Ball, L.M.
Citation
Ball, L. M. (2010, March 4). Clinical and laboratory features of mesenchymal
stromal cells in pediatric stem cell transplantation. Retrieved fromhttps://hdl.handle.net/1887/15035
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Chapter 4
Co-infusion of ex vivo expanded, parental mesenchymal stromal cells prevents life-threatening acute GvHD, but does not reduce the risk of graft failure in pediatric patients given allogeneic umbilical cord blood transplantation
L M Ball*
ME Bernado*
A Cometo H Roelofs M Zecca MA Avanzini A Bertaina L Vinti A Lankester R Maccario O Ringden K Le Blanc RM Egeler WE Fibbe**
F Locatelli**
\*;** . contributed equally to this work
BONE MARROW TRANSPLANTATION 2010; (In press)
Summary
Compared to bone marrow transplantation (BMT), umbilical cord blood transplantation (UCBT) is associated with lower rate of engraftment and delayed hematological/immunological recovery. This leads to increased risk of early post- transplantation mortality (TRM) due to infection. Acute graft-versus-host disease (aGvHD), although occurring less frequently in UCBT compared to BMT, is also significantly associated with increased rate of early TRM. Bone marrow mesenchymal stromal cells (MSCs) are known to support normal in vivo hematopoiesis and co- transplantation of MSCs has been shown to enhance engraftment of human, cord blood, hematopoietic cells in NOD/SCID mice. In 13 children with hematological disorders (median age 2 years) undergoing UCBT, we co-transplanted paternal, HLA- disparate MSCs with the aim of improving hematological recovery and reducing rejection. We observed no differences in hematological recovery or rejection rates compared to 39 matched historical controls, most of whom received G-CSF after UCBT. However, the rate of grade III-IV aGvHD was significantly decreased in the study cohort as compared to controls (P=0.05), thus resulting in reduced early TRM.
Although these data do not support the use of MSCs in UCBT to support hematopoietic engraftment, they suggest that MSCs, possibly thanks to their immunosuppressive effect, may abrogate life-threatening aGvHD and reduce early TRM.
Introduction
Since the first successful transplantation of hematopoietic stem cells (HSC) derived from umbilical cord blood (UCB) from an HLA-identical sibling in 1988,1 the number of patients receiving umbilical cord blood transplantation (UCBT), in particular from an unrelated donor (UD), has steadily increased.2-7
As compared with bone marrow transplantation (BMT), UCBT offers the clinical advantages of absence of risks for the donor, reduced risk of transmitting infections, reduced incidence and severity of graft-versus-host disease (GvHD) and, for transplants from UDs, rapid availability of cryopreserved cells, the median time for a successful donor search being less than 1 month.2,3,8-12
Results of UCBT from either an HLA-compatible sibling or from an UD have been reported to be comparable with those obtained in children transplanted with bone marrow (BM) cells.1,13-15 However, in comparison with pediatric patients transplanted with BM cells from a matched UD, children receiving an unrelated UCBT, most being HLA-disparate, have both delayed kinetics of hematopoietic recovery and increased risk of transplant-related mortality (TRM) in the early post-transplantation period, mainly attributable to a higher risk of infectious complications.16,17
Strategies aimed at improving engraftment and reducing TRM in UCBT recipients, such as infusion of two units in the same recipient, ex-vivo expansion of UCB HSC and intrabone injection of cord blood cells, have been recently proposed.18-20 Besides these approaches, the co-infusion of mesenchymal stromal cells (MSCs) together with UCB HSC could be an alternative option for optimizing the engraftment of donor cells.
Indeed, MSCs, which are multipotent cells capable of differentiation into several mesenchymal lineages,21,22 possess peculiar properties of immuno-modulation on all cells involved in the immune response.23-27 Moreover, Noort et al.28 demonstrated, in a NOD-SCID mouse model, that co-transplantation of MSCs facilitated, both in BM and peripheral blood, UCB HSC engraftment, especially when low numbers of UCB- derived CD34+ cells were infused.
MSCs have been already successfully used in the clinical setting to treat acute GvHD, as well as to accelerate hematopoietic recovery in patients given either BM or peripheral blood HSC.29-34 In particular, we have recently demonstrated that, in patients given a T-cell depleted peripheral blood stem cell transplantation (PBSCT)
from an HLA-haploidentical relative, co-infusion of donor-derived MSCs with HSCs was safe and able to reduce the risk of graft failure.34
We report on 13 children undergoing either a related or UD UCBT in whom we co- infused BM-derived, ex-vivo expanded, parental MSCs. Results in these patients were compared with those of a group of 39 historical controls consisting of children given either related or UD UCBT.
Patients and Methods
Patients
Thirteen pediatric patients (median age 2 years, range 0.8-14) with hematological disorders were enrolled in this study and received co-transplantation of UCB cells and parental-derived MSCs between May 2006 and January 2008. The results of these 13 patients were compared with those obtained in a group of 39 historical controls (median age 4 years, range 0.8-17) transplanted in one of the three centers (Pavia;
Leiden; Stockholm) and matched as closely as possible for original disease and type of UCB donor employed. Details on the study population and controls are reported in Table 1.
The study was approved by the ethical committees and written informed consent was obtained from all patients or from their parents/legal guardians and from MSC donors.
Donor-recipient histocompatibility was determined by serological typing for HLA-A and -B and by high-resolution DNA typing for HLA-DRB1.14 Two (15%) out of the 13 study patients received UCBT from an HLA-identical sibling, whereas the remaining 11 were transplanted from an UD who was perfectly matched in four (31%) cases, or had a single or double HLA-disparity in six (46%) and one (8%) case, respectively. In the control group, eight (20%) patients received UCBT from a related HLA-identical donor, whereas 31 underwent an UD UCBT, from a perfectly matched donor in five (13%) cases, a 5/6 matched donor in 25 (64%) and a 4/6 matched donor in one (3%) case, respectively.
Table 1. Characteristics of patients and controls
Patients (n = 13) Controls (n = 39) p value
Transplantation years (range) May 2006 – Jan. 2008 Feb. 1998- Dec. 2007
Median age (range) years at transplantation 2 (0.8-14) 4 (0.8-17) NS
Patient gender
Male 7 (54%) 24 (61%) NS
Female 6 (46%) 15 (39%) NS
Original disease
ALL AML MDS/JMML HLH
6 (46%) 1 (8%) 2 (15%) 4 (31%)
21 (54%) 5 (13%) 9 (23%) 4 (10%)
NS
Disease status at transplantation 1st CR
2nd CR Other CR Disease present
5 (38%) 3 (24%) 0 (0%) 5 (38%)
8 (21%) 14 (36%) 4 (10%) 13 (33%)
NS
UCB donor
Matched family donor Matched UD
Partially matched UD (5/6) Partially matched UD (4/6)
2 (15%) 4 (31%) 6 (46%) 1 (8%)
8 (20%) 5 (13%) 25 (64%) 1 (3%)
NS
Conditioning regimen
TBI-based vs. Chemotherapy-based 4/9 (31 vs. 69%) 15/24 (38 vs. 62%) NS
GvHD prophylaxis CsA CsA+steroids CsA+MTX
2 (15%) 11 (85%) 0 (0%)
9 (23%) 27 (69%) 3 (8%)
NS
Anti-thymocyte globulin No
Yes
2 (15%) 11 (85%)
8 (21%) 31 (79%)
NS
G-CSF No Yes
10 (77%) 3 (23%)
12 (31%) 27 (69%)
< 0.05
UCB unit characteristics
Number of TNC infused x 107/kg (median, range) Number of CD34+ cells infused x 105/kg (median, range)
5.7 (1.6 – 13.3) 2.2 (0.6-6.0)
6.6 (1.4 – 11.6) 2.6 (0.5-6.6)
NS NS Number of MSCs infused x 106/kg (median, range)
MSC donor (mother vs. father)
1.9 (1-3.9) 1/12 (8 vs. 92%)
NA NA Haematopoietic recovery
Number of days to PMN recovery (median, range) Number of days to PLT recovery > 20x109/l (median, range)
Number of days to PLT recovery > 50x109/l (median, range)
30 (17-42) 32 (14-85)
38 (33-113)
28 (13-44) 36 (18-91)
49 (20-110)
NS NS
NS
ALL = acute lymphoblastic leukemia; AML = acute myeloid leukemia; MDS/JMML = myelodisplastic syndrome/juvenile
myelomonocytic leukemia; HLH = hemophagocytic lymphohistiocytosis; CR = complete remission; UCB = umbilical cord blood; UD = unrelated donor; TBI = total body irradiation; TNC = total nucleated cells; PMN = polymorphonuclear neutrophils; PLT = platelets;
UCBT = umbilical cord blood transplantation; GvHD = graft-versus-host disease; CsA = cyclosporine; MTX = methotrexate; G-CSF = granulocyte-colony stimulating factor; NA = not applicable; MSC = mesenchymal stromal cells; NS = non-significant.
Preparative regimens varied according to patient age and disease, and the center protocols (see also Table 1). In both groups, GvHD prophylaxis was based on the use of Cyclosporine (CsA) alone in case of transplantation from an HLA-identical sibling, and CsA together with steroids for patients receiving UCBT from an UD,14 with the exception of three patients in the control group who received CsA and Methotrexate.
Pre-transplantation anti-thymocyte globulin was administered to all recipients of an UD UCBT. Details on patient and UCB unit characteristics, conditioning regimens, GvHD prophylaxis, as well as the median number of UCB cells infused are reported in Table 1. There was no significant difference between patients and controls in terms of age, gender, underlying disease and disease status at transplantation, UCB donor employed, conditioning regimen and type of GvHD prophylaxis administered, number of total nucleated cells (TNCs) and CD34+ cells infused (see also Table 1 for details).
In the control group, the majority of children (69%) received granulocyte-colony stimulating factor (G-CSF) after transplantation to facilitate neutrophil recovery, whereas only 23% of the study patients were administered the growth factor (P<0.05).
Preparation of MSCs
Approximately five weeks before the scheduled date of transplantation, MSCs were isolated and expanded ex-vivo from 50-70 mL parental BM, as previously described.34
Criteria for MSC release for clinical use were as follows: spindle shape morphology, absence of contamination by pathogens, viability ≥ 80%, and an immune phenotype characterized by the expression of CD73, CD90 and CD105 surface molecules and the absence of CD34, CD45, and CD31. MSCs were infused at passage (P) 2 or 3, to reduce the potential risk of in vitro transformation of the cells, and when at least 1 x106/kg of recipient body weight were available; the median dose of MSCs infused was 1.9 (range 1-3.9) x106/kg of recipient body weight, at a final concentration of 1-2x106 cells/mL.
Either conventional karyotype or molecular karyotype through array-comparative genomic hybridization (array-CGH) was performed on the cell products before infusion and resulted normal.
Co-transplantation of MSCs and UCB cells
On day 0, while strictly monitoring vital signs, the study patients received parental MSCs intravenously via a central venous catheter, followed by the infusion, four hours later, of UCB cells. The MSC donor was the father in 12 (92%) out of 13 patients enrolled. The median number of UCB total nucleated cells (TNCs) and CD34+ cells infused in the study patients was 5.7 (range 1.6-13.3) x107/kg recipient weight and 2.2 (range 0.6-6) x105/kg, respectively. The median number of UCB TNCs and CD34+ cells given to controls was 6.6 (range 1.4-11.6) x107/kg recipient weight and 2.6 (range 0.5- 6.6) x105/kg, respectively (see also Table 1 for further details).
Statistical analysis
The report date for the analysis was August 1, 2009, i.e. the day at which the centres locked up data on patient outcomes. Patients were censored at time of death or last follow-up. Definitions of outcome are detailed in Supplementary Information.
Probability of overall survival (OS), leukaemia-free survival (LFS) and event-free survival (EFS) were estimated by the Kaplan-Meier product-limit method and expressed as percentage and 95% confidence interval (95% CI).37 Probabilities of disease recurrence, acute and chronic GvHD, graft failure and TRM, were calculated as cumulative incidence curves (and 95% CI), in order to adjust the analysis for competing risks.38,39 For acute and chronic GvHD, death and relapse were the competing events. For relapse, non-relapse mortality was the competing event, while for TRM, relapse represented the competing event.
A Student t-test, Fischer exact test and Chi square with Yates correction were used to assess differences between study and historical control groups. The significance of differences between EFS curves was estimated by the log–rank test (Mantel–Cox), while the Gray’s test was used to assess differences in relapse risk, TRM, acute and chronic GVHD.40 A P value equal or less than 0.05 was considered significant.
Statistical analysis was performed using the STATA package41 and the R 2.5.0 software package42.
Results
In all parental donors, expansion of a sufficient number of BM-derived MSCs (>1x106/kg) was successful; study patients received a median number of 1.9x106 MSCs/kg (range 1-3.9). No MSC infusion-related toxicity was observed.
Neutrophil and platelet engraftment, and graft rejection
Two (15%) study patients, both transplanted with a 5/6 matched UD UCB unit, experienced primary graft failure. They were both re-transplanted. The first one, who was affected by juvenile myelomonocytic leukemia, received a second UCBT from a different 5/6 matched UD, whereas the second, suffering from hemophagocytic lymphohistiocytosis (HLH), underwent T-cell depleted peripheral blood stem cell transplantation from his HLA-haploidentical father. Both patients are currently alive without any sign of GvHD and are considered to be successfully cured of their original disorder. The number of TNCs, CD34+ cells and MSCs infused in patients with sustained engraftment and in those who experienced primary graft failure in the study group were super-imposable (data not shown).
The cumulative incidence of graft failure in the study group was 15% (95% CI, 4-55%), whereas that of controls was 3% (95% CI, 0-18%; P=N.S., see also Figure 1 for details) with one patient affected by acute myeloid leukemia (AML) and transplanted from a 5/6 matched UD experiencing primary graft failure in the control group.
For study patients who engrafted, the median time to reach neutrophil recovery was 30 days (range 17-42), whereas that of controls was 28 days (range 13-44; P=N.S.). The median time to reach a platelet count above 20x109/L and 50x109/L was 32 days (range 14-85) and 38 days (range 33-113) respectively in patients receiving MSCs, whereas that of controls was 36 days (range 18-91; P=N.S.) and 49 days (range 20-110;
P=N.S.), respectively.
Figure 1. Probability of rejection following UCBT (study patients vs. historical matched controls)
0.0 0.2 0.4 0.6 0.8 1.0
0 1 2 3 4 5
YEARS AFTER CBT
CUMULATIVE INCIDENCE (95% CI)
UCBT: 3% (0-18) UCBT+MSC: N = 13; E = 2
UCBT: N = 39; E = 1
UCBT+MSC: 15% (4-55)
P = N.S.
P = N.S.
No differences were observed between children receiving UCBT and MSc compared to historical controls.
Significantly more children received G-CSF in the controls group which may have influenced this observation
Acute and chronic GvHD
The cumulative incidence of grade II-IV acute GvHD in patients receiving MSCs was 31% (95% CI, 14-70%), whereas that of controls was 41% (95% CI, 28-60%; P=N.S.).
However, none of the study patients developed grade III-IV acute GvHD, whereas the cumulative incidence of the most severe forms of this complication in controls was 26%
(95% CI, 15-44%; P=0.05, see also Figure 2).
In the study group, four (31%) patients developed grade II acute GvHD; they were all transplanted from an UD. All four patients responded to additional either single (1 case) or multiple (3 cases) infusions of parental and/or third party MSCs performed at different times after UCBT, after failing first- or second-line immunosuppressive
treatment (see also Table 2 for details). In the control group, six (15%), seven (18%) and three (8%) patients developed grade II, III and IV acute GVHD, respectively. The three children with grade IV acute GvHD died because of GVHD progression, not responsive to several lines of immunosuppressive therapy (not including MSC treatment).
Figure 2.. Grade III-IV acute Graft versus Host disease (GvHD) following UCBT (study patients vs. historical matched controls)
0.0 0.2 0.4 0.6 0.8 1.0
0 20 40 60 80 100
DAYS AFTER UCBT
CUMULATIVE INCIDENCE (95% CI)
UCBT+MSC: 0%
UCBT: 26% (15-44) P = 0.058
UCBT UCBT+MSC
39 13
Evaluable
0 0 4 1 8 N.
(0%) (0%) (31%) (8%) (61%) (%)
3 7 6 5 18 N. (%)
(8%) Grade IV
(18%) Grade III
(15%) Grade II
(13%) Grade I
(46%) Absent
Grade
Significant reduction oF acute severe GvHD was observed in children receiving co transplantation of UCBT and MSCs.
Table 2. Characteristics of patients with grade II acute GvHD treated with MSCs
Pt = patient; UCB = umbilical cord blood; F = female; M = male; GvHD = graft-versus-host disease;
UD = unrelated donor; PDN = prednisolone; MMF = mofetil mycophenolate; MSC = mesenchymal stromal cells;
CR = complete response.
Ten study patients and 30 controls, surviving more than 100 days after transplantation, were evaluated for the occurrence of chronic GvHD. None of the MSC-treated patients at risk developed chronic GvHD, whereas three (10%) children experienced chronic GvHD in the control group which was extensive in two cases. All patients with chronic GvHD had previously had grade III acute GvHD. The cumulative incidence of chronic GvHD was 0% and 11% (95% CI, 4-31%; P=N.S.) in study patients and controls, respectively.
Transplantation-related morality
One patient in the MSC group died of transplantation-related complications (HHV6 encephalitis) before engraftment (day +33); he was affected by HLH and received an UD UCBT from a 5/6 matched unit. Eight patients in the control group experienced transplantation-related mortality at a median time of 51 days (range 28-159) after the allograft; they were all transplanted from an UD which was 5/6 matched in seven cases and 6/6 matched in the remaining one. The cumulative incidence of TRM was 8% (95% CI, 1-51%) and 21% (95% CI, 11-38%; P=N.S.) in the study patients and controls, respectively. Acute GvHD and infections, either bacterial or fungal, were the most common causes of transplantation-related death in controls.
Pt Sex UCB donor
aGvHD grading
Organ involve- ment
GvHD treatment before MSCs
Previous failed therapy
N° of MSC infusions
MSC donor
Total MSC dose/kg
GvHD Outcome
1 F Matched
UD
Grade II Skin, gut PDN ≥2 mg/Kg;
MMF
Second line
4 Father
(3); third party (1)
7 X 106 CR
2 F Matched
UD
Grade II Skin, gut PDN ≥2 mg/Kg;
MMF
Second line
3 Father
(2); third party (1)
6 X 106 CR
3 M Mismatched
UD (4/6)
Grade II Skin, gut PDN ≥2 mg/Kg
First line 4 Father (1);
third party (3)
9 X 106 CR
4 F Matched
UD
Grade II Gut
PDN ≥2
mg/Kg First line 1 Father 2 X 106 CR
Overall survival, event-free and leukemia-free survival
Ten study patients are alive, at a median follow-up of 28 months (range 19-38); in the control group, 25 patients are alive with a median follow-up of 42 months (range 16- 134). The Kaplan-Meier estimate of OS at three years is 63% (95% CI, 43-97%) and 64% (95% CI, 48-79%; P=N.S.) in study patients and controls, respectively. All surviving patients, both in the MSC and in the control group, have a Karnofsky/Lansky score of 100%.
Three (23%) study patients, affected by acute lymphoblastic leukaemia (ALL), experienced disease recurrence at 6, 8 and 16 months after transplantation. Two out of the three, transplanted from an HLA-identical sibling, have received a second transplantation using BM-derived HSC from the same donor. One of the two is currently alive, in remission, with sustained donor engraftment, whereas the other developed chronic GvHD with obstructive bronchiolitis which was the cause of death 15 months after the second transplantation. The third patient experiencing leukaemia relapse was affected by T-lineage ALL and was transplanted from a 5/6 matched UD.
He subsequently received a haploidentical T-cell depleted peripheral blood stem cell transplantation from his brother and died two months later due to grade IV acute GvHD and adenovirus disseminated infection. In the control group, nine (23%) patients, seven affected by ALL and two by AML, experienced relapse at a median follow-up of six months (range 2-17). Six of them died because of disease progression.
The cumulative incidence of relapse at three years was 25% (95% CI, 9-67%) and 23%
(95% CI, 13-42%; P=N.S.) in study patients and controls, respectively.
The Kaplan-Meier estimates for LFS and EFS at three years for study patients were 67% (95% CI, 41-94%) and 48% (95% CI, 19-78%), respectively; whereas those of controls were 56% (95% CI, 40-72%; P=N.S.) and 53% (95% CI, 37-69%; P=N.S.), respectively.
Chimerism analysis of ex vivo expanded MSCs derived from recipient BM at three- month intervals up to one-year after UCBT using PCR for informative donor recipient
Table 3. Patient follow-up data.
Pt Sex Age at UCBT
UCB donor Diagnosis Follow up
Hematopoietic chimerism analysis
Acute GvHD
Chronic GvHD
Time of last PB or BM chimerism
Outcome
1 F 1 y Mismatched
UD
BCP ALL +38 mo 100% donor Grade I NO +36 mo Alive; CR
2 F 8 y Matched
family
BCP- ALL
+25 mo 100% donor NO NO +25 mo
Relapsed 6 mo after UCBT, re- transplanted from same donor (BM cells). Died due to chronic GvHD 15 mo after 2nd Tx in CR
3 M 3.7 y Matched
family
BCP- ALL
+36 mo 100% donor NO NO +36 mo
Relapsed 8 mo after UCBT, re- transplanted from same donor (BM cells). Alive;
CR
4 M 4.3 y Matched
UD
BCP- ALL
+35 mo 100% donor NO NO +35 mo Alive and well.
CR
5 M 13.5 y Mismatched
UD
T-ALL +24 mo 100% donor NO NO +16 mo
Relapsed 16 mo after UCBT. Re- transplanted from his haploidentical brother; died due to acute GvHD 2 mo after 2nd Tx in CR
6 F 6.4 y Matched
UD
BCP- ALL
+29 mo 100% donor Grade II NO +24 mo Alive; CR
7 F 1.1 y Matched
UD
AML +27 mo 100% donor Grade II NO +24 mo Alive; CR
8 M 6.3 y Mismatched
UD
JMML +24 mo Primary graft failure
NE NE
+24 mo (100% cells of the 2nd donor)
Primary graft failure. Re- transplanted.
Alive; CR
9 M 1.2 y Mismatched
UD
JMML +19 mo 100% donor Grade II NO +18 mo Alive; CR
10 M 0.8 y Mismatched
UD
HLH +36 mo 100% donor NO NO +36 mo Alive; CR
11 F 1.8 y Matched
UD
HLH +19 mo 100% donor Grade II NO +18 mo Alive; CR
12 F 2.1 y
Mismatched
UD HLH +1 mo = NE NE NE NE
Died before engraftment
13 M 2.2 y Mismatched
UD
HLH +22 mo Primary graft failure
NE NE +22 mo
(100% cells of the 2nd donor)
Primary graft failure.
Autologous reinfusion. Alive.
Pt = patient; y = years; UCBT = umbilical cord blood transplantation; UD = unrelated donor; BM = bone marrow; PB = peripheral blood; M = male; F = female; mo = months; BCP-ALL= B-cell precursor acute lymphoblastic leukemia; T-ALL= T- lineage acute lymphoblastic leukemia AML= acute myeloid leukemia; MDS/JMML = myelodysplastic syndrome/juvenile myelomonocytic leukemia; HLH = hemophagocytic lymphohistiocytosis; NE = not evaluated; CR = complete remission; Tx = transplantation; = = dead. Full donor chimerism was defined as >98% donor cells in peripheral blood and bone marrow.
Chimerism analysis was routinely performed at 1-2 weekly intervals in the first months post-transplantation on peripheral blood mononuclear cells. Other serial controls of chimerism were subsequently performed at variable intervals. The last known analysis is shown for all study patients (+ months post UCBT).
polymorphisms43 did not show any evidence of MSC donor cells. Hematopoietic chimerism is detailed in Table 3.
Discussion
The stem cell niche in the bone marrow represents the most appropriate habitat for HSC, providing a spatial structure which enables both self-renewal and differentiation of hematopoietic progenitors. The cells forming the marrow niche consist of macrophages, fibroblasts, adipocytes, osteoprogenitors, endothelial cells, reticular cells and MSCs, which, together, contribute to support hematopoiesis through crosstalk with HSC.44 Preclinical studies indicate that co-transplantation of human MSCs is able to promote engraftment of UCB-derived HSC in NOD-SCID mice and in fetal sheep.
The enhancing effect involved cells of myeloid, lymphoid and megakaryocytic lineages, this finding demonstrating that the engraftment-promoting effect of MSCs was not lineage specific and it was particularly prominent when the dose of HSC was
low.28,45 Altogether, these data suggest that co-infusion of MSCs could be a promising
strategy to optimize the engraftment of UCB progenitors, especially in the presence of HLA-disparities between the donor and the recipient and have provided the rationale for testing the capacity of these cells to facilitate hematological recovery in human patients given allogeneic UCBT.
MSCs have been initially used in a single patient transplanted with UCB cells with the aim of improving the outcome of double unit UCBT.47 In this patient, MSCs were administered without clinical adverse effects and, interestingly, the single unit predominance described after multiple cord blood unit transplantation was abrogated.47 More recently, the results of a preliminary, phase I–II trial aimed at establishing the safety of ex-vivo culture-expanded allogeneic human MSCs from haploidentical related donors co-transplanted during UD UBCT were reported.48 Eight children were enrolled in the trial receiving a median MSC number of 2.1x106 (range, 0.9–5.0)/kilogram body weight. Infusion of ex-vivo culture-expanded haploidentical MSCs proved to be safe and patients had a neutrophil recovery at a median time of 19 days after the allograft.48 The results on our 13 children undergoing UCBT and treated with haploidentical BM-derived ex vivo expanded MSCs from a
parent support the safety of the approach, as we observed neither infusion toxicity, nor ectopic tissue formation.
Compared with UD-BMT recipients, children receiving UCBT have a reduced rate of sustained donor engraftment and delayed hematopoietic recovery. Slow neutrophil recovery, as well as impaired immune reconstitution, increases early TRM, 9,13,16,17, 49
and thus reduces the overall effectiveness of this approach. The infused cell dose is an important factor related to outcome, both in terms of engraftment and overall survival.2,8-10,16,17
Children receiving a higher cell dose are less likely to experience fatal complications.7,15-17,49,50
In particular, the Minnesota group demonstrated that recipients receiving a TNC dose before cryopreservation <2.5 x107/kg or CD34+ infused cell dose <1.7 x 105/kg, had inferior neutrophil engraftment and slower recovery.15 There were no differences in the median number of either TNCs or CD34+ cells infused between our patients given MSCs and control groups of children receiving UCBT, with the majority of children receiving doses greater than the minimum threshold suggested by the Minnesota recommendations.15 This may have obscured any positive effect of MSCs to accelerate neutrophil and platelet recovery, as in the animal model the positive effect of MSCs on hematopoietic recovery was more evident when co- infused with low number of CD34+ cells.28,45
The co-infusion of haploidentical MSCs did not improve the cumulative incidence of graft failure in the study group as compared to historical matched controls. This observation is in contrast to our recently published report of co-transplantation of haploidentical MSCs in children receiving T-cell depleted, HLA-disparate PBSCT from a relative.34 This difference may be related to the mechanisms underlying graft failure in the setting of haploidentical SCT versus CBT. Graft dysfunction in UCBT may be inherent to the low numbers of HSC infused and/or to altered homing mechanisms, whereas graft failure in the haploidentical setting may be mainly due to immune- mediated mechanisms. Moreover, any positive effect of MSCs in children undergoing UCBT in our study may have been also obscured by the use of G-CSF in historical controls. The result of our study would, however, indicate that the use of haploidentical MSCs to overcome delayed engraftment or to prevent graft rejection in children undergoing UCBT cannot be justified.
Several previously published reports aimed at comparing the outcome of UCBT and BMT from UD in children with hematological disorders showed reduced incidence of
GvHD in recipients of UCBT as compared to children given BMT.16,17,49,52-54
However, acute GvHD, even in the most severe form, can occur also in patients transplanted with UCB cells, significantly contributing to early TRM. In these series of UCBT performed in children, the incidence of grade I-II and grade III-IV acute GvHD ranged between 30-50% and 10-20%, respectively.16,17 The rates of acute GvHD observed in our historical control group was similar to that reported in these published data, obtained from large cohorts of children undergoing UCBT. In our children receiving MSCs no severe (i.e. grade III-IV) acute GvHD was observed in contrast to the historical matched controls (26%) and no patient died due to GvHD-associated TRM.
These findings suggest that the co-infusion of MSCs at time of transplantation sufficiently reduced donor T cell alloreactivity. Similarly, no study patient developed chronic GvHD compared to 10% of historical controls. In view of these observations, we conclude that MSC co-infusion may be an effective way of reducing GvHD-related TRM in UCBT recipients. This would be particularly useful in children undergoing UCBT for non-malignant disease, as well as in patients given double unit UCBT, in whom the rates of acute GvHD have been reported to be increased compared to single unit UCBT.18 Moreover, the observation that our four patients treated with parental MSCs who developed grade II acute GvHD responded to additional doses of MSCs suggests that infusion of ex-vivo expanded MSCs, regardless of the type of donor, is a possible effective treatment in steroid-resistant acute GvHD,31 although a greater number of patients should be studied to draw firm conclusions on this issue.
The 3-year cumulative incidence of leukaemia relapse was comparable in study patients and controls, this finding suggesting that the co-infusion of parental MSCs in UCBT recipients is not associated with an increased risk of disease recurrence.
Moreover, the incidence of viral infection/reactivation, as well as of other fungal or bacterial infections, did not differ between the two groups. This latter observation indicates that patients receiving MSC infusion do not seem to be exposed to an increased risk to develop infectious complications after transplantation.
In summary, our study shows that the co-infusion of haploidentical BM-derived MSCs is feasible and safe and significantly reduces the incidence of life-threatening acute GvHD, and GvHD-associated TRM.
Acknowledgements
The authors wish to acknowledge the cooperation of patients and families who have participated to date in this study, the medical and ancillary staff associated with the transplant centers for contributing to the overall excellent care of the patients and the data managers for assisting in the preparation of data for the manuscript.
This work has been partly supported by grants from the Istituto Superiore di Sanità (National Program on Stem Cells), European Union (FP6 program ALLOSTEM), Regione Lombardia (Research Project: ‘Trapianto di cellule staminali adulte per scopi di terapia cellulare sostitutiva, riparativa e rigenerativa’), Fondazione CARIPLO to F.L. and the Dutch Program for Tissue Engineering.
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