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Respiratory outcomes in Mucopolysaccharidosis Type I (MPS I), have mainly focused on upper airway obstruction, with the evolution of the restrictive lung disease being poorly documented. We report the long-term pulmonary function outcomes and examine the potential factors affecting these in 2 cohorts of MPS I patients, those who have undergone Haematopoietic Stem Cell Transplantation (HSCT) and those treated with Enzyme Replacement Therapy (ERT). The results were stratified using the American Thoracic Society (ATS) guidelines. 66 patients, capable of adequately performing testing, were identified by a retrospective case note review, 46 transplanted (45 Hurler, 1 Non-Hurler) and 20 having ERT (17 Non-Hurler and 3 Hurler diagnosed too late for HSCT). 5 patients died; 4 in the ERT group including the 3 Hurler patients. Overall 14% of patients required respiratory support (non-invasive ventilation (NIV) or supplemental oxygen)) at the end of follow up. Median length of follow-up was 12.2 (range = 4.9-32) years post HSCT and 14.34 (range = 3.89-20.4) years on ERT. All patients had restrictive lung disease. Cobb angle and male sex were significantly associated with more severe outcomes in the HSCT cohort, with 49% having severe to very severe disease. In the 17 Non-Hurler ERT treated patients there was no variable predictive of severity of disease with 59% having severe to very severe disease. During the course of follow up 67% of the HSCT cohort had no change or improved pulmonary function as did 52% of the ERT patients. However, direct comparison between therapeutic modalities was not possible. This initial evidence would suggest that a degree of restrictive lung disease is present in all treated paediatrically diagnosed MPS I and is still a significant cause of morbidity, though further stratification incorporating diffusing capacity for carbon monoxide (DLCO) is needed.

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.

A substantial number of individuals with bone marrow failure (BMF) present with one or more extra-hematopoietic abnormality. This suggests a constitutional or inherited basis, and yet many of them do not fit the diagnostic criteria of the known BMF syndromes. Through exome sequencing, we have now identified a subgroup of these individuals, defined by germline biallelic mutations in DNAJC21 (DNAJ homolog subfamily C member 21). They present with global BMF, and one individual developed a hematological cancer (acute myeloid leukemia) in childhood. We show that the encoded protein associates with rRNA and plays a highly conserved role in the maturation of the 60S ribosomal subunit. Lymphoblastoid cells obtained from an affected individual exhibit increased sensitivity to the transcriptional inhibitor actinomycin D and reduced amounts of rRNA. Characterization of mutations revealed impairment in interactions with cofactors (PA2G4, HSPA8, and ZNF622) involved in 60S maturation. DNAJC21 deficiency resulted in cytoplasmic accumulation of the 60S nuclear export factor PA2G4, aberrant ribosome profiles, and increased cell death. Collectively, these findings demonstrate that mutations in DNAJC21 cause a cancer-prone BMF syndrome due to corruption of early nuclear rRNA biogenesis and late cytoplasmic maturation of the 60S subunit.

Tay-Sachs disease is a rare metabolic disease caused by a deficiency of hexosaminidase A that leads to accumulation of GM2 gangliosides predominantly in neural tissue. Late-onset Tay-Sachs disease variant is associated with a higher level of residual HexA activity. Treatment options are limited, and there are a few described cases who have undergone haematopoietic stem cell transplantation (HSCT) with variable outcome.We describe a case of a 23-year-old male patient who presented with a long-standing tremor since 7 years of age. He had gait ataxia, a speech stammer and swallowing problems. His condition had had a static course apart from his tremor that had been gradually deteriorating. Because of the deterioration in his neurological function, the patient had an uneventful, matched-sibling donor bone marrow transplant at the age of 15 years. Eight years post-HSCT, at the age of 23, he retains full donor engraftment, and his white cell beta-HexA of 191 nmol/mg/h is comparable to normal controls (in-assay control = 187). He continues to experience some intentional tremor that is tolerable for daily life and nonprogressive since HSCT.HSCT is a potential treatment option which might arrest neurodegeneration in patients with LOTS.

The advances in hematopoietic cell transplantation (HCT) over the last decade have led to a transplant-related mortality below 15%. Hepatic sinusoidal obstruction syndrome/veno-occlusive disease (SOS/VOD) is a life-threatening complication of HCT that belongs to a group of diseases increasingly identified as transplant-related, systemic endothelial diseases. In most cases, SOS/VOD resolves within weeks; however, severe SOS/VOD results in multi-organ dysfunction/failure with a mortality rate > 80%. A timely diagnosis of SOS/VOD is of critical importance, given the availability of therapeutic options with favorable tolerability. Current diagnostic criteria are used for adults and children. However, over the last decade it has become clear that SOS/VOD is significantly different between the age groups in terms of incidence, genetic predisposition, clinical presentation, prevention, treatment and outcome. Improved understanding of SOS/VOD and the availability of effective treatment questions the use of the Baltimore and Seattle criteria for diagnosing SOS/VOD in children. The aim of this position paper is to propose new diagnostic and severity criteria for SOS/VOD in children on behalf of the European Society for Blood and Marrow Transplantation.

Allogeneic haemopoietic stem cell transplantation offers a potentially curative treatment option for a wide range of life-threatening malignant and non-malignant disorders of the bone marrow and immune system in patients of all ages. With rapidly emerging advances in the use of alternative donors, such as mismatched unrelated, cord blood and haploidentical donors, it is now possible to find a potential donor for almost all patients in whom an allograft is indicated. Therefore, for any specific patient, the transplant physician may be faced with a myriad of potential choices, including decisions concerning which donor to prioritize where there is more than one, the optimal selection of specific umbilical cord blood units and which conditioning and graft-versus-host disease prophylactic schedule to use. Donor choice may be further complicated by other important factors, such as urgency of transplant, the presence of alloantibodies, the disease status (homozygosity or heterozygosity) of sibling donors affected by inherited disorders and the cytomegalovirus serostatus of patient and donor. We report UK consensus guidelines on the selection of umbilical cord blood units, the hierarchy of donor selection and the preferred conditioning regimens for umbilical cord blood transplantation, with a summary of rationale supporting these recommendations.

In vivo T-cell depletion, using alemtuzumab therapy prior to SCT, can reduce the incidence of GVHD. This treatment has a potential to delay immune reconstitution resulting in increased morbidity due to viral illnesses. We retrospectively analyzed data on all pediatric patients with non-malignant disorders who received alemtuzumab-based conditioning regimens in our center over the last 10 yr (n = 91). Our data show an OS of 91.2%. The incidence of acute (grade 2-4) GVHD was 18.7% and that of chronic GVHD 5.5%. Viremia due to adenovirus, EBV and CMV was seen in 19.8%, 64.8% and 39.6% patients, respectively, with only two deaths attributed to viral infection (adenovirus). Chimerism level at three month was predictive of graft outcome. Nine patients, who had graft failure after first SCT, were salvaged with a second SCT using RIC and same donor (if available). Based on these results, we conclude that the use of in vivo T-cell depletion is safe, achieves good chimerism and does not lead to increased morbidity and mortality due to viral infections. It is associated with a reduced incidence of chronic GVHD.

Little information is currently available regarding the pharmacokinetics (PK) of busulfan in infants and small children to help guide decisions for safe and efficacious drug therapy. The objective of this study was to develop an algorithm for individualized dosing of i.v. busulfan in infants and children weighing ≤12 kg, that would achieve targeted exposure with the first dose of busulfan. Population PK modeling was conducted using intensive time-concentration data collected through the routine therapeutic drug monitoring of busulfan in 149 patients from 8 centers. Busulfan PK was well described by a 1-compartment base model with linear elimination. The important clinical covariates affecting busulfan PK were actual body weight and age. Based on our model, the predicted clearance of busulfan increases approximately 1.7-fold between 6 weeks to 2 years of life. For infants age

Hematopoietic stem cell gene therapy for neurological disorders relies on transmigration of donor-derived monocytes to the brain, where they can engraft as microglia and deliver therapeutic proteins. Many mouse studies use whole-body irradiation to investigate brain transmigration pathways, but chemotherapy is generally used clinically. The current evidence for transmigration to the brain after chemotherapy is conflicting. We compared hematopoietic donor cell brain engraftment after bone marrow (BM) transplants in busulfan- or irradiation-conditioned mice. Significantly more donor-derived microglial cells engrafted posttransplant in busulfan-conditioned brain compared with the irradiated, in both the short and long term. Although total Iba-1+ microglial content was increased in irradiated brain in the short term, it was similar between groups over long-term engraftment. MCP-1, a key regulator of monocyte transmigration, showed long-term elevation in busulfan-conditioned brain, whereas irradiated brains showed long-term elevation of the proinflammatory chemokine interleukin 1α (IL-1α), with increased in situ proliferation of resident microglia, and significant increases in the relative number of amoeboid activated microglia in the brain. This has implications for the choice of conditioning regimen to promote hematopoietic cell brain engraftment and the relevance of irradiation in mouse models of transplantation.

Hematopoietic stem cell transplantation (HSCT) is the standard of care in children with Hurler syndrome (HS) as it is the only therapy that can arrest disease progression. We examined the incidence, patterns and outcomes of graft failure in all HS children undergoing first HSCT at the Royal Manchester Children's Hospital or the University of Minnesota Children's Hospital from 1983 to 2016. Implementation of busulfan pharmacokinetic monitoring started in 2004 in both institutions. Two hundred and forty HS children were included in this analysis (historical era (pre-2004), n=131; current era (post 2004), n=109). The proportion of patients with graft failure was significantly lower in the current era compared with the historical era (37.2% vs 10.1%, respectively). Of 49 patients with graft failure in the historical era, 1 had aplasia and 48 had autologous reconstitution. All the 11 graft failures of the current era occurred in recipients of cord blood transplants (7 aplasia and 4 autologous reconstitution). The outcomes of second transplant in these patients has improved, with 89% of such patients alive and engrafted in the current era compared with 58% in the historical era. The pattern of graft failure has changed from autologous reconstitution, likely secondary to inadequate myelosuppression in the historical era, to aplasia in the current era, likely due to imperfect immunosuppression.

In order to support the clinical application of hematopoietic stem cell (HSC) gene therapy for mucopolysaccharidosis I (MPS I), biosafety studies were conducted to assess the toxicity and tumorigenic potential, as well as the biodistribution of HSCs and progenitor cells (HSPCs) transduced with lentiviral vectors (LV) encoding the cDNA of the alpha-iduronidase (IDUA) gene, which is mutated in MPS I patients. To this goal, toxicology and biodistribution studies were conducted, employing Good Laboratory Practice principles. Vector integration site (IS) studies were applied in order to predict adverse consequences of vector gene transfer and to obtain HSC-related information. Overall, the results obtained in these studies provided robust evidence to support the safety and tolerability of high-efficiency LV-mediated gene transfer and above-normal IDUA enzyme expression in both murine and human HSPCs and their in vivo progeny. Taken together, these investigations provide essential safety data to support clinical testing of HSC gene therapy in MPS I patients. These studies also underline criticisms associated with the use of currently available models, and highlight the value of surrogate markers of tumorigenicity that may be further explored in the future. Notably, biological evidence supporting the efficacy of gene therapy on MPS I disease and its feasibility on patients' HSCs were also generated, employing clinical-grade LVs. Finally, the clonal contribution of LV-transduced HSPCs to hematopoiesis along serial transplantation was quantified in a minimum of 200-300 clones, with the different level of repopulating cells in primary recipients being reflected in the secondary.

Mucopolysaccharidosis IIIB (MPS IIIB) is a lysosomal storage disorder characterized by severe behavioural disturbances and progressive loss of cognitive and motor function. There is no effective treatment, but behavioural testing is a valuable tool to assess neurodegeneration and the effect of novel therapies in mouse models of disease. Several groups have evaluated behaviour in this model, but the data are inconsistent, often conflicting with patient natural history. We hypothesize that this discrepancy could be due to differences in open field habituation and home cage behaviour. Eight-month-old wild-type and MPS IIIB mice were tested in a 1-h open field test, performed 1.5 h after lights on, and a 24-h home cage behaviour test performed after 24 h of acclimatization. In the 1-h test, MPS IIIB mice were hyperactive, with increased rapid exploratory behaviour and reduced immobility time. No differences in anxiety were seen. Over the course of the test, differences became more pronounced with maximal effects at 1 h. The 24-hour home cage test was less reliable. There was evidence of increased hyperactivity in MPS IIIB mice, however, immobility was also increased, suggesting a level of inconsistency in this test. Performance of open field analysis within 1-2 h after lights on is probably critical to achieving maximal success as MPS IIIB mice have a peak in activity around this time. The open field test effectively identifies hyperactive behaviour in MPS IIIB mice and is a significant tool for evaluating effects of therapy on neurodegeneration.

Norovirus infection is a major cause of nonbacterial gastroenteritis. In immunocompetent individuals the illness caused by norovirus is mostly self limiting. Excretion of norovirus has been reported to be prolonged in the immunocompromised including adult HSCT recipients. We report a case series of 13 children who received HSCT and required prolonged parenteral and enteral nutrition due to severe gut dysfunction accompanying protracted norovirus excretion that was monitored by RT-PCR. The median duration of viral excretion was 150 days (range 60-380) and the eventual clearance of norovirus from feces was closely associated with donor T cell recovery in the peripheral blood. There was no disease manifestation beyond the gut but the severity and length of norovirus associated illness suggests that HSCT should be delayed where possible in patients excreting the virus prior to conditioning therapy.

Early detection of mucopolysaccharidosis (MPS) is an important factor in treatment success; therefore, good disease biomarkers are vital. We evaluate heparin cofactor II-thrombin complex (HCII-T) as a biomarker in serum and dried blood spots (DBS) of MPS patients. Serum HCII-T and urine dermatan sulphate:chondroitin sulphate (DS:CS) ratio are also compared longitudinally against clinical outcomes in MPSI, II and VI patients following treatment. Samples were collected from MPS patients at the Royal Manchester Children's Hospital. DS:CS ratio was obtained by measuring the area density of spots from 2D electrophoresis of urinary glycosaminoglycans. Serum and DBS HCII-T was measured by sandwich ELISA. Serum HCII-T is elevated approximately 25-fold in MPS diseases that store DS, clearly distinguishing untreated MPSI, II and VI patients from unaffected age-matched controls. Serum HCII-T is also elevated in MPSIII, which leads to storage of heparan sulphate, with an increase of approximately 4-fold over unaffected age-matched controls. Urine DS:CS ratio and serum HCII-T decrease in response to treatment of MPSI, II and VI patients. HCII-T appears to respond rapidly to perturbations in treatment, whilst DS:CS ratio responds more slowly. HCII-T is a suitable biomarker for MPSI, II and VI, and it may also be informative for MPS diseases storing HS alone, such as MPSIII, although the elevation observed is smaller. In treated MPS patients, HCII-T and DS:CS ratio appear to measure short-term and long-term treatment outcomes, respectively. The potential value of HCII-T measurement in DBS for newborn screening of MPS diseases warrants further investigation.

There are a number of options for salvage treatment in children and adolescents with relapsed and refractory classical Hodgkin Lymphoma. These include salvage with standard dose chemotherapy, high dose chemotherapy with autologous stem cell transplant, allogeneic stem cell transplant or other novel approach. Radiotherapy has an important role in the salvage of some patients as part of a combined modality approach. This review outlines these salvage approaches and discusses whether the evidence from paediatric studies justifies a risk-adapted approach to salvage for individual patients or whether all patients should receive consolidation with high dose chemotherapy and autologous stem cell transplantation, which is often described as standard salvage management in adults. The important prognostic factors and how these may be used to allocate patients to standard versus high dose chemotherapy regimens are discussed. The role of allogeneic transplantation, novel agents and late effects will also be discussed.

Mucopolysaccharidosis IIIB (MPSIIIB) is a lysosomal storage disease characterised by progressive central nervous system degeneration in patients, with death usually in the late teens. Serious behavioural problems have been reported in children at the early stages of the disease, such as hyperactivity and severe sleep disturbances, which suggest alterations in circadian rhythms. We investigated the circadian rhythm of locomotor activity of young and old MPSIIIB mice, under a 24-h light-dark (LD) cycle and under constant darkness (DD), and also examined neuropeptide expression in the suprachiasmatic nucleus (SCN), site of the principal biological pacemaker. We show that MPSIIIB mice have higher activity levels during the light (resting) phase of the LD cycle, together with weaker circadian rhythms, and a longer active phase due to a late peak of activity, in both LD and DD. In addition, young MPSIIIB mice showed shorter phase delays in response to a light pulse in DD. Increased lysosomal storage, neuroinflammation and changes in the expression of Arginine Vasopressin and Vasointestinal Polypeptide, two circadian neuropeptides, were observed in the SCN, which may be in part responsible for the changes in circadian behaviour observed in MPSIIIB mice. These findings suggest an alteration of the circadian system in MPSIIIB mice, and may inform better clinical management of circadian, sleep and behavioural disturbances in patients with MPSIII.

Mucopolysaccharide (MPS) diseases are lysosomal storage disorders caused by deficiencies of enzymes catabolising glycosaminoglycans (GAGs). Abnormal GAG accumulation leads to symptoms including severe progressive neurological decline, skeletal deformities, organomegally, respiratory compromise and premature death. Treatment is available for some MPS diseases; enzyme replacement therapy for MPS I, II and VI, and haematopoietic stem cell transplantation for MPS I, VI and VII. These treatments are reliant on early diagnosis of the disease and accurate monitoring of treatment outcomes. Blood enzyme levels and total urinary GAGs are commonly used biomarkers in diagnosis of MPS but are not good measures of treatment outcome. Serum heparin cofactor II-thrombin complex (HCII-T), which is a GAG regulated serpin-protease complex, has recently been identified as a promising biomarker for MPS diseases. Here we present an assessment of the HCII-T biomarker in mouse models of MPS I, IIIA and IIIB, which suggests that HCII-T is a reliable marker for MPS I when measured in serum or dried blood spots stored for over a year at 4 degrees C, but that murine MPS IIIA and IIIB cannot be reliably detected using this biomarker. We also show that HCII-T formation in vivo is dependent on the presence of excess intravenous dermatan sulphate (DS), whilst intravenous heparan sulphate (HS), does not promote complex formation effectively. This suggests that HCII-T will prove effective as a biomarker for MPS I, II, VI and VII diseases, storing dermatan sulphate but may not be as appropriate for MPS III, storing heparan sulphate. With careful sample preparation, HCII-T ELISA could prove to be a useful biomarker for both newborn screening and measurement of treatment outcomes in selected MPS diseases.

Mucopolysaccharidosis type IIIB (Sanfilippo syndrome) is a lysosomal storage disease caused by a genetic defect in the production of alpha-N-acetylglucosaminidase. This results in lysosomal and extracellular accumulation of the undegraded glycosaminoglycan (GAG) substrate, heparan sulphate. Affected patients show progressive CNS degeneration characterised by mental retardation, hyperactivity and seizures, with death usually in the mid teens to early twenties. Visceral organ storage is also present but is relatively mild compared to other MPS diseases storing similar substrates. No treatments currently exist for MPS IIIB. Genistein is a broad spectrum protein tyrosine kinase inhibitor which acts on several different growth factor receptors, notably EGF and IGF receptors, both of which are important for proteoglycan synthesis. Recent work has shown that genistein can reduce GAG synthesis in patients' fibroblasts in vitro and there is evidence in patients to suggest that it may be an effective substrate reduction therapy agent for MPS III. Here we have tested the dose responses of MPS IIIB mice to daily sub-chronic dosing of genistein in half log increments compared to carrier over 8 weeks. We show clear reductions in liver lysosome compartment size in both sexes and significant dose dependent improvements in total liver GAGs and hair morphology in male MPS IIIB animals following genistein treatment. Male MPS IIIB mice exhibited considerably more liver storage than females and responded better to treatment. No changes in total GAGs, lysosomal size or reactive astrogliosis in the brain cortex were observed after 8 weeks of treatment despite evidence that genistein can cross the blood brain barrier. This is the first demonstration of genistein treatment in MPS models in vivo.

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.

A recent report, prepared in March 2003, regarding the paediatric transplantation activity registered between 1970 and 2002 in the European Bone Marrow Transplantation (EBMT) database showed a decrease in the number of registrations in 2001 and in 2002. In order to validate this observation, the Paediatric Diseases Working Party (PDsWP) secretariat distributed a questionnaire to 395 institutions participating in the EBMT Registry. Each institution was requested to check the number of transplants they reported and to confirm or to correct the figures. As of 15 March 2004, replies had been received from 135 centres reporting a median of 48 transplants per centre over the study period, total 17 891 (58% of the total number). Among them, 55 confirmed their original figures, while 80 corrected the numbers. The overall number of autologous and allogeneic SCTs performed and not reported were 461 and 692, respectively. Most of the teams that corrected their figures stated that their data managers could provide missing data to the EBMT; 260 other teams, each reporting a median of 15 transplants during the study period, total 12 866 (42% of the total number) chose not to reply. A report prepared in March 2004, following the PDsWP survey, showed an increasing number of transplants performed on patients below 18 years of age between 1973 and 2002 and reported to the EBMT Registry (328 autologous and 628 allogeneic) as compared to the 2003 report. This first PDsWP survey, reaching more than 50% of activity in the field, illustrates that the decrease in activity we observed in the 2003 report does not correspond to a decrease in the number of transplants that were actually performed. It demonstrates the compliance of most major paediatric institutions and confirms the important role of cooperation between National Registries and EBMT Registries.

Niemann Pick Type C2 (NPC2) is a rare autosomal recessive disease caused by mutations in the NPC2 gene (OMIM 601015). Clinically, NPC2 presents in most cases in the neonatal period with inflammatory lung disease, which may lead to death in the first year. If patients survive the neonatal period, they may develop a severe neurological disease. Here we present the developmental and neurological follow up at 5 years of age of a child with NPC2 successfully treated with allogenic bone marrow transplantation (BMT) at the age of 16 months. A homozygous p.E20X sequence variation previously associated with a severe phenotype was identified. In contrast to the previously reported patients with the same mutations, our patient has no respiratory compromise and has made some developmental progress (especially gross motor), though is significantly delayed (particularly in speech and language). Haematopoietic stem cell transplantation (HSCT) could be considered for patients with this mutation as long as performed early in the course of the disease.

Summary. Haemopoietic dysfunction, ranging from single-lineage cytopenia to severe aplasia and/or myelodysplasia (MDS), is prominent in Shwachman–Diamond syndrome (SDS). To assess haemopoietic stem cell proliferation in SDS, we compared leucocyte telomere length in 12 patients with SDS to that of 41 controls, using an in-gel hybridization technique. SDS patients had an age-adjusted mean telomere length 1·4 kilobase pairs (kbp) shorter than controls (P

Recent studies have demonstrated excessive telomeric shortening in peripheral blood leucocytes of bone marrow transplant (BMT) recipients. This finding has raised concerns about accelerated haemopoietic ageing that might predispose to clonal disorders and late graft failure. We studied the peripheral blood neutrophils and T cells of 14 fully engrafted long-term survivors of BMT. We found that in both neutrophils and T cells there was significant telomere shortening in the recipient (0.6 and 0.5 kb, respectively; P

Summary We retrospectively analysed the outcome of consecutive children with idiopathic severe aplastic anaemia in the United Kingdom who received immunosuppressive therapy (IST) or matched unrelated donor (MUD) haematopoietic stem cell transplantation (HSCT). The 6-month cumulative response rate following rabbit antithymocyte globulin (ATG)/ciclosporin (IST) was 32·5% (95% CI 19·3–46·6) (n = 43). The 5-year estimated failure-free survival (FFS) following IST was 13·3% (95% confidence interval [CI] 4·0–27·8). In contrast, in 44 successive children who received a 10-antigen (HLA-A, -B, -C, -DRB1, -DQB1) MUD HSCT there was an excellent estimated 5-year FFS of 95·01% (95% CI 81·38–98·74). Forty of these children had failed IST previously. HSCT conditioning was a fludarabine, cyclophosphamide and alemtuzumab (FCC) regimen and did not include radiotherapy. There were no cases of graft failure. Median donor chimerism was 100% (range 88–100%). A conditioning regimen, such as FCC that avoids total body irradiation is ideally suited in children. Our data suggest that MUD HSCT following IST failure offers an excellent outcome and furthermore, if a suitable MUD can be found quickly, MUD HSCT may be a reasonable alternative to IST.

Background: Neurodegenerative metabolic disorders such as mucopolysaccharidosis IIIB (MPSIIIB or Sanfilippo disease) accumulate undegraded substrates in the brain and are often unresponsive to enzyme replacement treatments due to the impermeability of the blood brain barrier to enzyme. MPSIIIB is characterised by behavioural difficulties, cognitive and later motor decline, with death in the second decade of life. Most of these neurodegenerative lysosomal storage diseases lack effective treatments. We recently described significant reductions of accumulated heparan sulphate substrate in liver of a mouse model of MPSIIIB using the tyrosine kinase inhibitor genistein. Methodology/Principal Findings: We report here that high doses of genistein aglycone, given continuously over a 9 month period to MPSIIIB mice, significantly reduce lysosomal storage, heparan sulphate substrate and neuroinflammation in the cerebral cortex and hippocampus, resulting in correction of the behavioural defects observed. Improvements in synaptic vesicle protein expression and secondary storage in the cerebral cortex were also observed. Conclusions/Significance: Genistein may prove useful as a substrate reduction agent to delay clinical onset of MPSIIIB and, due to its multimodal action, may provide a treatment adjunct for several other neurodegenerative metabolic diseases. © 2010 Malinowska et al.

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'.

Hurler Syndrome is corrected by allogeneic BMT by the action of donor enzyme on recipient tissue. In this paper, we describe monitoring of 39 patients transplanted in two centres to determine donor chimerism, enzyme level and residual substrate - expressed as dermatan sulphate to chondroitin sulphate ratio. We show that in fully engrafted recipients, the enzyme level, expressed as mumol/g total protein/h, post-transplant is 24.2 from an unrelated donor and 10.2 from a heterozygote family donor (P

The classical treatment algorythm of idiopathic severe aplastic anemia (SAA) forsees, if a sibling donor is not available in the family, a first line option represented by immunosuppressive therapy (IST) with ATG and Cyclosporine A (CsA). IST provides a very good survival rate but a fairly high rate of failures (no response, relapse, need for transplant). This is particularly important in childhood and adolescence where faulty hematopoiesis represents a relevant limitation of quality of life. The option of haematopoietic stem cell transplantation (HSCT) from matched unrelated donor (MUD) as front line treatment with no prior failed IST, has never been investigated so far. This study provides for the first time the outcome analysis of 29 consecutive SAA patients who received an upfront unrelated donor HSCT without prior IST, in 9 UK Centres and compares each of these patient with 3 matched controls from the data base of the SAAWP of the EBMT who, in a similar time-span, underwent Matched Sibling Donor (MSD) HSCT. Twenty four patients received HLA-A,-B,-C,-DQ-,-DRB1 matched MUD HSCTs whilst 5 received single antigen/allele Mismatched Unrelated Donor (MMUD) grafts. The conditioning regimen was FCC (Fludarabine, Cyclophosphamide and Alemtuzumab) with the addition of low dose (3cGy) TBI for the MMUD HSCTs. GVHD prophylaxis was with CsA +-Mycophenolate. The primary endpoints were overall survival (OS) and event free survival (EFS). Events were death, disease recurrence, second HSCT, clinical PNH and post-transplant malignancies. Complete remission (CR) was defined as Hb > 10 g/dl, neutrophil count > 1x 109/l and platelet count > 100x109/l. The 87 (3 for each MUD/MMUD HSCT) patients from the EBMT data base who underwent MSD HSCT as first line treatment were matched to the upfront MUD/MMUD cohort by age, gender, time from diagnosis to HSCT and source of stem cells. Their median follow-up was 1.6 years (range 0.01-9.39). In the upfront MUD/MMUD cohort there were 12 males (41%) and 17 females (59%). Median age at HSCT was 8.46 years (range 1.73-19.11), 72% was aged below and 28% above 12 years; median interval from diagnosis to HSCT was 0.39 years (range 0.19-1.35) with 72 % receiving bone marrow and 28% peripheral blood stem cells. The median follow-up was 1.7 years (range 0.19-8.49). The median time to neutrophil recovery (>0.5 x 109/l) was 18 days (range 9-29) and the median time to platelet recovery (>50 x 109/l) was 19 days (range 10-40). The 100 day cumulative incidence (CI) of grade II-IV acute GVHD was 10 ± 6% ; there was only one case of grade III-IV acute GVHD (frequency of 3.5%). The 1 year CI of chronic GVHD was 19 ± 8%. There were 5/29 cases (frequency of 17,2 %), 4 in the MUD and 1 the MMUD subset. Chronic GVHD was limited in all cases and restricted to skin. There were only 2 events in this cohort; one primary graft failure following a HLA-A MMUD HSCT who has now successfully received a second HSCT and one death due to idiopathic pneumonia syndrome. No post-treatment malignancies occurred at last follow-up. The other 27 patients are in CR at last follow-up. The median interval diagnosis-neutrophil recovery was similar in MUD/MMUD (0.39 years; range 0.19-1.35) vs MSD transplants (0,31 years; range 0,1- 0,45) (p=0.93). The 2 year OS was 96%±4% in the upfront MUD/MMUD cohort vs 91%±3% of the MSD cohort (p=0.30). The 2 year EFS was 92%±5% in the upfront cohort vs 87%±4% in MSD (p=0.37) (Fig 1). The 2 year CI of rejection was 4%±4% in the MUD/MMUD vs .1%±1% in the MSD group (p=0.48). This is the first controlled study indicating that in idiopathic SAA of childhood and adolecence upfront MUD/MMUD HSCT using FCC as conditioning regimen, has similar outcome to MSD HSCT. MUD HSCT may be considered a feasible and successful treatment option when children lack a MSD and a MUD is readily available. Figure 1 Figure 1. Comparison of EFS between upfront MUD/MMUD and front-line MSD HSCT. Events were: death, disease recurrence, second HSCT, clinical PNH and post-transplant- malignancies. Table 1 EFS N events 2-yrs Pr (%) (SE) p-value MUD/MMUD 29 2 92 (5) 0.37 MSD 87 11 87 (4) Disclosures Dufour: Pfizer: Consultancy.

Bone marrow transplantation (BMT) is increasingly used in an attempt to correct inborn errors of metabolism (IEM). However, little is known about effects of BMT from patients with IEM donating for non-affected recipients. We present data from a 8.5-year-old girl who underwent BMT in second remission for relapsed acute lymphoblastic leukaemia (ALL) at the age of 7 years from her HLA-identical brother who was severely affected by Hunter syndrome (Mucopolysaccharidosis type II, iduronate-2-sulphatase (IDS) deficiency). After BMT not only leukocyte but also plasma activity of IDS was absent. Mixing experiments and immunoadsorption suggest antibody-mediated enzyme inhibition. However, her urinary glycosaminoglycan excretion has not increased post BMT and clinical signs of mucopolysaccharidosis are absent 20 months after BMT. We conclude that patients with white cell enzyme deficiencies and other IEMs do not have to be excluded from bone marrow donation. Antibody production by the graft may occur and be reflected by a marked reduction in plasma enzyme levels but not tissue activity. Similar antibody responses resulting in enzyme inactivation might also affect other enzyme replacement strategies for individuals with IEM.

Worldwide more than 400 patients with Hurler’s syndrome (HS), characterized by severe neurodegeneration, cardiac disease, skeletal abnormalities and death in early childhood, have undergone allogeneic-HSCT since 1980. Although, long term follow up of successfully transplanted children is very encouraging, the engraftment and survival results are very variable between the various studies, ranging from less than 25% up to more than 85%. We retrospectively analyzed the results of 146 patients transplanted in Europe from 1994–2004, to assess: 1) the effect of conditioning regimen and grafts (-manipulation) used on the “alive and engrafted” rate and 2) the transplantation-related morbidity/mortality. HSCT with a family donor was performed in 52 patients. An unrelated donor was used in 94 patients. The majority of patients received marrow (n=103). The rest received cordblood (n=23) or peripheral blood (n=20). Twenty-eight patients received a T-cell depleted (TCD) graft. Conditioning regimens used were grouped as follows: busulfan-cyclofosfamide 200mg/kg (n=68), busulfan- with high dose cyclofosfamide (either 240mg/kg or 260mg/kg; n=41), fludarabine-based myelo-ablative (n=19) and reduced intensity conditioning regimens (RIC: n=18). Fourteen patients received dose-adjusted busulfan. Engrafted was defined as a donor chimerism of more than 10%, and an alpha-L-iduronidase level of more than the lower limit of normal for the heterozygote individuals (>4.5 nmol/hr/mg). The “alive and engrafted” rate after first transplantation and overall “alive and engrafted” rate after one to three transplantations was 83/146 (57%) and 111/146 (76%), respectively. The median follow up was 39 mth (5–120mths). Multivariate analysis (confounders: age, sex, heterozygote donor, unrelated donor, stem cell source, HLA-disparity, conditioning regimen, TCD and busulfan targeting) on the primary endpoint “alive and engrafted” showed that RIC (RR 13,4: 2,6–67,1) and TCD (RR 5,7: 1,14–28,4) are individual risk factor for graft failure. Busulfan targeting suggests to be an individual protective factor (RR 0,27; 0,04–1,8); 12/14 (86%) were “alive and engrafted” after 1st HSCT. Thirthy three patients received a 2nd graft, of whom 26/33 (82%) are alive and engrafted: 16/21 using the same, 10/12 using a different donor, and 16/19 after myeloabaltive, 10/14 after RIC. Two of the 3rd HSCTs were successful. After 1st HSCT moderate to severe aGvHD (grade ≥2) occurred in 23/146 (16%) patients. Extensive cGvHD was seen in 2/114 (1.4%) patients, only. IPS/DAH was seen in 4/134 (2.3%) patients and VOD in 12/134 (9%) patients. Main cause of death (n=28) was infectious (n=15: mainly viral). Other causes of death: GvHD (n=3), Cardiac ECI (n=2), VOD (n=2), DAH (n=1), unknown (n=1), Hurler (n=4). The majority of the “alive and engrafted” patients have a donor chimerism of >95% (91/111; 82%), 11/111 (9,9%) between 75–95%, 6/111 (5,4%) between 50–75% and 3/111 (2.7%) between 10–50%. In summary, no stem cell source (BM, cordblood and PBSC) is superior and no conditioning regimen used is superior. RIC and TCD results in inferior engraftment rates. Second HSCTs are successful in more than 80%. Relatively low morbidity rates are seen. The engraftment of HSCT for HS can be optimized by avoiding T-cell depletion, RIC and probably by busulfan-targeting.

Worldwide more than 400 patients with Hurler’s syndrome (HS), characterized by severe neurodegeneration, cardiac disease, skeletal abnormalities and death in early childhood, have undergone allogeneic-HSCT since 1980. Although, long term follow up of successfully transplanted children is very encouraging, graft-failure, mortality and morbidity can limit its success. Although ERT (laronidase) is available for HS, a HSCT is still needed to abort severe neurodegeneration. We analyzed 21 patients who underwent a HSCT with ERT in Europe between 12/2003 to 4/2005, to assess: the effect of ERT on the “alive and engrafted” rate and the transplantation-related morbidity/mortality. Twenty-one patients received ERT (100 U/kg weekly) from diagnosis until approximately 7 weeks (range 0–20 wks) after successful HSCT. Clinical condition was, except for 1 patient, moderate/good before start of ERT. Thirty-one HSCTs were performed in these patients: 8 patients received a 2nd graft because of graft-failure of whom 2 received a 3th graft. For the first HSCT, 8 patients received full-graft marrow or PBSC, the others T-cell depleted (marrow/PBSC: n=3) or cordblood (n=10). Sixteen unrelated donors and 5 family donors (4sibs/1haplo) were used. Conditioning regimens used were conventional myeloablative (n=17) or treosulfan / fludarabine (n=4). Engrafted was defined as a donor chimerism of more than 10%, and an alpha-L-iduronidase level >LLN for heterozygote individuals (>4.5 nmol/hr/mg). Effect of ERT on the “alive and engrafted” rate was analysed in a multivariate analysis with a historic (transplanted 1994–2004) control group (n=148). Clinical condition before HSCT was good for all patients. The “alive and engrafted” rate after first HSCT and overall “alive and engrafted” rate after one to three HSCTs was 12/21 (57%) and 17/21 (81%), respectively. The median follow up was 8 mth (3–17mths). Two patients died: 1 after 2nd and 1 after 3th HSCT. HSCTs with full-graft were successful in 5/8, and for TCD 0/3 and cordblood 8/10. Five of 8 patients receiving a 2nd graft are alive and engrafted and 1/2 after 3rd graft. Multivariate analysis (confounders: age, sex, heterozygote donor, unrelated donor, stem cell source, HLA-disparity, conditioning regimen, T-cell depletion and busulfan-targeting) on the primary endpoint “alive and engrafted” showed that ERT doesn’t significantly influence the primary endpoint (RR 0,54: 0,15–2). After 1st HSCT moderate to severe aGvHD (grade ≥2) occurred in 1/21 (4,8%) patients. Extensive cGvHD wasn’t seen. IPS/DAH was seen in 1/21 (4,8%) and VOD in 2/21 (9,6%) patients. Cause of death was extensive cGvHD after 3rd and candida sepsis after 2d HSCT. The majority of the overall “alive and engrafted” patients are having a donor chimerism of >95% (15/17), 2 patients are mixed chimeric (75–95%). ERT-infusion-related toxicity was limited to mild reactions. In patients with HS, ERT with HSCT is well tolerated, with low morbidity rates, but had no effect (neither positive nor negative) on the engraftment in this group. Specifically for patients in a poor clinical condition preHSCT, ERT might be of advantage. A prospective study is needed to determine the longer term outcome of ERT on HSCT outcomes.