Stem Cell Transplant

Pediatr Clin N Am 49 (2002) 989 – 1007

New approaches to hematopoietic cell transplantation for hematological diseases in children Paul Woodard, MD a,*, Bertram Lubin, MD b,c, Mark C. Walters, MD b,d a

Division of Stem Cell Transplantation, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, Memphis, TN, 38105, USA b University of California, San Francisco, CA, USA c Children’s Hospital Oakland Research Institute, 5700 Martin Luther King Jr Way, Oakland, CA, 94609, USA d Children’s Hospital and Research Center, 747 52nd. Street, Oakland, CA, 94609, USA

Introduction Bone marrow transplantation for nonmalignant hematological diseases For more than 30 years, selected malignant and nonmalignant diseases have been treated by hematopoietic cell transplantation (HCT) [1– 9]. The conventional application of HCT for hematological disorders has relied on myeloablative conditioning before human leukocyte antigen (HLA)-identical sibling bone marrow transplantation to correct the underlying hematological defect. Employing this strategy, HCT successfully replaced defective erythrocytes among patients with sickle cell anemia [7] and b-thalassemia [10] and restored normal hematopoiesis in patients with severe aplastic anemia [11] and other bone marrow failure disorders [12] (See box). Best results followed transplantation from HLAidentical sibling donors. Survival among young patients with severe aplastic anemia exceeded 90%[1] and event-free survival among children with sickle cell anemia was approximately 85% after HCT (Fig. 1) [7,13]. Similar results for b-thalassemia have been reported, although the probability of event-free survival was affected by the degree of iron overload and liver dysfunction that existed before transplantation (Fig. 2) [10,14]. Although applied less frequently for Diamond-Blackfan anemia [15], dyskeratosis congenita [16,17], chronic granulomatous disease [18,19], amegakaryocytic thrombocytopenia [20,21], and * Corresponding author: E-mail address: [email protected] (P. Woodard). 0031-3955/02/$ – see front matter D 2002, Elsevier Science (USA). All rights reserved. PII: S 0 0 3 1 - 3 9 5 5 ( 0 2 ) 0 0 0 2 6 - 3

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Kostmann’s disease [9], HCT also has curative potential for these disorders. HCT effectively eliminates the hematological manifestations of Fanconi’s anemia [4,22 – 24]. List of Nonmaligmant hematological diseases cured by HCT in pediatrics Severe Aplastic Anemia b-Thalassemia Sickle Cell Disease Fanconi Anemia Diamond-Blackfan Anemia Dyskeratosis Congenita Chronic Granulomatous Disease Amegakaryocytic Thrombocytopenia Kostmann’s Disease

The principal objective of allogeneic transplantation for hematological diseases is to replace defective recipient cells with healthy donor cells, a subtle distinction from the goals of HCT for malignancies, which are to harness the myeloablative activity of pretransplant conditioning therapy and the anti-tumor effect of graft versus host disease (GVHD) for the purpose of destroying resistant

Fig. 1. Overall survival, event-free survival, and rejection rates after matched sibling donor HCT for sickle cell disease. (From Walters MC, Storb R, Patience M, et al. Impact of bone marrow transplantation for symptomatic sickle cell disease: an interim report. Blood 2000;95:1918 – 24.)

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Fig. 2. Overall survival, rejection-free survival, and rejection rates after matched sibling donor HCT for beta thalassemia. (From Lucarelli G, Clift RA. Marrow transplantation in thalassemia. In: Thomas ED, Blume KG, Forman SJ, eds. Hematopoietic Cell Transplantation, 2nd edition. Malden: Blackwell Science; 1998, p. 1137 – 44; with permission.)

malignant cells. Unlike those who have recurrent or high-risk malignancies, many individuals with hematological disorders such as sickle cell anemia and thalassemia do not have immediately life-threatening problems, hence concerns about safety rather than impending relapse after HCT are heightened. Thus, the future of HCT for these nonmalignant hematological disorders is very likely to include a focus on accomplishing a successful outcome with reduced toxicity and on methods to expand the availability of HCT without adversely altering its safety profile. New approaches that might advance these aims are under development and are highlighted in this chapter.

Pretransplant conditioning: impact on outcome Pretransplant preparation typically consists of chemotherapy with or without total body irradiation (TBI) and accomplishes two requirements of allogeneic HCT: first, it eradicates host hematopoiesis and second, it suppresses the recipient’s immune system to permit engraftment of donor hematopoietic cells. A notable variation of this strategy is utilized in HCT for severe combined immunodeficiencies, which do not typically require pretransplant preparation because a state of immunosuppression exists naturally [25]. Alternatively, among those with severe aplastic anemia, ablation of host hematopoiesis is not a requirement for engraftment of donor cells, thus pretransplant preparation is focused on suppression of the host immune system. Thus, for HLA-identical sibling allografting, a combination of cyclophosphamide (200 mg/kg) and antithymocyte globulin (ATG) as pre-transplant therapy has proved adequate to ensure engraftment [26]. Hemoglobinopathies present yet another set of problems that must be considered before transplantation. The bone marrow of these patients is proliferative and hypercellular, a property caused in part by ineffective erythropoiesis.

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Thus, in most instances conventional myeloablative pretransplant therapy has been administered to clear sufficient space to support the engraftment of donor cells. A second consideration is that hemoglobinopathy patients undergo frequent, if not chronic exposures to blood products, thereby increasing the likelihood of immunological reactivity to minor histocompatibility antigens expressed on leukocytes that accompany these transfusions. It has been suggested that these exposures form the basis for the high frequency of disease recurrence that occurs among hemoglobinopathy transplant recipients. Thus, pretransplant therapy for these patients must be sufficiently immunosuppressive to overcome this propensity for immunological graft rejection. Unfortunately, delivering sufficiently intensive therapy carries risks of toxicity, as demonstrated by the experience of HCT for adult patients with sickle cell disease and b-thalassemia major [27,28]. The challenge here is to identify a preparative regimen that is sufficiently immunosuppressive to promote engraftment, yet not excessively toxic among high-risk recipients so as to cause excessive morbidity and transplant-related mortality. Toward this end, most patients with sickle cell disease and b-thalassemia major have received myeloablative doses of Busulfan (14 –16 mg/kg) and Cyclophosphamide (200 mg/kg) with or without horse ATG (90 mg/kg of ATGam1) [7,13]. However, there have been attempts to generate a risk-based dosing approach to pretransplant therapy, described below. Attenuation of pre-transplant preparation has been explored in patients with b-thalassemia major, where the rates of graft rejection and mortality have varied with liver size and hepatic fibrosis from hemosiderosis that existed before HCT (Lucarelli Risk Classification) [10]. Patients were classified based on compliance or noncompliance with regular chelation therapy, presence or absence hepatomegaly, and if there was evidence of portal fibrosis by liver biopsy. Class I patients had none of these risk factors, Class II patients had one or 2 risk factors, and Class III patients had all 3 risk factors. Busulfan and cyclophosphamide dosing was assigned at varying levels [10,28]. Four hundred sixty-nine children who were less than 17 years of age received busulfan 14 mg/kg and cyclophosphamide 200 mg/kg. In this group of 469 children there were 119 Class I patients, 297 class II patients, and 53 Class III patients. Patients who had Class I features fared best and had a disease-free survival of 94%. Patients who had Class III features experienced an inferior outcome, with a survival of approximately 60%, event-free survival of approximately 50%, with failures due to rejection (11%) or nonrejection mortality (35%). To mitigate the high mortality among class III patients, pretransplant preparation was modulated by cyclophosphamide dose reduction. Reductions to 120 to 160 mg/kg before transplantation for class III patients have resulted in improved survival (83% one year after HCT). However, the benefit of improved survival was balanced by a compensatory increase in graft rejection (28%), so that overall event-free survival was only modestly improved by these maneuvers. In contrast, the benefit of decreased cyclophosphamide dosing in adult patients was more pronounced. Among young adults who had predominantly class III characteristics, decreased cyclophosphamide dosing did not appreciably increase the risk of graft rejection/recurrent

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thalassemia [27,28]. Since 1997, the Pesaro team has augmented pre-transplant immunosuppression with fludarabine, azathioprine and hydroxyurea followed by conventional preparation with busulfan and cyclophosphamide (160 mg/kg) for thalassemics with class III features who were less than 17 years of age. This modification successfully improved disease-free survival to 96% [10]. These data support the idea that reduced intensity conditioning before transplantation in high-risk patients might not be required for engraftment of donor cells. In addition, it is possible that a reduction in pre-transplant dose intensity will translate into improved survival. Fanconi anemia represents another clinical model where reducing pretransplant dose intensity was translated into improved survival. This disorder presents unique challenges for transplantation due in part to excessive sensitivity to alkylating agents such as busulfan and cyclophosphamide that significantly increases the morbidity of HCT. In general, pretransplant conditioning for Fanconi anemia has included cyclophosphamide, anti-T cell antibodies with or without reduced dose irradiation delivered to limited fields that include lymphoid tissue [4,12,22]. Here too, the benefits of dose reduction have been observed in several studies. De-escalation of dose intensity involving cyclophosphamide and limited-field irradiation demonstrated survival rates that varied from 58% to 76% with an incidence of graft rejection that was not significantly higher when compared to more intensive transplant regimens. There was, however, a higher rate of graft failure among older patients who had a lower pretransplant platelet count. As in thalassemia, these data are consistent with the notion that selected high-risk patients with hereditary hematological disorders might benefit from reduced dose-intensity preparation for HCT.

Stable mixed chimerism While replacement by donor hematopoietic stem cells is the goal of HCT, another outcome after delivering modulated pretransplant conditioning therapy is the emergence of donor-host hematopoietic chimerism. This occurs in part due to pretransplant therapy that is not sufficiently ablative to destroy all host immune cells. When donor-host tolerance supports a stable co-existence of donor and host hematopoiesis after HCT, generally between 2.5 and 97.5% residual host cells, this state is termed stable mixed hematopoietic chimerism. Conventionally, lymphohematopoietic tolerance is established after HCT in response to ablative pretransplant therapy to destroy host T-cells, and to a short-lived period of postgrafting immunosuppression to suppress donor T-cells. The consequences of immunological intolerance are graft rejection (caused by surviving alloreactive host T-lymphocytes) and GVHD (caused by donor T-cell alloreactivity). Several mechanisms including clonal deletion, clonal anergy, and active suppression have been explored as putative models of T-cell tolerance [29]. The thymus is the site of central clonal deletion, whereby the ab T-cell receptor expressed in immature thymocytes interacts with major histocompatiblity complex (MHC) self-peptides

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expressed on the surface of antigen-presenting cells. T-cells are deleted if the avidity of the cellular interaction is high. A second mechanism termed clonal anergy, may occur if the interaction of T-cells with peptide-MHC complexes are not accompanied by co-stimulatory signals. Thus, a T-cell becomes anergic to the presented antigen. Active suppression of alloimmunity also may be caused by suppressor T-cells, ‘‘veto’’ cells, natural suppressor cells, or other mechanisms to suppressive alloreactive T-cells. In malignant disorders, the emergence of stable mixed chimerism after HCT has been correlated with the delivery of less-intensive pretransplant conditioning therapy and with T-cell depletion of donor grafts [30 –32]. Those patients who developed mixed chimerism benefited from a decreased risk of GVHD [31,33,34]. While mixed chimerism was not universally predictive of disease recurrence [2,31,33 – 36], abrogation of the graft-versus-leukemia (GVL) activity with donor-host T-cell chimerism was predictive of relapse in certain clinical settings (eg, T-cell depleted transplantation for chronic myelogenous leukemia) where the GVL effect remains an important factor for eliminating minimal residual disease [31,37,38]. Thus, the benefits of residual host hematopoiesis and lymphoid chimerism after HCT for malignant disorders remain somewhat uncertain. Among 116 patients with severe aplastic anemia (SAA) and 197 patients with chronic myeloid leukemia (CML) who received HLA-identical sibling HCT [31], there was no significant increase in the rate of rejection associated with mixed chimerism. However, the incidence of grade II to IV acute GVHD was lower and survival was improved in SAA patients with mixed chimerism receiving cyclosporine alone as GVHD prophylaxis. These studies demonstrate the potential benefit of mixed chimerism, particularly in severe SAA where there was a benefit from a reduction in the GVHD risk. Among those who undergo HCT for nonmalignant disorders, the development of stable mixed donor-host hematopoietic chimerism has the potential for considerable ameliorative effect, an observation that has been particularly well documented for b-thalassemia major [34,39], but also in other hereditary disorders [2,7,40]. Approximately 10% of children with sickle cell disease and thalassemia major developed stable mixed chimerism after conventional HLAidentical sibling HCT [7,33]. Among those with b-thalassemia major, stable mixed chimerism persisted for 2 to 11 years after HCT [34]. These patients remained transfusion-independent with hemoglobin levels that varied from 8.3 to 14.7 g/dl. The level of donor chimerism ranged from 25% to 90%. One child with sickle cell anemia who had 11% donor cells in the bone marrow (hemoglobin SAA donor) currently has a hemoglobin S fraction of < 10% and remains transfusion-independent, free from sickle-related events more than 6 years after HCT. Other patients with higher levels of donor chimerism (30 to 40%) have hemoglobin S fractions that mirror donor HbS levels and also experienced a significant clinical benefit. These observations are consistent with the idea that chimerism even with a minority of donor cells might have a curative effect, and that full engraftment of donor cells is not a requirement for the benefit of transplantation.

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Novel methods of pre-HCT conditioning therapy If, in fact, the induction of stable mixed chimerism has curative potential for selected hematological disorders, it might be possible to replace toxic ablative pretransplant therapy with agents chosen for their immunosuppressive effect as a means to establish stable mixed chimerism. To explore this, several groups have employed nonmyeloablative preparative regimens before allogeneic HCT for malignant and nonmalignant diseases. When applied in the setting of hematological malignancies, investigators have exploited a graft-versus-tumor (GVT) effect to elicit clinical responses. The goal is to reduce the toxicity of HCT and thereby expand its availability by extending its availability to older individuals and other who have comorbid medical conditions. Two variations on the theme of nonmyeloablative transplantation have been explored. The first relies on reduced intensity preparation that is associated with hospitalization and accompanied by a risk of regimen-related toxicity, albeit at a reduced level. The reduced intensity pretransplant regimen is used to suppress the host-versus-graft (HVG) reaction and promote engraftment. A second variation utilizes pretransplant preparation that causes minimal myelosuppression, and thus can be administered in the outpatient setting. This approach relies on post-grafting immunosuppression to prevent GVH and HVG reactions, and thereby promote engraftment of donor cells. Both types of non-myeloablative investigations have used the nucleoside analog fludarabine, which has anti-leukemic and immunosuppressive properties, in combination with other chemotherapeutic agents or low-dose TBI to facilitate engraftment of donor cells. A regimen of fludarabine, reduced-dose busulfan and ATG was tested by Slavin et al [41] in the treatment of adults deemed high-risk for traditional myeloablative conditioning. Their purpose was to elicit a GVT effect that might also act as an immunological platform for donor lymphocyte infusions (DLI) in lieu of conventional myeloablative chemotherapy or radiotherapy. Investigators observed 85% survival and 81% disease-free survival in a group of adult patients with malignant and nonmalignant disorders that included 4 patients with non-malignant hematological conditions. While transplant-related toxicity was perhaps reduced, 4 cases of sepsis and 2 cases of severe venoocclusive disease of the liver were observed after administration of this reduced-intensity regimen. Minimal toxicity regimens have been modeled after preclinical large animal studies where a single fraction of TBI (200 cGy) followed by post-grafting immunosuppression with cyclosporine and mycophenolate mofetil (MMF) was sufficient to support donor hematopoietic engraftment from MHC-identical littermates in dogs [42]. This experience was successfully translated to patients with malignancies where a regimen of TBI (200 cGY) with or without fludarabine followed by post-grafting immunosuppression resulted in fewer than 3% of recipients having graft rejection after PBSC transplantation from HLAidentical sibling donors [42]. Of interest, 57% of patients treated in this manner completed the allogeneic transplant procedure without hospitalization.

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To provide a less toxic alternative for those with hereditary immunodeficiency syndromes, investigators reasoned that minimal or even no pretransplant conditioning might be sufficient for full or even partial engraftment of donor lymphocytes [25]. This prediction was based in part on observations that donor lymphoid chimerism among severe combined immunodeficiency (SCID) recipients was accomplished in the majority of cases without any pre-transplant conditioning, although for the most part, donor chimerism was restricted to the lymphoid compartment. However, unlike those with SCID, other forms of severe immunodeficiency have sufficient host alloimmunity to pose a barrier to allogeneic engraftment. Thus, myeloablative preparation before transplantation has been a requirement for a successful outcome, even though transplant-related toxicity has been problematic. To avert this toxicity, investigators in Seattle have studied transplantation without pretransplant conditioning in 2 patients with SCID and 4 with other immunodeficiency syndromes who received post-grafting immunosuppression with MMF and CSP after HLA-identical sibling or HLAmatched unrelated marrow allografting [43]. Evidence of T- and B-lymphocyte reconstitution was demonstrated in five patients, and four of six are surviving 9 to 38 months after transplantation, with 2 dying of infection or complications from a second myeloablative transplantation. This early experience suggests that minimally toxic preparation was sufficient for engraftment in these immunodeficient recipients, and that stable mixed donor-host chimerism was sufficient for a significant clinical benefit. To test this further, several trials employing nonmyeloablative transplantation to induce stable mixed chimerism as a curative treatment for children with symptomatic sickle cell disease have been initiated. One investigation has targeted children with less severe disease, before they develop end-organ toxicity that increases the risk of transplant-related morbidity. This is particularly attractive in SCD where selected individuals who have high-risk features might be spared the often devastating complications of this disease if intervention by HCT is employed earlier. This clinical trial for children with sickle cell disease will utilize the minimal toxicity conditioning regimen of low dose of TBI (200 cGy) and fludarabine followed by postgrafting immunosuppression by mycophenolate mofetil and cyclosporine to achieve mixed chimerism. This approach will be applied to children with HLA-identical sibling donors who have recurrent ACS or VOC but low transfusion exposure to maximize the potential for stable mixed chimerism. It is hoped that successful engraftment will prevent the onset of end-organ toxicity due to SCD. If successful, these techniques might also be applied for HLA-matched sibling donor transplantation for b-thalassemia major or bone marrow failure syndromes. Alternate sources of allogeneic hematopoietic stem cells There have been considerable efforts to extend the successful experience of HLA-identical sibling HCT partially matched HLA-related, and HLA-matched unrelated donors for hematological disorders. The goal of these efforts is to

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expand the clinical application of transplantation for the majority of patients who lack an HLA-identical sibling donor. While the experience with alternate donors for hematological disease is limited, these efforts have been challenged by the problems of GVHD and graft rejection (Table 1) [86 – 88]. This is perhaps best illustrated by the use of alternate donors for severe aplastic anemia where an increased incidence of graft rejection and GVHD have combined to impact negatively on outcome. In this setting the disease-free survival is approximately 30% to 50% [44 –46]. Based in part on these observations, transplantation from alternate donors for severe aplastic anemia is generally reserved for those who do not respond to an initial course of immunosuppressive therapy. This is illustrated by a recent analysis by Deeg et al [45]. The results of HCT from unrelated donors among 141 patients with severe aplastic anemia who had failed one or more courses of immunosuppressive therapy were reviewed. Seventyfour percent received HLA-matched marrow allografts from unrelated donors and 26% received grafts mismatched at one or more HLA-antigens. Thirty-two percent received T-cell depleted grafts. Among 131 evaluable patients, 89% had engraftment of donor cells. Among these, 52% developed grade II-IV acute GVHD and 31% of those at risk for developing chronic GVHD had clinical features of extensive disease. Patients who were HLA-matched by serology and by allele-level HLA-DRB1 typing had a superior survival to those who were DRB1-mismatched, with 56% of DRB1-matched compared to 15% of DRB1mismatched recipients surviving 3 years after HCT ( P = 0.001). In this analysis, minimizing HLA disparity yielded a superior outcome. Utilizing an alternative strategy to reduce the risk of GVHD associated with tissue damage after administration of intensive conditioning with TBI [47,48], a trial to study the impact of lower-dose total body irradiation after unrelated donor HCT is ongoing [48]. The experience of alternate donor HCT for thalassemia major and sickle cell anemia is somewhat limited and dispersed over several decades. A recent survey of HLA-mismatched related donor HCT described actuarial survival and graft failure rates of 75% and 55%, respectively, among 64 patients with b-thalassemia major [49]. The results of HLA-matched unrelated donor HCT were somewhat Table 1 Donor source and GVHD Donor

Grade II – IV acute GVHD

Grade III – IV acute GVHD

Chronic GVHD

HLA-ID sibling BM [85,86]

10 – 38%

3.4 – 15%

11 – 32%

Umbilical Cord Blood [55,60,62] Related Unrelated

22 – 25%

3 – 5% 9 – 20%

Matched Unrelated Donor BM [44,86 – 88]

25 – 64%

5.1 – 32%

Rates of acute and chronic GVHD by donor source Abbreviations: GVHD, Graft versus host disease

6 – 14% 0 – 9% 28 – 57%

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better in a contemporary group of 23 patients, who had a disease-free survival of 61% after pre-transplant preparation with Busulfan and cyclophosphamide, alone (N = 5) or in combination with thiotepa (N = 18) [50]. These preliminary results suggested a benefit of increased immunosuppression by thiotepa before HCT, which may represent a method to improve engraftment rates after unrelated donor transplantation.

New approaches to alternate donor sources and graft characteristics Recently, evidence that peripheral blood stem cells (PBSC) mobilized by cytokines such as granulocyte colony stimulating factor (G-CSF) or granulocytemacrophage colony stimulating factor (GM-CSF) might replace bone marrow as a source of hematopoietic stem cells has been accumulating. PBSC have largely replaced bone marrow (BM) for autologous HCT due to several important observations. These include easy accessibility for collection by apheresis and storage, observations of faster neutrophil and platelet engraftment after HCT that conferred shorter hospitalizations with reduced blood product utilization, fewer infections [51], and perhaps a reduced risk of tumor contamination in the PBSC collections. These findings were explored further in studies of allogeneic HCT comparing BM and PBSC transplantation from HLA-identical sibling donors. In a prospective phase III investigation, PBSC mobilized from HLA-identical donors resulted in more rapid myeloid recovery and comparable rates of acute graft versus host disease [52,53]. Among those with advanced malignant diseases, PBSC recipients had significantly improved outcomes. Additional reports suggested a higher rate of chronic graft versus host disease after PBSC transplantation, perhaps due to increased numbers of T-cells in the PBSC inoculum [52]. The role of PBSC transplantation from HLA-identical sibling donor for non-malignant hematological disorders remains undefined. Related and unrelated cord blood The blood remaining in the placenta following the birth of a child, referred to as umbilical cord blood, contains sufficient numbers of early and committed hematopoietic progenitor cells to be used for transplantation [54]. To date, over 2000 transplants have been performed using umbilical cord blood (UCB) and the number of UCB transplants increases each year. The majority of these have been performed for malignant conditions, metabolic diseases, congenital immunodeficiencies, and bone marrow failure states [2,55 –62]. UCB has several unique properties that make it an important source of hematopoietic stem cells for transplantation. The number and proliferative rate of colony-forming unit-granulocyte-macrophage (CFU-GM) is increased in cord blood compared to mobilized adult peripheral blood. Studies of CD34+ cells isolated from UCB demonstrate enhanced generation of committed hematopoietic progenitor cells compared to similar cells isolated from bone marrow [63]. UCB is

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immunologically naive compared to adult peripheral blood, a factor that facilitates its consideration for transplants where HLA incompatibility exists. The lower incidence of severe graft-versus-host disease associated with HLA-mismatched UCB transplantation may be related to lower levels of hematopoietic cytokines and lymphokines produced by UCB-derived T cells compared to adult peripheral blood [64], as well as decreased T-cell alloreactivity [65]. On a very practical level, UCB stored in public banking programs is immediately available for use and does not require an extended waiting period often associated with volunteer unrelated marrow donations. Invasive CMV disease should occur less frequently after UCB transplantation because UCB is rarely infected by CMV [66]. Finally, telomerase activity in cord blood cells suggests a theoretical benefit of prolonged life span compared to bone marrow derived cells from adult donors [65]. Cell dose and HLA compatibility are important factors that contribute to engraftment and successful outcome following UCB transplantation [55,62,67]. Recent experience of unrelated cord blood transplantation in adults suggest that cell dose considerations can be overcome by careful selection of candidate units, thus making UCB an important alternate stem cell source for pediatric and adult recipients [66,67]. While unrelated UCB transplantation has been performed a number of genetic conditions, unrelated cord blood transplantation has only been attempted in few children with sickle cell anemia. However, early results demonstrate the potential for a successful outcome [55]. Early reports of successful UCB transplantation for hemoglobinopathies using related UCB donors have been extended and confirmed by recent experience from the EUROCORD registry [68]. Forty-four patients (median age 5 years, range 1 to 20) with thalassemia (n = 33) or SCD (n = 11) received UCB transplantation (CBT) from a related donor. All survived and 36 of 44 children survive disease-free, with a median follow-up of 24 months (range 3 to 76) after transplantation. One patient with SCD compared to 7 out of the 33 patients with thalassemia experienced disease recurrence. Three of these 8 patients had sustained donor engraftment after a second transplant with bone marrow from the same sibling donor. The 2-year probabilities of event-free survival were 79% and 90% among patients with thalassemia and SCD, respectively. These results suggest that outcomes after UCB transplantation from sibling donors for hemoglobinopathies are similar to observations after bone marrow transplantation with the potential for reduced rates of graft-versus-host disease. These preliminary results warrant further investigation of this stem cell source for hematological disorders [16,57,69]. Based in part on these observations, there is growing interest in facilitating UCB collection and storage from families who currently have a child with sickle cell anemia or thalassemia and are having another child. A national program to support cord blood collection for families who might benefit from UCB transplantation has been developed at the Children’s Hospital Oakland Research Institute (CHORI). To date, this program has collected over 600 UCB donations from remote sites (primarily at community hospitals) in 42 states. High-quality UCB units with a low bacterial contamination rate (less than 3%) that have a cellular content sufficient to support transplantation ( > 2.5  107 cells/kg

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recipient weight collected in 85% of the cases) have been collected and processed by this program. These results demonstrate the value of a national cord blood banking resource for families with children who might benefit from UCB transplantation, which is being used to support prospective investigations of UCB transplantation [66].

Prevention and treatment of graft versus host disease and Haploidentical transplantation New pharmacologic agents that may prove effective in combination therapy to prevent GVHD have been developed. These include mycophenolate mofetil [70], tacrolimus [71,72], and sirolimus [73,74]. In addition, post-grafting immunosuppression with anti-T lymphocyte antibodies may lead to reductions in GVHD and graft failure and support the development of alternate donor stem cell sources [75,76]. T-cell depletion and positive selection processes that enrich for hematopoietic stem cells also have been studied to decrease the incidence of severe GVHD after transplantation from related but HLA-mismatched (haploidentical) donors. Potential benefits of transplantation from related haploidentical donors include: (i) donors are readily available, hence resulting in minimum delays in transplantation; (ii) it is possible to collect a large stem cell dose by pooling multiple collections; and (iii) real-time availability of fresh lymphocytes for DLI or PBSC for tandem HCT procedures. The disadvantages of haploidentical donor transplantation include: (i) historically high rates of GVHD, (ii) high rates of infection associated with GVHD and delayed immune reconstitution, and (iii) increased rates of graft rejection due to the histocompatibility barrier caused by HLA disparity [77,78]. Positive selection of hematopoietic stem cells Improvements in the prevention of GVHD and graft rejection coupled with minimal delays in immune reconstitution are necessary if haploidentical transplantation is to become widely applicable. Methodologies to purify HSCs have improved so that recovery rates of 60% to 80% are now possible [79]. In 77 procedures utilizing the CliniMACS stem cell selection device (Amcell Corporation) performed at St. Jude Children’s Research Hospital, a median purity of 97% was achieved with a median stem cell recovery of 75% (R Handgretinger, personal communication, 2002). Thus, current technology supports the collection of purified allogeneic stem cell grafts, engineered with CD34 cell purity that exceeds 97% and that has minimal T-cell contamination. These advances in stem cell selection now offer the option of haploidentical SCT for patients who otherwise lack suitable donors. In the past decade, Aversa and colleagues reported the use of highly purified stem cells from haploidentical donors among adults with hematological malignancies [80]. There was a dramatic reduction in acute graft versus host disease (GVHD) due to the profound T-cell depletion that

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resulted from positive selection of hematopoietic stem cells (CD34+ ) cells. Another strategy to overcome the histocompatibility barrier is to infuse a very large dose of hematopoietic stem cells. Preclinical animal data support the ability of megadoses of CD34+ cells to overcome engraftment resistance caused by HLA disparity [81]. This may in part result from a ‘‘veto’’ effect in which purified stem cells downregulate antidonor alloreactivity. Handgretinger extended these preclinical findings in a phase I clinical trial involving 39 children who had advanced malignant and nonmalignant diseases [82]. Purified samples of stem cells from haploidentical parental donor PBSC collections were isolated by the CliniMACS stem cell selection device (Amcell Corporation). Patients were prepared for transplantation with a myeloablative combination of thiothepa, cyclophosphamide, ATG, with busulfan or TBI. The 7 initial patients received CSP to prevent GVHD, but the subsequent 32 received no post-grafting immunosuppression. Primary engraftment was observed in 36 patients after PBSC transplantation from haploidentical donors, followed by late rejection in two. Ultimately, five patients received second PBSC infusions for graft rejection after receiving preinfusion anti-T cell antibody and corticosteroids, resulting in durable engraftment for four of five patients. No Grade III-IV acute GVHD or extensive chronic GVHD was observed in the absence of DLI. GVHD was limited to 6 of 21 high-risk leukemia patients who received DLI that was instituted to reduce the risk of relapse. The 3-year disease-free survival among children with non-malignant disorders was 75% (Fig. 3). These encouraging results are being tested in larger prospective studies. Delayed immune reconstitution accompanied by serious infections and posttransplant lymphoproliferative disease is another potential consequence of T-cell depleted HCT from haploidentical donors [83,84]. This problem might be addressed by maximizing the stem cell dose, a notion supported by observations in patients who received >20  106 CD34 + cells/kg and had more rapid T-lymphocyte recovery than those who received a lower CD34 + cell dose [82].

Fig. 3. Probability of disease-free survival after purified CD34+ HCT from HLA-mismatched parental donors. (Adapted from Handgretinger R, Klingebiel T, Lang P, et al. Megadose transplantation of purified peripheral blood CD34+ progenitor cells from HLA-mismatched parental donors in children. Bone Marrow Transplant 2001;27:777 – 83; with permission.)

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Natural killer cells (NK cells) recovered rapidly, while T- lymphocyte recovery of > 0.1  109 CD3+ cell/l required a longer period of time (median, 72 days). This approach will require vigilant monitoring for opportunistic infections from pathogens such as cytomegalovirus (CMV) and adenovirus. This can be accomplished with highly sensitive techniques such as polymerase chain reaction (PCR) to detect viral DNA. The successful and safe use of haploidentical donors utilizing stem cell selection technology might expand the availability of transplantation to most patients with nonmalignant diseases who might benefit from HCT.

Summary Hematopoietic cell transplantation (HCT) has been used for more 30 years for the treatment of selected malignant and nonmalignant diseases. Traditionally, HCT for hematological disorders has relied on myeloablative conditioning before HLA-identical sibling bone marrow transplantation to correct the underlying hematological defect. Most children with hematological diseases who are referred to HCT have features that portend significant morbidity and early mortality. Among SAA patients who have HLA-identical sibling donors, younger patients with profound pancytopenia might be considered early for HCT. For others who lack sibling donors, patients who receive HCT from alternate sources have generally failed one or more courses of intensive immunosuppressive therapy and remain transfusion-dependent, some with hemosiderosis, red cell alloimmunization, and platelet transfusion refractoriness [44,46,48]. Currently, HCT for SCD is generally restricted to those who have experienced a significant sickle-related complication such as stroke, recurrent acute chest syndrome, or recurrent painful episodes [7,13]. In contrast, most reserve HCT in thalassemia for younger, Lucarelli class I, good-risk patients who have HLA-identical sibling donors, and veer away from older, high-risk thalassemics for whom transplantation is a riskier clinical intervention. For groups such as young adults with thalassemia major, HCT might become more widely applicable if its toxicity was reduced. Several approaches undergoing development include reduced-intensity conditioning and attempts to prevent GVHD. New methods to reduce the intensity and toxicity of conditioning as well as to use highly purified stem cells with the reduction in graft versus host disease may allow for the use of matched unrelated donors or haploidentical donors. This would serve to provide potentially more children who could benefit from stem cell transplantation with donors. These advances will hopefully lead to benefits for the majority of children who lack HLA-identical donors.

Acknowledgments We acknowledge the support of the NIH (no. 5M01RR01271) and NHLBI (2 U24 HL61877-03A1).

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