Mini-review Umbilical cord blood for allogeneic transplantation in children and adults

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1 (2001) 27, Nature Publishing Group All rights reserved /01 $ Mini-review Umbilical cord blood for allogeneic transplantation in children and adults Department of Medicine, Case Western Reserve University, University Hospitals Ireland Cancer Center, Cleveland, Ohio, USA Summary: Early clinical reports outlining outcomes for primarily pediatric patients undergoing UCB transplantation, point to delayed time to hematopoietic recovery, and favorable incidence and severity of graft-versus-host disease. Intensive clinical and laboratory research is ongoing focused on strategies to foster UCB allogeneic donor engraftment, thereby allowing wider application of this stem cell source for patients requiring allogeneic transplantation. Bone Marrow Transplantation (2001) 27, 1 6. Keywords: umbilical cord blood; allogeneic transplantation Allogeneic blood and bone marrow stem cell transplantation is limited by the availability of suitable HLAmatched related donors. In 1988, umbilical cord blood (UCB) hematopoietic stem cells (HSC) from a related sibling were transplanted successfully into a child with Fanconi anemia. 1 Subsequently, UCB from HLA-mismatched related 2 and unrelated donors has been shown to successfully engraft pediatric 3 8 and a few reported adult patients 6,9 11 with hematologic malignancies, immunodeficiency syndromes, inborn errors of metabolism, or marrow failure syndromes. This article will review the use of UCB as a suitable alternative for allogeneic transplantation. Tables 1 and 2 outline the advantages and disadvantages for the application of UCB as a new source of HSC for patients requiring allogeneic transplantation. UCB collection and banking Since the first successful unrelated UCB transplant was performed by J Kurtzberg and the pediatric transplant team at Duke University in 1993, 7 clinical and laboratory studies have burgeoned and standard operating procedures have been developed for the collection, processing, and storage of UCB units UCB collection is performed during the final phase of labor or, more commonly, after delivery of Correspondence: Dr, Case Western Reserve University, University Hospitals Ireland Comprehensive Cancer Center, Euclid Avenue, Wearn 433, Cleveland, Ohio , USA Table 1 Advantages of UCB as hematopoietic stem cells for allogeneic transplantation 1 Placental or umbilical cord blood is an abundantly available source of stem cells which can be harvested at no risk to the mother or infant. 2 Ethnic balance in a cord blood repository can be maintained automatically in heterogeneous populations or can be controlled via collection from birthing centers representing targeted minority populations. 3 There is low viral contamination of UCB including cytomegalovirus and Epstein Barr virus. 4 UCB, cryopreserved and banked, is available on demand particularly for patients with unstable disease, eliminating delays and uncertainties that now complicate marrow collection from unrelated donors. 5 The amplification of allogeneic responses including Th1-associated cytokine production by neonatal T lymphocytes has been shown to be less than that of adult T cells, which may underlie UCB reduced graft-versus-host reactivity. 50,52,53 6 Frozen UCB can be easily shipped and thawed for use when needed, compared to freshly donated BM which has a limited shelflife, necessitating coordination between harvesting physicians, transportation personnel and transplantation teams. 7 There is an undistorted accumulation of HLA genotypes acquired in a UCB bank because stored UCB suffers no attrition except by clinical use, unlike volunteer unrelated adult donor registries in which donors are lost due to advancing age, new medical conditions, or geographic relocation. 8 As yet to be determined, although intriguing given the emerging understanding of age-dependent telomerase activity and important contributions of genetic HSC regulation with advancing age of the donor 71,72 to hematopoietic and immune function in the recipient, pediatric patients transplanted with newborn HSC would expectedly maintain normal hematopoietic and immune function during advancing decades, compared with HSC grafts received from adult donors Table 2 Disadvantages of UCB as hematopoietic stem cells for allogeneic transplantation 1 The limited number of hematopoietic progenitor cells contained in collected UCB units may contribute to failed and delayed kinetics of donor hematologic engraftment and restrict its use in adult recipients. 2 Future development of potential abnormalities of the newborn donor s HSC into adult life and their effects on the recipient is unknown at the time of transplant. 3 It is not feasible to collect additional donor HSC for patients experiencing graft failure, nor donor lymphocytes for recipients who relapse after initial UCB allografting.

2 2 the placenta, generally only after delivery of full-term normal infants. UCB HLA typing and infectious agent testing is performed following regulatory agency standards. 16 During prenatal care or immediately post-partum, a careful medical and family history is obtained from the mother. UCB units considered high risk for inherited HSC defects or infectious agents are either not collected, or are not released from quarantine to the clinical bank until results of infectious and genetic disease assays are available. Current procedures for UCB processing and thawing do not differ significantly from those for conventional HSC except that hetastarch is a component of the cryopreservation solution, and UCB units are thawed in an albumin/dextran solution to minimize cell lysis. 13,17 19 Computer-based unrelated UCB bank registries allow the clinician to search for potential grafts using the patient s weight for UCB graft nucleated cell dose calculation, and HLA genotype for degree and type of matching. Clinical reports: UCB pediatric recipients Seven peer-reviewed clinical reports have been published over the past 4 years, summarizing transplant outcomes for approximately 840 patients undergoing UCB related and unrelated allogeneic transplantation. 2 8,20 These studies focused primarily on pediatric recipients (median age ranging from 3 to 7 years) with high risk or recurrent hematologic malignancies or non-malignant hematologic disorders. The probability of donor myeloid engraftment in these UCB recipients in all series was 82% and in five reports was 90%. Preparative regimens administered were myeloablative, either total body irradiation-based or busulfanbased, and in a number of patients also included infusion of anti-lymphocyte globulin as further host immunosuppression. Median day to donor myeloid engraftment with an absolute neutrophil count (ANC) 500/ l was delayed compared to that observed after allogeneic transplantation with conventional grafts, and ranged from 22 to 30 days in these clinical reports. The majority of patients received Filgrastim (Amgen, Thousand Oaks, CA, USA) 5 10 g/kg subcutaneous or i.v. daily from day 0 until durable neutrophil recovery was achieved. The role of optimal combinations of hematopoietic growth factors administered in vivo after UCB transplantation is yet to be fully elucidated. Acute graft-versus-host disease (GVHD) grades II IV in these clinical reports ranged from 35 to 40%, despite the majority of recipients receiving grafts disparate at two or more HLA loci. Event-free survival covered a broad range (22 62%), likely reflective of patient selection in these phase I institutional pilot trials, coupled with high peri-transplant mortality attributed in part to delayed donor myeloid recovery. Transplant outcomes for patients allografted with related UCB were favorable when compared with those receiving unrelated UCB grafts. UCB allogeneic donor engraftment Banked unrelated UCB differs from conventional allogeneic grafts from HLA-matched siblings or unrelated adult donors in that greater HLA mismatching is not associated with an unacceptably high incidence of severe acute or chronic GVHD. However, patients undergoing unrelated UCB transplantation have generally received preparative regimens of increased intensity to facilitate engraftment, resulting in higher treatment-related mortality particularly in adult recipients. 9,21 Graft characteristics known to be of importance in producing rapid donor engraftment in recipients of conventional allografts include cell dose, CD34 + cell content, and HLA matching The primary cause underlying higher primary graft failure rates and delayed donor myeloid recovery in UCB recipients is the reduced HSC dose, with infusion of at least a log less nucleated and CD34 + cells, compared with conventional allogeneic grafts UCB graft variables which have predictive value for time to donor myeloid engraftment include cryopreserved and reinfused total nucleated cells, and infused CFU. 2,5,6 Rates of donor hematopoietic reconstitution are lower after UCB grafting, and kinetics of engraftment are delayed, compared with HLA-matched sibling allogeneic bone marrow grafts. 3 Although UCB graft analyses which have predictive value for hematopoietic engraftment include nucleated cell dose, and HLA matching using serologic class I typing and high resolution molecular typing at class II DRB1 loci, these graft characteristics fall short in reproducibly predicting the incidence and rates of donor engraftment. In recipients receiving allografts from unrelated adult donors, previous studies point to HLA class I disparity, including HLA class C disparity being correlated with higher rates of graft failure 26,27 and severe acute GVHD. 28 Moreover, minor histocompatibility disparity in unrelated allogeneic transplantation may underlie graft rejection. 29 Effects of the number and type of HLA disparities on UCB donor engraftment have not been fully elucidated. 30 HLA disparity in conventional related and unrelated blood and marrow grafting can result in graft rejection and graft-versus-host disease. However, improved clinical outcomes have been facilitated by the development of accurate, reproducible high-resolution DNA-based HLA typing methods. The further application of these DNAbased HLA typing methods to class I typing would be expected to further improve successful engraftment after unrelated UCB transplantation. Although the lower rates of donor engraftment and delays in rates of neutrophil, platelet and red cell recovery may be attributable to reduced total nucleated UCB cell dose, further factors yet to be carefully studied in vivo include possible unique UCB progenitor cell characteristics affecting homing and maturation in the recipient. These factors may serve to reduce the strength of correlations between UCB cell dose and HLA matching, with rates and kinetics of donor engraftment, including adhesion molecule expression on UCB HSC, 31 their homing characteristics, 32 the maturational stage of UCB progenitor cells, and/or altered allo-reactivity between UCB graft lymphocytes and recipient antigen-presenting cells. 36 Mechanisms for the graft facilitating activity of donor lymphocytes include inhibition or elimination of residual recipient immune cells capable of mediating graft rejection. 37 These graft HSC and immune factors may be of particular importance in the set-

3 ting of adults transplanted with UCB, receiving even lower graft CD34 + and nucleated cell doses compared with pediatric UCB recipients. CD34 + all quantification has thus far not been consistently predictive of time to donor hematopoietic engraftment in UCB recipients. The lack of consistent correlation between CD34 + content of infused UCB grafts and time to hematopoietic engraftment may be related to the quantification of CD34 + in UCB grafts post thaw rather than prior to cryopreservation, and/or reduced surface epitope density of CD34 + on UCB progenitor cells. 18,25 In vitro analyses of UCB CD34 + progenitors, moreover, compared with adult marrow and peripheral blood stem cells, point to a less mature phenotype 38,39 compared to marrow and peripheral blood grafts collected from adult donors. Nevertheless, the durability of long-term donor engraftment after transplantation of UCB is evident. To date in all series reported, there have been only four late graft failures observed, and in each case gancyclovir was administered for documented cytomegalovirus infection, likely contributing to secondary graft failure. 16 UCB in adult recipients The number of UCB HSC required to provide durable engraftment in an ablated adult recipient is not firmly established. Accepted practice limits infused UCB cell dose to nucleated cells/kg actual recipient body weight. While published reports have included some adult recipients transplanted with UCB, 6,10,11 an important question is whether a single UCB unit contains sufficient numbers of HSC to predictably engraft full stature adults. We have reported our preliminary experience in use of banked unrelated UCB in adult recipients. 9,21,40 These preliminary analyses indicate that UCB contains sufficient HSC to provide durable engraftment in full stature adult recipients over 40 kg in weight, although the observed primary graft failure rate approximates 10% and median day to attain donor neutrophil engraftment with an ANC 500/ l is 27 days (range days). Although significant delays in time to hematopoietic recovery is observed in adult recipients, this value does not differ significantly when compared with that observed in children grafted with banked unrelated UCB. 5,7,8 There are case reports for donor engraftment after infusion of more than one UCB unit into adult patients 41 to provide an increased HSC and progenitor cell dose, although clinical outcomes for these case reports await further clinical experience to determine the feasibility of this approach. Reduced GVHD after UCB transplant HLA disparity between the donor and recipient in allogeneic transplantation is an important determinant of GVHD. 28,42 Nevertheless, a higher incidence of acute GVHD has been observed in patients transplanted with HLA-matched unrelated grafts when compared with matched sibling grafts, despite class II HLA matching at high resolution molecular tissue typing. This may be attributable to reactivity of donor T cells with recipient minor histocompatibility antigens presented within the MHC complex Minor histocompatibility antigen disparity is expectedly greater between unrelated individuals. Despite infusion of HLA class I and II disparate grafts, the incidence and severity of GVHD observed in recipients of UCB grafts is favorable when compared with that in recipients of unrelated grafts from adult donors (MUD) or HLA mismatched family member grafts. 49 UCB T lymphocytes are typically CD45RA + and express low levels of activation markers, both of which are consistent with a naive Th0 phenotype. 50,51 It is unclear whether the reduced GVHD observed after UCB transplantation is due to reduced graft lymphocyte numbers, altered recognition of recipient self antigens by UCB donor T cells interacting with APC, and/or reduction in the amplification response of these naive donor T cells activated by recipient alloantigen presented within the context of MHC which serve to limit the cytokine and cellular cascade necessary to amplify donor alloreactivity to recipient antigens. 36,52 Alternatively, the low incidence of GVHD observed in recipients of HLA mismatched UCB may be related to the extensive immunosuppression from the myeloablative preparative regimens provided to ensure donor engraftment, and/or the lower dose of UCB graft T cells infused. Several in vitro studies of UCB point to the inherent lack of full expression of immunomodulatory cytokines by alloreactive T cells contained in UCB grafts. 36,50,53 In primary mixed leukocyte culture UCB T cells demonstrate proliferative responses to allogeneic stimulation, but little generation of cytotoxic effector function. In addition, restimulation of primary UCB cultures results in a state of proliferative unresponsiveness. 52,54 Therefore, the reduced GVHD summarized in clinical reports after UCB may be related to these in vitro observations, that immunologically competent cells contained in an UCB graft although capable of recognizing non-inherited antigens, do not elicit the normal cascade of events to expand these alloreactive lymphocytes. Reduced expression of nuclear factor of activated T cells-1 (NFAT1) may be one important molecular mechanisms underlying this reduced cytokine production by UCB T cells. 53 The incidence and severity of acute and chronic GVHD observed in UCB recipients, the majority of whom are children, has thus far been lower than that previously reported in recipients of MUD or partially HLA-mismatched family member grafts. The degree and/or type of HLA disparity effects on UCB transplant outcomes is currently not well understood. Initial reports pointed to a lack of correlation between HLA disparity and incidence of GVHD. 55 More recent data point to HLA disparity as an important indicator of UCB transplant outcomes. 6 It is yet to be determined whether UCB grafting from unrelated donors into adult patients will be associated with the reduced incidence and severity of GVHD observed thus far in pediatric recipients. Future initiatives: UCB transplantation Attempts to increase UCB cell dose has laid the basis for laboratory 56 and phase I clinical trials focused on ex vivo expansion of UCB grafts. Although early clinical trials 3

4 4 reported thus far do not point to more rapid hematopoietic recovery in UCB recipients, there is improved day 100 survival compared with historical controls. Laboratory studies reveal that UCB primitive hematopoietic stem and progenitor cells differ from those collected from adult donors. 35 Cord blood contains hematopoietic progenitor cells at a higher frequency, and these UCB progenitors also have a higher proliferative capacity. Short-term in vitro cultures of UCB CD34 + progenitor cells in the presence of cytokines generate a two- to three-fold expansion of these progenitors. 56 There is concern that ex vivo expansion of UCB may result in differentiation of primitive stem cells, thereby increasing the risk of late graft failure. However, laboratory studies point to the presence of immature progenitors which have limited proliferative response to cytokines, 60 thereby maintaining the stem cell component in expanded UCB grafts, and further, that observed cell expansion is derived from committed progenitor cells. 61 These preclinical studies identify further UCB graft engineering questions including the role of accessory lymphoid populations in ex vivo expanded allogeneic grafts, as well as the role of stromal elements in maintaining immature stem cells with self-renewal capacity during expansion In summary, banked unrelated umbilical cord blood (UCB) has emerged as a substitute allogeneic stem cell source, due to the lack of available HLA-matched related donors for patients requiring allogeneic transplantation, and the observed increased incidence and severity of acute and chronic GVHD when alternative HLA-matched unrelated or partially mismatched family member grafts are utilized. Early clinical reports of UCB transplantation have focused primarily on pediatric or small stature adult recipients. Clinical reports of UCB transplantation thus far point to slower rates of hematopoietic engraftment, and importantly, a low incidence of severe (grade III/IV) acute GVHD even when HLA-disparate grafts are infused. Cellular and molecular mechanisms underlying this reduced GVHD in UCB grafting have yet to be elucidated. Also, despite reduced acute and chronic GVHD observed in these UCB recipients, relapse rates remain low. Preliminary observations point to the fact that UCB from unrelated donors is a feasible alternative source of stem cells for transplantation in adults, resulting in durable although delayed hematopoietic reconstitution, with low incidence and severity of GVHD despite HLA disparity. Further studies utilizing partially HLAmatched banked unrelated UCB in pediatric and adult recipients to improve kinetics of hematopoietic recovery are warranted. Acknowledgements MJL is a Stephen Birnbaum Translational Research Investigator of the Leukemia and Lymphoma Society of America and Leukemia Scholar in Clinical Research (Grant No ). References 1 Gluckman E, Broxmeyer HE, Auerbach AD. Hematopoietic reconstitution in a patient with Fanconi s anemia by means of umbilical-cord blood from an HLA-identical sibling. New Engl J Med 1989; 17: Wagner JE, Kernan NA, Steinbuch M et al. Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease. Lancet 1995; 346: Rocha V, Wagner JE, Sobocinski KA et al. Graft-versus-host disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. Eurocord and International Bone Marrow Transplant Registry Working Committee on Alternative Donor and Stem Cell Sources. New Engl J Med 2000; 342: Locatelli F, Rocha V, Chastang C et al. 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