ALBERT

Description: Dyskeratosis congenita (DC) is a congenital disorder characterized by very short telomeres. Clinical presentation includes a diagnostic triad (lacey reticular pigmentation, nail dystrophy, and leukoplakia), aplastic anemia (the main cause of premature death), myelodysplastic syndrome/leukemia and solid tumors. Allogeneic HCT is the only curative option for the hematologic complications of DC but has been associated with a high risk of peri-transplant morbidity and early death. Only about fifty HCT for DC have been performed to-date, and the five year survival after related donor HCT has been about 75%, but only approximately 35% when an unrelated donor was used. To improve survival in DC patients by decreasing transplant mortality, we introduced a reduced intensity regimen including cyclophosphamide (50 mg/kg), fludarabine (200 mg/kg), low dose total body irradiation (200 cGy), and (in patients 3 and 4) campath 1H (1 mg/kg). To decrease the risk of graft rejection, grafts were not T-cell depleted. We report outcomes in four consecutive patients, two adults and two children, all of whom engrafted with donor hematopoiesis: Age (years) Sex Graft and HLA match NC dose (×108/kg) CD34 dose (×108/kg) Follow-up (months) Donor chimerism 24 M URD dUCB 4/6, 4/6 0.55, 0.39 0.5, 0.43 1 (dead*) 83% 29 F REL PBSC 6/6 13.93 4.43 20 (alive) 100% 5 F URD BM 7/8 1.38 1.55 16 (alive) 100% 2 M URD BM 8/8 5.92 2.31 3 (alive) 100% Legend: M, male; F, female; NC, nucleated cell; URD, unrelated donor; REL, related donor; BM, bone marrow; dUCB, double umbilical cord blood; PBSC, peripheral blood stem cells; *Patient had autologous recovery after the first dUCB and died of sepsis 1 month after the second dUCB; HLA matching is reported for antigen level HLA-A, B and allele level DRB1 for cord blood, and allele level typing for HLA-A, B, C, DRB1 for PBSC or BM. The most recent donor chimerism is reported. To decrease the risk of graft rejection and prevent graft versus host disease (GvHD) patients received cyclosporine and mycophenolate mofetil. Patient 2 developed limited chronic GvHD and patient 4 developed grade III skin acute GvHD. Both were treated successfully with systemic and topical steroids. Our data suggest that this conditioning regimen results in a low rate of transplant related complications without compromising engraftment. Critically, early fatal pulmonary and vascular complications, common in post-transplant courses in DC patients, were not observed. This highlights the need to avoid drugs that are associated with pulmonary toxicity such as busulfan, and to limit radiation to the lung in patients with DC. This new less intensive conditioning regimen appears to result in a low rate of transplant related complications, and yet has adequate immunosuppressive activity to permit engraftment from alternative donors in DC patients.

Description: Metachromatic Leukodystrophy (MLD) is a rare demyelinating disease caused by deficient lysosomal arylsulfatase-A (ARSA) activity and resulting pathologic sulfatide excess. Signs and symptoms vary with the age of disease onset. In early phenotypes (“late infantile” and “early juvenile”; onset 〈 6 years; E-MLD) rapid neurologic regression and death are observed. In late disease (“late juvenile” and “adult”; onset ≥ 6 years; L-MLD) a neuropsychiatric prodrome often precedes frank neurologic decline. For over 30 years, allogeneic hematopoietic stem cell transplantation (HSCT) has been used to halt or curb MLD progression. Yet published outcome data are scant and mixed. We report characteristics of and outcomes for 42 MLD patients following HSCT at the University of Minnesota, the largest such report to our knowledge. From 1984 to 2008, 17 E-MLD and 25 L-MLD patients were transplanted. The median age at HSCT in the E-MLD group was 5 years (range, 0 – 8 years); the median age at transplant in the L-MLD group was 20 years (range, 6 – 44 years). Twelve (29%) patients were male. Preparative regimens varied. Twenty-seven (64%) patients received myeloablative busulfan/cyclophosphamide conditioning; 11 (26%) received a cyclophosphamide/TBI regimen; 4 (10%) underwent reduced-intensity conditioning. Allograft sources also varied. 10 (24%) patients received an HLA-matched sibling marrow graft; 16 (38%) had an unrelated marrow donor (HLA-mismatched = 6); 16 (38%) received UCB grafts (HLA-mismatched = 14). Thirty-three (79%) patients were symptomatic at HSCT. The median time from symptom onset to diagnosis was 17 months in E-MLD and 50 months in L-MLD patients. Pre-HSCT brain MRI data was available for 37 patients; 36 (97%) demonstrated white-matter abnormalities. Of 40 patients with evaluable pre-HSCT nerve conduction velocity (NCV) studies, 37 (93%) had measurable peripheral neuropathy. Non-neuropsychological clinical involvement was scored per a neurologic function scale (NFS, 0 = no neurologic findings, 25 = maximal neurologic findings) spanning the domains of auditory processing, vision, motor, swallowing, incontinence, and seizures. Of 40 patients evaluable for pre-HSCT NFS, 25 (63%) had a non-zero score (range, 1 - 4) and 15 (37%) a zero score. Five-year survival was 58%. Greatest survival (70%) was seen in recipients of HLA-matched, sibling marrow. Among alternative graft recipients (n = 32), favorable survival followed UCB transplantation compared to unrelated marrow (62% versus 47%). Ten patients (24%) died of transplant-related causes by day 180 (3 from hepatic veno-occulsive disease; 2 each from graft-versus-host disease (GvHD) and sepsis; 1 each from graft failure, thrombotic thrombocytopenic purpura, and multiple organ failure syndrome). Four additional patients died within 1 year of transplant, all from infection. Four patients (all E-MLD) died from disease progression (range, 1.5 – 7.1 years post-HSCT). Fourteen patients (33%) developed grade II-IV acute GvHD; 6 (14%) developed chronic GvHD. The median survivor follow-up was 10 years; 74% demonstrated ≥ 80% donor engraftment at most recent observation. Of 30 evaluable patients, only 9 (31%) demonstrated progression of white-matter disease at median MRI follow-up of 1.26 years. For 22 patients with post-HSCT NCV studies, 17 (77%) evidenced worsening peripheral neuropathy at median follow-up 2.9 years. Twenty-four patients had evaluable post-transplant NFS scores. In E-MLD, the median increase in NFS score was 9 (range, 1 - 10) at median follow-up of 3.4 years. In L-MLD, the median increase was 3 (range, 0 - 22) at median follow-up of 3 years. Symptom status (present versus absent) at the time of HSCT neither impacted survival nor post-transplant NFS. Of 15 L-MLD patients surviving past one year, 7 had an evaluable verbal IQ (VIQ, average range 80 - 120) on both pre- and post-HSCT neuropsychological evaluations. In this subgroup, the mean pre- and post-HSCT VIQ were 83 and 75, respectively; mean follow up was 4.1 years post-HSCT. In summary, we report outcomes following HSCT for a large MLD cohort. Preliminary analysis suggests favorable clinical neurologic and neuropsychological performance following transplant for L-MLD. Ongoing analysis of the entire cohort, including comparisons against untreated siblings, aims to further define the neurological and neuropsychological benefit of HSCT for MLD. Disclosures: No relevant conflicts of interest to declare.

Description: Mucopolysaccharidosis type I (MPS IH; Hurler syndrome) is a congenital deficiency of α-L-iduronidase, leading to lysosomal storage of glycosaminoglycans that is ultimately fatal following an insidious onset after birth. Hematopoietic cell transplantation (HCT) is a life-saving measure in MPS IH. However, because a suitable hematopoietic donor is not found for everyone, because HCT is associated with significant morbidity and mortality, and because there is no known benefit of immune reaction between the host and the donor cells in MPS IH, gene-corrected autologous stem cells may be the ideal graft for HCT. Thus, we generated induced pluripotent stem cells from 2 patients with MPS IH (MPS-iPS cells). We found that α-L-iduronidase was not required for stem cell renewal, and that MPS-iPS cells showed lysosomal storage characteristic of MPS IH and could be differentiated to both hematopoietic and nonhematopoietic cells. The specific epigenetic profile associated with de-differentiation of MPS IH fibroblasts into MPS-iPS cells was maintained when MPS-iPS cells are gene-corrected with virally delivered α-L-iduronidase. These data underscore the potential of MPS-iPS cells to generate autologous hematopoietic grafts devoid of immunologic complications of allogeneic transplantation, as well as generating nonhematopoietic cells with the potential to treat anatomical sites not fully corrected with HCT.

Description: Relapse is more frequent after autologous than allogeneic bone marrow transplantation (BMT), due in part to lack of T-lymphocyte mediated allogeneic graft-versus-leukemia (GVL) effects. Infusions of leukemia-reactive T cells to patients after autologous BMT may be a means for providing a GVL effect. Costimulation of T cells by binding of the CD28 receptor on T cells with B7-counter receptors on antigen presenting cells amplifies antigen-specific T-cell responses. To enhance generation of leukemia reactive cytotoxic T lymphocytes (CTL), the murine B7-1– and B7-2–costimulatory molecule cDNAs were introduced into the MHC class I+, class II−, murine meyloid leukemia cell line C1498. B7-1 expression greatly enhanced the ability of the leukemia cells to generate and expand leukemia reactive CTL in vitro. A highly cytolytic and C1498 specific CD8+ CTL line was generated by B7-1 costimulation. This CTL line proliferated autonomously and produced interleukin-2 when provided B7-1 or B7-2 costimulation by C1498 leukemia cells. To test the in vivo antileukemia properties of this CTL line, irradiated syngeneic BMT recipients were given graded doses of leukemia cells on day 0, followed by CTL infusions beginning on day 1 post-BMT. Recipients of 107 CTL had a 3 log reduction in leukemia burden such that 100% of mice were protected from a supralethal leukemic cell dose. Sustained immune responses were detectable up to 3 months postinfusion of the CTL line. B7-1 or B7-2 costimulation in vivo did not augment antileukemia effects of infused CTL post BMT. These results suggest that B7 costimulation of leukemia reactive CTL may be important for their ex vivo generation and expansion for use in human adoptive immunotherapy of leukemia.

Description: Preclinical models have shown that transplantation of marrow mesenchymal cells has the potential to correct inherited disorders of bone, cartilage, and muscle. The report describes clinical responses of the first children to undergo allogeneic bone marrow transplantation (BMT) for severe osteogenesis imperfecta (OI), a genetic disorder characterized by defective type I collagen, osteopenia, bone fragility, severe bony deformities, and growth retardation. Five children with severe OI were enrolled in a study of BMT from human leukocyte antigen (HLA)–compatible sibling donors. Linear growth, bone mineralization, and fracture rate were taken as measures of treatment response. The 3 children with documented donor osteoblast engraftment had a median 7.5-cm increase in body length (range, 6.5-8.0 cm) 6 months after transplantation compared with 1.25 cm (range, 1.0-1.5 cm) for age-matched control patients. These patients gained 21.0 to 65.3 g total body bone mineral content by 3 months after treatment or 45% to 77% of their baseline values. With extended follow-up, the patients' growth rates either slowed or reached a plateau phase. Bone mineral content continued to increase at a rate similar to that for weight-matched healthy children, even as growth rates declined. These results suggest that BMT from HLA-compatible donors may benefit children with severe OI. Further studies are needed to determine the full potential of this strategy.

Description: MPS IH (Hurler syndrome) is caused by severe mutations in a-L-iduronidase gene, leading to accumulation of complex sugars, termed glycosaminoglycans (GAG), in tissues. The disorder is lethal unless treated with hematopoietic stem cell transplantation (HSCT). When compared to other recipients of HSCT, MPS I patients are well-known to have an increased incidence of bleeding complications in peri-transplant period. To investigate whether this bleeding propensity is related to the enzyme deficiency and GAG accumulation we evaluated five MPS IH patients (2 females and 3 males, mean age: 1.1 years) prior to any therapy. We observed that three of the five patients had an elevated activated partial thromboplastin time (aPTT, normal range 22–35 seconds). As no heparin was used in any of these patients, we reasoned that the accumulation of heparan sulfate, a hallmark of MPS IH, may serve as a heparin-like anticoagulant. Should this assumption be correct, higher levels of GAG would be expected in the three patients with the elevated aPTT. This, in fact, was what we have observed (Figure), suggesting that dose - effect relationship exists between whole body GAG load (assessed as milligrams of urinary GAG normalized to grams of creatinine; x-axis) and level of anticoagulation (aPTT in seconds; y-axis). Figure Figure In this context, it is important to note that the patient with the second highest value of urinary GAG and aPTT experienced pulmonary bleeding on day 12 after HSCT requiring intubation and ventilatory support for 25 days. We hypothesized that endogenous GAG heparan sulfate exerts its antithrombotic effect through antithrombin III-mediated inhibition of factor Xa, as in clinically used low molecular weight heparinoid, danaparoid. To test this hypothesis, we have assessed anti-Xa levels in mice with a-L-iduronidase deficiency, an animal model of MPS IH. As expected, the anti-Xa level were not significantly different in wild type C57Bl/6 mice (N=3) and heterozygous mice (N=6): 0.11±0.005 IU/cc plasma versus 0.10±0.004 IU/cc plasma (p=0.2). Anti-Xa values of MPSI mutant mice (N=14), however, were significantly elevated when compared to the age-matched sex-matched wild type controls (0.19±0.05 IU/cc plasma versus 0.11±0.005 IU/cc plasma; p

Description: MPS IH (Hurler syndrome) is caused by severe mutations in a-L-iduronidase gene, leading to accumulation of complex sugars, termed glycosaminoglycans (GAG), in tissues. The disorder is lethal unless treated with hematopoietic stem cell transplantation (HSCT). When compared to other recipients of HSCT, MPS I patients are well-known to have an increased incidence of bleeding complications in peri-transplant period. To investigate whether this bleeding propensity is related to the enzyme deficiency and GAG accumulation we evaluated five MPS IH patients (2 females and 3 males, mean age: 1.1 years) prior to any therapy. We observed that three of the five patients had an elevated activated partial thromboplastin time (aPTT, normal range 22–35 seconds). As no heparin was used in any of these patients, we reasoned that the accumulation of heparan sulfate, a hallmark of MPS IH, may serve as a heparin-like anticoagulant. Should this assumption be correct, higher levels of GAG would be expected in the three patients with the elevated aPTT. This, in fact, was what we have observed (Figure), suggesting that dose - effect relationship exists between whole body GAG load (assessed as milligrams of urinary GAG normalized to grams of creatinine; x-axis) and level of anticoagulation (aPTT in seconds; y-axis). Figure Figure In this context, it is important to note that the patient with the second highest value of urinary GAG and aPTT experienced pulmonary bleeding on day 12 after HSCT requiring intubation and ventilatory support for 25 days. We hypothesized that endogenous GAG heparan sulfate exerts its antithrombotic effect through antithrombin III-mediated inhibition of factor Xa, as in clinically used low molecular weight heparinoid, danaparoid. To test this hypothesis, we have assessed anti-Xa levels in mice with a-L-iduronidase deficiency, an animal model of MPS IH. As expected, the anti-Xa level were not significantly different in wild type C57Bl/6 mice (N=3) and heterozygous mice (N=6): 0.11±0.005 IU/cc plasma versus 0.10±0.004 IU/cc plasma (p=0.2). Anti-Xa values of MPSI mutant mice (N=14), however, were significantly elevated when compared to the age-matched sex-matched wild type controls (0.19±0.05 IU/cc plasma versus 0.11±0.005 IU/cc plasma; p