 reactivation. Non-toxic, specific treatment or prevention of HHV6 reactivation might improve the safety and efficacy of the HSCT procedure.

[Umbilical cord blood from an unrelated donor as source for stem cell transplantations in inborn errors of metabolism]. / Navelstrengbloed van een onverwante donor als bron voor stamceltransplantaties bij aangeboren stofwisselingsziekten.

In certain inborn errors of metabolism, an allogeneic stem cell transplantation is able to prevent disease progression. This is only possible when the stem cell transplantation (SCT) is performed early in life, before cerebral involvement has occurred. In addition to bone marrow and peripheral blood, unrelated umbilical cord blood appears to be an effective stem cell source as well. Important advantages of umbilical blood as stem cell source are: the time between diagnosis and SCT can be considerably reduced; there is a greater chance of finding a suitable donor and the risk of graft-versus-host disease and viral transmission is decreased. By far the most common disease in the group of inborn metabolic errors for which SCTs are performed is Hurler's disease. In these patients, the percentage of successful transplantations is considerably higher after the use ofunrelated cord blood than when bone marrow or peripheral blood is used as a stem cell source. In addition, donor chimerism occurred significantly more often in those patients who had received unrelated umbilical cord blood. There are also potential disadvantages attached to the use of umbilical blood as stem cell source: the possibility of only one donation per donor and less adaptive immunity following umbilical blood SCT with an increased risk of reactivation of a previous viral infection. However, these disadvantages are less applicable to young children with inborn errors of metabolism. The improvement of transplantation techniques and the availability of this new stem cell source could improve the success rate of this procedure and consequently the prognosis of these severely affected patients.

Outcomes of hematopoietic stem cell transplantation for Hurler's syndrome in Europe: a risk factor analysis for graft failure.

Hurler's syndrome (HS), the most severe form of mucopolysaccharidosis type-I, causes progressive deterioration of the central nervous system and death in childhood. Allogeneic stem cell transplantation (SCT) before the age of 2 years halts disease progression. Graft failure limits the success of SCT. We analyzed data on HS patients transplanted in Europe to identify the risk factors for graft failure. We compared outcomes in 146 HS patients transplanted with various conditioning regimens and grafts. Patients were transplanted between 1994 and 2004 and registered to the European Blood and Marrow Transplantation database. Risk factor analysis was performed using logistic regression. 'Survival' and 'alive and engrafted'-rate after first SCT was 85 and 56%, respectively. In multivariable analysis, T-cell depletion (odds ratio (OR) 0.18; 95% confidence interval (CI) 0.04-0.71; P=0.02) and reduced-intensity conditioning (OR 0.08; 95% CI 0.02-0.39; P=0.002) were the risk factors for graft failure. Busulfan targeting protected against graft failure (OR 5.76; 95% CI 1.20-27.54; P=0.028). No difference was noted between cell sources used (bone marrow, peripheral blood stem cells or cord blood (CB)); however, significantly more patients who received CB transplants had full-donor chimerism (OR 9.31; 95% CI 1.06-82.03; P=0.044). These outcomes may impact the safety/efficacy of SCT for 'inborn-errors of metabolism' at large. CB increased the likelihood of sustained engraftment associated with normal enzyme levels and could therefore be considered as a preferential cell source in SCT for 'inborn errors of metabolism'.

Haematopoietic cell transplantation (HCT) in combination with enzyme replacement therapy (ERT) in patients with Hurler syndrome.

Hurler syndrome (MPS 1H) is the severe form of mucopolysaccharidosis type 1 (MPS 1). Haematopoietic cell transplantation (HCT) is the treatment of choice, but carries a high incidence of graft failure and morbidity. The use of enzyme replacement therapy (ERT) might improve the clinical signs and symptoms before HCT, resulting in less transplantation-related complications. Moreover, clearance of glycosaminoglycans (GAG's) from the bone marrow might improve engraftment. Twenty-two patients with MPS 1H received one or more HCT procedures in combination with ERT. One patient with severe cardiomyopathy improved significantly after ERT. All children were in a relatively good clinical condition before HCT. Of patients 59, 82 and 86% were alive and engrafted after one, two and three HCT procedures, respectively. Two patients died after repetitive HCT. No serious ERT-infusion-related toxicity occurred. ERT with HCT was well tolerated. Neither a positive nor a negative effect on the number of patients who are alive and engrafted after receiving ERT before HCT as compared to a historic cohort was noted. However, patients in a poor clinical condition before HCT might benefit from ERT.

Individualized conditioning regimes in cord blood transplantation: Towards improved and predictable safety and efficacy.

INTRODUCTION: The conditioning regimen used in cord blood transplantation (CBT) may significantly impact the outcomes. Variable pharmacokinetics (PK) of drugs used may further influence outcome. Individualized dosing takes inter-patient differences in PK into account, tailoring drug dose for each individual patient in order to reach optimal exposure. Dose individualization may result in a better predictable regimen in terms of safety and efficacy, including timely T cell reconstitution, which may result in improved survival chances. AREAS COVERED: Conditioning regimens used in CBT varies significantly between and within centres. For busulfan, individualized dosing with therapeutic drug monitoring has resulted in better outcomes. Anti-thymocyte globulin (ATG), used to prevent rejection and GvHD, significantly hampers early T-cell reconstitution (IR). Timely IR is crucial in preventing viral reactivations and relapse. By individudalizing ATG, IR is better predicted and may prevent morbidity and mortality. EXPERT OPINION: Individualization of agents used in the conditioning regimen in CBT has proven its added value. Further fine-tuning, including new drugs and/or comprehensive models for all drugs, may result in better predictable conditioning regimens. A predictable conditioning regimen is also of interest/importance when studying adjuvant therapies, including immunotherapies (e.g. cellular vaccines or engineered T-cell) in a harmonized clinical trial design setting.

Paediatric reduced intensity conditioning: analysis of centre strategies on regimens and definitions by the EBMT Paediatric Diseases and Complications and Quality of Life WP.

The aim of this analysis was to explore the diversity of reduced intensity conditioning (RIC) in paediatric allo-SCT in daily practice across Europe. Data from the European Group for Blood and Marrow Transplantation (EBMT) Promise database from 1994 to 2008 were supplemented by a survey of EBMT centres performing paediatric allo-SCT on the current policy asking for the underlying diseases and for the drug combinations. Records from 161 centres from 30 countries were analysed and 139 various RIC regimens were reported. More centres applied RIC for malignant rather than for non-malignant diseases. In general, fludarabine (FLU)-based regimens predominated except for BU-based regimens in myeloid malignancies and haemoglobinopathies. Treosulfan (TREO) was mainly applied for unspecified malignant diseases and for haemophagocytic diseases. FLU-based regimens revealed the greatest number of different combinations. Correlating the number of regimens with the number of treating centres revealed the lowest variety in FLU and the highest variety in TBI and TREO. FLU/melphalane and FLU/CY were the most frequent combinations. This extreme heterogeneity in RIC may influence both the efficacy and the safety of the procedures, which requires further investigation. Optimization and standardization of RIC is the final goal to provide a platform for future prospective studies.

Biomarker profiling of steroid-resistant acute GVHD in patients after infusion of mesenchymal stromal cells.

We performed a prospective phase II study to evaluate clinical safety and outcome in 48 patients with steroid-refractory grade II-IV acute graft-versus-host disease (aGVHD) treated with mesenchymal stromal cells (MSCs). Clinical outcomes were correlated to comprehensive analyses of soluble and cellular biomarkers. Complete resolution (CR) of aGVHD at day 28 (CR-28) occurred in 12 (25%) patients, CR lasting >1 month (CR-B) occurred in 24 (50%) patients. One-year overall survival was significantly improved in CR-28 (75 versus 33%, P=0.020) and CR-B (79 versus 8%, P<0.001) versus non-CR patients. A six soluble biomarker-panel was predictive for mortality (HR 2.924; CI 1.485-5.758) when measured before MSC-administration. Suppression of tumorigenicity 2 (ST2) was only predictive for mortality 2 weeks after but not before MSC-administration (HR 2.389; CI 1.144-4.989). In addition, an increase in immature myeloid dendritic cells associated with decreased mortality (HR 0.554, CI 0.389-0.790). Patients had persisting T-cell responses against defined virus- and leukemia-associated antigens. In conclusion, our data emphasize the need to carefully assess biomarkers in cohorts with homogeneous GVHD treatments. Biomarkers might become an additional valuable component of composite end points for the rapid and efficient testing of novel compounds to decrease lifecycle of clinical testing and improve the success rate of phase II/III trials.