Pilot trial of risk-adapted cyclophosphamide intensity based conditioning and HLA matched sibling and unrelated cord blood stem cell transplantation in newly diagnosed pediatric and adolescent recipients with acquired severe aplastic anemia.

BACKGROUND: Cyclophosphamide-based conditioning regimens and allogeneic hematopoietic stem cell transplantation (AlloHSCT) from matched related donors (MRD) has resulted in the highest survival rates in children and adolescents with acquired severe aplastic anemia (SAA). Time to transplant has consistently been associated with decreased overall survival. Reduced toxicity conditioning and AlloHSCT has been used successfully in other pediatric non-malignant diseases. PROCEDURE: We piloted a risk-adapted AlloHSCT approach, using fludarabine and anti-thymocyte globulin based conditioning with high (200 mg/kg) and low (60 mg/kg) dose cyclophosphamide as upfront treatment in newly diagnosed pediatric patients with acquired SAA incorporating alternative donor sources, including cord blood. Average risk for non-engraftment patients with <10 transfusions received low dose cyclophosphamide (60 mg/kg); High Risk, those with ≥10 transfusions received conditioning regimen with higher intensity cyclophosphamide (200 mg/kg). RESULTS: Seventeen patients were enrolled and underwent AlloHSCT including 12 males and 5 females with mean age of 8 years (range 3-16), and median follow-up time of 39 months (range 1-135). Donor sources included MRD BM (6/6 [n = 9], 5/6 [n = 2]) and unrelated CB (5/6 [n = 4], 4/6 [n = 2]). Five year OS was 67.6% (37.9-85.4). Three secondary graft failures (17.6%) occurred in the low dose cyclophosphamide arm. CONCLUSIONS: Upfront treatment with risk-adapted cyclophosphamide conditioning AlloSCT is well tolerated for the management of newly diagnosed pediatric and adolescent patients with acquired SAA. However, the increased risk of graft rejection in the lower dose arm warrants additional research regarding the optimal intensity of cyclophosphamide-based conditioning regimen to reduce toxicity without increasing graft failure.

An age-dependent pharmacokinetic study of intravenous and oral mycophenolate mofetil in combination with tacrolimus for GVHD prophylaxis in pediatric allogeneic stem cell transplantation recipients.

Acute graft-versus-host disease (aGVHD) still remains a major limiting factor following allogeneic stem cell transplantation (AlloSCT) in pediatric recipients. Mycophenolate mofetil (MMF), an uncompetitive selective inhibitor of inosine monophosphate dehydrogenase, is a new immunosuppressant agent without major mucosal, hepatic, or renal toxicity compared to other prophylactic aGVHD immunosuppressant drugs. Although there has been an extensive pharmacokinetic (PK) experience with MMF administration following solid organ transplantation in children, there is a paucity of PK data following its use in pediatric AlloSCT recipients. We investigated the safety and PK of MMF as GVHD prophylaxis following intravenous (i.v.) and oral (p.o.) administration (900 mg/m(2) every 6 hours) in conjunction with tacrolimus, after myeloablative (MA) and nonmyeloablative (NMA) conditioning and AlloSCT in 3 distinct age groups of pediatric AlloSCT recipients (0-6 years, 6-12 years, and 12-16 years). Mycophenolic acid (MPA) in plasma samples was measured either by high-performance liquid chromatography (HPLC) or liquid chromatography/mass spectrometry (LC/MS/MS) as we have previously described. Plasma samples were obtained at baseline and at 0.5, 1, 2, 3, 4, and 6 hours after i.v. dosing on days +1, +7, +14, and at 2 time points between day +45 and +100 after p.o. administration post AlloSCT. MPA PK analysis included AUC (0-6 hours), C(max), T(max), C(ss), V(ss), C trough (C(0)), CL, and T((1/2).) Thirty-eight patients, with a median age of 8 years (0.33-16 years), 20/18 M:F ratio, 21/17 malignant/nonmalignant disease, 17/21 MA: NMA conditioning, 16 of 22 related/unrelated allografts. Median time to myeloid and platelet engraftment was 18 and 31 days, respectively. Mean donor chimerism on day +60 and +100 was 83% and 90%, respectively. Probability of developing aGVHD grade II-IV and extensive chronic GVHD (cGVHD) was 54% and 34%, respectively. There was significant intra- and interpatient MMF PK variability. There was a significant increase in i.v. MPA area under the curve (AUC)(0-6 hour) and C(max) (P < .0003) and a significant decrease in CL(ss) (P < .002) and V(ss) (P < .001) on day +14 versus day +7. Children <12 years of age had a significant increase in i.v. MPA T(max) (P = .01), V(ss) (P = .028), and CL(ss) (P < .001) compared to the older age group. There was a trend in increased i.v. MPA CL(ss) following MA versus NMA conditioning (P < .054); i.v. and p.o. MMF administration (900 mg/m(2) every 6 hours) in combination with tacrolimus was well tolerated in pediatric AlloSCT recipients. There was a significant increase in MPA exposure on day +14 versus day +7, suggesting improved enterohepatic recirculation at day +14 post-AlloSCT. Children <12 years of age appear to have a significantly different MPA PK profile compared to older children and adolescents and may require more frequent dosing.

Incidence of Viral and fungal infections following busulfan-based reduced-intensity versus myeloablative conditioning in pediatric allogeneic stem cell transplantation recipients.

Reductions in the duration and nadir of neutropenia have translated into a significant decrease in bacteremia in adult recipients of allogeneic stem cell transplantation (allo-SCT) with reduced-intensity conditioning (RIC) during the first 30 days after transplantation. It remains to be determined whether RIC allo-SCT also will result in a decrease in systemic viral infections (SVIs) and invasive fungal infections (IFIs), which are more dependent on alterations in cellular immunity. We compared the incidence of SVIs and IFIs in children receiving busulfan-based RIC allo-SCT and in children receiving myeloablative conditioning (MAC) allo-SCT for various malignant and nonmalignant diseases. Allo-SCT recipients at risk for cytomegalovirus (CMV) received ganciclovir/foscarnet, and most of the patients received antifungal prophylaxis with liposomal amphotericin B until day +100. Eighty-six patients (median age, 7.5 years; 70% with malignant disease, 30% with nonmalignant disease; 80% average risk, 20% poor risk) were evaluated. The probability of developing grade II-IV acute graft-versus-host disease (aGVHD) was 29.1% (95% confidence interval [CI]=16.7%-41.6%) in RIC allo-SCT versus 40.3% (95% CI=23.9%-56.6%) in MAC allo-SCT (P=.23), and that of chronic GVHD (cGVHD) was 28.9% (95% CI=14.7%-43.0%) in RIC allo-SCT versus 28.4% (95% CI=10.5%-46.3%) in MAC allo-SCT (P=.73). The overall probability of developing an SVI was 58%, and that of developing an IFI was 15%. These probabilities did not differ significantly by conditioning intensity. In a multivariate Cox regression model, the following were identified as independent risk factors for invasive fungal infection: older age (hazard ratio [HR]=1.3; 95% CI=1.1-1.6; P=< .01), poor risk status (HR=6.5; 95% CI =1.1-37.4; P=.03), and CMV-positive recipient (high vs low CMV risk group, HR=26.7; 95% CI=3.4-210.8; P=< .01). Overall infection-related mortality was only 1.1% (1/86) for SVIs and 2.3% (2/86) for IFIs. Our data indicate that RIC allo-SCT does not carry a lower risk of SVIs and IFIs than MAC allo-SCT in pediatric recipients.

Reduced intensity and non-myeloablative allogeneic stem cell transplantation in children and adolescents with malignant and non-malignant diseases.

Allogeneic hematopoietic stem cell transplant (AlloSCT) from related or unrelated histocompatible donors has been well established as potentially curative therapy for children and adolescents with selected malignant and non-malignant diseases. In the malignant setting non-myeloablative (NMA)/reduced intensity (RI)-AlloSCT eradicates malignant cells through a graft versus malignancy effect provided by alloreactive donor T-lymphocytes and/or natural killer cells. In patients with non-malignant diseases NMA/RI AlloSCT provides enough immunosuppression to promote engraftment and correct underlying genetic defects. In children, myeloablative AlloSCT is not only associated with acute short-term toxicities but also long-term late complications such as growth retardation, infertility, and secondary malignancies. NMA/RI-AlloSCT in children may be associated with reduction in use of blood products, risk of infections, transplant-related mortality, and length of hospitalization. Despite the success of RI-AlloSCT in adults, large prospective and/or randomized multicenter studies are necessary in children and adolescent recipients to define the appropriate patient population, optimal conditioning regimens, cost-benefits, survival and differences in short-term and long-term effects compared to conventional myeloablative conditioning.

The potential of umbilical cord blood multipotent stem cells for nonhematopoietic tissue and cell regeneration.

Stem cells have been isolated from human embryos, fetal tissue, umbilical cord blood (UCB), and also from "adult" sources. Adult stem cells are found in many tissues of the body and are capable of maintaining, generating, and replacing terminally differentiated cells. A source of pluripotent stem cells has been recently identified in UCB that can also differentiate across tissue lineage boundaries into neural, cardiac, epithelial, hepatocytic, and dermal tissue. Thus, UCB may provide a future source of stem cells for tissue repair and regeneration. Its widespread availability makes UCB an attractive source for tissue regeneration. UCB-derived stem cells offer multiple advantages over adult stem cells, including their immaturity, which may play a significant role in reduced rejection after transplantation into a mismatched host and their ability to produce larger quantities of homogenous tissue or cells. While research with embryonic stem cells continues to generate considerable controversy, human umbilical stem cells provide an alternative cell source that has been more ethically acceptable and appears to have widespread public support. This review will summarize the in vitro and in vivo studies examining UCB stem cells and their potential use for therapeutic application for nonhematopoietic tissue and cell regeneration.

A low incidence of nontuberculous mycobacterial infections in pediatric hematopoietic stem cell transplantation recipients.

Hematopoietic stem cell transplantation (HSCT) is being used to treat a wide spectrum of clinical disorders but opportunistic infection remains an important factor determining outcomes for these patients. Nontuberculous mycobacterial (NTM) infections are being reported more frequently in HSCT recipients and the incidence of NTM infections in adult recipients is reported to be 0.4%-4.9%. However, the incidence and severity of NTM infections are less well described in pediatric HSCT recipients. Centers for Disease Control and Prevention guidelines were used to define definite and probable NTM infection among 132 children undergoing 169 HSCT between January 2000 and December 2004 at our institution. NTM infection was diagnosed in 5 of 132 pediatric recipients (3.8%). There were no NTM infections diagnosed in the autologous HSCT recipients and the incidence of NTM in allogeneic HSCT recipients was 6.4% (95% confidence interval, 0.8-11.9). The mean age of the HSCT recipients who developed NTM infections was 8 years (range, 2-19 years); 3 were male and 2 were female. Four conditioning regimens included alemtuzumab and 3 had antithymocyte globulin. Of the 5 patients with NTM infections, 2 met the criteria for definite infection and 3 for probable infection. Of the 2 patients with definite NTM infection, 1 had disseminated disease with Mycobacterium avium complex and the other had Mycobacterium chelonae catheter-related bloodstream infection. The probable NTM infections were 1 skin infection with Mycobacterium kansasii and 2 lower respiratory tract infections with M avium complex. Median time to NTM infection was 115 days (range, 14-269 days) after HSCT. Two patients had graft-versus-host disease at the time of NTM infection. All 5 patients received 3-4 antimycobacterial drugs and all NTM infections resolved. In summary, the incidence of NTM infection in pediatric HSCT recipients appears similar to that described in adult HSCT recipients and the outcome appears to be excellent with the proper antibiotic therapy. The increased use of anti-T cell antibodies appears to be associated with an increased risk of NTM infections in pediatric HSCT recipients. Multicenter studies are needed to identify the risk factors, early diagnostic criteria, and optimal therapy.

Cord blood immunology and stem cell transplantation.

Allogeneic stem cell transplantation can be curative in a variety of malignant and nonmalignant disorders. Unfortunately, more than 75% of potential recipients lack a matched family donor. Although 50% of these recipients may find a matched unrelated adult stem cell donor from one of the worldwide registries, the other 50% have had no other viable donor alternatives. Cord blood cellular immunity is immature at birth and allows for a greater human leukocyte antigen disparity between a cord blood donor and recipient after an unrelated cord blood transplant. More than 25 cord blood banks have been developed worldwide to support the growing clinical needs of unrelated cord blood transplantation. Standard operating procedures have been developed for maternal donor screening and consent, cord blood collection, processing, cryopreservation, characterization, shipping, and thawing. Cord blood transplantation after myeloablative and reduced-intensity conditioning has been successfully demonstrated, resulting in long-term full donor chimerism, decreased Grade 3/4 acute graft-versus-host disease and improvements in overall survival. Several areas of ongoing research include ex vivo expansion of cord blood hematopoietic progenitor cells to enhance the rapidity of engraftment and isolation and activation of select immune cell populations for prevention or treatment of acute graft-versus-host disease, infectious complications, and tumor reoccurrence.

A pilot study of tacrolimus and mycophenolate mofetil graft-versus-host disease prophylaxis in childhood and adolescent allogeneic stem cell transplant recipients.

Tacrolimus (FK506)/mycophenolate mofetil (MMF) has been demonstrated to be an effective salvage therapy for steroid-resistant chronic graft-versus-host disease (GVHD), but its effectiveness as prophylaxis for acute GVHD (aGVHD) is unknown. We investigated the safety and efficacy of FK506/MMF in preventing aGVHD and sparing the use of methotrexate and methylprednisolone in childhood and adolescent allogeneic stem cell transplant (AlloSCT) recipients. Thirty-four childhood and adolescent patients (median age, 7 years; range, 0.5-21 years; 24 males and 10 females) undergoing 37 AlloSCTs for malignant (n = 22) and nonmalignant (n = 12) disorders received FK506 (0.03 mg/kg/d by continuous intravenous infusion) and MMF (15 mg/kg per dose orally or intravenously twice daily). Stem cell sources included 22 umbilical cord blood donors (21 unrelated and 1 related), 6 related bone marrow donors, and 9 related peripheral blood donors. Malignant diagnoses included 7 acute lymphoblastic leukemias, 3 acute myeloid leukemias, 1 acute promyelocytic leukemia, 2 non-Hodgkin lymphomas, 4 Hodgkin diseases, 3 chronic myeloid leukemias, and 2 neuroblastomas; nonmalignant diagnoses included 2 beta-thalassemias, 1 sickle cell disease, 4 aplastic anemias, 1 Wiskott-Aldrich syndrome, 1 Hurler syndrome, 2 hemophagocytic lymphohistiocytoses, and 1 myelodysplastic syndrome. The probability of developing grade > or =II aGVHD was 45.4% +/- 9.7% (7 related bone marrow/related peripheral blood; 5 umbilical cord blood), and for chronic GVHD it was 38.1% +/- 19.7%. FK506/MMF was well tolerated. Three patients had grade III to IV neurotoxicity (disorientation and leukoencephalopathy); 4 patients developed grade III to IV nephrotoxicity (all received concomitant nephrotoxins). Patients who achieved target mycophenolic acid levels (1.0-3.5 microg/mL) before day +30 had a significantly reduced incidence of developing grade >/=II aGVHD (16.7% +/- 15.2% versus 100%; P <.02). These results suggest that FK506/MMF is well tolerated and may be a safe and effective methotrexate- and methylprednisolone-sparing alternative GVHD prophylaxis regimen after AlloSCT. Further pharmacokinetic and pharmacodynamic studies are ongoing in pediatric and adolescent AlloSCT recipients to define optimal MMF dosing.

Correction of phenotype in a thalassemia mouse model using a nonmyeloablative marrow transplantation regimen.

Gene therapy, the replacement of normal human beta- or gamma-globin genes into the hematopoietic stem cells of patients with homozygous beta-thalassemia, is a promising therapy for the future. High-level lineage-specific stable globin expression in transduced cells reinfused into patients in an autologous transplantation setting could be curative, if successful. Previous studies have shown high-level donor chimerism in nonmyeloablated non-thalassemic hosts. We have now studied the conditions for stable long-term engraftment of normal cells into a thalassemia mouse model that lead to high-level donor chimerism and correction of the abnormal phenotype. Thalassemic female mice treated with 0 to 300 cGy whole-body irradiation received transplantations of donor cells harvested from wild-type males. Engraftment of male cells was quantitated by Y-chromosome polymerase chain reaction analysis of blood and marrow progenitors, and changes in hemoglobin levels, red cell morphology, and spleen size were measured at various times posttransplantation. High-level stable donor cell engraftment was achieved in mice given 200 cGy and receiving transplants of 2 x 10(7) or more donor cells. The anemia, abnormal peripheral blood smears, and splenomegaly improved in the thalassemic mice that had successful engraftment. These studies demonstrate that stable and successful levels of engraftment of normal cells can correct the thalassemic phenotype without fully myeloablating the host. This animal model should allow us to test the amount of cytoreduction required and the level of engraftment and beta-globin expression needed in autologous transplantation of beta-globin gene-transduced cells to correct the abnormal phenotype in thalassemic mice, and it may be relevant to human clinical trials, as well.