Hypomorphic RAG deficiency: impact of disease burden on survival and thymic recovery argues for early diagnosis and HSCT.

Patients with hypomorphic mutations in the RAG1 or RAG2 gene present with either Omenn syndrome or atypical combined immunodeficiency with a wide phenotypic range. Hematopoietic stem cell transplantation (HSCT) is potentially curative, but data are scarce. We report on a worldwide cohort of 60 patients with hypomorphic RAG variants who underwent HSCT, 78% of whom experienced infections (29% active at HSCT), 72% had autoimmunity, and 18% had granulomas pretransplant. These complications are frequently associated with organ damage. Eight individuals (13%) were diagnosed by newborn screening or family history. HSCT was performed at a median of 3.4 years (range 0.3-42.9 years) from matched unrelated donors, matched sibling or matched family donors, or mismatched donors in 48%, 22%, and 30% of the patients, respectively. Grafts were T-cell depleted in 15 cases (25%). Overall survival at 1 and 4 years was 77.5% and 67.5% (median follow-up of 39 months). Infection was the main cause of death. In univariable analysis, active infection, organ damage pre-HSCT, T-cell depletion of the graft, and transplant from a mismatched family donor were predictive of worse outcome, whereas organ damage and T-cell depletion remained significant in multivariable analysis (hazard ratio [HR] = 6.01, HR = 8.46, respectively). All patients diagnosed by newborn screening or family history survived. Cumulative incidences of acute and chronic graft-versus-host disease were 35% and 22%, respectively. Cumulative incidences of new-onset autoimmunity was 15%. Immune reconstitution, particularly recovery of naïve CD4+ T cells, was faster and more robust in patients transplanted before 3.5 years of age, and without organ damage. These findings support the indication for early transplantation.

Persistence of HIV reservoir following successful haematopoietic stem cell transplant for juvenile myelomonocytic leukaemia in a child with perinatally acquired HIV.

This report describes a case of juvenile myelomonocytic leukaemia (JMML) on a background of both perinatally acquired HIV infection and congenital cytomegalovirus, and management of antiretroviral therapy during haematopoietic stem cell transplant. Peripheral blood HIV viral load remained below the lower limit of detection throughout and following transplant and is currently <20 RNA copies/mL. The child is currently in remission from JMML, but HIV DNA remains detectable despite myeloablative conditioning and sustained plasma HIV viral suppression.

A mass spectrometry method for sensitive, specific and simultaneous detection of bovine blood meal, blood products and milk products in compound feed.

Feed sustainability is one of the biggest challenges for the next few years. Solutions have to be found that take feed quality and safety into account. Animal by-products are one valuable source of proteins. However, since the bovine spongiform encephalopathy (BSE) crisis, their use has been strictly regulated. The objective of this study was to propose a routine, sensitive and specific method using ultra-high performance liquid chromatography coupled to tandem mass spectrometry for the detection of blood-derived products and milk powder in feed. Contaminated aquafeeds were analysed in order to evaluate the sensitivity and specificity of the method. This new method meets both selectivity and sensitivity (0.1% (w/w)) requirements imposed by the European Commission for animal proteins detection methods. It offers an innovative and complementary solution for the simultaneously identification of authorised and unauthorised animal by-products such as processed animal proteins (PAPs).

Allogeneic stem cell transplantation for refractory acute myeloid leukemia in pediatric patients: the UK experience.

We report outcomes for 44 children who underwent stem cell transplantation (SCT) for refractory AML in the UK between 2000 and 2012. Median age at SCT was 11.5 years. Twenty-three patients had primary refractory and 21 relapsed refractory AML. Refractory disease was confirmed by cytogenetics/molecular genetics in 24 cases. Median follow-up of the whole cohort is 6.8 years (2.1-14.9 years). Thirty patients (68%) achieved a CR following SCT. Transplant-related mortality at 1 year was 18%. Acute GVHD incidence was 52% (grade ⩾III 19%), chronic 7%. Relapse was the major cause of treatment failure and occurred in 32% of patients at a median of 61 days post SCT. Five-year overall survival and leukemia-free survival (LFS) were 43% (95% CI 31-61%). All patients with favorable cytogenetics (n=6) are alive in CR. Outcomes in patients with primary refractory disease were equivalent to those with relapsed refractory AML. Blast percentage ⩽30% in the BM pre-SCT, myeloablative conditioning and acute GVHD proved to be favorable prognostic features. We could stratify patients according to age ⩾10 years and >30% blasts in BM pre-SCT. Patients with none/one of these risk factors were highly salvageable (5 years LFS 53%) whereas those with both factors had a very poor prognosis (5 years LFS 10%). This may facilitate decision making on whether it is appropriate to consider transplant in such patients.

Vaccination to improve the persistence of CD19CAR gene-modified T cells in relapsed pediatric acute lymphoblastic leukemia.

Trials with second generation CD19 chimeric antigen receptors (CAR) T-cells report unprecedented responses but are associated with risk of cytokine release syndrome (CRS). Instead, we studied the use of donor Epstein-Barr virus-specific T-cells (EBV CTL) transduced with a first generation CD19CAR, relying on the endogenous T-cell receptor for proliferation. We conducted a multi-center phase I/II study of donor CD19CAR transduced EBV CTL in pediatric acute lymphoblastic leukaemia (ALL). Patients were eligible pre-emptively if they developed molecular relapse (>5 × 10-4) post first stem cell transplant (SCT), or prophylactically post second SCT. An initial cohort showed poor expansion/persistence. We therefore investigated EBV-directed vaccination to enhance expansion/persistence. Eleven patients were treated. No CRS, neurotoxicity or graft versus host disease (GVHD) was observed. At 1 month, 5 patients were in CR (4 continuing, 1 de novo), 1 PR, 3 had stable disease and 3 no response. At a median follow-up of 12 months, 10 of 11 have relapsed, 2 are alive with disease and 1 alive in CR 3 years. Although CD19CAR CTL expansion was poor, persistence was enhanced by vaccination. Median persistence was 0 (range: 0-28) days without vaccination compared to 56 (range: 0-221) days with vaccination (P=0.06). This study demonstrates the feasibility of multi-center studies of CAR T cell therapy and the potential for enhancing persistence with vaccination.

Predicting CD4 T-Cell Reconstitution Following Pediatric Hematopoietic Stem Cell Transplantation.

Hematopoietic stem cell transplantation (HSCT) is an increasingly common treatment for children with a range of hematological disorders. Conditioning with cytotoxic chemotherapy and total body irradiation leaves patients severely immunocompromised. T-cell reconstitution can take several years due to delayed restoration of thymic output. Understanding T-cell reconstitution in children is complicated by normal immune system maturation, heterogeneous diagnoses, and sparse uneven sampling due to the long time spans involved. We describe here a mechanistic mathematical model for CD4 T-cell immune reconstitution following pediatric transplantation. Including relevant biology and using mixed-effects modeling allowed the factors affecting reconstitution to be identified. Bayesian predictions for the long-term reconstitution trajectories of individual children were then obtained using early post-transplant data. The model was developed using data from 288 children; its predictive ability validated on data from a further 75 children, with long-term reconstitution predicted accurately in 81% of the patients.

Identification of specific bovine blood biomarkers with a non-targeted approach using HPLC ESI tandem mass spectrometry.

Animal by-products are valuable protein sources in animal nutrition. Among them are blood products and blood meal, which are used as high-quality material for their beneficial effects on growth and health. Within the framework of the feed ban relaxation, the development of complementary methods in order to refine the identification of processed animal proteins remains challenging. The aim of this study was to identify specific biomarkers that would allow the detection of bovine blood products and processed animal proteins using tandem mass spectrometry. Seventeen biomarkers were identified: nine peptides for bovine plasma powder; seven peptides for bovine haemoglobin powder, including six peptides for bovine blood meal; and one peptide for porcine blood. They were not detected in several commercial compound feed or feed materials, such as blood by-products of other animal origins, milk-derived products and fish meal. These biomarkers could be used for developing a species-specific and blood-specific detection method.

Treosulfan-based conditioning regimens for allogeneic haematopoietic stem cell transplantation in children with non-malignant diseases.

An increasing number of children with non-malignant diseases can be cured by allogeneic haematopoietic stem cell transplantation (HSCT). Treosulfan (L-treitol-1,4-bis-methanesulfonate) is being used more frequently for conditioning, owing to its' lower toxicity profile compared with conventional myeloablative regimens. A retrospective analysis was performed of children registered in the EBMT database, who received treosulfan before HSCT between January 2005 and 2010, to identify possible dose-related toxicity and determine the incidence of engraftment, treatment-related mortality and overall survival (OS). Results from 316 transplants from 11 different countries are presented. Ninety-five (30%) were under 1 year of age at the time of transplant. OS was 83% and event-free survival was 76%; 3-year OS and event-free survival of infants below 1 year were 79% and 73%, respectively. No association was found with age at transplant, dose of treosulfan given, other agents used in combination with treosulfan, donor type, stem cell source, or second or subsequent transplant. In this report of the largest number of children to date receiving treosulfan for non-malignant diseases, treosulfan is shown to be a safe and effective agent even for those under 1 year of age at the time of transplant. Further prospective studies are needed using precisely defined protocols with pharmacokinetic monitoring and detailed chimerism analysis. In addition, long-term studies will be vital to determine long-term effects, for example, on fertility in comparison with other regimens.

No evidence of transfusion transmission of Adenovirus and Epstein-Barr virus infections in paediatric recipients post-bone marrow transplant.

Adenovirus and Epstein-Barr virus can cause significant morbidity and mortality in paediatric patients post-bone marrow transplant. The source of infection is thought to be either reactivation of latent viruses or primary infection. We have investigated whether transfusion of blood components from viraemic donors could provide a route of primary infection in these patients and sought the prevalence of viraemia in the blood donor population from England. In 32 linked donor/recipient samples and 300 unselected blood donors, we found no evidence to suggest that these infections in paediatric bone marrow transplant recipients had been acquired from transfused blood components.

Single-donor granulocyte transfusions for improving the outcome of high-risk pediatric patients with known bacterial and fungal infections undergoing stem cell transplantation: a 10-year single-center experience.

Bacterial and fungal infections remain a significant cause of transplant-related mortality following allogeneic stem cell transplantation (SCT). Granulocyte transfusions (GTs) may reduce the neutropenic period after SCT and prevent further progression of the existing infection (that is, therapeutic GT) in addition to standard antibacterial and antifungal therapy. A retrospective analysis was performed on 28 consecutive pediatric SCT recipients who received at least one dose of GT between March 2003 and November 2013 at a single institution. All donors were conditioned with G-CSF+dexamethasone with harvest performed 12-18 h later. Indications for SCT were acute leukemia in 46% (13/28) and severe aplastic anemia in 21% (6/28). The main indications for GT were invasive fungal disease in 50%, bacterial infection in 21% and co-morbidities with predicted reduced tolerance to sepsis in 18% (5/28). The median number of GT was 6 (range 1-14) with a median dose of 3.56 × 10(10) granulocytes infused. The median increment in ANC was 1.06 × 10(9)/L and correlated with the granulocyte dose infused. Adverse reactions observed were mild and infrequent. Sixty-four percent of patients (18/28) are alive with only 2 of the 10 deaths being related to progression of infection. In addition there was a low overall incidence of grade 3-4 acute mucositis and a very low incidence (7%) of acute GvHD grade 3-4. Single-donor GTs afford protection to children undergoing SCT at additional risk of infection and may reduce the overall incidence of severe GvHD.

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.

Determination of the ruminant origin of bone particles using fluorescence in situ hybridization (FISH).

Molecular biology techniques such as PCR constitute powerful tools for the determination of the taxonomic origin of bones. DNA degradation and contamination by exogenous DNA, however, jeopardise bone identification. Despite the vast array of techniques used to decontaminate bone fragments, the isolation and determination of bone DNA content are still problematic. Within the framework of the eradication of transmissible spongiform encephalopathies (including BSE, commonly known as "mad cow disease"), a fluorescence in situ hybridization (FISH) protocol was developed. Results from the described study showed that this method can be applied directly to bones without a demineralisation step and that it allows the identification of bovine and ruminant bones even after severe processing. The results also showed that the method is independent of exogenous contamination and that it is therefore entirely appropriate for this application.

Retrospective study of alemtuzumab vs ATG-based conditioning without irradiation for unrelated and matched sibling donor transplants in acquired severe aplastic anemia: a study from the British Society for Blood and Marrow Transplantation.

This retrospective national study compared the use of alemtuzumab-based conditioning regimens for hematopoietic SCT (HSCT) in acquired severe aplastic anemia with antithymocyte globulin (ATG)-based regimens. One hundred patients received alemtuzumab and 55 ATG-based regimens. A matched sibling donor (MSD) was used in 87 (56%), matched unrelated donor (MUD) in 60 (39%) and other related or mismatched unrelated donor (UD) in 8 (5%) patients. Engraftment failure occurred in 9% of the alemtuzumab group and 11% of the ATG group. Five-year OS was 90% for the alemtuzumab and 79% for the ATG groups, P=0.11. For UD HSCT, OS of patients was better when using alemtuzumab (88%) compared with ATG (57%), P=0.026, although smaller numbers of patients received ATG. Similar outcomes for MSD HSCT using alemtuzumab or ATG were seen (91% vs 85%, respectively, P=0.562). A lower risk of chronic GVHD (cGVHD) was observed in the alemtuzumab group (11% vs 26%, P=0.031). On multivariate analysis, use of BM as stem cell source was associated with better OS and EFS, and less acute and cGVHD; young age was associated with better EFS and lower risk of graft failure. This large study confirms successful avoidance of irradiation in the conditioning regimens for MUD HSCT patients.

T cell depletion in paediatric stem cell transplantation.

Haematopoietic stem cell transplantation (HSCT) can be a curative procedure for a growing number of paediatric diseases, but as the indications for HSCT grow, so does the need to find suitable stem cell donors. When the preferred option of a genoidentical sibling donor is not available alternative donors, including unrelated adult or umbilical cord blood donors, or haploidentical related donors may be considered. Outcome following alternative donor HSCT has improved over the past 20 years but graft-versus-host disease (GvHD) remains a significant obstacle. T cell depletion (TCD) for non-genoidentical grafts aims to reduce the morbidity and mortality associated with GvHD, but this intervention has not led directly to improved survival due to delayed immune reconstitution and increased infections, graft rejection and increased rates of disease relapse. Limited data from the paediatric population, however, suggest some encouraging results for children undergoing haploidentical HSCT: a move from positive selection of CD34(+) haematopoietic stem cells towards negative depletion of specific cell subsets in order to retain useful accessory cells within the graft appears to enhance immune reconstitution and improve disease-free survival. Here we review recent paediatric outcome data for T cell-depleted HSCT, explore the role of serotherapy in conditioning regimens and look at future possibilities to improve outcome, including novel allodepletion techniques, suicide gene therapy and pathogen-specific immunotherapy.

Alternative donor SCT for the treatment of MHC class II deficiency.

MHC Class II deficiency is a rare primary immunodeficiency disease characterized by absent HLA Class II expression resulting in CD4 lymphopenia, lack of Ag-specific responses and recurrent infection. Without successful allogeneic SCT, most children succumb to infection within the first decade of life. To date, alternative donor transplants for this disorder have been inferior to SCT for other forms of combined immunodeficiency disease due to an increased incidence of graft rejection, GVHD and death from infections generally acquired before haematopoietic cell transplantation. This study details the transplant outcome of 16 affected children consecutively transplanted at four centers since 1990, 8 of whom required mechanical ventilation pretransplant. Stem cells were derived from an HLA-mismatched family member (n=10), an HLA-matched unrelated adult donor (n=4), or an unrelated cord blood donor (n=2). Graft failure occurred in five children, all of whom underwent a second SCT. Six patients developed acute GVHD although no patient developed chronic GVHD after primary transplantation. CD4 T-cell reconstitution remained below the normal range for age, suggesting defective thymopoiesis after allo-SCT. Nonetheless, 69% of children survive without GVHD at a median follow-up of 5.7 years, indicating improved outcomes compared with previous studies.

Impact of viral reactivations in the era of pre-emptive antiviral drug therapy following allogeneic haematopoietic SCT in paediatric recipients.

While pre-emptive rituximab therapy for EBV has substantially reduced the incidence of post-transplant lymphoproliferative disorder, following allogeneic haematopoietic SCT (HSCT), cytomegalovirus (CMV) and adenovirus (ADV) still contribute to significant morbidity and mortality after HSCT. We therefore aimed to identify high-risk children who could benefit from recent advances in virus-specific immunotherapy, define the impact of viral reactivations on survival and estimate the economic burden of pre-emptive antiviral drug therapy. Between 2005 and 2010, prospective monitoring of 291 paediatric HSCT procedures revealed that reactivation of CMV (16%), ADV (15%) and EBV (11%) was frequent during period of CD4 T-cell lymphopenia (0.15 × 10(9) L(-1); P<0.05). We report significant risk factors for reactivation, most notably the use of serotherapy and development of GVHD (grade II) in the presence of pre-existing infection (ADV) or donor and/or recipient seropositivity (CMV, EBV). Most interestingly, CMV and ADV viraemia were the major independent predictors of mortality (P<0.05). CMV, ADV or EBV viral reactivation caused prolonged hospitalization (P<0.05), accounted for 15% of all mortality and substantially increased the cost of transplantation by ∼£22 500 ($34 000). This provides an economic rationale for targeting high-risk HSCT recipients with interventions such as virus-specific cell therapy.