Bone Marrow Transplantation (2009) 43, 245–251 & 2009 Macmillan Publishers Limited All rights reserved 0268-3369/09 $32.00
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ORIGINAL ARTICLE
Treatment of refractory acute GVHD with third-party MSC expanded in platelet lysate-containing medium M von Bonin1, F Sto¨lzel1, A Goedecke1, K Richter1, N Wuschek1, K Ho¨lig1, U Platzbecker1, T Illmer1, M Schaich1, J Schetelig1, A Kiani1, R Ordemann1, G Ehninger1, M Schmitz2 and M Bornha¨user1 1 Medizinische Klinik und Poliklinik I, University Hospital Carl-Gustav-Carus, Dresden, Germany; and 2Institute for Immunology, Technical University of Dresden, Dresden, Germany
Mesenchymal stem cells have been shown to mediate immunomodulatory effects. They have been used in patients with steroid-refractory acute GVHD (aGVHD), but their relevance as a therapeutic agent targeting aGVHD has still to be defined. In this case series, we report 13 patients with steroid-refractory aGVHD who received BM-derived MSC expanded in platelet lysatecontaining medium from unrelated HLA disparate donors. MSC were characterized by their morphological, phenotypical and functional properties. All tested preparations suppressed the proliferation of in vitro activated CD4 þ T cells. MSC were transfused at a median dosage of 0.9 106/kg (range 0.6–1.1). The median number of MSC applications was 2 (range 1–5). Only two patients (15%) responded and did not require any further escalation of immunosuppressive therapy. Eleven patients received additional salvage immunosuppressive therapy concomitant to further MSC transfusions, and after 28 days, five of them (45%) showed a response. Four patients (31%) are alive after a median follow-up of 257 days, including one patient who initially responded to MSC treatment. In our patient cohort, response to MSC transfusion was lower than in the series reported earlier. However, our experience supports the potential efficacy of MSC in the treatment of steroid-refractory aGVHD. Bone Marrow Transplantation (2009) 43, 245–251; doi:10.1038/bmt.2008.316; published online 29 September 2008 Keywords: allogeneic hematopoietic stem cell transplantation; acute GVHD; MSC
transplantation (HCT) or donor lymphocyte infusion and remains a major cause of morbidity and mortality. Firstline treatment is based on corticosteroids, and the rate of durable responses varies between 50 and 80%, depending on initial severity.1 In case of failure after corticosteroid treatment, different therapeutic options have been introduced as second- or third-line strategies. According to the pathophysiology of aGVHD, most pharmacological compounds try to inhibit T-cell activation and thereby aim to restore donor–host immunotolerance. Several studies have shown efficacy for cytostatic drugs preferentially targeting lymphocytes (for example, MTX, pentostatin), for antibodies targeting the cytokine effector pathway (for example, infliximab, etanercept) or specific T-cell antigens (for example, antithymocyte globulin and alemtuzumab).2 However, prognosis for corticosteroid non-responders remains disappointing with a 1-year survival of only 30%.1 Mesenchymal stem cells are multipotent adult stem cells capable of generating osteoblasts, myoblasts, chondroblasts, adipocytes and stromal cells.3 MSC have recently been shown to mediate immunomodulatory properties in vitro, interacting with cellular components of the immune system and inducing a shift from pro- to anti-inflammatory cytokines.4 By inhibiting T-cell proliferation after stimulation by alloantigens and mitogens and by preventing the activity of cytotoxic T cells, MSC may become a useful tool in steroid-refractory aGVHD.5 So far, MSC have been clinically applied to facilitate engraftment in HCT and to prevent and treat aGVHD.5–15
Materials and methods Introduction Acute graft-versus-host-disease (aGVHD) is a frequent complication after allogeneic hematopoietic stem cell
Correspondence: Professor M Bornha¨user, Medizinische Klinik und Poliklinik I, University Hospital, Fetscherstrasse 74, Dresden 01307, Germany. E-mail:
[email protected] Received 23 April 2008; revised 20 August 2008; accepted 22 August 2008; published online 29 September 2008
Patient characteristics Between March and December 2007, 13 consecutive patients with steroid-refractory aGVHD after allogeneic HCT received third-party MSC within a compassionate use program. Patient characteristics are summarized in Table 1. Median age at transplantation was 58 years (range 21–69). All patients received PBSC (4 from a sibling donor, 9 from an unrelated donor) after varying reduced-intensity conditioning regimes. All patients with multiple myeloma, CLL, non-Hodgkin’s lymphoma, ALL and one patient (UPN 1513) with AML were transplanted because of
Treatment of acute GVHD with MSC M von Bonin et al
246 Patient’s characteristics
Table 1 UPN
Gender
816 994 1145 1353 1434 1453 1485 1486 1493 1508 1513 1524 1581
M F F F M F M M F M F M M
Age (years) 60 56 55 62 65 38 69 64 21 65 44 38 58
Diagnosis
Regimen (all RIC)
Donor
HLA-disparity
GVHD prophylaxis
MM MM MM CLL MDS NHL MPS/MDS CLL AML SAA AML ALL NHL
Flu/Mel Flu/Mel Flu/Mel/ATG Flu/BU 188Rhe/Flu/BU/Alem Flu/Mel/ATG Flu/BU/ATG Flu/BU Flu/Mel Flu/TBI Flu/Mel/ATG CY/TBI 90Y/Flu/TBI
Unrelated Sibling Unrelated Unrelated Unrelated Sibling Unrelated Unrelated Sibling Unrelated Unrelated Unrelated Sibling
HLA-B
CsA/MTX CsA/MTX CsA/MTX CsA/MTX CsA CsA/MTX CsA/MTX CsA/MTX CsA CsA/MMF CsA Tacro/MMF CsA/MMF
HLA-B
HLA-Cw
CD34+ ( 106/kg)
CD3+ ( 108/kg)
6.4 8 8 7 6.2 5.1 7.5 5.8 6 8 8.5 2 9.7
4.2 2.0 3.1 5.8 4.3 0.3 2.0 2.6 1.3 1.2 4.4 4.6 2.4
Abbreviations: 90Y ¼ yttrium-90 ibritumomab tiuxetan; 188Rhe ¼ rhenium 188-labeled anti-CD66 monoclonal antibody; Alem ¼ Alemtuzumab; ATG ¼ antithymocyte globulin; Flu ¼ fludarabin; MDS ¼ myelodysplastic syndrome; Mel ¼ melphalan; MM ¼ multiple myeloma; MMF ¼ mycophenolate mofetil; MPS/MDS ¼ myeloproliferative/myelodysplastic syndrome; NHL ¼ non-Hodgkin’s lymphoma; RIC ¼ reduced-intensity conditioning; SAA ¼ severe aplastic anemia; Tacro ¼ tacrolimus.
relapse of the underlying disease after completing standard therapy (including autologous SCT in patients with multiple myeloma). Patients with ALL (UPN 1524) and AML (UPN 1513), respectively, had received re-induction chemotherapy directly before allogeneic HCT. One patient with acquired severe aplastic anemia had failed to respond to an initial course of immunosuppressive therapy with CsA and antithymocyte globulin. Patients with high-risk (defined by either cytogenetic changes or course of the disease) myelodysplastic syndrome and myeloproliferative syndrome/myelodysplastic syndrome received allogeneic HCT without preceding therapy. The second patient with AML (UPN 1493) received early allogeneic HCT (immediately after the first induction chemotherapy) because of high-risk disease defined by cytogenetic changes and insufficient response to the preceding therapy. In vivo T-cell depletion was conducted in five patients (four with antithymocyte globulin and one with alemtuzumab, respectively). Three patients had received grafts from an HLA-mismatched donor. All of them, except patient UPN 816, had received antithymocyte globulin. The median number of transfused CD34 þ cells per kg body weight was 7 106 (range 2–9.7). The grafts contained median of 2.6 108 CD3 þ T cells per kg (range 0.3–5.8).
GVHD grading and response criteria Nine patients developed aGVHD directly after allogeneic HCT, three after donor lymphocyte infusion and one after cessation of immunosuppressive therapy. Grading and staging of aGVHD was performed according to criteria defined by the Consensus Workshop.16 The median time to the first symptoms of aGVHD was 29 days (range 0–61) after the causative event: 17 days after the cessation of immunosuppression (n ¼ 1), 23 days (range 0–49) after the last donor lymphocyte infusion (n ¼ 3) or 31 days (range 13–61) directly after allogeneic HCT (n ¼ 9). Eleven patients suffered from grade IV and two suffered Bone Marrow Transplantation
from grade III aGVHD by the time of MSC infusion. Skin, liver and gastrointestinal (GI) tract were affected in 6, 10 and 11 patients, respectively. All patients had failed at least two immunosuppressive regimes, including steroids, as an established first-line treatment. Previous systemic treatment for aGVHD included prednisolone (n ¼ 12), methylprednisolone (n ¼ 9), infliximab (n ¼ 6), MTX (n ¼ 3) and pentostatin (n ¼ 1). All patients with GI and skin involvement received budesonide and steroid-containing externa, respectively. Further details are given in Table 2. Progressive or persistent symptoms despite 5 days of treatment with steroids (42 mg/kg per day prednisolone equivalent) or 48 h after escalation of steroid-concomitant immunosuppressive therapy (that is, MTX, infliximab, pentostatin) were defined as therapy-refractory aGVHD. Additional immunosuppresssive therapy was given if symptoms of aGVHD persisted or progressed 1 week after the first MSC infusion. In case of amelioration of aGVHDrelated symptoms within a week after first MSC infusion, patients were defined as MSC responders and immunosuppression was not further escalated. Re-evaluation of global response, including all immunosuppressive medication administered, was performed 28 days after first MSC transfusion. Global response criteria were defined as follows: Progressive disease in cases of increase in staging or number of affected organs and no response in cases of no changes in staging or organ involvement. Mixed response (MR) was defined as improvement in staging of one organ with no change in others; partial response (PR) was defined as a decrease in staging but no resolution of all signs; complete response (CR) included cases of resolution of all signs, and overall response (OR) included CR, PR and MR. Risks associated with MSC treatment were fully explained to all patients and informed consent was obtained in all cases. Vital signs were monitored during and after intravenous bolus infusion of MSC. All patients received 4 mg dimetinden and 50 mg ranitidine before MSC transfusion.
Treatment of acute GVHD with MSC M von Bonin et al
247 Patient’s response to MSC treatment
Table 2
UPN Onset of Escalated IS aGVHD d after Before During Tx MSC MSC infusion applicationa 816
16
P MP Inf P MP PS P MP Inf P MP Inf P Inf
994
31
1145
85c
1353
476c
1434
145c
1453
29
P MP
1485
51
1486
61
1493
82e
P MP Inf P MP MTX P MP
1508
44
MP Inf
1513
13
P MTX
1524
36
P Inf
1581
25
P MTX
First MSC infusion
After MSC applicationa
Outcome
Grade Affected Stage Organ Global organs response response
d after Tx
Before first MSC infusion
d after Tx
d after onset of aGVHD
Grade
Affected organs
Stage
41
12
IV
50
19
IV
Inf
434
20
IV
Inf
498
22
IV
158
13
IV
38
9
IV
PS P
55
4
IV
MP
92
31
IV
Inf PS MP ECP P Inf
99
17
III
Skin Liver GI tract Skin Liver GI tract Skin Liver GI tract Skin Liver GI tract Skin Liver GI tract Skin Liver GI tract Skin Liver GI tract Skin Liver GI tract Skin Liver GI tract
0 4 4 0 2 4 4 3 0 1 2 4 2 4 4 0 4 4 0 2 4 0 3 3 0 0 4
63
19
III
16
IV
0 0 4 4 0 4
NA
29
Skin Liver GI tract Skin Liver GI tract
48
12
IV
10
IV
3 3 4 4 2 0
NA
35
Skin Liver GI tract Skin Liver GI tract
P Inf
P MP Alem Inf PS ECP Inf PS P Inf ECP
IV
II
II
IV
NA
0
II
III
III
IV
NA
MR
60b
PR
197b
MR
692d
PD
545b
NA
176b
CR
299d
MR
147b
MR
257b
NR
215d
NA NA NA CR NC NR
NA
85b
MR
185d
NA
NA
55b
NA
NA
61b
Skin Liver GI tract Skin Liver GI tract Skin Liver GI tract Skin Liver GI tract Skin Liver GI tract Skin Liver GI tract Skin Liver GI tract Skin Liver GI tract Skin Liver GI tract
0 4 0 0 1 1 0 1 0 0 3 4 NA NA NA 0 0 0 0 0 1 0 2 3 0 0 4
NC NR CR NC PR PR CR PR NC CR PD NR NA NA NA NC CR CR NC CR PR NC PR NR NC NC NR
Skin Liver GI tract Skin Liver GI tract
NA NA NA 0 0 4
Skin Liver GI tract Skin Liver GI tract
NA NA NA NA NA NA
Abbreviations: aGVHD ¼ acute GVHD; Alem ¼ Alemtuzumab; CR ¼ complete response; d ¼ days; DLI ¼ donor lymphocyte infusion; ECP ¼ extracorporeal photopheresis; GI ¼ gastrointestinal; Inf ¼ infliximab; IS ¼ immunosuppression; MP ¼ methylprednisolone; MR ¼ mixed response; NA ¼ not available (patient died during MSC period); NC ¼ no change (organ at the time point of first MSC infusion not affected and no worsening at the end of MSC period); NR ¼ no response; P ¼ prednisolone; PD ¼ progressive disease; PS ¼ pentostatin; PR ¼ partial response; Tx ¼ Transplantation. a Period of 28 days after the first MSC infusion. b Death. c Patients received DLI (onset of aGVHD after last DLI: UPN 1145—0 days, UPN1353—49 days, UPN 1434—23 days). d patients are still alive. e Patient developed aGVHD 17 days after cessation of immunosuppression.
MSC isolation and GMP grade expansion MSC were obtained from BM aspirates of 12 healthy donors after informed consent and approval by the local institutional review board. MSC were designated nos. 5, 7, 8, 9, 13, 14, 15, 16, 17, 18, 19 and 25 and additionally marked with the number of passage. Only MSC after first or second passage were used for therapy and in vitro experiments, as immunomodulatory function has been
predominantly described in MSC batches from early passages. MSC were isolated and expanded according to a validated production protocol within a certified clean room unit. Bone marrow aspirates were diluted about 1:5 with PBS (Invitrogen, Karlsruhe, Germany). The mononuclear cell fraction was isolated by density gradient centrifugation Bone Marrow Transplantation
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248
(900 g, 30 min, room temperature) using Biocoll solution (Biochrom, Berlin Germany; density 1077 g/l) and seeded at a density of 6 104–1 105 per cm2 into T175 cell culture flasks (Greiner, Frickenhausen, Germany). The first passage was completed 2–3 weeks after isolation. The resulting fibroblastoid adherent cells were cultivated at 37 1C at a humidified atmosphere containing 8% CO2. The expansion medium consisted of DME-low glucose (Invitrogen, Karlsruhe, Germany) containing human platelet lysate at a concentration of 10%.17 The medium was replaced twice weekly. Cells were harvested at subconfluence using TrypZean (Sigma Aldrich, Steinheim, Germany; GMP grade equivalent of Trypsin). Cells at passage 1 (and thereafter) were plated at a mean density of 5 103/cm2 in T175 cell culture flasks (Greiner, Frickenhausen, Germany) and were frozen in part in medium containing 10% DMSO (WAK Chemie, Steinbach, Germany) and 5% human serum albumin (HSA, Baxter, Unterschleisheim, Germany). For clinical use or in vitro experiments, either frozen aliquots of the MSC were thawed or MSC were used directly after isolation. Additional information concerning the growth kinetics during ex vivo expansion is provided as Supplementary Table 1. Platelet lysates were derived from single-donor platelet apheresis concentrates collected at our transfusion department. The platelet concentration of each product was 1 109 platelets per ml. Aliquots of the apheresis product were frozen at 20 1C, thawed overnight and heat inactivated at 56 1C for 30 min in a water bath. For cultivation, platelet lysates of two donors were mixed after elimination of remaining platelets by centrifugation (5000 g, room temperature, 45 min) (Supplementary Table 2). Comparative data concerning GMP grade MSC expansion in FCS vs platelet lysate have been previously reported.18
In vitro analysis of MSC characteristics All MSC preparations were characterized by FACS. Standard staining and analysis protocols were applied using commercial FITC- or phycoerythrin-conjugated antibodies against CD105, CD166, CDw90, CD73, CD45, CD14 and CD34 (Becton Dickinson, San Jose, CA, USA). The potential for osteogenic and adipogenic differentiation was proven by standard differentiation media (Miltenyi Biotec, Bergisch Gladbach, Germany). Clonogenicity was tested by a standard 14 d CFU-F assay (Miltenyi Biotec). Besides sterility, the parameters mentioned had to suggest viability of the individual lot before a preparation was released for an individual patient. Only MSC from CMVseronegative healthy donors were used in CMV-seronegative patients. All donors had undergone infectious disease marker testing according to the National Marrow Donor Program (NMDP) criteria.19 Thymidine incorporation assay Blood samples were obtained from healthy donors with informed consent. PBMCs were prepared by FicollHypaque (Biochrom, Berlin, Germany) density centrifugation. Subsequently, CD4 þ T cells were isolated from PBMCs by negative depletion using immunomagnetic Bone Marrow Transplantation
separation according to the manufacturer’s instructions (Miltenyi Biotec). The purity of the obtained CD4 þ T cells was 493% as assessed by flow cytometric analysis. To determine whether MSC influence T-cell proliferation, irradiated MSC (1 104 cells per well) were co-cultured with allogeneic CD4 þ T cells (1 105 cells per well) in the presence of 1 mg per ml PHA (Sigma Aldrich, Taufkirchen, Germany) for 4 days in round-bottomed 96-well plates. Culture medium consisted of RPMI 1640 (Biochrom) supplemented with 2 mM L-glutamine, 10 mM sodium pyruvate, 1% non-essential amino acids, 100 mg per ml penicillin, 100 mg per ml streptomycin (all from Biochrom) and 10% human serum (CC pro, Neustadt, Germany). 3 H-thymidine (1 mCi, Hartmann Analytic, Braunschweig, Germany) was added to each well for the last 18 h of culture. Cells were harvested and incorporation was determined in a beta counter (Wallac, Freiburg, Germany).
Results In vitro characteristics of MSC All MSC lines showed osteogenic and adipogenic differentiation potential and were proven to be viable in CFU-F assays. A homogenous phenotype was confirmed by FACS with a median purity for CD105/CD166 and Cdw90/CD73 of 96.9 and 99.8%. Negativity for CD34, CD45 and CD14 was confirmed in all cases before the product was released. To investigate the impact of MSC on the proliferation of CD4 þ T cells, irradiated MSC lines derived from four healthy donors were co-cultured with allogeneic CD4 þ T cells in the presence or absence of PHA. As expected, PHA stimulation led to an increase in incorporated radioactivity, expressed as c.p.m. (counts per minute), from 1303±113 to 22 403±2918 c.p.m. Co-cultivation with MSC did not fully reverse the proliferation induced by PHA, but approximately reduced by half the incorporated radioactivity to 10 880±1795 c.p.m. Furthermore, co-cultivation with MSC 5.1, 7.1, 8.1 and 9.1 decreased 3Hthymidine incorporation to 11 306±1332, 9029±572, 12 742±925 and 10 443±840 c.p.m., respectively. All experiments were carried out in triplicates. As summarized in Figure 1, all MSC lines efficiently impaired the proliferation of PHA-stimulated CD4 þ T cells. Clinical effects Clinical course of therapy and response are summarized in Table 2 and Figure 2. The first MSC infusion was given at a median of 41 days (range 20–91) after transplantation, donor lymphocyte infusion or cessation of immunosuppression and 16 days after the onset of aGVHD (range 4–31). The median number of MSC transfusion was 2 (range 1–5), the median interval between each MSC application was 7 days (range 3–19) and the average MSC dose given was 0.9 106 per kg body weight (range 0.6–1.1 106 per kg body weight) (Table3 and Figure 3). No infusion-related toxicity was observed during or immediately after the administration of MSC. Concomitant immunosuppressive treatment because of therapy-refractory aGVHD included infliximab (n ¼ 8),
Treatment of acute GVHD with MSC M von Bonin et al
249
prednisolone (n ¼ 5), pentostatin (n ¼ 4), methylprednisolone (n ¼ 3), extracorporeal photopheresis (n ¼ 3) and alemtuzumab (n ¼ 1). Only two patients (15%) did not need further escalation of immunosuppression after the first MSC transfusion. Four patients (31%) died within a period of 28 days after the first MSC transfusion. Reasons for death were pneumonia (n ¼ 2), diffuse alveolar hemorrhage (n ¼ 1) and liver failure (n ¼ 1), the latter probably related to GVHD. During the whole observation period, five more patients died: two patients died because of organ toxicity
and infection (liver toxicity and encephalitis), and three patients succumbed to GVHD-associated problems (liver failure, microangiopathy and paralytic ileus). At the time of the report, four patients (31%) are still alive: two without any evidence of GVHD and the other two with chronic GVHD of the GI tract. The median follow-up for all patients is 185 days (range 55–692) after allogeneic HCT and 92 days (range 7–261) after first MSC infusion, respectively. The median follow-up for the patients who are still alive is 257 days (range 185–692) after allogeneic HCT and 207 days (range 116–261) after first MSC infusion. Until now, none of the surviving patients has experienced relapse of the underlying disease. Global overall response, 28 days after first MSC infusion was 54% (n ¼ 7), including the two patients who responded immediately within a week after first MSC infusion. Global OR was composed of one CR, one PR and five MR (39%). With regard to initially affected organs, improvement (CR and PR) in skin, liver or GI tract involvement was observed in three (50%), five (50%) and four (36%) cases, respectively. In patients with primary skin, liver and/or GI tract involvement, the global OR (CR, PR and MR) was 33% (n ¼ 2), 60% (n ¼ 6) and 64% (n ¼ 7), respectively. The results are shown in Table 4. Three of the survivors (n ¼ 4) responded to the initial treatment, including one CR and two MR. Within this small-sized cohort, no correlation between underlying disease, graft source, donor type or HLA-match grade and the response to MSC therapy could be identified.
25 000
c.p.m.
20 000
15 000
10 000
5 000
0 4+ T
T
T
T
4+
4+
4+
CD
CD
CD
CD T
T
4+
4+
CD
CD
lls
lls
lls
lls
lls
ce
ce
ce
ce
ce
lls
ce
+
+
+
+
+
A
A
A
A
PH
PH
PH
PH
A
PH
+
+
+
+
SC
SC
SC
SC
M
M
M
M
1
9.
1 8.
1
7.
1
5.
Figure 1 Suppression of T-cell proliferation. MSC efficiently impair the proliferation of CD4 þ T cells. Irradiated MSC (1 104 cells per well) were co-incubated with allogeneic CD4 þ T cells (1 105 cells per well) in the presence of PHA for 4 days in round-bottomed 96-well plates. For comparison, CD4 þ T cells were cultured alone in the presence or absence of PHA. 3H-thymidine was added to each well for the last 18 h of culture. Cells were harvested and incorporation was determined in a beta counter. The results of MSC derived from four healthy donors are presented as mean±s.e. of triplicate determinations.
Discussion In our cohort, only two patients (15%) responded immediately and did not require re-escalation of immunosuppression. OR, 28 days after first MSC infusion, including all immunosuppression administered, was 54%.
UPN
Organ involvement before MSC period
994
1145
1353
1434
1453
1485
1486
1493
1508
1513
1524
1581
2
2
2
2
3
2
5
1
3
2
4
2
2
yes
no
yes
yes
no
no
yes
yes
yes
yes
yes
yes
yes
NA
Skin Liver GI tract
No. of MSC infusions Escalated IS#
Organ involvement after MSC application
816
Skin
NA
NA
NA
Liver
NA
NA
NA
NA
GI tract
NA
NA
NA
NA
Red = grade III-IV, blue = grade I-II, gray = no involvement, NA = not available (patient died during MSC period), # = further escalation of immunosuppressive therapy in a period of 28 days after the first MSC infusion
Figure 2 Organ involvement before first MSC infusion and 28 days after first MSC infusion. Red ¼ grade III–IV, blue ¼ grade I–II, gray ¼ no involvement, NA ¼ not available (patient died during MSC period), # ¼ further escalation of immunosuppressive therapy in a period of 28 days after the first MSC infusion.
Bone Marrow Transplantation
Treatment of acute GVHD with MSC M von Bonin et al
250 Table 3 UPN
816 994 1145 1353 1434 1453 1485 1486 1493 1508 1513 1524 1581
Cell count per infusion ( 106/kg)
No. of MSC infusions 2 2 2 2 3 2 5 1 3 2 4 2 2
Interval between MSC infusions (days)
Mean
Minimum
Maximum
Median
0.9 0.6 0.8 0.9 0.9 0.9 0.9 1.0 0.9 1.0 0.8 0.9 0.8
0.8 0.6 0.6 0.8 0.6 0.8 0.6
0.9 0.7 1.1 1.0 1.0 1.0 1.0
8 3 10 7 7 4 7
0.9 1.0 0.7 0.9 0.6
1.0 1.0 1.0 0.9 0.9
6 5 7 7 19
Minimum
Response to MSC treatmenta
Maximum
3
11
6
8
6
6
5
8
No Yes No No No Yes No No No No No No No
Amelioration of aGVHD-related symptoms within a week after first MSC infusion.
UPN
a
MSC treatment
1581 1524 1513 1508 1493 1486 1485 1455 1434 1353 1145 994 816
0.9 0.9 0.8 1.0 1.0 1.0 0.7 1.0 1.0 0.8 0.6 0.7 0.8
0.6 0.9 0.8
0.7 1.0
1.0
0.9
0.9
1.0
1.0
1.0
1.0
0.8 0.6
0.9 1.0 1.1
0.6 0.9
0
5
10
15
20
25
30
Days after first MSC infusion
Figure 3
Table 4
Dose and time point of MSC infusion.
Initial organ involvement, organ-specific response and global response 28 days after first MSC infusion
Organ response (no./%) Affected organ Skin (n ¼ 6) Liver (n ¼ 10) GI tract (n ¼ 11) Global response (no./%) n ¼ 13
CR
PR
NR
PD
NA
3 (50%) 2 (20%) 2 (18%)
0 3 (30%) 2 (18%)
0 1 (10%) 4 (36%)
0 1 (10%) 0
3 (50%) 3 (30%) 3 (27%)
CR
PR
MR
NR
PD
NA.
1 (8%)
1 (8%)
5 (38%)
1 (8%)
1 (8%)
4 (31%)
Abbreviations: CR ¼ complete response; GI ¼ gastrointestinal; NA ¼ not available (death before ending of 28-day period after first MSC infusion); NC ¼ no change (organ at the time point of first MSC infusion not affected and no worsening at the end of the MSC period); NR ¼ no response; OR ¼ overall response; PD ¼ progressive disease; PR ¼ partial response.
Four patients (31%) died during this evaluation time period. At this time, four patients are still alive (median 257 days after allogeneic HCT, range 185–692). One of them was primarily assessed as an MSC responder. Between 2000 and 2006, the 1- and 2-year survival of patients with grades III–IV steroid-refractory GVHD not receiving MSC at our center was 28% (11/40) and 20% (8/40), respectively. The largest series reporting MSC as therapy for GVHD, which has been fully published, contained nine patients, including one with extensive chronic GVHD.12 OR in Bone Marrow Transplantation
patients with aGVHD was 75%, including five patients with CR. The improved response when compared with our cohort can not only be explained in part by differences in the patient’s characteristics, but might be also related to differences in MSC application. In both groups, BM derived MSC were used but the donors differed. All our patients received MSC from unrelated mismatched donors, whereas only four patients of the other series obtained third-party MSC (four patients received MSC at least once from haploidentical donors, twice from HLA-identical
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siblings). Because anti-FCS antibodies with uncertain clinical significance could be detected in patients after receiving MSC that were expanded in vitro using FCS,20 we decided for safety reasons to replace FCS by platelet lysates.17 In addition, compared with our series, the MSC dosage tended to be higher with a median of 1 106/kg (range 0.7–9 106 per kg) and nine patients received only one or two MSC infusions in our cohort. Another publication reported a complete disappearance of GVHD symptoms in five of six patients (83%) after receiving adipose tissue-derived MSC.7 Although one might speculate that the origin of MSC and the serum source for expansion could impact on the anti-inflammatory and immunosuppressive effects of MSC, in vitro data suggest that the cells used in our study have functional activity. On the other hand, there is a large interindividual variability between MSC from different donors, and in part the variations between the studies can also be explained by differences in the time point of application and number of transfused cells. Further controlled studies will hopefully define the optimal source, appropriate timing, cell dosing and potential response predictors in patients receiving MSC as a salvage therapy for refractory aGVHD.
Acknowledgements This work was supported by the Centre for Regenerative Therapy Dresden (CRTD). MB and GE received additional support from the Deutsche Forschungsgemeinschaft (SFB 655; from cells to tissues).
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Bone Marrow Transplantation
