Umbilical Cord Blood Transplantation Supported by Third-Party ... - Core

Biol Blood Marrow Transplant 19 (2013) 682e691

Review

Umbilical Cord Blood Transplantation Supported by Third-Party Donor Cells: Rationale, Results, and Applications

ASBMT

American Society for Blood and Marrow Transplantation

Koen Van Besien 1, *, Hongtao Liu 2, Nitin Jain 2, Wendy Stock 2, Andrew Artz 2 1 2

Weill Cornell Medical College, New York, New York Section of Hematology/Oncology, University of Chicago, Chicago, Illinois

Article history: Received 9 September 2012 Accepted 2 November 2012 Key Words: Umbilical Cord Blood Transplant Third party donor cells Haplo Cord NIMA IPA

a b s t r a c t Low incidence of graft-versus-host disease provides the major rational for pursuing umbilical cord blood (UCB) stem cell transplantation (SCT). Considerable evidence also suggests a lower rate of recurrence after UCB SCT than after transplantation from adult donors. Recent advances in understanding of the human fetal immune development provide a rational underpinning for these clinical outcomes. The fetal immune system is geared toward maintaining tolerance to foreign antigens, particularly to the maternal antigens to which it is exposed throughout gestation. To this purpose it is dominated by a unique population of peripheral T regulatory cells that actively maintain tolerance. This and other features of the UCB lymphoid system explains the low incidence of graft-versus-host disease and superior outcomes of UCB SCT with noninherited maternal antigenematched grafts. At the same time, highly sensitized maternal microchimeric cells are frequently detected in UCB and likely contribute to superior graft-versus-leukemia effects and low rates of disease recurrence in inherited paternal antigenematched UCB recipients. However, historically erratic and slow hematopoietic recovery after UCB SCT leads to increased early morbidity and mortality, excessive hospitalization, and increased costs. This has held up the widespread utilization of UCB SCT in adults. Here we summarize recent data on UCB SCT with an emphasis on studies of co-infusion of adult CD34 selected hematopoietic stem cells with UCB SCT. This procedure, through transient engraftment of adult hematopoietic stem cells, largely overcomes the problem of delayed engraftment. It also results in predictable engraftment of a UCB with the desired characteristics. We also briefly discuss unresolved issues and possible future applications of this technology. Ó 2013 American Society for Blood and Marrow Transplantation.

INTRODUCTION Attempts at allogeneic transplantation were reported as early as 1957 (and among the small group of early recipients, at least 1 received fetal rather than adult bone marrow) [1]. These early attempts faltered because of graft rejection and graft-versus-host disease (GVHD). It was not until the discovery of the human leukocyte antigen (HLA) system and the recognition of its pivotal role in GVHD and graft acceptance that allogeneic transplantation became a feasible procedure [2]. Initially restricted to HLA-identical sibling pairs, it was rapidly expanded to unrelated donor transplantation. Refinements in HLA typing over the past decade have led to the recognition that HLA-identical donors are lacking for many [3]. It is estimated that only 20% of African Americans, approximately 70% of Caucasians, and intermediate percentages of subjects of other ethnicities have access to HLA-identical unrelated donors [4]. The development of transplant methods that obviate the need for HLA identity is therefore imperative. Umbilical cord blood (UCB) transplantation has considerable promise, because of its tolerance to the host environment and its potent graft-versusleukemia (GVL) effects, features that are briefly summarized in the light of current understanding of the fetal immune system. When supported by co-infusion of third-

Financial disclosure: See Acknowledgments on page 689. * Correspondence and reprint requests: Koen Van Besien, MD, PhD, Weill Cornell Medical College, Section of Hematology/Oncology, 520 East 70th Street, New York, NY 10021. E-mail address: [email protected] (K. Van Besien).

party cells, hematological reconstitution after UCB stem cell transplantation (SCT) is fast and reliable, removing one of the largest hurdles to successful UCB SCT. BIOLOGICAL CHARACTERISTICS OF UCB AND IMPLICATIONS FOR TRANSPLANTATION The cellular composition of the UCB graft reflects the functional status of the fetus at full-term gestation. UCB contains lymphoid and dendritic cells as well as cells of hematopoietic lineages [5,6]. In addition, many if not all UCB units contain variable percentages of cells of maternal origin, a phenomenon called maternal microchimerism [7-11]. Important studies over the past years (reviewed by Mold and McCune [12,13]) have led to a new understanding of the organization and function of the human fetal immune system. Fetal Immune System Is Geared Toward Tolerance to Foreign Antigens From an immunological standpoint, pregnancy represents an extraordinary situation in which both fetus and mother are exposed to an immunologically foreign organism [12]. In this process, the fetus develops profound and longlasting tolerance to antigens to which it was exposed during gestation. Owen was the first to observe this, reporting on the immunological behavior of Freemartin cattle, genetically females, but in whom there is lifelong male chimerism due to transmission of cells from a male twin during pregnancy [14]. Subsequent reports on tolerance to tissue grafts between fraternal twins, both in humans and other large mammals, established that tolerance to major

1083-8791/$ e see front matter Ó 2013 American Society for Blood and Marrow Transplantation. http://dx.doi.org/10.1016/j.bbmt.2012.11.001

K. Van Besien et al. / Biol Blood Marrow Transplant 19 (2013) 682e691

histocompatibility complex antigens can occur upon intrauterine exposure between fraternal twins who share an intrauterine blood supply [12]. Owen and colleagues were again the first to show that tolerance to noninherited maternal antigens (NIMAs) can occur during gestation when they studied the effects of fetal exposure to Rhesus antigens [15]. During pregnancy, rhesus-negative women often develop anti-rhesus antibodies, the cause of hemolytic disease of their newborns. But those women who were daughters of rhesus-positive mothers had been rendered tolerant during fetal life and rarely developed rhesus antibodies during their own pregnancies. Claas et al. extended this concept in a landmark 1988 article reporting that heavily transfused and multiply sensitized adults failed to raise antibodies to foreign HLA antigens that were present in their own mothers (NIMA). Presumably, they had been rendered tolerant during fetal life to these antigens [16]. The mechanisms of fetal tolerance remained long poorly understood and were attributed to immaturity of the fetal immune system [17,18]. But recent evidence has shown that the fetal immune system undergoes extensive development during gestation with waves (or so-called layers) of cells of different phenotypic profile and/or function succeeding each other [13] (Figure 1). This model of layered immune system development is reminiscent of the better known model of development of the erythroid system where early embryonic cells are replaced by cells containing fetal hemoglobin followed by another switch at the time of

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delivery toward adult hemoglobin development. The transcription factor Lin28 was recently shown to be a master regulator of the this swith [19]. A complete description of this rapidly developing area is well beyond the scope of this review, but data with potential relevance to UCB SCT are summarized below. The 2 beststudied cell populations in this regard are CD4 T cells and B1 type B cells. T regulatory cells (CD4þ, CD25þ, FoxP3þ), dominate the fetal immune system during midgestation, with numbers declining toward adult levels by the time of delivery [5,20,21]. These T regulatory cells are thought to be essential in the development of fetal tolerance to maternal cells [12,22]. Recent work has shown that although fetal T regulatory cells are phenotypically similar to adult cells, they are functionally quite different and have a unique gene expression profile. Similarly, CD4þ and CD25 negative fetal T cells are functionally different from their adult counterparts. Complex transplant experiments support the model of fundamentally different T cell populations in the fetus and the adult [23]. How many fetal T cells persist at the time of delivery and possibly even throughout childhood is still unknown. But it is likely that the powerful suppressive effect of fetal T regulatory cells contributes at least partially to the suppression of GVHD reactions after UCB SCT. B cell immunogenesis is similarly characterized by successive generations of different B cell types. In mice, 2 types of B cells are recognized, labeled B1 and B2 cells. B1 cells (CD5þ in mice) predominate during neonatal life and

Figure 1. Model for transition from fetal to adult hematopoiesis (reproduced from Mold and McCune [13]). Several potential scenarios could account for the layering of the adaptive immune system during development. Based on the linear decline in the frequency of T regulatory (Treg) cells in umbilical cord blood across the third trimester of development, we propose that a shift in hematopoietic stem cell (HSC) identity from fetal to adult occurs at some point during this time. Whether this occurs through the de novo generation of adult hematopoietic stem cells from an upstream progenitor cell or from a direct conversion of fetal hematopoietic stem cells to adult-type hematopoietic stem cells remains unknown. Examples of different hematopoietic lineages that have been shown to arise during fetal development and after birth are listed below the figure. BM indicates bone marrow; HSPC, hematopoietic stem and progenitor cells.

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appear to derive from unique precursors [13]. B cells in human UCB constitute about 11% of lymphocytes (as opposed to about 4% in adults), and a very high percentage of these B cells are CD5þ and CD27þ [24,25]. These CD27þ cells in human UCB have recently been identified as the counterpart of murine B1 cells [25]. B1 cells are important in innate immunity through secretion of “natural Ig” and through their ability to stimulate T cells. It is likely that they contribute to innate resistance to infections [25]. The rapid proliferation of CD5 þ B cells after UCB SCT may therefore be an important, though still incompletely studied, component of effective immune reconstitution. Less is known about the ontogenesis of other components of the fetal immune system. Umbilical cord dendritic cells are hyporeactive upon stimulation with limited up-regulation of surface receptors, limited signaling, and a bias against inducing CD4 Th1 responses [5,12,26]. The preferential Th2 responses may further diminish GVHD. Fetal natural killer (NK) cells have reduced function and cytolytic activity compared with adult NK cells [5]. Tolerance induction is therefore the hallmark of the fetal immune system, and at least partial persistence of the fetal immune system in the full-term UCB contributes to the unusually low rate of GVHD after UCB SCT despite significant HLA disparity and to the tolerance of NIMA antigens that has been shown to occur after UCB SCT as was previously shown in solid organ transplant [27]. In an initial preliminary report, Van Rood et al. had access to a set of UCB SCT recipients with complete HLA typing of the infant donor of the UCB unit and of the mother of the infant [28]. Most UCB recipients were HLA mismatched with their donors at one or several antigens. In some cases, the HLA mismatch was identical to an HLA antigen present in the mother of the infant donor (NIMA match). In other cases, the mismatched HLA antigen was not present in the donor’s mother (no-NIMA match) (for an example, see Table 1). They found that treatment-related mortality and overall mortality was significantly lower in the recipients of NIMA-matched transplants, particularly among recipients older than 10 years of age. Acute and chronic GVHD were reduced among NIMA-matched transplant recipients, but this reduction was not statistically significant. Neutrophil engraftment was more rapid among NIMA-matched recipients, and, somewhat surprisingly, there was also a trend toward lower relapse rate in the NIMAmatched patients. The effect of NIMA matching on transplant-related mortality (TRM) was recently confirmed in a study where

Table 1 Examples of NIMA Match versus NIMA Mismatch Recipient NIMA match CB unit 1 Mother of unit 1 NIMA mismatch CB unit 2 Mother of unit 2

A1

A24

B7

B65

DR0102

DR1501

A2 A1

A24 A24

B7 B57

B65 B65

DR0102 DR0102

DR1501 DR1305

A2 A3

A24 A24

B7 B7

B65 B65

DR0102 DR0102

DR1501 DR1501

In this example, 2 identical 5/6 HLA-matched cord blood units are evaluated for use in a particular recipient. The mismatch between UCB recipient and donor is in the HLA-A locus. UCB recipient HLA-A1 and CBU HLA-A2. Case 1 NIMA match: The mother of CBU1 happens to have HLA-A1. During gestation, CBU was therefore exposed to HLA-A1 and has become tolerant to NIMA HLA-A1. Case 2 NIMA mismatch: The mother of CBU2 does not have HLA-A1. This CBU was therefore never exposed to HLA-A1 and has not become tolerant to it. All things being equal, transplantation with CBU1 is expected to have superior outcome.

the outcome of 48 NIMA-matched UCB SCTs was compared with that of 116 noneNIMA-matched UCB SCTs in a casecontrol fashion [29]. The groups were matched for important covariates. No effect of NIMA matching on relapse rate, acute or chronic GVHD, or neutrophil recovery was found in this more recent analysis. But TRM was lower after NIMAmatched UCB SCTs compared with NIMA-mismatched UCB SCTs (relative risk, .48; P ¼ .05; 18% versus 32% at 5 years). Consequently, overall survival was higher after NIMAmatched UCB SCT. The 5-year probability of overall survival was 55% after NIMA-matched UCB SCTs versus 38% after NIMA-mismatched UCB SCTs (P ¼ .04). Of note, the importance of NIMA matching may also extend to the setting of adult haploidentical transplantation. Two groups have shown that transplant from a NIMA-matched haploidentical donor is associated with improved outcome, mainly because of a lower incidence of GVHD [30,31]. Maternal Microchimerism in the UCB May Be Responsible for GVL Several observations point to a reduced rate of disease recurrence after allogeneic cord blood transplantation compared with adult-related or -unrelated donor transplantation. This was initially revealed in double UCB SCT, which has been shown to have lower recurrence rates compared with adult transplant [32,33] (though the benefit of reduced rates of recurrence was largely offset by increased early TRM after UCB SCT). In some studies, similar low relapse rates have been observed after single UCB SCT [32,34]. Some have attributed the increased GVL effects after UCB SCT to increased mismatching [35,36]. Indeed, preliminary evidence in children shows that more mismatching decreases the risk of disease recurrence. This mechanism could explain why relapse rates after UCB SCT are lower than those after transplant from HLA-identical donors. But haploidentical adult grafts are even more HLA mismatched than UCB. Yet, in several studies, haploidentical SCT is associated with higher rates of disease recurrence [37,38]. Others have proposed that killer cell immunoglobulin-like receptor (KIR)ligand mismatching after UCB SCT may be associated with reduced relapse risk [39]. Although this may be correct, KIR antigen mismatching is also frequent in matched related and unrelated donor SCT, and KIR mismatching does not explain the differential relapse rates with these graft sources. More recently, Van Rood et al. proposed a hypothesis based on the observation of frequent maternal microchimerism in the graft [40]. It has been known for decades that maternal cells can cross the placenta and colonize the fetus and vice versa (for review see Mold and McCune [12]). These cells are most prevalent during midgestation but can also be frequently detected in UCB. In contrast to the fetal immune system that invariably becomes tolerant to NIMAs, the maternal immune system becomes sensitized to the paternal HLA antigens inherited by the fetus (the inherited paternal antigens [IPA]). This phenomenon is the frequent cause of anti-HLA antibodies in parous women that can complicate platelet transfusions and contribute to graft rejection in stem cell and organ transplantation [41-43]. Because sensitized maternal cells are present in virtually all UCB, van Rood et al. reasoned that the small number of maternal cells rather than the majority of fetal lymphoid cells are primarily responsible GVL effects after cord blood transplant. In an ingenious analysis, they compared the outcome of two sets of patients undergoing UCB SCT. In most cases, there was an HLA mismatch between the mother of


the UCB donor and the transplant recipient to which the mother was sensitized. They labeled this donorerecipient combination “shared IPA transplants.” In some cases, labeled “no-shared IPA transplants,” there was no IPA target in the recipient. This can happen when father and mother of the fetus share an HLA haplotype (for an example, see Table 2); this fetus can be HLA identical or nearly HLA identical to the mother. In other cases, the HLA-mismatch between recipient and cord blood was such that the IPA targets were absent in the recipient. The recurrence rate was significantly lower in shared IPA transplants than in no-shared IPA transplants, presumably because of the antiIPA mediated GVL effects in the IPA transplants. This study, although retrospective and with several limitations, provides a powerful and rational explanation for maternally mediated GVL effects in UCB SCT. It will be up to future investigators to prospectively validate these observations. Interestingly, in pediatric recipients of parental haploidentical T celledepleted SCT, there is a much better outcome in patients receiving SCT from the mother compared with those receiving paternal SCT, probably because mothers have been previously sensitized to the child’s IPAs [44]. Hematopoietic Potential of UCB: Comparison with Adult Stem Cells Stem cells and progenitor cells are present in UCB, at a much higher concentration than in adult peripheral blood and in concentrations that may exceed even those of adult bone marrow [6,45]. The phenotypic characteristics of UCB and adult bone marrow cells are remarkably similar however [46], with the exception of a higher density of expression of CD34 on the cells and an increased expression of HLA DR on neonatal cells. Functionally, UCB has a higher proportion of cells with stem cell characteristics, including the ability to replate and expand while maintaining primitive characteristics. They are also more proliferative in long-term cultures and have increased migration capacity. Finally, in contrast to adult bone marrow cells, they are less dependent on exogenous cytokines for establishing growth in SCID mouse models [47]. The mechanisms underlying the proliferative and expansive advantage of UCB over adult hematopoietic stem cells include longer telomere length, more rapid exit from

Table 2 Example of IPA Targeting versus No IPA Targeting by Maternal Microchimerism Recipient IPA targeted CB unit 1 Mother of unit 1 Not IPA targeted CB unit 2 Mother of unit 2

A1

A24

B7

B65

DR0102

DR1501

A2 A1

A24 A24

B7 B57

B65 B65

DR0102 DR0102

DR1501 DR1305

A2 A3

A24 A24

B7 B7

B65 B65

DR0102 DR0102

DR1501 DR1501

In this example, the same identical 5/6 HLA-matched CBUs are evaluated for use in the same recipient. The mismatch between UCB recipient and donor is in the HLA-A locus. UCB recipient HLA-A1 and CBU HLA-A2. Case 1 IPA targeting: During gestation, microchimeric maternal cells of CBU1 were sensitized to paternal HLA-B7 and HLA-DR 1501. These same IPA antigens are present in the recipient and are therefore targets of GVL activity. Case 2 No IPA targeting: The mother of CBU2 happens to be homozygous to her child in HLA-B and HLA-DR. Therefore, exposure to HLA-B7 and HLADR1501 did not, in this case, lead to sensitization of maternal microchimeric cells. All things being equal, transplantation with CBU1 is expected to have superior outcome due to more potent GVL effects. This also illustrates how one can simultaneously select UCB units to be NIMA matched and IPA targeted.

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G0/G1 into the cell cycle, increased cytokine sensitivity, and/or paracrine cytokine effects. Gene expression analysis shows considerable differences in gene expression patterns between adult bone marrow and UCB, with fetal gene expression regulated by the transcription factor Sox17 [48]. In murine competitive repopulation experiments, fetal or newborn blood tends to outcompete adult bone marrow competitors [49]. Rosler et al. directly compared the hematopoietic potential of human adult and fetal hematopoietic stem cells in an SCID HU model [50]. For this purpose, immunodeficient disease (SCID) mice were implanted with human fetal bones. HLA-mismatched CD34 selected cells from 2 human donors were injected simultaneously and 8 to 10 weeks later engraftment of myeloid and lymphoid cells was analyzed. Both adult bone marrow CD34 cells and cord blood CD34 cells could routinely engraft and differentiate into myeloid and B-lymphoid cells in this assay. After 8 to 10 weeks, they accounted for approximately 50% of cells circulating in the animals. When cord blood and adult bone marrow were injected simultaneously, the engraftment of cord blood cells was markedly superior to that of adult bone marrow cells. Cord blood engraftment was detected in 10 of 10 animals. Adult cells were detected in only 5 of 10 animals, and even in those, the adult cells constituted only a small fraction, less than 10% of all myeloid and B cells. Although hematopoietic stem cell characteristics of UCB favor long-term dominance, other work has shown that rate of engraftment is determined by the number of hematopoietic stem cells transplanted [51]. In clinical practice, the usual measure of hematopoietic stem cells is the number of CD34þ cells in the graft. UCB grafts contain at least 1 log fewer CD34þ cells than adult CD34þ stem cell grafts. The low number of hematopoietic stem cells in UCB grafts explains the often slow and erratic hematopoietic recovery. CLINICAL UCB SCT: OVERCOMING LIMITATIONS The first UCB SCT was reported in 1989, and since then extensive experience has accrued with UCB SCT using both related and unrelated donors. Multiple aspects of this experience have been reviewed extensively by others [52-54]. Here, we only briefly summarize those data with an emphasis on novel developments and areas of controversy. It is now well accepted that UCB SCT can result in durable engraftment and cure of leukemia. The incidence of acute and chronic GVHD is relatively low despite extensive HLA mismatching between donor and recipient [35]. Still, even though mismatching is much better tolerated with UCB SCT compared with adult mismatched SCT, matching cannot be completely ignored. Better matching grafts tend to produce better long-term outcomes, and recent data suggest that HLA-C matching further improves outcomes, particularly among recipients that are 5/6 or 6/6 matched at HLA A, B, and DR [55]. There is also considerable evidence that UCB SCT, particularly double UCB SCT, has a lower recurrence rate than adult SCT. For example, the group from Minnesota compared the outcomes of myeloablative conditioning followed by double UBC SCT, matched related, matched unrelated, and mismatched unrelated transplants for adults with hematological malignancies [33]. The risk of relapse was only 15% of recipients of double UCB (95% confidence interval, 9% to 22%), significantly lower than in recipients of matched related donor SCT (43%; 95% confidence interval, 35% to 52%), adult matched unrelated donor SCT (37%; 95% confidence interval, 29% to 46%), or adult mismatched unrelated donor SCT (35%; 95% confidence interval, 21% to 48%). Similarly, in

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parallel studies of haploidentical versus double UCB SCT, recurrence rates were considerably lower after double UCB SCT [32]. Verneris et al. [32] suggested that the reduced rate of recurrence was limited to recipients of double UCB SCT versus single UCB SCT. Other investigators did not confirm this. A recently reported randomized study failed to show an advantage to double UCB SCT over single UCB SCT in children [33]. The limitations of UCB SCT are also increasingly clear and relate mostly to graft failure/rejection or delayed engraftment. Closely associated with this are the prolonged length of stay, increased expense, and increased TRM after UCB SCT [56,57]. Several groups reported an association between the presence of donor-specific antibodies (DSA) and graft rejection in HLA-mismatched transplant and particularly in cord blood transplant [42,43,58-60], but there is considerable debate over the relative importance of this finding [61], over the exact mechanisms by which this occurs, and over the management of transplant patients with positive DSA [62,63]. For many transplant programs, there is now sufficient evidence to avoid grafts that are targeted by DSA [64]. Another area of debate centers on UCB quality and its assessment. As best studied by the Sloan-Kettering group, it is clear that occasional cases of graft failure are due to poor graft quality [65,66]. Further investigations are required to understand the best determinants and measures of cord quality to ensure engraftment. For an extensive discussion of this topic, we refer to the recent review by Barker et al. [65]. Less widely debated is the issue of study endpoints and determinants of success. UCB studies typically focus on engraftment, usually defined as the first of 3 consecutive days of neutrophil recovery of absolute neutrophil count (ANC) > 0.5 109/L. This may not be the best endpoint for studies. In consecutive studies, the Seattle group has shown that time to achievement of an ANC > 0.1 109/L is better correlated with survival after allogeneic transplant, regardless of graft type, and that TRM rapidly increases if this milestone is not reached by day 16 [67,68]. Rather than focusing on the median time to neutrophil recovery, it may therefore be advisable in future studies to report the percentage of patients achieving an ANC > 0.1 109/L by day 16. Unfortunately, this measure is not readily available in US labs, because differential counts are not usually performed in patients with ANC < 0.5 109/L. Platelet recovery is another very important determinant of long-term outcome. Delayed platelet recovery is associated with increased length of stay and increased expense, and in some studies it is an important correlate of TRM [56,57,69]. Platelet recovery after UCB SCT tends to be much more delayed than that after adult transplantation. ENHANCING HEMATOPOIETIC RECOVERY AFTER UCB SCT Initial efforts at improving outcomes of UCB SCT addressed the issue of cell dose by infusing several products [70,71]. Before double UCB, many patients were ineligible for UCB SCT because of lack of UCB units that were both well enough matched and had an acceptable cell number. The problem was particularly dire among African American patients. There is obviously some genetic clustering; as a result UCB from African American newborns tends to be the best match for African American recipients, but UCB from African American infants on average contains considerably fewer nucleated cells [72]. By combining UCB units, an acceptable cell number could be reached. Though hematopoietic recovery remains unpredictable and often slow,

transplantation could now be offered most patients in need. It is now well established that in most cases only one of the UCB units ensures durable engraftment. What biological characteristic, if any, determines the winner graft remains a topic of debate [73,74]. Much effort has been devoted to expansion of UCB stem cells or subsets of such cells. This field has the promise of tissue engineering with generation of cell types of particular interest that potentially could be further manipulated in vitro to generate improved antileukemic effects [75]. As far as cord blood is concerned, the primary goal of most trials is to enhance hematopoietic recovery. Several excellent reviews have summarized this field [76,77]. Although faster than expected hematopoietic recovery has been observed, this does not occur reliably. In some studies, expanded UCB has been infused with nonexpanded cells from another donor; in such cases, durable engraftment is almost universally derived from the nonexpanded cord. No study or expanded UCB has yet been able to show any impact on platelet recovery. An additional hurdle for UCB expansion is the time required for generating a sufficient number of cells to ensure rapid recovery. This is expected to take several weeks, a time period not always permissible because of the aggressive nature of many hematological malignancies. To circumvent this problem, efforts are ongoing to develop “off the shelf” products that might provide transient hematopoietic support. CO-INFUSION OF ADULT PROGENITOR CELLS WITH UCB STEM CELLS A technically less-demanding approach consists of the infusion of UCB cells with cytokine mobilized CD34þ selected donor peripheral blood stem cells from an adult, usually a mismatched relative. Fernandez et al. reported 3 patients with advanced hematological malignancies who underwent UCB SCT after myeloablative conditioning supported by co-infusion of adult haploidentical CD34þ cells [78]. In all patients, adult donor hematopoiesis was transiently detected and caused rapid count recovery but disappeared over time. The same group has since performed 87 such transplants, and detailed results were reported on the 55 initial patients [79,80]. Four other groups have reported outcomes of cord blood transplant supported by co-infusion of adult, usually haploidentical cells [81-84]. As of July 2012, data on 206 patients have been presented and are summarized in Table 3. In all studies, the adult donor cells were T cell depleted, usually by a CD34 selection process. In all studies, antithymocyte globulin (ATG) was part of the conditioning regimen, further T cell depleting the adult donor and, as a secondary effect, also depleting the cord blood unit. But there were also major differences. The National Institutes of Health study was restricted to young patients with severe aplastic anemia [82], They used a nonmyeloablative conditioning regimen. They were the only group to use equine ATG as part of the conditioning regimen (as opposed to rabbit ATG in all other studies). The Utrecht group in the Netherlands treated children, most of whom had metabolic disorders [83]. The largest experiences come from the Madrid group and from the University of Chicago [80,84,85]. Both studies treated adults with hematological malignancies. The patients in Chicago were 15 years older (median age, 51 versus 34 years) and more often had advanced hematological malignancies. The Spanish group used myeloablative conditioning, and the Chicago group used a fludarabine melphalan regimen. GVHD

52 (10-69) Liu et al., updated from [84]

rATG indicates rabbit antithymocyte globulin; CI, cumulative incidence; eATG, equine ATG; ext, extensive; Fail, graft failure; Flu, fludarabine; HBV, hepatitis B virus; IQR, interquartile range; lim, limited; Mel, Melphalan; MFU, median follow-up; Mod, moderate; OS, overall survival; PFS, progression-free survival; PTLD, posttransplantation lymphoproliferative disease; R, related; Treo, treosulfan; U, Unrelated.

42% @ 1 yr 53% @ 1 yr 5 PTLD 1 adeno 1 CMV dx

39 (28-49) Kwon 20 2012 [81]

59

12 Non-malig 8 (0.2-24) malign 3

AML 11, ALL 6, other 3 AML/MDS 39 Flu Mel rATG ALL 10 NHL 7 CLL/MPD 3

R

11 (IQR 10-14)

21 (IQR 16-36)

3 prim 4 sec

6

3

6%

28%

29%

20 (2-54)

47% 8% 44% @ mod/sev 1 yr 0 27 (9-84) 14 (9-28)

1 29 (14-300)

Bu Flu rATG (9), R Treo, flu Campath (2) TBI coþrATG 2, R (19) U (1) BuCyFlu rATG 11

12(9-15)

1

2

0

4 mild, 1 mod 0 2 0 10 (10-38) R SAA 19 (7-27)

Flu Cy, TBI 2 Gy eATG

36(IQR 19-75)

73% (8/11) 11 (.5-46) 2

14 (3-42) 1

44%

CMV disease, Toxoplasma, HBV PTLD 47% @ 5 yr 56% @ 5 yr 7 4 4 1 11/12 11/12 CI 35% CI 17% (8-35) 9 lim, 3 ext 6 3 1 32 (13-98) 10 (9-36) R (38) U (17) Cy TBI rATG (41) BuCY rATG 11 AML 30%, ALL 40%, others 30% 34 (14-60)

Bautista, 55 2009 [80] Sebrango 2010 [94] Gormley 12 2011 [82] Childs 2012 [95] Lindemans 11 2012 [83]

Author

Table 3 Haplocord Summary of Studies

Diagnosis Age, Median (range) N

Conditioning

Adult Donor Relation R versus U

Median Time to ANC

Time to Plat

Fail Both Grafts

Fail Cord aGVHD Only III-IV

cGVHD

Relapse TRM Rate

MFU (mo) PFS

OS

Major Complications


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prophylaxis consisted of calcineurin inhibitors combined with steroids in Spain and combined with mycophenolate mofetil in Chicago. Infection prophylaxis varied, with very intensive cytomegalovirus (CMV) prophylaxis routinely used in Chicago [86,87]. There were also differences in donor selection procedures. Most adult donors were haploidentical relatives, but approximately one-third of the Spanish patients received cells from completely mismatched unrelated donors. Cord blood selection procedures were similar, although the Chicago group allowed a lower UCB cell dose (average of 1.6 107 nucleated cells/kg recipient weight). Regardless of the patient and donor selection or procedural differences, all groups observe rapid neutrophil recovery with a median time of 10 to 11 days (Figure 2). This early hematopoietic recovery is almost invariably generated by the adult graft. Median time to platelet recovery was also faster than what is generally reported after UCB SCT and ranged from 19 days in Chicago to 32 days in Madrid (Figure 3) (compared with 38 days after reduced-intensity conditioning and double UCB SCT in Minnesota) [57]. In most patients, initial hematopoiesis is over time replaced by UCB-derived hematopoiesis (Figure 4). Occasional failures of the UCB graft or of both grafts were observed in all series. In some cases, graft failure was associated with DSA. UCB graft failures were occasionally attributed to poor cord blood viability [85]. Of note, in the Chicago experience, an excessive dose of adult haploidentical CD34 cells was associated with decreased UCB engraftment. The Madrid group addressed cord failures by infusion of a second UCB unit, which tended to engraft and outcompete the residing adult graft. In other centers, the adult graft was left unchallenged after cord blood failure. Such patients, a minority, had a high incidence of relapse and opportunistic infections [84,85]. The data from the National Institutes of Health are particularly provocative. Until recently, the outcome of UCB SCT was disappointing for patients with severe aplastic anemia. In their series, 11 of 12 heavily transfused patients engrafted and had durable responses [82]. Other outcomes varied considerably; the incidence of opportunistic infections, particularly CMV disease and toxoplasmosis, was considerable in the Spanish cohort but much less in other cohorts. This difference may be due to differences in infection prophylaxis, particularly CMV prophylaxis and in GVHD prophylaxis and treatment. The routine use of steroids for GVHD prophylaxis as practiced in Madrid may result in an increased propensity for infectious complications. Cases of Epstein-Barr virus (EBV) reactivation and EBV posttransplantation lymphoproliferative disease were observed in all series, and close monitoring and preemptive treatment for this complication is warranted. Incidence and severity of GVHD was generally low. In the Madrid series, there were 6 cases of grade III to IV acute GVHD out of 55 recipients. None was observed in the other centers. There were 3 cases of extensive chronic GVHD in Spain but none in the other centers. Despite this low incidence of severe GVHD, the relapse rates for those with malignant disease were moderate. The cumulative incidence of recurrence was 17% at 1 year in Spain and 28% in Chicago. Long-term immune reconstitution was studied by the Chicago group and by the Madrid group. The Chicago group assessed lymphocyte subsets, T cell diversity, Cylex Immuknow assay ([Cylex, Columbia, MD] a measure of T cell responsiveness), and serological response to pneumococcal vaccination [88]. NK cell and B cell reconstitution were

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Figure 2. Time to neutrophil engraftment: results from (A) Madrid and (B) Chicago (reproduced from Sebrango et al. [94] and Liu et al. [84]). ANC-500 indicates time to neutrophil count of 500; CB ANC-500, time to achieve 500 neutrophils from the UCB graft; CB full chimerism, time to full cord blood chimerism.

extremely rapid, occurring at 1 month and 3 months, respectively. T cell recovery was delayed with a gradual increase in the number of T cells, starting around 6 months posttransplantation, and was characterized by a diverse polyclonal Tcell repertoire. Recovery of immunoglobulins and responsiveness to pneumococcal vaccination was observed. T cell spectratype was often remarkably diverse. The authors concluded that immune reconstitution after haplocord transplantation was similar to that seen after cord blood transplantation, despite infusion of much lower cord blood cell doses. The Madrid group reported similar observations in their patients [89]. NK and B cells recovered to normal or higher than normal by 1 and 2 months, respectively. Recovery of T cells was slower. Serial analyses of signal joint T cell receptor excision circles showed very low levels by the third month after CBT, followed by recovery to levels persistently similar or higher than those observed before transplantation and in normal control subjects. In both the Chicago (Figure 4) and

Madrid series, early T cell recovery was derived from the adult donor followed by gradual replacement by cells of UCB origin. It is likely that most of the early B cells after haplocord transplant are also UCB derived. Of interest, the National Institutes of Health group observed immediate UCB-derived T cell reconstitution, without detectable adult donorederived T cells at any time point. This strikingly different kinetic profile of immune reconstitution may be due to the use of a different formulation of ATG. Nobody has evaluated the origin of NK and B cells. CONCLUSION, OPPORTUNITIES, AND UNRESOLVED ISSUES The characteristics of the umbilical cord grafts include potent hematopoietic potential and an immune system dominated by effectors of peripheral tolerance, but also by the presence of microchimeric maternal cells that are routinely sensitized to the recipient. These biological features help to explain the ability of UCB cells to outcompete adult

Figure 3. Time to platelet recovery. (A) Madrid cumulative incidence of platelet count >20,000/mL and >50,000/mL. (B) Chicago cumulative incidence of platelet count >20,000/mL (reproduced from Sebrango et al. [94] and Liu et al. [84]).


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Figure 4. Evolution of chimerism in the Chicago series. (A) Unfractionated peripheral blood cells. (B) CD3 cells (reproduced from Liu et al. [84]).

grafts and cause minimal GVHD while exerting GVL effects that are most likely due to maternal cells. Future research may provide further evidence of these mechanisms, particularly of the GVL effects exerted by maternal cells. At present, these features provide a compelling rationale to continue clinical research to optimize the use of this unique stem cell source. Slow and erratic hematopoietic recovery is the Achilles heel of UCB SCT and has prevented more widespread use in adults. It causes a heightened incidence of early mortality, prolonged length of hospital admissions, and significant costs. Co-infusion of adult cells as described here is the first method that is readily applicable and results in reliable and clinically relevant improvements in hematopoietic recovery. Highly encouraging results are now available from 4 centers with over 200 patients undergoing transplantation. There are many opportunities for further development of this technology. For example, cord blood selection algorithms are currently heavily weighted toward maximizing cell doses at the expense of HLA matching. But our data suggest that for haplocord SCT, UCB cell dose has no effect on outcome. It should thus be possible to select cords based on other criteria that may be more important for long-term outcomes, such as HLA matching, NIMA matching, IPA targeting, or even KIR mismatching. In contrast to double UCB SCT, the selected graft will reliably be the “winner”. This concept will require prospective validation but, if confirmed, will have significant potential to improve long-term outcomes. There is also the possibility of using haplocord SCT to support the infusion of rare cell populations that have important therapeutic implications. For example, haplocord transplant has already been used in the transplantation of an HIV-positive leukemia patient, where the UCB donor was CCR5 delta 32 mutated and therefore resistant to HIV [90]. At least in theory, this approach could also be used for transplanting genetically modified UCB cells or to support transplantation for sickle cell disease. Many questions and issues remain to be resolved, some of which are briefly discussed. Graft rejection or failure has occurred in each series and constitutes a considerable risk. The quality and assessment of cord blood products is under continuing debate, and standards are developing.

Adhering to rigorous standards of viability as proposed by the Sloan-Kettering group may prevent some issues. DSA have also recently been identified as risk factors for graft rejection, and exposure of the graft to DSA should be avoided if possible. Finally, very little is known about the interplay between adult hematopoietic and UCB cells. The group from Chicago found a correlation between adult graft CD34 dose and persistence of the adult graft. Though very preliminary, these data are intriguing and suggest that the balance between UCB and adult graft could be manipulated by varying the adult cell dose (and possibly its composition). Unresolved questions also linger around the choice of the adult donor and its fate. Is there a benefit to using related donors? Or might a totally mismatched adult, as the Spanish group has utilized in one-third of their patients, be preferred for a temporary graft as this may enhance the likelihood of the cord blood unit predominating? Do such donors cause more complications or, to the contrary, could they be selected in a fashion to optimize GVL effects (eg, by KIR mismatching)? Is T cell depletion necessary for the adult donor or would it be outcompeted by the umbilical cord, even if were infused without manipulation? Data in this regard are extremely limited. Is the adult graft destined to irreversibly and completely disappear, or are there residual adult donor cells that persist in the blood or other recipient organs? And if so, what is their function, if any? In the Chicago series, a small fraction of residual adult donor T cells were often detected in the blood. Other questions pertain to the conditioning regimen and particularly to the use of ATG, which has been part of every protocol but also contributes to some of the side effects, particularly to EBV reactivation [91]. One wonders whether ATG could be omitted or replaced by other agents with less risk for EBV reactivation, for example, by alemtuzumab [92]. The further development of this and other related transplantation procedures, combined with parallel advances in diagnostics and supportive care, may finally fulfill the promise of a donor for all [93]. ACKNOWLEDGMENTS Financial disclosure: Drs. Van Besien and Stock received research funding from Genzyme Inc and participated in advisory board meetings for Genzyme Inc.

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