Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis.

Osteopetrosis includes a group of inherited diseases in which inadequate bone resorption is caused by osteoclast dysfunction. Although molecular defects have been described for many animal models of osteopetrosis, the gene responsible for most cases of the severe human form of the disease (infantile malignant osteopetrosis) is unknown. Infantile malignant autosomal recessive osteopetrosis (MIM 259700) is a severe bone disease with a fatal outcome, generally within the first decade of life. Osteoclasts are present in normal or elevated numbers in individuals affected by autosomal recessive osteopetrosis, suggesting that the defect is not in osteoclast differentiation, but in a gene involved in the functional capacity of mature osteoclasts. Some of the mouse mutants have a decreased number of osteoclasts, which suggests that the defect directly interferes with osteoclast differentiation. In other mutants, it is the function of the osteoclast that seems to be affected, as they show normal or elevated numbers of non-functioning osteoclasts. Here we show that TCIRG1, encoding the osteoclast-specific 116-kD subunit of the vacuolar proton pump, is mutated in five of nine patients with a diagnosis of infantile malignant osteopetrosis. Our data indicate that mutations in TCIRG1 are a frequent cause of autosomal recessive osteopetrosis in humans.

Combination of enzyme replacement and hematopoietic stem cell transplantation as therapy for Hurler syndrome.

Hurler syndrome (mucopolysaccharidosis type I, MPS IH) is characterized by a deficiency of alpha-L-iduronidase resulting in progressive multiorgan dysfunction. We sought to determine whether enzyme replacement therapy (ERT) with iduronidase in the peritransplant period affects outcome of hematopoietic stem cell transplantation (HSCT) for MPS IH. Seven children with MPS IH at a median age of 1.5 years at the time of myeloablative HSCT were eligible. All patients had null mutations in IDUA gene. Iduronidase (0.58 mg/kg per dose) was administered intravenously in 11-14 weekly doses before HSCT and 8 weekly doses after HSCT. The infusions were well tolerated. All patients developed antibodies to iduronidase but all engrafted with >90% donor hematopoiesis. A majority of patients had significant pulmonary complications before ERT and HSCT but all are alive and well with a median follow-up of more than 1 year after HSCT. This suggests that ERT prior to HSCT is unlikely to alter engraftment. In addition, morbidity was acceptable, despite a previous history of pulmonary difficulties that suggested that these patients were high risk for these complications. Therefore, we recommend treatment of MPS IH patients with combination of ERT and HSCT therapy to further investigate its potential to enhance outcomes with HSCT.

Growth and endocrine function in patients with Hurler syndrome after hematopoietic stem cell transplantation.

Short stature is characteristic of Hurler syndrome, or mucopolysaccharidosis type IH (MPS IH). Hematopoietic stem cell transplantation (HSCT) is used to treat children with MPS IH. While HSCT corrects some of the metabolic features of MPS IH, its effects on growth are not well delineated. We investigated growth in patients with MPS IH after HSCT and described accompanying endocrine abnormalities. A cohort of 48 patients with MPS IH who had received HSCT between 1983 and 2005 were included. The prevalence of short stature (height <-2 s.d. score, SDS) before HSCT was 9%, and increased to 71% at last follow-up (6.9+/-5.1 years after HSCT). Short stature was positively associated with increased age at HSCT (P=0.002) and TBI (P=0.009). In total, 23% had growth hormone deficiency and/or low insulin-like growth factor-1, one female patient had premature adrenarche, one precocious puberty and 27% had clinical or subclinical hypothyroidism. Growth failure is highly prevalent in children with MPS IH after HSCT. Children who had no TBI exposure and were younger at the time of HSCT had a better height outcome.

N-acetyl-L-cysteine improves outcome of advanced cerebral adrenoleukodystrophy.

Hematopoietic stem cell transplantation as a treatment for childhood cerebral adrenoleukodystrophy (ALD) has historically only been successful in early disease. As ALD is associated with oxidative damage, we reasoned that adjunctive therapy with an antioxidant agent, N-acetyl-L-cysteine (NAC), may provide protection from rapid neurologic decline in boys with advanced cerebral disease. We report three boys with advanced ALD, whose neurologic status and brain radiographic findings were stabilized by treatment including NAC 8-11 months after hematopoietic stem cell transplantation. These results contrast with previous survival data in cerebral ALD patients who had a similar degree of brain involvement, all of whom died within 1 year of stem cell infusion despite a full donor engraftment. Thus, NAC merits investigation as a therapeutic strategy for patients with advanced ALD as an intervention that could change this lethal disease to a condition amendable to treatment with hematopoietic stem cell transplantation.

Analysis of factors affecting development of carpal tunnel syndrome in patients with Hurler syndrome after hematopoietic cell transplantation.

Children with Hurler syndrome (mucopolysaccharidosis type IH (MPSIH)) have skeletal, joint and soft tissue abnormalities that may persist or progress after hematopoietic stem cell transplantation (HSCT). We report our single center experience with development of carpal tunnel syndrome (CTS) in 43 children with MPSIH after HSCT. Twenty-three children (59%) developed CTS following HSCT; 19 of the 39 children with enzyme activity in the normal or heterozygous range developed CTS (49%), whereas all four children with low heterozygous or absent enzyme activity developed CTS after HSCT. Fourteen of 19 related donor marrow recipients, eight of 19 of those receiving an unrelated donor graft and one of five unrelated cord blood recipients developed CTS. The mean age at surgical release was 4.8 years. With each year increase in age at HSCT, there was a 55% increased risk. Age and enzyme activity after HSCT were significant factors in the development of CTS. Transplantation by 2 years of age reduced the risk of developing CTS by 46%; higher enzyme activity led to a 78% reduction in the risk of developing CTS. However, children transplanted for MPSIH remain at risk for the development of CTS, and should be monitored on an ongoing basis by nerve conduction velocity testing.

Non-myeloablative conditioning for second hematopoietic cell transplantation for graft failure in patients with non-malignant disorders: a prospective study and review of the literature.

Allogeneic hematopoietic cell transplantation (HCT) effectively treats several non-malignant disorders such as selected lysosomal disorders, cerebral adrenoleukodystrophy and hemoglobinopathies. However, rates of graft failure (GF) in non-malignant populations exceed those of patients with malignant indications for HCT. Salvage conditioning regimens and outcomes for second HCT for GF vary immensely in the literature. We report 17 consecutive pediatric patients with non-malignant disorders who underwent a second allogenic HCT for GF using a non-myeloablative, low-dose busulfan-based regimen. Graft sources for the second transplant included umbilical cord blood, unrelated bone marrow and unrelated PBSCs. Median age at time of second HCT was 6.6 years (1.1-14.6 years). Fourteen of seventeen patients (82%) achieved engraftment, with a 3-year overall survival of 82% (95% CI, 54-94%). Day 100 transplant-related mortality was 12% (95% CI, 0-27%). CMV and adenovirus reactivation occurred in 30% and fungal infections in 18%. The incidence of grade II-IV acute GvHD disease was 35% (95% CI, 13-58%) with only 6% grade III-IV (95% CI, 0-17%). In summary, we illustrate excellent overall survival and acceptable toxicity using a non-myeloablative conditioning regimen for second HCT as salvage therapy for first GF in patients with non-malignant conditions.

Changes in the incidence, patterns and outcomes of graft failure following hematopoietic stem cell transplantation for Hurler syndrome.

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.

Intensity of MRI Gadolinium Enhancement in Cerebral Adrenoleukodystrophy: A Biomarker for Inflammation and Predictor of Outcome following Transplantation in Higher Risk Patients.

BACKGROUND AND PURPOSE: Outcomes following hematopoietic stem cell transplantation for higher risk childhood-onset cerebral adrenoleukodystrophy are variable. We explored whether a brain MR imaging gadolinium intensity scoring system improves prediction of neurologic outcome. MATERIALS AND METHODS: We developed a 4-point scale of gadolinium intensity relative to the choroid plexus: 0 = no enhancement; 1 = hypointense; 2 = isointense; 3 = hyperintense. The interobserver concordance of the scale was assessed on 30 randomly chosen studies. Scores were generated for 64 evaluable patients and compared with CSF chitotriosidase levels, a known inflammatory marker correlating with outcomes following transplantation. For 25 evaluable higher risk patients (Loes ≥10), the gadolinium intensity score was compared with longer term posttransplantation clinical change. RESULTS: The gadolinium intensity scoring system showed good interobserver reproducibility (κ = 0.72). Of 64 evaluable boys, the score positively correlated with average concomitant CSF chitotriosidase activity in nanograms/milliliter/hour: 0: 2717, n = 5; 1: 3218, n = 13; 2: 6497, n = 23; and 3: 12,030, n = 23 (P < .01). For 25 evaluable higher risk patients, more intense pretransplantation brain MR imaging gadolinium enhancement predicted greater average loss on the adrenoleukodystrophy neurologic function scale following transplantation: 0/1: adrenoleukodystrophy neurologic function scale score difference = 4.3, n = 7; 2/3: adrenoleukodystrophy neurologic function scale score difference = 10.4, n = 18 (P = .05). CONCLUSIONS: Gadolinium enhancement intensity on brain MR imaging can be scored simply and reproducibly for cerebral adrenoleukodystrophy. The enhancement score significantly correlates with chitotriosidase. In boys with higher risk cerebral disease (Loes ≥10), the enhancement score itself predicts neurologic outcome following treatment. Such data may help guide treatment decisions for clinicians and families.

Long-Term Cognitive and Functional Outcomes in Children with Mucopolysaccharidosis (MPS)-IH (Hurler Syndrome) Treated with Hematopoietic Cell Transplantation.

The long-term cognitive and functional outcomes of children with mucopolysaccharidosis type I (MPS-IH) post-hematopoietic cell transplant (HCT) are not well documented, and the role of genetic and treatment factors in these outcomes has yet to be defined. In this multi-site, international study, we (1) characterize the cognitive and functional status of 47 individuals (ages 2-25, mean of 10.6 years) with MPS-IH who are 1-24 years post HCT (mean = 9 years) and (2) examine contributions of genotype, transplant characteristics, and sociodemographic factors to cognitive ability, adaptive behavior, and quality of life. The overall cognitive ability of our sample was mildly impaired, more than two standard deviations below general population norms. Parent reported adaptive behaviors (i.e., communication, daily living, and motor skills) were similarly impaired with a relative strength in socialization. Quality of life, as reported by parents, fell more than two standard deviations below population norms for physical functioning; however, psychosocial quality of life (emotional well-being) approximated population norms. In linear regression analysis, adjusted for demographic and treatment factors, mutation severity was associated with lower cognitive ability (p = 0.005) and adaptive functioning (p = 0.004), but not parent ratings of children's quality of life. Older age at HCT was associated with poorer physical quality of life (p = 0.002); lower socioeconomic status (p = 0.028) and unrelated bone marrow HCT (p = 0.010) were associated with poorer psychosocial quality of life. Implications for screening and early intervention for children at risk for poorer cognitive and functional outcomes are described.

Unrelated donor bone marrow transplantation for children with acute leukemia.

PURPOSE: To test the use of unrelated donor bone marrow transplantation (URD BMT) to cure children with high-risk acute leukemias. PATIENTS AND METHODS: Between June 1985 and December 1994, 50 children with acute leukemia (15 acute myelogenous leukemia [AML], 35 acute lymphoblastic leukemia [ALL]; 22 greater than second complete remission [CR]) received BMT from a URD at the University of Minnesota. Ages ranged from 0.9 to 17.5 years (median, 8.8). Median follow-up is 2.1 years (range, 1 to 7.3). Thirty patients (60%) received bone marrow fully matched at HLA-A,B and DRB1; 20 (40%) received bone marrow with a major or minor mismatch at a single HLA-A or B locus. RESULTS: The median time to neutrophil engraftment was day 24 (range, 14 to 42 days) in those receiving matched and day 25 (range, 15 to 32 days) in those receiving mismatched marrow (P = .35). The incidence of grades III to IV graft-versus-host disease (GVHD) was 23% (95% confidence interval [CI], 7% to 39%) in matched and 32% (95% CI, 8% to 52%) in HLA-mismatched patients (P = .57). The incidence of chronic GVHD was 50% (95% CI, 28% to 72%) in matched and 57% (95% CI, 23% to 91%) in mismatched patients (P = .80). Disease-free survival for patients with ALL is 37% (95% CI, 21% to 53%) at 1 year and 30% (95% CI, 15% to 46%) at 2 years; for patients with AML, 53% (95% CI, 28% to 78%) at 1 year and 33% (95% CI, 6% to 60%) at 2 years. CONCLUSION: URD BMT is an effective treatment for children with poor-prognosis acute leukemia and should be considered for all high-risk patients. Early referral of patients is strongly recommended.

Circulating macrophage colony-stimulating factor is not reduced in malignant osteopetrosis.

Malignant osteopetrosis is a disorder characterized by a deficiency in osteoclast number or function. In one animal model of osteopetrosis, the op/op mouse, macrophage colony-stimulating factor (M-CSF) is absent, and the administration of M-CSF corrects the defects. We evaluated the serum of 13 patients with malignant osteopetrosis by an M-CSF radioimmunoassay to determine if a quantitative M-CSF deficiency existed in these patients. All patients had M-CSF present in levels equal to or higher than control serum. In addition, serum from 6 osteopetrotic patients was tested in a bioassay to determine if the M-CSF present is biologically active, and in all cases there was demonstrable activity in these samples. We provide evidence that deficiency of circulating M-CSF is unlikely to be a major contributor to the etiologic basis for the majority of children with malignant osteopetrosis.

Endogenous IL-2 production by natural killer cells maintains cytotoxic and proliferative capacity following retroviral-mediated gene transfer.

Interleukin (IL)-2 therapy given at tolerable doses is insufficient to induce maximum activation of natural killer (NK) cells. We recently demonstrated that NK cells expanded in vivo can be maximally activated by short-term ex vivo incubation with 1000 U/mL IL-2. However, IL-2 withdrawal, which would occur with reinfusion, may lead to a rapid loss of cell viability and function. We hypothesized that retroviral transduction could provide an endogenous source of IL-2 to maintain NK function as measured by proliferation and cytotoxicity. Enriched NK cells were transduced with supernatants containing an MFG-based retrovirus designed to express murine IL-2 cDNA. Several supernatant transduction strategies were evaluated. NK cells were initially cultured in 1000 U/mL of huIL2 for 7-8 days, harvested, and replated prior to transduction (4 hours at 37degrees C); this proved insufficient to sustain NK proliferation or maintain cytotoxicity after exogenous human IL-2 (huIL-2) withdrawal. An alternative transduction procedure using phosphate-depleted medium, centrifugation, and transduction for 16 hours at 32degrees C was then evaluated. NK cells transduced under these conditions maintained significant NK proliferation in the absence of exogenous IL-2 compared with sham-transduced controls. Two consecutive daily transductions resulted in less proliferation, suggesting that several exposures to retroviral supernatant may inhibit subsequent NK proliferation. Cytotoxicity of the transduced NK cells against K562 and Raji was maintained under these conditions without exogenous IL-2. Sham-transduced NK cells produced 8.3+/-2.6 U/mL of murine IL-2 (muIL-2) by ELISA (background) after 7 days without exogenous IL-2. In contrast, 109+/-23 U/mL muIL-2 was produced by NK cells transduced with supernatant from the MFG/muIL-2 producer line. These experiments demonstrate that NK cells can be successfully transduced with retroviruses and induced to express sufficient IL-2 to maintain their proliferative and cytotoxic functions. Transduction of IL-2 genes into NK cells may offer advantages over exogenous IL-2 administration in maintaining maximum function for use in antitumor immunotherapy.

Retroviral-mediated transfer of the murine interleukin-3 gene engineered for intracellular retention results in a myeloproliferative syndrome but is associated with circulating interleukin-3 levels.

Myeloid leukemias have been shown to secrete as well as respond to cytokines such as interleukin-3 (IL-3) with an increased growth rate and may therefore become self-stimulatory through an external autocrine mechanism. In vitro evidence that IL-3 is functional within the intracellular compartment has been obtained through modification of the murine IL-3 gene to encode for the amino acids SEKDEL on the carboxyl terminus of the protein, resulting in preferential intracellular retention. The ability of bone marrow-derived hematopoietic progenitor cells to increase their proliferative capacity through intracellular mechanisms was investigated in vivo using retroviruses containing the wild-type or SEKDEL-modified IL-3 gene, transcriptionally regulated by the retroviral long terminal repeat (LTR) or by the SV40 early promoter, in lethally irradiated, bone marrow-reconstituted mice. Bone marrow cells exposed to the N2KDEL virus containing the SEKDEL-modified IL-3 gene were shown by bioassay to retain large amounts of IL-3 intracellularly, and the presence of an integrated provirus containing the SEKDEL sequences was demonstrated by polymerase chain reaction (PCR) in the spleen and bone marrow of these animals. Transduction with all four types of IL-3 viruses resulted in dramatic increases in the circulating white blood cell (WBC) count; this myeloproliferative state occurred within several weeks following bone marrow transplantation (BMT), when viruses expressing the IL-3 or modified gene were under transcriptional regulation of the viral LTR, and approximately 2 months post-BMT, when they were under control of the SV40 internal promoter. Serum levels of IL-3 were measured in transplanted animals and found to be markedly increased in each case in which WBC elevation was observed, including mice receiving marrow transduced with constructs containing the IL-3 gene modified for intracellular retention. No animals were observed in which myeloproliferation occurred without secretion. From these experiments, it seems unlikely that exclusively intracellular mechanisms are a major contributor to the development of the myeloproliferative syndrome observed in these animals.

Flt3 ligand antitumor activity in a murine breast cancer model: a comparison with granulocyte-macrophage colony-stimulating factor and a potential mechanism of action.

We have shown that Flk2/Flt3 ligand (Flt3L)-transduced tumor vaccine induces transferable T cell protection against a murine breast cancer cell line, but a direct comparison with the potent effector GM-CSF, the activity against preestablished tumors, and the mechanism of antitumor response in this breast cancer model are not known. We compared vaccination with C3L5 cells expressing Flt3L (C3Lt-Flt3L) and GM-CSF (C3L5-GMCSF) by injecting 1 x 10(4) cells subcutaneously into the chest wall and then, after 4 weeks, challenging the contralateral chest of tumor-free mice with parental C3L5 cells. C3L5-Flt3L and C3L5-GMCSF had reduced in vivo growth rates (25% tumor formation each) compared with 100% tumor formation of C3L5 cells expressing only neomycin phosphotransferase (C3L5-G1N). However, when tumor-free animals were challenged with parental C3L5 cells, C3L5-Flt3L vaccination was significantly better at preventing tumor growth (p < 0.05) than C3L5-GMCSF vaccination (33% of C3L5-Flt3L-vaccinated animals developed tumor compared with 77% of C3L5-GMCSF-vaccinated animals). Adoptive transfer of immunity for both vaccines was demonstrated; splenic T cells from tumor-free mice protected naive mice from parental tumor challenge. To simulate minimal disease, parental C3L5 cells at two concentrations (high, 5 x 10(3) cells; or low, 1 x 10(3) cells) were injected into the contralateral chest wall 4 days prior to treatment with C3L5-G1N or C3L5-Flt3L. C3L5-Flt3L treatment decreased contralateral parental tumor formation (high, 67% tumor free; low, 90% tumor free) compared with C3L5-G1N treatment (high and low, 0% tumor free). Immunodepletion of activated natural killer cells with anti-asialo-GM1 blocked C3L5-Flt3L- and C3L5 plus soluble Flt3L-mediated antitumor activity. Thus, Flt3L-transduced tumor cells manifest potent antitumor activity, apparently mediated, at least partially, by natural killer cells.

B7-1 expression decreases tumorigenicity and induces partial systemic immunity to murine neuroblastoma deficient in major histocompatibility complex and costimulatory molecules.

Neuroblastoma may escape an immune attack by virtue of its low expression of surface accessory molecules essential in the antitumor response. Murine neuroblastoma, neuro-2a, was transduced with the retroviral vector LB7-1SN to examine the influence of B7-1 expression on the immune response directed against a low major histocompatibility class (MHC) I and class II negative, B7-2, and ICAM-1 negative tumor. Using a retroperitoneal model for implantation of neuroblastoma in its natural site, we demonstrated that expression of B7-1 by neuro-2a reduces its tumorigenicity. Coinjection of B7-1-positive and -negative cells improved survival compared with mice receiving B7-1-negative cells alone. This was dependent on the ratio of B7-1+ to B7-1- neuro-2a cells injected. CD8+ and not CD4+ T-cell depletion significantly increased tumor-induced mortality in syngeneic A/J mice, indicating that B7-1 decreases tumorigenicity primarily by direct constimulation of CD8+ T cells. Rejection of N-2a/B7-1 tumors or preimmunization with irradiated N-2a/B7-1 cells die not increase protection to challenge with unmodified neuro-2a cells over mice vaccinated with N-2a/neo. Furthermore, cytotoxic T lymphocyte (CTL) precursor frequencies were not significantly higher after in vivo priming and in vitro stimulation with irradiated N-2a/B7-1 compared with N-2a/neo, indicating that B7-1 costimulation by the tumor, in the absence of adequate antigen presentation by MHC molecules, may limit the generation of effective CTLs.

Irradiation of singly and doubly transduced murine neuroblastoma cells expressing B7-1 and producing interferon-gamma reduces their capacity to induce systemic immunity.

We have previously reported that immunization with low major histocompatibility complex (MHC) class I expressing murine neuroblastoma (neuro-2a) transduced with B7-1 fails to induce significant protection to wild-type tumor challenge. In this study we investigated whether B7-1 expressing neuro-2a cells can stimulate an effective T-cell response if they were cotransduced with the interferon-gamma (IFN-gamma) gene to upregulate MHC class I. Transfer of both the IFN-gamma and B7-1 genes into neuro-2a (N-2a/B7-1/IFN) almost completely abrogated the tumorigenic potential of this tumor and improved survival when compared with mice receiving the single transductants, N-2a/IFN and N-2a/B7-1. Rejection of N-2a/B7-1/IFN was mediated primarily by CD8+ T cells. When irradiated tumor cells were tested, IFN-gamma gene transfer into neuro-2a significantly increased immunogenicity, but transfer of the B7-1 gene did not. However, nonirradiated N-2a/B7-1, N-2a/IFN, and N-2a/B7-1/IFN cells were significantly more effective in eliciting systemic immunity against subsequent wild-type tumor challenge than their irradiated counterparts. N-2a/B7-1/IFN was more immunogenic than N-2a/B7-1 but not more than N-2a/IFN, indicating that B7-1 does not further increase immunogenicity of neuro-2a over that induced by IFN-gamma transduction. These findings should be considered when designing gene modified tumor vaccines for use in human trials.

Hematopoietic stem cell transplantation for infantile osteopetrosis.

Infantile osteopetrosis is a lethal disorder resulting from a severe defect in the ability of osteoclasts to resorb bone. The only therapy shown to be capable of providing lasting benefit is allogeneic hematopoietic stem cell transplantation (HCT). We report the outcome of 10 patients with infantile malignant osteopetrosis treated with HCT from an HLA A, B, DRB1 matched (n=6) or A or B locus mismatched (n=4) family member or unrelated donor at the University of Minnesota between 1978 and 1997. Eight of 10 patients achieved primary engraftment; secondary graft failure was seen in two patients. Five of 10 patients survive; three with full or partial donor chimerism and two with autologous hematological recovery. Transient or partial donor chimerism can be sufficient to correct the hematological manifestations of osteopetrosis. We recommend early referral for consideration of HCT with a related or unrelated donor as neurosensory manifestations of osteopetrosis are generally not reversible. Donor engraftment may be easier to achieve early in the course of the disease.

Graft-versus-leukemia is sufficient to induce remission in juvenile myelomonocytic leukemia.

It has been unclear whether a graft-versus-leukemia (GVL) effect assists in the control of juvenile myelomonocytic leukemia (JMML) following allogeneic bone marrow transplant. We describe a patient with JMML who relapsed early after an unrelated donor transplant, and following withdrawal of immunosuppression developed graft-versus-host disease (GVHD). Associated with GVHD the proportion of donor cells measured by variable nucleotide tandem repeat (VNTR) analysis increased, and peripheral blasts and cutaneous disease were eliminated. These findings strongly suggest that GVL has a role in the control of JMML.

Unrelated donor bone marrow transplantation for acute leukemia in patients with Down's syndrome.

The incidence of hematologic disorders in patients with Down's syndrome (DS) is significantly increased, and includes neonatal transient abnormal myelopoiesis and acute leukemias. Treatment of children with DS and leukemia has been controversial because of toxicity and associated congenital cardiac and other abnormalities. The role of BMT, particularly from an unrelated donor (URD), remains undefined in this population. We report two children with DS and acute leukemia successfully treated with intensive chemotherapy and matched URD bone marrow transplantation. One child was transplanted in third remission of ALL and has been disease free for 8 months. A second child with AML was transplanted in second remission and is disease free 15 months post-BMT.

Outcome of second hematopoietic cell transplantation in Hurler syndrome.

Hurler syndrome (HS) is an autosomal recessive, inherited metabolic storage disorder due to deficiency of lysosomal alpha-L-iduronidase (IDU) enzyme activity. Untreated patients develop progressive mental retardation and multisystem morbidity with a median life expectancy of 5 years. Allogeneic hematopoietic cell transplantation (HCT) can achieve stabilization and even improvement of intellect, with long-term survival. However, children with HS have an increased incidence of graft failure, usually with concomitant autologous marrow reconstitution. Between 1983 and 2000, 71 Hurler children underwent HCT at the University of Minnesota. Of these 71, 19 (27%) experienced graft failure. We report HCT outcomes in all 11 Hurler patients receiving a second HCT at the University of Minnesota. Median age at second HCT was 25 months (range, 16 to 45 months); median time from first HCT was 8 months (range, 4 to 18.5 months). The conditioning regimen consisted of cyclophosphamide/TBI/ATG (n = 8) or busulfan/cyclophosphamide/ATG (n = 3). The source of bone marrow was an unrelated donor in six, matched sibling in four, and mismatched related in one. Five of the 11 grafts were T cell depleted prior to infusion. Overall, 10 of 11 patients showed donor-derived engraftment, of whom three developed grade 3 to 4 acute GVHD. Five of 11 patients are surviving a median of 25 months (range, 2 months to 12 years) with an overall actuarial survival of 50% (95% CI, 27% to 93%) at 4 years. All five show sustained donor engraftment with normalization of IDU activity levels. Three of five evaluable patients demonstrated stabilization of neuropsychological function after second HCT. Currently, allogeneic donor-derived hematopoiesis provides the only chance for long-term survival and improved quality of life in Hurler patients. While graft failure in Hurler patients requires further investigation, a timely second HCT can be well-tolerated and beneficial.