Treat or Prevent Graft-versus-Host Disease

Biol Blood Marrow Transplant 22 (2016) 1416e1423

Biology of Blood and Marrow Transplantation journal homepage: www.bbmt.org

Heterogeneity in Studies of Mesenchymal Stromal Cells to Treat or Prevent Graft-versus-Host Disease: A Scoping Review of the Evidence Mina Rizk, Madeline Monaghan, Risa Shorr, Natasha Kekre, Christopher N. Bredeson, David S. Allan* Blood and Marrow Transplantation, Department of Medicine, The Ottawa Hospital and University of Ottawa and Ottawa Hospital Research Institute, Ottawa, Ontario, Canada

Article history: Received 19 January 2016 Accepted 11 April 2016 Key Words: Mesenchymal stromal cell Graft-versus-host disease Hematopoietic cell transplantation Systematic review Treatment Prevention

a b s t r a c t Effective treatments are lacking for the treatment of steroid-refractory graft-versus-host disease (GVHD), a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. Mesenchymal stromal cells (MSCs) have demonstrated promise but there is uncertainty regarding their clinical effectiveness. A systematic scoping review of the literature was performed to characterize the heterogeneity of published studies and identify opportunities for standardization. Thirty studies were identified, including 19 studies (507 patients) addressing the treatment of acute or chronic GVHD and 11 prevention studies (277 patients). Significant heterogeneity was observed in the age and diagnoses of study subjects, intensity and specifics of the conditioning regimens, degree of HLA matching, and source of hematopoietic cells. MSCs were derived from bone marrow (83% of studies), cord blood (13%), or adipose tissue (3%) and were cryopreserved from third-party allogeneic donors in the majority of studies (91% of prevention studies and 63% of treatment studies). Culture conditions and media supplements were highly variable and characterization of MSCs did not conform to all International Society for Cellular Therapy criteria in any study. MSCs were harvested from cell culture at passage 1 to 7 and the dosage of MSCs ranged from 0.3 to 10 106/kg, using varying schedules of administration. Treatment response criteria were not standardized and effectiveness in controlled treatment studies (5 studies) was unconvincing. Details of actively recruiting trials suggest heterogeneity still persists with only 53% of registered trials describing the use of standard GVHD response criteria and few detailing methods of MSC manufacturing. Future studies will need to make substantial coordinated efforts to reduce study heterogeneity and clarify the role of MSCs in GVHD. Ó 2016 American Society for Blood and Marrow Transplantation.

INTRODUCTION Graft-versus-host disease (GVHD) is a significant complication after allogeneic hematopoietic cell transplantation (HCT) and is associated with increased morbidity and mortality [1]. It is caused by incompatibilities in major histocompatibility complex genes between recipient and donor and by immunocompetent T cells in the graft recognizing minor antigens on host cells [2]. GVHD may be subdivided into an acute form, classically defined as occurring within the first 100 days after transplantation, and a chronic form, which normally occurs after 100 days after transplantation. It is estimated that between 40% and

Financial disclosure: See Acknowledgments on page 1422. * Correspondence and reprint requests: David S. Allan, MSc, MD, 501 Smyth Rd, Box 704, Ottawa ON Canada, K1H 8L6. E-mail address: [email protected] (D.S. Allan).

http://dx.doi.org/10.1016/j.bbmt.2016.04.010 1083-8791/Ó 2016 American Society for Blood and Marrow Transplantation.

70% of patients undergoing allogeneic HCT will develop grade II to IV acute GVHD (aGVHD), with many factors influencing the probability of developing the condition as well as its severity, including donor HLA matching, the patient’s conditioning regimen, and the source of the graft (ie, bone marrow, granulocyte colonyestimulating factoreprimed peripheral blood or cord blood) [1]. Initial treatment of GVHD typically involves immune suppression with glucocorticoids; however, many patients do not respond to initial therapy [3]. The American Society of Blood and Marrow Transplantation recently published treatment guidelines for aGVHD [4]. The guidelines highlight deficiencies in study design, the lack of wellestablished response criteria, and small sample size as major limitations to identifying new treatments for aGVHD. No clear second-line treatment recommendation can be made for steroid-refractory cases.

M. Rizk et al. / Biol Blood Marrow Transplant 22 (2016) 1416e1423

Mesenchymal stromal cells (MSCs) represent a treatment option with significant promise and were not considered in the American Society of Blood and Marrow Transplantation guidelines as they were not commercially available at the time in the United States. MSCs are pluripotent cells that may be expanded from bone marrow, adipose tissue, umbilical cord, or placental tissues and can differentiate into multiple cell lineages of mesenchymal origin [5]. The immunomodulatory function of mesenchymal stromal cells has been explored in the last 10 years as a potential treatment for GVHD. This emerged after the observation that MSCs could attenuate alloreactivity in mixed lymphocyte assays and prevent rejection of skin allografts in baboons [6]. MSCs have been shown to inhibit T and B lymphocyte activation, block the function of antigen-presenting cells, and inhibit NK cells [7]. Moreover, MSCs can increase the number of regulatory T lymphocytes and attenuate inflammation and induce tolerance [8]. Cell-tocell contact appears to be important; however, paracrine functions also appear relevant. Contact-dependent mechanisms of immune modulation include activation of the PD-1 (programmed cell death protein 1) pathway, Fas-1emediated apoptosis, engagement of VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1), and upregulation of CD39 and adenosine production [9-12]. The importance of paracrine mechanisms of immune modulation has been demonstrated in vitro [13] and suggested by successful treatment of aGVHD using exosomes derived from MSCs [14]. Many phase I and II clinical trials examining the use of MSCs for GVHD have been performed, but the extent to which MSCs can improve patient outcomes remains unclear. Uncertainty regarding the clinical effectiveness of MSCs is likely due to significant heterogeneity across these studies with regard to patient populations, concomitant treatments and GVHD prevention strategies, and variability in the methods used to prepare MSCs and tools used in the diagnosis and assessment of GVHD. Although the International Society for Cellular Therapy has established minimum criteria for defining MSCs [15], it is unclear how many published clinical studies applied these criteria. Likewise, it is unclear how patient populations and details of MSC administration varied in these studies. A systematic scoping review is needed to characterize the heterogeneity of published studies and identify opportunities for standardization of methods for future studies that seek to clarify the role of MSCs in the management of GVHD. METHODS Eligibility Criteria for Systematic Search We included all interventional and retrospective clinical studies describing the use of MSCs in human patients for the treatment or prevention of aGVHD and/or chronic GVHD (cGVHD). Only articles where GVHD was reported as a primary outcome were included for analysis. Review articles, editorials, and preclinical studies were excluded. Conference abstracts were also excluded as we sought to extract methodological details that would generally be provided in full-length publications only. Articles written in languages other than English or French were excluded. Search Strategy, Study Selection, and Data Extraction A systematic search of the literature was performed according to recommendations by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses [16]. A search strategy was developed to identify studies in MEDLINE, EMBASE, and PUBMED databases. The search strategy was peer reviewed by a medical information specialist. Databases were searched for records dated from January 1946 to June 2015. The electronic search strategy is presented in Supplemental Table S1. Reference lists of relevant studies were also examined manually to identify studies that may have been missed by the electronic search. Titles and abstracts of studies identified by the systematic

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search were screened for relevance by 2 independent investigators. Relevant articles identified through the screen were reviewed for complete assessment of eligibility criteria. Discrepancies were resolved through consensus. Data were manually extracted using Distiller SR software. We also searched www.clinicaltrials.gov and the International Clinical Trials Registry Platform of the World Health Organization on December 4, 2015 using the terms “mesenchymal” and “graft versus host disease” to identify the breadth of ongoing activity and to review registered protocol information of active studies to address aspects of study heterogeneity in comparison to published reports.

RESULTS A total of 1310 citations were identified through the systematic search. After initial screening of titles for relevance, 538 studies underwent comprehensive review. A total of 467 studies were subsequently excluded for the following reasons: only an abstract was available (130 records), review article (120 records), preclinical study (115 records), did not report GVHD-related outcomes (48 records), MSCs not administered (48 records), and letters to the editor (6 records). A summary of the study selection process is provided in Figure 1. GVHD as Primary Outcome versus Secondary Outcome Among 70 studies identified in our search, 32 identified GVHD as a secondary outcome and were excluded. In these studies, MSCs were given to examine MSC safety, toxicity, and pharmacology (12 studies), to treat nonmalignant hematologic disorders (10 studies), and to accelerate hematopoietic engraftment after transplantation (10 studies). Of the 38 studies where GVHD was the primary outcome, 8 studies were classified as case reports or case series and were excluded from analysis (defined as studies with a study population of 5 patients or fewer). The 30 remaining studies included 19 studies where MSCs were given for the treatment of GVHD [17-35] and 11 studies where MSCs were given for the prevention of GVHD [36-46]. The main characteristics of these studies are summarized in Table 1. Patient heterogeneity The 11 studies using MSCs as prevention for GVHD included a total of 277 patients with a mean age of 23.8 years (range, 5 to 58). The proportion of male (54%) and females (46%) was not different. Although all of these patients underwent allogeneic HCT, significant heterogeneity in the indication for transplantation was observed, with 69% of subjects having a range of malignant disorders and 31% with nonmalignant disorders (ie, aplastic anemia and others). The source of cells used for transplantation in the prevention studies was a combination of bone marrow and peripheral blood progenitor cells (PBPC) from the same donor in 42% of patients (owing to several large studies using haploidentical donors with combined marrow and granulocyte colonyeforming factorestimulated PBPCs as the source of cells), PBPC alone in 21% of patients, bone marrow in 9%, and cord blood in 8% of patients. In 21% of cases, the source of cells was not clearly reported. Most transplantations used HLA-mismatched donors (57%), including related haploidentical donors, whereas 22% of transplantations were from matched related or unrelated donors. The degree of HLA matching was not provided in 21% of transplantations in these prevention studies. Conditioning regimens also varied with 46% of patients undergoing myeloablative treatment and 27% undergoing nonmyeloablative or reduced-intensity regimens [47]. Details of the conditioning regimen were not explicitly stated for 27% of patients. The 19 studies using MSCs as treatment for GVHD included a total of 507 patients with a mean age of 30.9

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Figure 1. Flow diagram of systematic search results and study selection.

(range, 6 to 58) years (Table 1). More males were enrolled than females (63% and 37%, respectively) in the treatment studies, including 399 patients with aGVHD and 106 patients with cGVHD (Table 2). The underlying condition was malignant disorders in 83% of these patients and the source of cells transplanted was granulocyte colonyeforming factorestimulated PBPC (52% of patients), bone marrow (27%), cord blood (13%), bone marrow combined with PBPC (2%), and donor leukocyte infusions (1%). The source of hematopoietic cells was not stated in 5% of patients. In patients with GVHD treated with MSCs, donors were HLA matched (65% of patients, including 125 related donors, 111 unrelated donors, and 92 cases where the donor relation was not reported), HLA mismatched (30% of patients, including related and unrelated), or not clearly stated (5% of patients) (Table 1). Patients receiving MSCs for the treatment of aGVHD had predominantly grade III or IV disease (84% of patients with aGVHD) with multiorgan involvement: 72% had skin involvement, 81% had gastrointestinal involvement, and 46% had liver involvement. Of the patients receiving treatment for cGVHD, 12% had 1 organ involved, 57% had 2 organs involved, and 31% had 3 or more organs involved. MSC heterogeneity There was significant heterogeneity in cell culture conditions for MSC products administered to patients, whether for prevention or treatment of GVHD. The most common tissue source for MSC growth was bone marrow (7 of 11 prevention

studies and 18 of 19 treatment studies). Four prevention studies used umbilical cordederived MSCs and 1 treatment study used adipose-derived MSCs. There was also heterogeneity in the conditions under which the MSCs were cultured. The most common culture media used to support MSC growth was DMEM and alpha-modified MEM in 50% and 20% of the studies, respectively. FBS was the media supplement of choice for most studies (57%), followed by human platelet lysate (23%). Infusion of freshly cultured MSCs that had not been cryopreserved was reported in only 1 prevention study using HLA-matched MSC products expanded from related donors [40], whereas thirdparty cryopreserved MSCs were reported in the remaining prevention studies and in 10 of 19 treatment studies. Noncryopreserved MSCs were infused in 6 treatment studies, fresh or cryopreserved MSCs were reported in 2 studies, and details of MSC storage were not provided in 1 treatment study. We also examined the degree of characterization of MSC products in each study and compared them to the internationally accepted criteria of the International Society for Cellular Therapy (ISCT) [15]. Although only 2 studies were published before the publication of the ISCT standard criteria for MSCs, no studies reported on all of the ISCT criteria and there was wide disparity in the degree of characterization of MSCs. The most commonly met criteria were expression of the cell surface markers CD105 (84% of studies), CD73 (81%), and CD90 (75%), and lack of expression of CD45 (87%) and CD34 (84%). Only 1 study met the criteria of lacking expression of the marker CD79 alpha or CD19 and only 7


Table 1 Summary of Patient Populations Examined in Clinical Trials

Table 3 Summary of MSCs Administered to Patients in Studies

Transplant Characteristic

MSCs as Treatment

MSCs as Prevention

Total

Studies, n References Total patients, n Age*, median (range), yr Gender (M/F) Diagnosis, n (%) Malignant Nonmalignant Not stated Stem cell product, n (%) BM PBPC CB BMþPBPC DLI Not stated Donor Matched, related Matched, unrelated Matched, relation not stated Mismatched Not stated Conditioning MA NMA/RICT Not stated

19 [1-19] 507 30.9 (6-58) 318/189

11 [20-30] 277 23.8 (5-58) 149/128

30 784 28.7 (5-58) 467/317

420 (82.8) 69 (13.6) 18 (3.6)

192 (69.3) 85 (30.7) 0 (0.0)

612 154 18

136 265 66 8 5 27

(26.8) (52.3) (13.0) (1.6) (1.0) (5.3)

24 56 22 117 0 58

(11.6) (20.2) (7.9) (42.2) (0.0) (20.9)

160 321 88 125 5 85

125 111 92 153 26

(24.7) (21.9) (18.1) (30.2) (5.1)

48 4 10 157 58

(17.3) (1.4) (3.6) (56.7) (20.9)

173 115 102 310 84

242 (47.7) 109 (21.5) 156 (30.8)

362 214 208

120 (43.3) 105 (37.9) 52 (18.8)

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M indicates male; F, female; BM, bone marrow; CB, cord blood; DLI, donor leukocyte infusion; MA, myeloablative; NMA, nonmyeloablative; RICT, reduced-intensity conditioning transplantation. * Mean of median values reported in studies.

studies (23%) demonstrated cellular differentiation to osteogenic, adipogenic, and chondrogenic lineages (Table 3). Wide disparity in cell dosage and replicative status of MSCs (reported as culture passage number) was reported. Almost all studies (97%) presented the median dosage of MSCs administered, whereas only 23 studies (74%) reported the dosage range. The data are summarized in Figure 2. The dose of MSCs administered ranged from 0.3 to 10 106 cells/kg to 1.0 107 cells/kg. Only 17 of 31 studies (55%) presented information on the passage number of MSC cultures (range, 1 to 7) or number of cell divisions occurring in culture of the MSCs administered to patients (Figure 3). Effectiveness of MSCs A total of 167 patients out of 277 developed grade I to IV aGVHD (59%) in prevention studies and 75 patients (31%)

MSC Characteristic

Prevention

Treatment

All Studies

Studies, n MSC source BM Adipose Umbilical cord Same as HSC donor, (no. patients) Expansion media DMEM Alpha-MEM Other Not stated Media supplement FBS Platelet lysate Serum free Not stated Infusion Fresh, no. patients Thawed, no. patients Not stated ISCT criteria met, no. studies (%) CD105þ CD73þ CD90þ CD45 CD34 CD79á or CD19 CD14 or CD11b HLA DR Differentiation to osteoblasts Differentiation to adipocytes Differentiation to chondroblasts All ISCT criteria met, no. studies (%)

11

19

30

7 0 4 1

18 1 0 1 (7)

25 1 4 2

4 2 1 4

11 4 1 3

15 6 2 7

4 2 1 4

13 5 0 1

17 7 1 5

1 10 0

7 11 1

8 21 1

9 8 7 9 8 0 6 7 4 3 1 0

17 17 16 18 18 1 13 6 9 9 6 0

(82) (73) (64) (82) (73) (0) (55) (64) (36) (27) (9) (0)

(89) (89) (84) (95) (95) (5) (68) (32) (47) (47) (32) (0)

26 25 23 27 26 1 19 13 13 12 7 0

(87) (83) (77) (90) (87) (3) (63) (43) (43) (40) (23) (0)

ISCT indicates International Society for Cellular Therapy.

developed grade II to IV aGVHD (Figure 4). In treatment studies of aGVHD and/or cGVHD, a total of 364 patients (72%) responded (complete or partial response) (Figure 5). Definitions for the diagnosis of GVHD and response to treatment were variable. Of the 30 clinical trials included for analysis, 7 were controlled. The effectiveness of MSCs compared with controls is summarized in Figure 6. Only 1 treatment study compared MSCs to standard treatment and the other controlled treatment study compared doses of MSC.

Table 2 Summary of GVHD Characteristics of Patients Treated with MSCs Characteristic

MSCs as Treatment

Studies, n Total patients, n aGVHD, n (%) Grade I Grade II Grade III Grade IV Grade III/IV Organ involvement Skin GI Liver cGVHD 1 Organ 2 Organs 3þ Organs

19 507 399 3 61 109 168 58

(100.0) (0.8) (15.3) (27.3) (42.1) (14.5)

288 323 186 106 13 62 33

(72.2) (81.0) (46.6) (100.0) (12.2) (58.4) (31.1)

GI indicates gastrointestinal. Grade of aGVHD according to the referenced study.

Figure 2. Dosage of MSCs (cells per kg) administered to patients with acute or chronic GVHD (median reported value and range). Horizontal hatched line (bold) represents average value (light hatched line is range) of reported medians for acute and chronic GVHD.

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Figure 3. Median passage number and range of MSC cell cultures reported for each study enrolling patients with acute or chronic GVHD.

Pooling data from these studies was not performed. Five controlled studies of MSCs to prevent GVHD incorporated standard GVHD prophylaxis in the control arm. None of the studies demonstrated a convincing benefit for MSCs. Although 1 study reported a significant reduction in the incidence of grades II to IV aGVHD with MSCs [40], reanalysis of their published data using Fisher’s exact test for comparing rates of categorical outcome measures yielded a P value > .05 (Figure 6B). Meta-analysis of these studies has been recently reported and was not repeated [48]. Registered Trials Recruiting Patients After a search of actively enrolling registered trials, a total of 42 unique records were identified, including 10 ongoing trials actively recruiting patients with aGVHD and 6 trials recruiting patients with cGVHD. Only 1 study that was enrolling patients for the prevention of aGVHD using MSCs was identified. The status of 9 registered trials was unknown with no formal update in the 2 years before our search, 9 trials were completed, 2 were no longer available, 2 trials were suspended and/or withdrawn, and 3 trials did not address GVHD. Among actively recruiting studies, 6 were randomized controlled trials (RCTs) addressing the treatment of aGVHD (3 studies) or cGVHD (3 studies). The characteristics of the registered trials that are actively

Figure 4. Numbers of patients with acute GVHD (grade 0 or 1 in light grey and grades 2 to 4 in black) for each study of prophylactic MSC administration. Overall incidence of acute GVHD provided.

Figure 5. Number of patients with no response (grey) or a complete or partial response (black) for each study of MSC treatment of acute or chronic GVHD. Overall incidence of patients who responded (complete or partial response) or did not respond is provided.

enrolling patients are summarized in Table 4. With regard to cell manufacturing details, the source of cells was derived from bone marrow (4 studies), umbilical cord blood (2 studies), adipose tissue (1), amniotic membranes (1), or was not reported (9). The doses of MSCs for treatment and/or prevention of GVHD were highly variable both in terms of dose and frequency and/or not reported (Table 4). More specific details regarding manufacturing were not available, including specific criteria for donor selection, culture duration and media composition, cell characterization, method of cryopreservation and/or transportation of the cells, whether potency assays were used, and whether ISCT criteria were used to characterize the final product. Patients included in the studies were typically adult patients, ranging in age from 12 to 80 and only 1 study restricted enrollment to a particular site of GVHD involvement (study of mucosal injection of MSCs for oral GVHD, NCT02055625). No study explicitly limited enrollment to specific factors related to the conditioning treatment or type of donor. The use of standardized or National Institutes of Health guidelines for the diagnosis of GVHD and response to treatment was described for the majority of studies and is summarized in Table 4.

Figure 6. (A) Forest plot of controlled studies of MSC treatment of GVHD and (B) for GVHD prevention. Study results are not pooled due to heterogeneity.


Table 4 Characteristics of Actively Recruiting Registered Clinical Trials Characteristic

Trials (n)

Treatment of aGVHD* Treatment of cGVHDy Prevention of aGVHDz RCT aGVHD treatment cGVHD treatment Prevention of aGVHD MSC source BM Umbilical CB Adipose tissue Amniotic membrane Not reported MSC dose, aGVHD treatment 1 million/kg weekly 4 4 million/kg 1 Not reported MSC dose, cGVHD treatment 1 million/kg, q 2 weeks 4 2 million/kg, 6 doses 4 weeks 1-2 million/kg, monthly 6-9 2.5-4.0 million, mucosa 1 Not reported MSC dose, GVHD prevention 1 million/kg 1 dose GVHD criteria Acute, CR at 28 days Acute, other or not reported Chronic, NIH criteria Chronic, other or not reported

10 6 1 3 3 0 4 2 1 1 9 3 1 6 1 2 1 1 1 1 6 5 3 3

CR indicates complete response; NIH, National Institutes of Health. * Registered trials of aGVHD: NCT01765634, NCT00603330, NCT02241018, NCT01754454, NCT01589549, NCT02032446, JPRN-UMIN000015017, EUCTR2012-004915-30-NL, IRCT2014072618603N1, and EUCTR2007004310-14-BE. y Registered trials of cGVHD: NCT01222039, NCT01765660, NCT00972660, NCT02055625, NCT02291770, and NCT01522716. z Registered trial of prevention of aGVHD: NCT02270307.

DISCUSSION Our systematic scoping review of clinical studies using MSCs for the treatment or prevention of GVHD highlights the marked heterogeneity that complicates interpretation and clinical translation of MSC-based therapy. Although response rates for the prevention and treatment of GVHD appear promising, the paucity of controlled studies and the lack of adherence to standard cell product specifications, including dosage and treatment details, are major issues identified in our review. Moreover, most studies were performed before more widespread adoption of standard GVHD diagnostic and response criteria, making it difficult to understand the full extent and duration of responses. Heterogeneity in patient populations enrolled in the studies and the use of concomitant therapies are additional barriers to identifying the optimal use of MSCs. Ongoing registered trials appear to adopt GVHD response criteria more often but heterogeneity in MSC manufacturing persists. Overcoming ambiguity related to MSC effectiveness in the management of GVHD will require the design of studies that address factors contributing to this marked heterogeneity and could include standardized MSC manufacturing. GVHD remains 1 of the greatest challenges in transplantation care and contributes significantly to the morbidity and mortality of allogeneic transplantation. Many patients have steroid-refractory disease and second-line options remain largely ineffective. Relatively few randomized controlled studies have been performed and no clear option for secondline therapy has emerged. Moreover, improvements in

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strategies to prevent GVHD have largely focused on improved HLA matching [49] and the use of antithymocyte globulin [50,51]. Although MSCs were first used to enhance hematopoietic engraftment, reduced rates of GVHD in the allogeneic setting were observed, leading to the first reports of MSC therapy for aGVHD [52]. Regarding the safety of MSC treatment, a recent metaanalysis of prospective studies summarized toxicity outcomes and adverse events related to MSC treatment for a range of conditions, including Crohn’s disease, stroke, cardiomyopathy, and GVHD [53]. A total of 36 studies were identified (1012 patients), including 8 RCTs (321 patients). There was no increase in infusional toxicity, organ toxicity, infection, death, or malignancy; however, MSC infusions were associated with an increase in transient fever. Taken together, MSC infusion appears safe and well tolerated despite broad heterogeneity in cell product manufacturing conditions. A recently published meta-analysis on the effectiveness of MSCs to prevent GVHD only included published RCTs and reported on 3 studies involving 117 patients [48]. Rates of aGVHD and cGVHD were not reduced with MSCs and rates of relapse and cytomegalovirus reactivation were not different compared with controls, although the authors state that interpretation of their meta-analysis was limited by small numbers of patients. Two recent systematic reviews of MSCs to treat GVHD have been conducted. Hashmi et al. [54] recently identified 18 reports and extracted complete response rates and rates of survival at 6 months in a subset of studies. No direct comparison with other therapies was performed in their analysis; however, no association was observed using a random effects model between rates of survival at 6 months and patient age, type of MSC culture supplement (fetal bovine serum versus human platelet lysate), or the dose of MSCs administered. Overall response rates, however, were increased in patients receiving MSCs cultured using fetal bovine serum and responders had a 4-fold greater overall survival at 6 months (63% versus 16%). The authors conclude that RCTs of MSC versus non-MSC therapy are needed. In another recent meta-analysis [55], controlled clinical studies of MSC cotransplantation at the time of stem cell infusion were identified but many studies were performed with the primary goal of enhancing hematopoietic engraftment and GVHD prevention was reported as a secondary outcome. It is unclear whether these studies were powered to detect differences in rates of GVHD. In their pooled analysis of 9 studies involving 309 patients, there was no significant difference in the incidence of aGVHD or cGVHD between patients who received MSCs and controls. Importantly, 2 RCTs of MSCs have been conducted but remain unpublished regarding the treatment of aGVHD using a cell product manufactured by OSIRIS Therapeutics and were not included in the meta-analysis because 6-month survival data were not available. We also excluded these studies from our analysis as we sought to analyze methodological details that are only available in full publications. A second meta-analysis addressing the use of MSCs to treat aGVHD was recently published [56] and identified 13 published studies (301 patients). Overall, they reported a significant association with complete response rates and greater overall responses in pediatric patients compared with adults, in patients with grade II versus higher grade GVHD, and in patients receiving extended courses of treatment compared with induction treatment only. Heterogeneity in cell product manufacturing

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highlighted in our analysis may explain some of the marked variability in results observed in the individual studies included in these recent meta-analyses. The 2 randomized studies addressing the use of a commercially available MSC product, remestemcel-L (Prochymal; Osiris Therapeutics, Columbine, MD, USA), for steroid-refractory or up-front therapy of aGVHD have not yet been published but have been presented in abstract form [57,58]. There was no significant difference in the primary endpoint of durable complete responses (complete response at 28 days) in either study (35% versus 30%, n ¼ 260 [P > .05] and 45% versus 46%, n ¼ 192 [P > .05], respectively). In subgroup analysis, patients with steroid-refractory aGVHD of the liver, MSC therapy was associated with improved response (76% versus 47%, P ¼ .026, n ¼ 61) and durable complete responses (29% versus 5%, P ¼ .046). Rationalizing the results of the Prochymal (Osiris Therapeutics) studies with other studies of MSC treatment is ongoing [59,60] and the transplantation community awaits the full publication of these important RCTs to provide greater insight regarding specific details of the studies. In addition, future meta-analyses and scoping reviews can be updated with data from these 2 prospective RCTs. Adaptations to MSC production may be relevant for future consideration, including the isolation of extracellular microvesicles derived from MSCs. Given that some biological activities of MSCs may be independent of cell-to-cell contact, investigators have characterized exosome-like microvesicles that are released by MSCs and contain bioactive molecules such as proteins and microRNA that may be active in modulating GVHD, as described in a recent case report [14]. Better defined cell-based product preparations will be appealing from a regulatory perspective. The development of autologous-based products may also be appealing and has been associated with some promise [61]. Our scoping review highlights significant heterogeneity in several areas, including the patient populations that have been enrolled, variations in the diagnostic and response criteria used to evaluate GVHD, and in the product preparation. Future studies, however, can take advantage of recently published criteria for better defining GVHD [62], reporting consistent clinical outcomes [63], and better defining MSC preparations that will yield more informative results. The field appears well positioned to embrace these recently developed clinical trial tools and conduct controlled studies that will help to address the ambiguity surrounding MSCbased interventions for GVHD. We acknowledge that our scoping review has limitations. Although we used broad search criteria for our scoping review, it is possible that some studies were overlooked. It was reassuring to note, however, that studies included in a recent metaanalysis were also identified in our search (and 2 additional studies were also identified). Excluding abstracts from our analysis meant we excluded data from at least 2 large RCTs; however, the extraction of methodological details from full publications was highly instructive and will be most useful in the interpretation of these RCTs when they are finally published. Taken together, our systematic review identifies the need for rigor and consistency between studies of MSCs for the management of GVHD. Although MSCs may be most promising for the treatment of steroid-refractory aGVHD, future studies should focus on the use of well-characterized MSC products that have undergone a low number of cell divisions and use an effective dosage of cells, which remains to be defined, for specific patient populations. Additional controlled trials are needed in clearly defined groups of patients using cell products

that are more potent if MSC-based treatment is to emerge as an effective option in the management of GVHD. ACKNOWLEDGMENTS The authors thank the Ottawa Hospital Foundation for financial support of a summer student stipend (M.R.), the Canadian Blood Services for a Summer Student Internship (M.M.), and the Department of Medicine, University of Ottawa, for support (D.S.A., N.K., C.N.B.). Financial disclosure statement: D.S.A. is the medical director of the OneMatch Stem Cell & Marrow Network and a paid medical consultant for Canadian Blood Services. Conflict of interest statement: There are no conflicts of interest to report. SUPPLEMENTARY DATA Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.bbmt.2016.04.010. REFERENCES 1. Jagasia M, Arora M, Flowers MED, et al. Risk factors for acute GVHD and survival after hematopoietic cell transplantation. Blood. 2012;119: 296-307. 2. Sung AD, Chao NJ. Concise review: acute graft-versus-host disease: immunobiology, prevention, and treatment. Stem Cells Transl Med. 2013;2:25-32. 3. MacMillan ML, Weisdorf DJ, Wagner JE, et al. Response of 443 patients to steroids as primary therapy for acute graft-versus-host disease: comparison of grading systems. Biol Blood Marrow Transplant. 2002;8:387-394. 4. Martin PJ, Rizzo JD, Wingard JR, et al. First and second-line systemic treatment of acute graft-versus-host disease: recommendations of the American Society of Blood and Marrow Transplantation. Biol Blood Marrow Transplant. 2012;18:1150-1163. 5. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143-147. 6. Bartholomew A, Sturgeon C, Siatskas M, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol. 2002;30:42-48. 7. Yagi H, Soto-Gutierrez A, Parekkadan B, et al. Mesenchymal stem cells: mechanisms of immunomodulation and homing. Cell Transplant. 2010; 19:667-679. 8. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune responses. Blood. 2005;105:1815-1822. 9. Augello A, Tasso R, Negrini SM, et al. Bone marrow mesenchymal progenitor cells inhibit lymphocyte proliferation by activation of the programmed death 1 pathway. Eur J Immunol. 2005;35:1482-1490. 10. Akiyama K, Chen C, Wang D, et al. Mesenchymal-stem-cell-induced immunoregulation involves FAS-ligand-/FAS-mediated T cell apoptosis. Cell Stem Cell. 2012;10:544-555. 11. Ren G, Zhao X, Zhang L, et al. Inflammatory cytokine-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in mesenchymal stem cells are critical for immunosuppression. J Immunol. 2010;184:2321-2328. 12. Saldanha-Araujo F, Ferreira FI, Palma PV, et al. Mesenchymal stromal cells up-regulate CD39 and increase adenosine production to suppress activated T-lymphocytes. Stem Cell Res. 2011;7:66-74. 13. Rasmusson I, Ringden O, Sundberg B, Le Blanc K. Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells. Transplantation. 2003;76:1208-1213. 14. Kordelas L, Rebmann V, Ludwig A-K, et al. MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus host disease. Leukemia. 2014;28:970-973. 15. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8:315-317. 16. Moher D, Liberati A, Tetzlaff J, Altman DG. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med. 2009;6:e1000097. 17. Ringden O, Uzunel M, Rasmusson I, et al. Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease. Transplantation. 2006;81:1390-1397. 18. Fang B, Song Y, Liao L, et al. Favorable response to human adipose tissue-derived mesenchymal stem cells in steroid-refractory acute graft-versus-host disease. Transplant Proc. 2007;39:3358-3362. 19. Le Blanc K, Frassoni F, Ball L, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet. 2008;371:1579-1586.


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