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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 conven-
tional 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. Employ-
ing 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 HLA-
identical 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 gran-
ulomatous disease [18,19], amegakaryocytic thrombocytopenia [20,21], and

* Corresponding author:
E-mail address: paul.woodard@stjude.org (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
990 P. Woodard et al / Pediatr Clin N Am 49 (2002) 989–1007

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 dis-


eases 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.)
P. Woodard et al / Pediatr Clin N Am 49 (2002) 989–1007 991

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 anti-
thymocyte 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 prolif-
erative and hypercellular, a property caused in part by ineffective erythropoiesis.
992 P. Woodard et al / Pediatr Clin N Am 49 (2002) 989–1007

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 fre-
quent, 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 hepato-
megaly, 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 cyclo-
phosphamide 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 approxi-
mately 60%, event-free survival of approximately 50%, with failures due to re-
jection (11%) or nonrejection mortality (35%). To mitigate the high mortality
among class III patients, pretransplant preparation was modulated by cyclophos-
phamide 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 compen-
satory 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 cyclophospha-
mide dosing did not appreciably increase the risk of graft rejection/recurrent
P. Woodard et al / Pediatr Clin N Am 49 (2002) 989–1007 993

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 pretrans-
plant 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 post-
grafting 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
994 P. Woodard et al / Pediatr Clin N Am 49 (2002) 989–1007

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 HLA-
identical 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.
P. Woodard et al / Pediatr Clin N Am 49 (2002) 989–1007 995

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 hemato-
logical 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 veno-
occlusive 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 HLA-
identical sibling donors [42]. Of interest, 57% of patients treated in this manner
completed the allogeneic transplant procedure without hospitalization.
996 P. Woodard et al / Pediatr Clin N Am 49 (2002) 989–1007

To provide a less toxic alternative for those with hereditary immunodeficiency


syndromes, investigators reasoned that minimal or even no pretransplant con-
ditioning 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) recipi-
ents 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 HLA-
matched 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 min-
imally 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
P. Woodard et al / Pediatr Clin N Am 49 (2002) 989–1007 997

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. Seventy-
four 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 DRB1-
mismatched 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
Grade II – IV Grade III – IV
Donor acute GVHD acute GVHD Chronic GVHD
HLA-ID sibling BM [85,86] 10 – 38% 3.4 – 15% 11 – 32%

Umbilical Cord Blood [55,60,62]


Related 3 – 5% 6 – 14%
Unrelated 22 – 25% 9 – 20% 0 – 9%

Matched Unrelated Donor BM [44,86 – 88] 25 – 64% 5.1 – 32% 28 – 57%


Rates of acute and chronic GVHD by donor source
Abbreviations: GVHD, Graft versus host disease
998 P. Woodard et al / Pediatr Clin N Am 49 (2002) 989–1007

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 granulocyte-
macrophage 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 immunode-
ficiencies, 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
P. Woodard et al / Pediatr Clin N Am 49 (2002) 989–1007 999

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
1000 P. Woodard et al / Pediatr Clin N Am 49 (2002) 989–1007

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 immunosup-
pression 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. Poten-
tial 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 trans-
plantation 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 Corpora-
tion) 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
P. Woodard et al / Pediatr Clin N Am 49 (2002) 989–1007 1001

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 post-
transplant 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.)
1002 P. Woodard et al / Pediatr Clin N Am 49 (2002) 989–1007

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 accom-
plished 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 alloimmuniza-
tion, 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 under-
going 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).
P. Woodard et al / Pediatr Clin N Am 49 (2002) 989–1007 1003

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