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Blood First Edition Paper, prepublished online June 9, 2011; DOI 10.1182/blood-2010-11-319376
Original Article
Scientific category: Transplantation
Long-term outcome and lineage-specific chimerism in 194 patients with WiskottAldrich syndrome treated by hematopoietic cell transplantation in the period 19802009: an international collaborative study
Running title: HCT for WAS: clinical outcome and chimerism
Daniele Moratto1, Silvia Giliani1, Carmem Bonfim2, Evelina Mazzolari3, Alain Fischer4,15, Hans
D. Ochs5, Andrew J. Cant6, Adrian J. Thrasher7, Morton J. Cowan8, Michael H. Albert9, Trudy
Small10, Sung-Yun Pai11, Elie Haddad12, Antonella Lisa13, Sophie Hambleton6, Mary Slatter6,
Marina Cavazzana-Calvo4,15, Nizar Mahlaoui15, Capucine Picard4,15,16, Troy R. Torgerson5,
Lauri Burroughs17, Adriana Koliski2, Jose Zanis Neto2, Fulvio Porta3, Waseem Qasim7, Paul
Veys18, Kristina Kavanau8, Manfred Hönig14, Ansgar Schulz14, Wilhelm Friedrich14§, Luigi D.
Notarangelo19§
1
“A. Nocivelli” Institute for Molecular Medicine, Pediatric Clinic, University of Brescia, and
Laboratory of Genetic Disorders of Childhood, Spedali Civili, Brescia, Italy
2
Bone Marrow Transplantation Unit, Federal University of Parana, Curitiba, Brazil
3
Department of Haematology/Oncology, Spedali Civili, Brescia, Italy
4
Necker Medical School and Paris Descartes University, France, EU.
5
Center for Immunity and Immunotherapies, Seattle Children's Research Institute, University
of Washington, Seattle, WA, USA
6
Children's Bone Marrow Transplant Unit, Great North Children’s Hospital, Newcastle, UK
7
Centre for Immunodeficiency, Institute of Child Health, London, UK
8
Division of Blood and Marrow Transplantation, UCSF Children's Hospital, San Francisco,
CA, USA
9
Department of Pediatric Haematology/Oncology, Dr. von Haunersches Kinderspital, Munich,
Germany
10
Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY, USA
11
Department of Pediatric Hematology-Oncology, Children's Hospital, Boston, MA, USA
12
Division of Immunology, Department of Pediatrics, CHU Sainte-Justine, Université de
Montréal, Montreal, Quebec, Canada
1
Copyright © 2011 American Society of Hematology
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13
Institute of Molecular Genetics - CNR - National Research Council of Italy, Pavia, Italy
14
Department of Pediatrics, University of Ulm, Ulm, Germany
15
Pediatric Hematology-Immunology Unit, Necker Hospital, Assistance Publique Hôpitaux de
Paris, Paris, France, EU.
16
Study Center of Primary Immunodeficiencies, Assistance Publique Hôpitaux de Paris,
Necker Hospital, Paris, France, EU.
17
Fred Hutchinson Cancer Research Center and University of Washington, Seattle WA USA
18
Department of Bone Marrow Transplantation, Great Ormond Street Children's Hospital
NHS Trust, London, United Kingdom
19
Division of Immunology and The Manton Center for Orphan Disease Research, Children's
Hospital, Harvard Medical School, Boston, MA 02115, USA
§
These authors share the senior authorship.
Corresponding author:
Luigi D. Notarangelo, M.D.
Division of Immunology and The Manton Center for Orphan Disease Research
Children’s Hospital Boston
Karp Building, Room 9210
1 Blackfan Circle
Boston, MA 02115
USA
Tel: (617)-919-2276
FAX: (617)-730-0709
Email: [email protected]
2
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Abstract
In this retrospective collaborative study, we have analyzed long-term outcome and donor cell
engraftment in 194 patients with the Wiskott-Aldrich syndrome who have been treated by
hematopoietic cell transplantation (HCT) in the period 1980-2009. Overall survival was
84.0%, and was even higher (89.1% 5-year survival) for those who received HCT since the
year 2000, reflecting recent improvement of outcome following transplants from mismatched
family donors and for patients who received HCT from an unrelated donor at more than 5
years of age. Patients who went to transplant in better clinical conditions had a lower rate of
post-HCT complications. Retrospective analysis of lineage-specific donor cell engraftment
showed that stable full donor chimerism was attained by 72.3% of the patients who survived
for at least one year after HCT. Mixed chimerism was associated with an increased risk of
incomplete reconstitution of lymphocyte count and post-HCT autoimmunity, and myeloid
donor cell chimerism <50% was associated with persistent thrombocytopenia. These
observations indicate continuous improvement of outcome after HCT for WAS, and may have
important implications for the development of novel protocols aiming to obtain full correction
of the disease and reduce post-HCT complications.
3
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Introduction
Wiskott-Aldrich syndrome (WAS, OMIM 301000) is a severe X-linked disorder
characterized by microthrombocytopenia, eczema, and immunodeficiency,1-4 and is caused
by hemizygous mutations in the WAS gene, that encodes the WAS protein (WASp).5 WASp is
expressed in hematopoietic cells and mediates rearrangement of the actin cytoskeleton in
response to cell activation.2,6 A functional deficit of WASp is often associated with
immunological defects, including reduced number and function of T lymphocytes, impaired
antibody production (especially to polysaccharide antigens), defective Natural Killer cell
function, reduced chemokinesis of phagocytes and dendritic cells, functional defects of
regulatory T cells (Tregs) and abnormal induction of apoptosis (reviewed in3,4). A clinical
scoring system has been developed to reflect the variability of the clinical phenotype
associated with WAS mutations.1,7 Patients with a typical WAS phenotype (score 3 to 5) are
highly susceptible to severe bacterial, viral and opportunistic infections. Furthermore, a
significant proportion (24% to 72% in various series) develop autoimmune and inflammatory
complications,8-11 and there is an increased risk of hematological malignancies, mainly
lymphoma and leukemia. In contrast, a score of 1-2 is attributed to patients with a milder
phenotype, X-linked thrombocytopenia (XLT), that is characterized by reduced and delayed
occurrence of infections, autoimmunity and malignancies, and prolonged survival.12 These
differences in disease severity correlate, albeit imperfectly, with the amount of residual
expression of WASp.9,13 While the WAS scoring system has limited value in infants and
young children (who may not yet have developed the full disease phenotype), it can be used
to reflect the patients’ clinical history at the time of HCT.14
In spite of advances in clinical care, patients with classic WAS have a poor prognosis,
and the median life expectancy is only 15 years, unless hematological and immune
reconstitution is achieved by hematopoietic cell transplantation (HCT).15 In various series,
4
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HCT from HLA-matched related donors (MRD) has consistently resulted in survival rates
>80%.16-20 However, MRDs are available for only a minority of patients. Experience with Tcell-depleted HCT from HLA-mismatched family donors (MMFD) has been less satisfactory,
with survival rates between 37% and 55%.16,17,19 More recently, HCT from HLA-matched
unrelated donors (URD) and partially matched unrelated cord blood (UCB) have been
increasingly used to treat patients with severe primary immunodeficiencies, including WAS. A
large collaborative study of the International Bone Marrow Transplant Registry and the
National Marrow Donor Program showed that the five-year survival rate for WAS patients
after MUD-HCT was 71%,16 and comparable or superior results (with survival rates between
80% and 86%) have been reported subsequently in other studies.17-20 In spite of this
progress, several problems remain to be addressed.
Age at HCT has been reported to impact overall survival after HCT for WAS. In
particular, two separate groups reported that five-year survival was significantly worse for
patients who were transplanted after five years of age,16,17 with none of the patients
undergoing URD-HCT at ≥5 years of age surviving 5 years or more after HCT.16
In most cases, HCT for WAS was performed using a fully myeloablative conditioning
regimen, in order to permit stable donor stem cell and multilineage engraftment, which is
expected to result in full correction of the hematological and immunological defects. However,
with this approach approximately 10% of the patients reject the graft, and even more develop
mixed or split chimerism.1 In a European study of 96 patients who survived at least 2 years
after HCT, Ozsahin et al. estimated that as many as 20% of the long-term survivors
developed autoimmunity independent of chronic graft-versus-host disease (cGvHD);
furthermore, the risk of autoimmunity was significantly higher for patients who developed
mixed and split chimerism after receiving MUD-HCT.14
5
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Analysis of lineage-specific chimerism and correlation with correction of the disease
after HCT for WAS has received relatively little attention in the literature. Here we report the
results of a retrospective collaborative study on 194 patients who have received HCT for
WAS. We analyzed outcome, and the effects of clinical status and age at HCT, donor type,
and lineage-specific chimerism with respect to survival and complications of HCT.
Patients and Methods
Patients
Data were collected on 194 patients with a clinical diagnosis of Wiskott-Aldrich
syndrome who received HCT in twelve European and American centers between 1980-2009
(see Supplemental Methods for details). Eight and 66 of the patients included in this study
have been reported previously by Filipovich et al.16 and Ozsahin et al.14 respectively. Clinical
and laboratory information collected before and after HCT were entered by each center in a
de-identified manner in a common electronic spreadsheet (see supplemental methods for
details). Informed consent in accordance with the Declaration of Helsinki was obtained from
the parents of all children. The study was approved by the local Institutional Review Board or
Ethical Committee at all centers.
Hematopoietic cell transplantation
HCT was performed according to the protocols in use at the time at each of the
participating Institutions. Transplantation was done using bone marrow (BM), cord blood (CB)
or peripheral blood stem cells (PBSC) as the hematopoietic stem cell (HSC) source. Donor
cells were obtained from HLA-matched sibling donors (MSD), other matched family donors
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(MFD), mismatched family donors (MMFD), unrelated donors (URD) and partially matched
unrelated cord blood (UCB). Donor and recipient HLA typing was performed by serology for
earlier patients and by molecular DNA typing in more recent years; methods were dependent
on each center’s practice.
Patients underwent either myeloablative or reduced-intensity conditioning (RIC).
Busulfan and Cyclophosphamide were used most frequently as myeloablative regimen, while
Melphalan or Treosulfan in combination with Fludarabine were most commonly selected for
RIC (see supplemental methods for details). For transplantation from MMFD, the graft was Tcell depleted by E-rosetting and soybean lectin agglutination, in vitro treatment with Campath1M monoclonal antibody (mAb) and complement, or more recently by positive selection of
CD34+ cells.
Graft-versus-host disease (GvHD) prophylaxis was performed with methotrexate or
cyclosporine A (CsA) or both. Anti-thymocyte globulin (ATG) or Alemtuzumab were included
in the conditioning for GvHD prophylaxis for non-depleted URD-HCTs and for graft rejection
prevention of T- depleted transplants. Acute and chronic GvHD were graded as previously
reported.21,22 Occurrence and resolution of acute GvHD (aGVHD) grade 3-4 and/or extensive
chronic GvHD (cGVHD) were annotated in the database spreadsheet. Precautions to reduce
the risk of infection were based on reverse isolation, antimicrobial prophylaxis, and
immunoglobulin replacement therapy, according to the policies adopted at each center.
Mutation analysis and WASp expression
Mutation analysis at the WAS locus was performed by polymerase chain reaction
(PCR) based amplification of genomic DNA using primers spanning exon-intron boundaries,
followed by direct sequencing.13 WASp expression was analyzed using Western blot13 or,
more recently, flow cytometry.23
7
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Chimerism analysis
Donor cell chimerism was assessed by the methods in use at the time in each centre,
including HLA-typing, molecular analysis of Short Tandem Repeats (STR), Fluorescent In
Situ Hybridization (FISH) or cytogenetics for X/Y chromosomes, as well as by flow-cytometric
analysis of WASp expression in combination with other lineage-specific surface markers.
Data on lineage-specific chimerism at the time of last follow-up visit were collected when
possible for patients who survived at least twelve months after HCT. Longitudinal data of
lineage-specific chimerism were available for 92 patients who survived at least 24 months
after HCT. Within each lineage, four categories of chimerism were established. Full chimerism
was defined by the presence of >95% donor cells, high chimerism as a percentage of donor
cells ranging from >50% to 95%, low chimerism as a percentage of donor cells ranging from
5% to 50% and null chimerism as <5% donor cells.
Statistical analysis
Means were compared by the Student’s t test. Survival curves were plotted by the
method of Kaplan and Meier and log-rank p values were determined for differences in
survival. Spearman's rank correlation coefficient was estimated to test the association
between myeloid donor engraftment and platelet count. Univariate analyses were performed
using the Wilcoxon-Mann-Whitney test. Multivariate analysis was performed using the Cox
proportional hazards regression models. Data analysis was performed using the JMP
Software, version 5.1.2 (SAS Institute, Cary, NC). The level of significance was considered to
be p< 0.05.
8
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Results
Recipient, donor and transplant characteristics
Among the 194 transplanted patients, 27 (13.9%) had been given a WAS clinical score
<3 at the time of HCT, indicating that they had not experienced any of the following: severe
infections, difficult to treat eczema, autoimmunity or malignancy (Table 1). In contrast, the
majority of patients (n=167; 86.1%) had already developed severe clinical features of WAS
and hence had a score ≥3. In particular, 139 patients (71.6%) had a history of recurrent
and/or severe infections, while 55 patients (28.3%) suffered from autoimmune disease,
especially autoimmune hemolytic anemia (AIHA; n=24) and vasculitis (n=16). Four patients
had developed Epstein-Barr virus-mediated lymphoproliferative disease (LPD), and one
patient each had juvenile myelomonocytic leukemia (JMML) or embryonal carcinoma of the
testis. Twenty-nine patients (14.9%) had been splenectomized prior to HCT, and ten of them
remained with a platelet count <50x109/L even after splenectomy.
Data on WAS gene mutations were available for 135 patients (69.6% of the entire
cohort). As shown in Table 1, the majority (n=87) carried severe mutations (frameshift, non
sense mutations or large deletions). Twenty-six patients carried missense mutations, with 14
of them located in exons 1-3, where XLT-associated mutations are clustered. Pre-transplant
data on WASp expression were available for 92 patients; 73 of them had undetectable WASp,
whereas 19 showed residual WASp expression (data not shown). As compared to patients
with residual WASp expression, those who lacked protein expression had a higher clinical
score at the time of HCT (mean ± s.d. = 3.82 ± 1.02 vs.3.16 ± 1.29 vs., p<0.05) and received
HCT at a younger age (29.3 ± 39.4 vs. 35.6 ± 36.5 months, p<0.05).
The median age at HCT was 34.6 months (range, 2-240). On average, patients with a
clinical score <3 received HCT at a younger age than those with a score ≥3 (23.4±28.2 vs.
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36.4±39.7 months; p<0.05). The majority of the patients (n=119; 61.3%) received HCT at less
than two years of age; in particular, 19 out of 27 patients with a clinical score <3 received
HCT at <2 years of age. On the other hand, 32 of the 194 patients (16.5%) were older than
five years (median, 108 months; range, 63-240 months) at the time of transplantation.
Transplant characteristics are reported in Table 2. The 194 patients received a total of
204 transplants; ten patients (six of whom had received a MMFD-HCT) required a second
transplant because of graft loss or rejection. The majority of the transplants (62.9%) were
performed in the period 2000-2009, reflecting wider access to transplantation from URD and
UCB. In particular, all 25 UCB-HCT and 71 of 93 (76.3%) URD-HCT were performed in 2000
or later. Bone marrow was the primary source of hematopoietic stem cells in 78.4% of all
transplants. The vast majority of the patients (88.1%) received a myeloablative conditioning
regimen; reduced-intensity conditioning was used more often in recent years (20 of the 23
HCT with RIC were performed since 2000).
Survival
Of the 194 patients transplanted, 159 (82%) were alive at the time of the study, with a
median follow-up of 76.8 months (range: 12-346). As shown in Figure 1A, both 5- and 8-year
survival was significantly better for patients transplanted since 2000 (89.9% and 83.3% vs.
74.9% and 73.4% for HCT performed up to 1999, p<0.005 and p<0.05, respectively).
Improved survival was observed for all donor types in the last decade (Figure 1B), but was
particularly significant (p<0.05) for recipients of MMRD-HCT, whose overall survival rate
increased from 52.2% to 91.7%.
There was a tendency for good clinical status at the time of HCT to result in better
survival. In particular, 5-year survival was 92.4% for patients with clinical score <3 vs. 79.3%
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for patients with a score of 5 (Figure 1C), but this difference did not reach statistical
significance. As shown in Fig. 1D, younger age at HCT was associated with higher 5-year
survival in patients treated with URD-HCT (91.9% vs. 73.3% for patients transplanted at <2
years of age vs. those >5-year-old; p<0.05). Age at HCT did not significantly affect survival in
patients treated by MSD- or MMFD-HCT (data not shown).
Multivariate analysis (Table 3) showed that better survival was associated with HCT
performed since the year 2000 (relative risk, RR=0.29, 95% C.I.: 0.11-0.77; p=0.017). In
contrast, use of MMFD or of UCB as source of stem cells was associated with reduced
survival (RR=10.5, p=0.003; and RR=9.98, p=0.028, respectively).
Thirty-five of the 194 patients (18%) have died, and causes of death are listed in Table
S1. Most deaths (27/35; 77.1%) occurred within the first year, with half of them (n=17) during
the first three months after transplantation. Of note, three of the eight patients who died more
than 12 months after HCT, underwent pre- (n=2) or post-transplant (n=1) splenectomy and
developed fulminant meningococcal (n=1) and pneumococcal (n=2) sepsis at 22, 62 and 72
months after HCT, respectively. Overall, infections (in the absence of GVHD) accounted for
death in 15 patients, and were more common among recipients of MMFD- and URD-HCT.
Fatal lymphoproliferative disease (n=2) and lymphoma (n=1) were observed in three patients
who were treated with MMFD-HCT before the year 2000. GVHD was reported as cause of
death in seven patients, four of whom had also developed infections.
Complications
Complications were common within the first year after HCT, affecting 45.9% of the
patients, but were observed more rarely (16.8% of the surviving patients) thereafter. Primary
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graft failure or graft rejection was observed in 13 patients (7%), in spite of the fact that 11 of
them had received myeloablative conditioning. Eight of these 13 patients were given T-cell
depleted MMFD-HCT. Patients with graft failure/rejection were equally distributed in the
periods 1980-1999 (n=6) vs. year 2000 or later (n=7).
Autoimmune manifestations, predominantly cytopenias and endocrinopathies, were
observed in 27 patients (13.9%), nine of whom had reported episodes of autoimmunity also
before HCT. Acute GvHD grade >2 was observed in 22 patients (11.3%) and progressed to
cGvHD in 11 of them. Infections requiring hospitalization occurred in 55 patients (28.4% of
the entire cohort). Five patients developed tumors, and one of them died with relapse of
testicular carcinoma.
Of 135 patients who survived at least 2 years after HCT, 39 (28.9%) suffered from
active complications at the time of last follow-up visit (median follow-up: 88.9 months; range:
24-346) or died (Table 4).
The risk of developing significant complications after HCT was higher among recipients
of UCB- or MMFD-HCT than of MSD-HCT (Figure 2A; 71.0% and 66.0% vs. 33.6%, p<0.005
and p<0.05, respectively). In contrast, the proportion of patients who developed complications
after URD-HCT (46.7%) was not significantly different from that observed after MSD-HCT.
Within the cohort of 115 patients who received URD- or UCB-HCT, survival was
comparable among those who were transplanted from fully matched vs. >1-antigen
mismatched (>1Ag-mm) donors (57/67 vs. 16/21, respectively; p=NS) (Table S2). However,
the proportion of patients who survived without complications was significantly higher among
recipients of transplants from fully matched vs >1Ag-mm (39/67 vs. 5/21, respectively;
χ2=4.26, p<0.05).
The risk of complications was also significantly influenced by the severity of the
disease, as assessed by clinical score at the time of HCT (Figure 2B). Only 32.4% of patients
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with a clinical score <3 developed complications, as compared to 53.6% of patients with a
score 3 to 4, and 56.1% of patients with score 5 (p<0.05 in both cases).
There was a trend for patients with residual WASp expression to result in lower
complication rate (26.3% vs. 41.1% for patients lacking WASp; data not shown), but the
difference was not statistically significant, possibly reflecting the low number of patients with
residual expression of WASp.
In a multivariate analysis, only transplantation from MMFD was identified as a
significant risk factor for the development of complications (RR: 2.45, CI: 1.15-5.20), mainly
reflecting the high incidence of graft failure/rejection observed in this group, especially before
2000 (Table 5).
Immunological reconstitution
Information on the absolute number of CD3+ and CD19+ lymphocytes at the time of last
follow-up was available in 145 and 143 patients respectively, who survived at least twelve
months after HCT. Normalization of the absolute count of T and B lymphocytes and of T
lymphocyte subsets (CD3+ >1000, CD4+ >600, CD8+ >300 and CD19+ >200 cells/μl) was
observed in 68.3% of the patients. Inability to attain normalization of the lymphocyte count
was associated with a higher incidence of mixed chimerism and/or autoimmune
manifestations (Table S3).
Information on immunoglobulin replacement therapy was available for 153 patients, 20
of whom (13.1%) required intravenous immunoglobulins (IVIG) at more than 12 months after
HCT. Data on antibody responses following immunization with tetanus toxoid (TT) and
pneumococcal polysaccharide (PnPS) antigens were available for a limited number of
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patients (72 and 53 patients, respectively), and protective titers of specific antibodies were
detected in 95.8% and 73.6% of the cases, respectively.
Platelet recovery
Data on pre- and post-HCT platelet number were available in 152 patients, including
25 of the 29 patients who had received pre-HCT splenectomy. Platelet count of these 25
patients was significantly higher than in non-splenectomized patients at the time of
transplantation (mean 95.5x109/L; 95% CI: 53.6-137.5x109/L vs. mean 29.0x109/L; 95% CI:
24.6-33.4x109/L); (p<0.001). HCT resulted in a significant increase of the mean platelet count
at last FU (>12 months after HCT) among both non-splenectomized (mean 235.6x109/L; 95%
CI: 213.3-257.9x109/L) and splenectomized (mean 318.3x109/L; 95% CI: 272-364.6x109/L)
patients (p<0.001 vs. pre-HCT values in both cases). However, 36 patients (23.7%) did not
achieve normalization of the platelet count (<150x109/L), and 14 of them showed persistent
severe thrombocytopenia (<50x109/L; range: 10-42x109/L). Among these, one patient
developed severe bleeding episodes requiring multiple platelet transfusions and eventually
received a second HCT 14 months after the primary transplantation; the remaining 13
patients had mild hemorrhagic manifestations, limited to petechiae. Nonetheless, because of
persistent thrombocytopenia, 2 of them received a stem cell boost at 40 and 84 months after
HCT, and 5 patients received post-HCT splenectomy, attaining a mean platelet count of
172.8x109/L (range: 106-231x109/L). Pre-HCT platelet count and clinical score were not
significantly different in patients who normalized the platelet count after HCT and those who
remained thrombocytopenic (data not shown).
Lineage-specific chimerism
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Longitudinal data on donor cell chimerism were available in 92 out of the 135 patients
who survived at least 24 months after HCT. Analysis of lineage-specific chimerism within this
cohort showed that chimerism was relatively unstable in the first year after HCT, with a
significant proportion of patients (<20%) changing chimerism group, but became more stable
thereafter (Figure 3A). At each time point, donor chimerism was more robust in T than in B
and myeloid cells (Figure 3B).
Ten patients received a second transplant within 17 months from the first HCT due to
primary graft failure or graft rejection; six of them are alive with full donor chimerism (Table
S4). Seven additional patients have received stem cell boosts (n=5) or donor lymphocyte
infusions (n=2) as a result of poor donor chimerism; although all seven patients are alive, only
two have achieved full donor engraftment.
Figure 4A illustrates the results of cross-sectional analysis of lineage-specific
chimerism at the time of last FU (median: 72.3 months; range: 12-346 months) in 154 of 167
patients (92.2%) who survived at least 12 months after HCT. Multilineage full donor
chimerism was observed in 111 patients (72.1%). The remaining 43 patients (27.9%) showed
mixed chimerism in at least one of the cell lineages tested. Low or null donor chimerism was
more common within myeloid cells (16.5%) than in B cells (7.4%) or T lymphocytes (3.2%).
A significant fraction of the 82 patients treated with URD-HCT attained low or null
donor myeloid chimerism (Figure 4B). In this group of patients, no correlation was identified
between lack of full donor chimerism and type of conditioning, year of HCT, age and clinical
score at HCT, pre-HCT WASp expression, or source of stem cells (data not shown).
However, when the analysis was restricted to 36 patients treated by URD-HCT since year
2000 (and for whom data on the number of CD34+ cells infused was available), poor (i.e.,
<50%) engraftment of donor myeloid cells was correlated with lower stem cell dose
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(4.14±1.71x106cells/Kg vs. 7.18±4.39x106cells/Kg for patients with ≥50% of donor myeloid
chimerism, p<0.05).
The degree of donor chimerism in the myeloid compartment had a significant impact
on the reconstitution of the platelet number (Figure 5A). In particular, higher platelet counts
were observed among patients who attained full vs. mixed/null donor myeloid cell engraftment
both for the cohort of 127 patients who were not splenectomized pre-HCT (274.5±111.3x109/L
vs. 104.2±79.7x109/L, p<0.001) and for the 25 patients who underwent pre-transplant
splenectomy (370.5±59.7x109/L vs. 240.0±129.1x109/L; p<0.005). Nonetheless, a significant
increase in the platelet count following HCT was observed also in patients with mixed/null
myeloid engraftment (p<0.001 for both groups as compared to platelet counts pre-HCT).
Statistical analysis using the non-parametric Spearman’s test in the cohort of nonsplenectomized patients confirmed, with few exceptions, the tight correlation between degree
of myeloid chimerism and platelet count (Figure 5B; r=0.5755; p<0.0001). Overall, these data
indicate that robust (>50%) and stable myeloid chimerism after HCT is associated with
normalization of the platelet count.
Low donor chimerism in T, B and myeloid cells was associated with subnormal
lymphocyte counts (Figure 6A).
It has been previously reported that mixed chimerism following HCT for WAS is
associated with increased risk of autoimmunity, particularly in patients with URD-HCT,14 but
the specific contribution of mixed chimerism in lymphoid or myeloid cells was not investigated.
In our series, the 25 patients who developed autoimmune manifestations after transplantation
and for whom data on lineage specific chimerism were available, showed a lower degree of
chimerism in each (T, B, myeloid) of the lineages tested as compared with patients who did
not develop autoimmunity (Figure 6B; T: 87.5 vs. 93.8%, p<0.05; B cells: 70.8 vs. 87.6%,
p<0.05; myeloid: 63.6 vs. 81.2%, p<0.01).
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Discussion
In this study, we analyzed the outcome of HCT in 194 WAS patients, treated in twelve
centers between 1980 and 2009. The large number of patients enrolled and the long duration
of the study allowed us to demonstrate that there is progressive improvement of outcome
after HCT for WAS. In particular, comparable survival rates were observed after MSD- and
after URD-HCT performed since 2000, and markedly improved survival rates have been
achieved with MMFD-HCT during the same period as compared to earlier years. This
improved outcome probably reflects larger availability and better selection of unrelated donors
(based on high-resolution HLA typing), advances in prevention and treatment of infections
and of EBV-related lymphoproliferative disease, use of less toxic conditioning regimens and
the introduction of more effective immunosuppressive drugs resulting in reduced risk of graft
rejection.
Ten years ago, Filipovich et al. had reported excellent survival after URD-HCT for WAS
in patients younger than five years at the time of transplantation, but older patients had a poor
outcome.16 Older age at transplantation has been identified as a significant risk factor also for
patients with other congenital immunodeficiencies, reflecting increased occurrence of
complications and progressive organ damage, reactivation of viral infections, and higher
incidence of GvHD in older recipients.18,20,24,25 Although age >5 years at HCT was associated
with less favorable survival after URD-HCT also in this study, 5-year survival for this group of
patients was 73.3%, indicating marked improvement compared to a previous study.16 These
data indicate that URD-HCT should no longer be restricted to WAS patients younger than five
years of age, especially if clinical conditions are good. However, it should be noted that most
(12 out of 15) of the patients who received URD-HCT at an age >5 years were less than 10
17
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years old. Therefore, it is not possible to assess outcome of URD-HCT for WAS, when
performed in adolescence or early adulthood.
While the degree of HLA matching did not affect survival following URD- or UCB-HCT
(likely due to the relatively small sample size), survival without complication was better in
patients transplanted from fully matched donors.
Most complications occurred within the first year after HCT. The occurrence of
autoimmunity early after HCT may reflect inadequacy of central and peripheral mechanisms
of T cell tolerance in the setting of T-cell lymphopenia26 Reactivation of viral infections (CMV,
EBV) is common, but careful monitoring of viremia and pre-emptive treatment has resulted in
significantly decreased infection-related mortality in recent years.
Although early complications resolved without sequelae in the majority of the patients,
approximately 30% of them had active complications at the time of last visit. In particular,
extensive cGvHD, neurological problems and autoimmune disease at the time of the last
follow-up visit were reported in 20, 10 and 12 patients, respectively. Similar data have been
previously reported by Ozsahin et al.,14 who found that 7-year event-free survival for WAS
patients surviving at least 2 years after HCT was 75%, and that 20% of the patients had
developed irreversible damage leading to sequelae. Overall, the Incidence of late
complications and sequelae after HCT for WAS is lower as compared to that observed after
HCT for SCID. 27
One of the main aims of our study was to characterize the stability of chimerism and
long-term function of the graft. To establish full donor chimerism, the eradication of hostderived hematopoietic and lymphoid cells is required. Incomplete deletion of host cells may
result either in primary graft failure due to rejection, with complete and early loss of the graft,
or may allow for the development of variable degrees of mixed chimerism. In some cases,
progressive loss of donor chimerism may follow, resulting in complete autologous
18
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reconstitution. While graft rejection reflects a host vs. donor immune response, mixed
chimerism with co-existence of both autologous and donor blood cells implies mutual
tolerance. In a recent study by Ozsahin et al.,14 a large fraction of WAS patients who
underwent HCT were reported to develop mixed or split chimerism, especially after URD- or
MMFD-HSCT, and this condition was associated with increased risk of late autoimmune
complications. However, the kinetics and distribution of lineage-specific chimerism were not
analyzed.
In the present study, we sought to investigate in greater detail the kinetics and stability
of lineage-specific chimerism, to identify risk factors that may favor development of mixed
chimerism, and to assess the impact of chimerism on disease correction and post-transplant
complications. Multilineage full donor chimerism was reported in 72.1% of patients who
survived at least twelve months after HCT. Retrospective sequential analysis of lineagespecific chimerism in a sub-cohort of 92 patients who survived at least two years after HCT,
demonstrated that mixed/split chimerism or autologous reconstitution usually becomes
apparent within the first year after HCT, and levels of mixed chimerism tend to remain stable
thereafter. Lower levels of donor chimerism were observed more often in myeloid than in
lymphoid cells; in particular, the highest degree of donor chimerism was detected in T
lymphocytes. A similar pattern has been observed in patients with Severe Combined
Immunodeficiency after transplantation following a mild conditioning regimen.28-31 These
observations are consistent with the notion that autologous stem cell reconstitution does not
necessarily affect development of T cells in the thymus once this organ is populated by donor
lymphoid progenitor cells. Alternatively, it is possible that expression of the WAS protein
confers a stronger and selective advantage in the lymphoid compartment (and especially in T
lymphocytes) than in myeloid cells. Data from heterozygous Was+/- mice32,33 and from carrier
females of X-linked thrombocytopenia34,35 support this hypothesis. However, patients with
19
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poor myeloid donor chimerism often failed to attain normalization of the lymphocyte count.
Furthermore, persistent thrombocytopenia after HCT was strongly associated with low or null
myeloid chimerism, suggesting that robust and stable engraftment of donor-derived myeloid
cells is required to correct this defect because of the lack of selective advantage for WASppositive cells in the myeloid compartment.32 Alternatively, it is possible that autoimmune
thrombocytopenia may contribute to persistence of thrombocytopenia in patients who fail to
attain full myeloid chimerism. Indeed, we found that patients with autoimmune manifestations
after HCT show a lower degree of donor chimerism. We have also shown that splenectomy
may induce normalization of the platelet count in patients who remain significantly
thrombocytopenic after HCT. However, the benefits of this strategy have to be weighed
against the risk of severe, potentially fatal infections, as confirmed by the finding that three
patients developed sepsis and died, among the 28 who received pre- or post-HCT
splenectomy.
In conclusion, this study confirms that HCT is an effective form of treatment for WAS,
and should be considered not only for patients <5 years of age, but also for those >5 years of
age with a matched related or unrelated donor, especially if in good clinical conditions.
However, robust and stable multilineage donor cell engraftment is required to fully correct the
disease, a goal that may be facilitated by infusion of a higher dose of donor stem cells.
Previous data favor myeloablative regimens to minimize the chance of autologous
reconstitution and recurrence or persistence of the WAS phenotype, and therefore for
patients in good conditions this remains the standard regimen. As less toxic regimens, such
as those based on targeted levels of busulfan (or treosulfan) and fludarabine become
available, their efficacy in the treatment of WAS should be tested.
20
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ACKNOWLEDGMENTS
We thank the patients and their families for their trust and cooperation. We thank
Alexandra Arnold, Chantal Harre, Corinne Jacques, Arnalda Lanfranchi, Stéphanie N’Daga
and Qili Zhu for manipulation of the samples, chimerism and immunological studies.
This work was partially supported by NIH grant 2P01HL059561-11-A1 (to LDN),
5P01HL059561-12 (to SG), P01 HL036444 (to LB), U54 AI082973-02 (to LDN and MC),
HD17427-43 (to HDO), by the Manton Foundation, Fondazione “Angelo Nocivelli” (to SG), the
Jeffrey Modell Foundation and the Dejoria Wiskott-Aldrich Research Fund (to HDO), and by
Cariplo grant 2010/0253 (to AL).
21
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AUTHORSHIP
Contribution: D.M., S.G. and L.D.N. designed research; A.L. performed statistical analysis;
D.M., S.G., W.F. and L.D.N. wrote the manuscript; C.B., E.M., A.F., A.J.C., A.J.T., H.D.O.,
M.J.C., M.H.A., T.S., S.-Y.P., E.H., M.H., A.S., F.P., M.C.-C., C.P., A.K., J.Z., N.M., W.Q.,
P.V., T.T., L.B., K.K., S.H., M.S., W.F., D.M., S.G. and L.D.N. took care of the patients and
collected clinical and laboratory data.
Conflict-of-interest disclosure: none of the authors declare any conflicting financial interests.
22
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Borghans JA, Bredius RG, Hazenberg MD, et al. Early determinants of long-term T-
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Cavazzana-Calvo M, Carlier F, Le Deist F, et al. Long-term T-cell reconstitution
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Mazzolari E, Forino C, Guerci S, et al. Long-term immune reconstitution and clinical
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Sarzotti-Kelsoe M, Win CM, Parrott RE, et al. Thymic output, T-cell diversity, and T-
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Westerberg LS, de la Fuente MA, Wermeling F, et al. WASP confers selective
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Meyer-Bahlburg A, Becker-Herman S, Humblet-Baron S, et al. Wiskott-Aldrich
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De Saint-Basile G, Schlegel N, Caniglia M, et al. X-linked thrombocytopenia and
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Tables
Table 1. Clinical presentation and genotypical classification of 194 WAS patients who
underwent HCT from 1980 to 2009
Patient characteristics
Clinical score at HSCT
(N=194)
Mutations Identified
(N=135)
Age at transplantation
(N=194)
Group
<3
3-4
5
Frameshift/Nonsense
Missense
Splice sites
Large deletions
In frame ins/del
<2 y.o.
2-5 y.o.
>5 y.o.
N
27
110
57
81
26
19
6
3
119
43
32
%
13.9
56.7
29.4
60.0
19.3
14.1
4.4
2.2
61.3
22.2
16.5
27
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Table 2. Characteristics of hematopoietic cell transpantations performed on 194 WAS
patients
Transplant
characteristics
Year of HCT
Donor type
Graft source
Conditioning
regimen
Group
Up to 1999
Since 2000
MSD
MFD
MMFD
URD
UCB
BM
PBSC
CB
BM+PBSC
Myeloablative
Reduced intensity
N. of first
(repeated)
transplantations
72 (4)
122 (6)
39 (1)
5
35 (6)
91 (2)
24 (1)
152 (3)
15 (4)
27 (1)
0 (2)
171 (4)
23 (6)
%
37.1
62.9
20.1
2.6
18.0
46.9
12.4
78.4
7.7
13.9
…
88.1
11.9
28
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Table 3. Multivariate analysis of risk factors possibly associated with death in a cohort of
194 patients treated by HCT
Variable
Year
Age
Clinical score
Donor
Conditioning
Autoimmunity
Acute GVHD
(grade III or IV)
Extensive chronic
GVHD
Group
Up to 1999
Since 2000
<2 y.o.
2-5 y.o.
>5 y.o.
<3
3-4
5
MSD
MMFD
MFD
URD
UCB
Myeloablative
Reduced intensity
No
Yes
No
Yes
No
Yes
Relative Risk of
death
1.00
0.29
1.00
0.73
2.46
1.00
0.86
1.28
1.00
10.5
7.15
4.56
9.98
1.00
1.12
1.00
0.25
1.00
1.32
1.00
0.92
95% CI
P
(0.11-0.77)
0.013
(0.25-2.11)
(0.97-6.19)
0.562
0.056
(0.17-4.30)
(0.25-6.41)
0.815
0.769
(2.20-50.4)
(0.59-87.3)
(0.91-22.6)
(1.29-77.1)
0.003
0.123
0.064
0.028
(0.31-3.99)
0.863
(0.033-1.91)
0.182
(0.38-4.58)
0.666
(0.25-3.33)
0.899
29
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Table 4. Outcome at last FU in the 135 WAS patients who survived at least two years after
transplant
Status
Active complications1
(observed in 39 patients)
Group
Alive
Deceased
Chronic GVHD
Fatal sepsis2
Autoimmune
manifestations3
Neurological sequelae4
N
130
5
20
3
12
10
%
96.3
3.7
14.8
2.2
8.9
7.4
1
Defined as residual complications still active at the time of last follow-up
Two of them were splenectomized patients
3
One patient deceased in a car accident
4
Comprising neurological delay, epilepsy, spastic displasia, glaucoma and various degree of
visual, hearing and speech deficits
2
30
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Table 5. Multivariate analysis of risk factors possibly associated with post-transplant
complications in a cohort of 187 patients treated by HCT
Variable
Year
Age
Clinical score
Cell Source
Donor
Conditioning
Group
Up to 1999
Since 2000
<2 y.o.
2-5 y.o.
>5 y.o.
<3
3-4
5
BM
PBSC
UCB
MSD
MMFD
MFD
URD
UCB
Myeloablative
Reduced intensity
Relative Risk of
complications
1.00
0.66
1.00
1.53
1.39
1.00
1.26
1.30
1.00
1.45
0.95
1.00
2.45
1.41
1.56
5.18
1.00
1.80
95% CI
P
(0.39-1.13)
0.130
(0.90-2.58)
(0.78-2.45)
0.115
0.262
(0.57-2.78)
(0.56-3.01)
0.574
0.543
(0.69-3.04)
(0.12-7.48)
0.324
0.960
(1.15-5.20)
(0.30-6.58)
(0.77-3.17)
(0.60-42.0)
0.019
0.662
0.219
0.124
(0.97-3.31)
0.060
31
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Legends
Figure 1. Outcome of 194 patients affected by Wiskott-Aldrich syndrome after
hematopoietic cell transplantation. Panel A) Probability of survival for all patients
according to year of transplant. Panels B-D) Five-year overall survival for patients
transplanted up to 1999 and since 2000 and grouped by donor type (B); clinical status
before transplant, as measured by clinical score (C); or for 91 patients receiving URD-HCT
and divided in three groups according to their age at transplant (D). *=p<0.05, ***=p<0.005
Figure 2. Probability of clinical and immunological complications for 194 WAS
patients who received hematopoietic cell transplantation. Panel A: Percentage of
patients who developed any complication (graft failure/rejection, acute GVHD grade III or
IV, extensive chronic GVHD, severe infections, autoimmune manifestations, tumors and
post-HCT sequelae) up to 5 years after HCT, according to donor type; Panel B:
Percentage of patients who developed any complication up to 5 years after HCT,
according to clinical status (measured by clinical score) at the time of HCT. *=p<0.05,
****=p<0.001
Figure 3. Longitudinal analysis of lineage specific chimerism after hematopoietic
cell transplantation. Data were collected for 92 WAS transplanted patients with at least
24 months of follow up after HCT. Chimerism in the T- and B-lymphocyte and in the
myeloid compartment was categorized according to the percentage of donor cells in four
different groups ranging from full (defined by the presence of >95% donor cells), high
(from >50% to 95%), low (from 5% to 50%) to null chimerism (<5%). These data are
reported for each cell type in panel A) to show the longitudinal profile of donor chimerism
variations, defined as changes in chimerism group, or in panel B) to display the distribution
of lineage-specific chimerism groups at various time points after HCT.
Figure 4. Quantitative analysis of lineage specific chimerism at the time of last
follow-up in 154 WAS transplanted patients who had at least 12 months of follow-up
after HCT. The percentage of donor-derived T, B, and myeloid cells is reported for each
patient. In panel A, data of lineage-specific chimerism at the time of last follow-up visit are
shown. Patients are grouped according to follow-up interval, and the number of patients
studied at each interval is indicated in parenthesis. In panel B, patients are grouped
according to donor type, and the number of patients receiving HCT from a specific type of
donor is indicated in parenthesis. MSD: matched sibling donor; MFD: other matched family
donor; MMFD: mismatched family donor; URD: unrelated donor; UCB: unrelated cord
blood *=p<0.05
Figure 5. Influence of the degree of myeloid cell engraftment on platelet count.
Platelet counts before and after HCT were reported for 152 WAS transplanted patients,
who had at least 12 months of follow up after HCT and for whom quantitative analysis of
donor cell engraftment on myeloid cells was available. Pre-transplant splenectomized
patients were separated from non-splenectomized patients. In panel A), patients of both
groups were further divided according to the degree of donor myeloid cell engraftment (full
or mixed/null). For each of them, platelet counts at diagnosis and at last follow up are
shown, with pre-transplant values of splenectomized patients reported both at diagnosis
and after splenectomy. **=p<0.01, ***=p<0.005, ****=p<0.001. In panel B), correlation
32
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between the platelet count of non-splenectomized patients and the percentage of donor
myeloid cell engraftment is shown. A significant correlation between these two parameters
was observed according to the non-parametric Spearman test (r=0.584, p<0.001).
Figure 6. Influence of the degree of donor cell engraftment on the reconstitution of
lymphocyte counts and autoimmunity after hematopoietic cell transplantation. Data
are shown for WAS-transplanted patients who had at least 12 months of follow-up after
HCT and for whom data of lineage-specific chimerism were available. In panel A), the
percentage of donor chimerism for each cell lineage at the time of last follow-up is shown
for patients who attained (Y) or did not attain (N) normalization of T- and B-cell counts
(defined as CD3+ >1000 cells/μL, CD4+ >600 cells/μL, CD8+ >300 cells/μL and CD19+
>200 cells/μL. Horizontal bars represent mean value; **=p<0.01, ***=p<0.005. In panel B),
the percentage of donor lineage-specific chimerism is shown for patients who developed
(Y) or did not develop (N) autoimmunity; *=p<0.05, **=p<0.01.
33
Figure 1
B
A
***
*
(8 years)
(5 years)
C
MSD
50
34
42 34
7
12
5
MMRD
3
D
*
Subjects at risk
Up to 1999
72
Since 2000
122
URD
UCB
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*
Patients who developed
complications (%)
A
****
Patients who developed
complications (%)
Figure 2
* *
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B
Figure 3
Patients changing
chimerism group (%)
Variation of donor chimerism group
T lymphocytes
B lymphocytes
Myeloid cells
Time after transplantation (months)
B
B lymphocytes
<6
6
12
24
36
48
60
Last
FU
Myeloid cells
<6
6
12
24
36
48
60
Last
FU
T lymphocytes
<6
6
12
24
36
48
60
Last
FU
Group distribution (%)
Longitudinal distribution of donor chimerism groups
Time after transplantation (months)
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A
Months of FU
(N. of patients)
*
Transplant type
(N. of patients)
Donor cell chimerism (%)
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Donor cell chimerism (%)
A
Figure 4
B
**
****
****
****
****
****
****
Non-splenectomized
Before HCT
Full engraftment
Splenectomized
After splenectomy, before HCT
Mixed/null engraftment
B
Plt count (number x 109/L)
From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
Plt count (number x 109/L)
A
Myeloid
donor chimerism (%)
Figure 5
Normalization of lymphocyte count
A
**
**
Normal count
(N. of patients)
Post-HCT autoimmunity
B
*
Autoimmunity
(N. of patients)
*
**
From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
Donor cell chimerism (%)
**
Donor cell chimerism (%)
Figure 6
From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
Prepublished online June 9, 2011;
doi:10.1182/blood-2010-11-319376
Long-term outcome and lineage-specific chimerism in 194 Wiskott-Aldrich
Syndrome patients treated by hematopoietic cell transplantation between
1980−2009: an international collaborative study
Daniele Moratto, Silvia Giliani, Carmem Bonfim, Evelina Mazzolari, Alain Fischer, Hans D. Ochs, Andrew
J. Cant, Adrian J. Thrasher, Morton J. Cowan, Michael H. Albert, Trudy Small, Sung-Yun Pai, Elie
Haddad, Antonella Lisa, Sophie Hambleton, Mary Slatter, Marina Cavazzana-Calvo, Nizar Mahlaoui,
Capucine Picard, Troy R. Torgerson, Lauri Burroughs, Adriana Koliski, Jose Zanis Neto, Fulvio Porta,
Waseem Qasim, Paul Veys, Kristina Kavanau, Manfred Hönig, Ansgar Schulz, Wilhelm Friedrich and
Luigi D. Notarangelo
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