Systematic review with meta-analysis: the efficacy and safety of stem cell therapy for Crohn’s disease

Stem cell therapy (SCT) for the treatment of Crohn’s disease (CD) is still in its infancy, and whether SCT is associated with improved outcomes is unclear. We performed a meta-analysis to evaluate the efficacy and safety of patients receiving SCT. Electronic databases were searched for studies that reported the use of stem cells for the treatment of patients with CD. Raw data from included studies were pooled for effect estimates. Subgroup analyses were performed for exploration of heterogeneity regarding all outcomes. We analyzed 21 studies comprising 514 patients with active CD. A random-effects meta-analysis of studies of SCT as systemic infusion showed 56% (95% confidence interval (CI) 33–76, n = 150) of patients achieved clinical response. Similarly, random-effects pooled rates of clinical or endoscopic remission were 46% (95% CI 25–69, n = 116) and 15% (95% CI 0–50, n = 48), respectively. A random-effects meta-analysis of all perianal CD studies showed that 57% (95% CI 44–69%, n = 251) of patients had healed fistula with SCT, with an odds ratio of 3.83 (95% CI 1.06–13.86, n = 121, P = 0.04) versus control. The pooled rate of clinical recurrence was high at 16% (95% CI 4–34, n = 101) with follow-up >12 months. The pooled rates of severe adverse events (SAEs) and SAEs related to SCT were 12% (95% CI 6–23, n = 378) and 8% (95% CI 3–18, n = 378), respectively. The Egger test suggests no publication bias existed for fistula healing (P = 0.36), but did for clinical response (P = 0.003). SCT seems potentially effective and may serve as an alternative treatment for refractory active CD. Toxicity will remain the most significant barrier to systemic SCT in patients with CD.


Background
Crohn's disease (CD) is a chronic relapsing inflammatory condition of the gastrointestinal tract that can result in lifelong ill health [1]. Immunosuppressive drugs, including biologicals, are the standard of care for CD, but some patients do not respond or lose response to treatment [2,3]. Therefore, alternative treatments for refractory CD remain an unmet need.
Hematopoietic stem cell transplantation (HSCT) might play a role in some of these treatment-resistant cases [4]. Previous studies have indicated that allogeneic HSCT may reset the immune system at a genetic level [5,6], and autologous HSCT eliminates aberrant clones by immunoablation and replacement with uncommitted stem cells (SCs), leading to de novo generation of an altered T-cell repertoire [7]. Case reports and series describe long-term treatment-free disease regression with autologous [8][9][10] and allogeneic [5,6] HSCT in some [6,11,12] but not all patients [8][9][10] with CD. However, the recent Autologous Stem Cell Transplantation International Crohn Disease (ASTIC) trial [13] failed to demonstrate a statistically significant improvement in sustained disease remission at 1-year of autologous HSCT compared with conventional therapy. These findings raise doubt about HSCT for patients with refractory CD.
Perianal fistulas are very common complications, appearing in 25% of CD patients. Of these, approximately half are complex. The only approved drug that has shown efficacy in a randomized clinical trial is infliximab [14,15]. Few treatment options exist for drug treatment-refractory patients, and repeated surgical options are associated with a significant risk of permanent stoma. These findings emphasize the need for novel treatment options for treatment-refractory complex perianal fistulas in patients with CD. Initial clinical results [16,17] suggested mesenchymal stem cells (MSCs) might have therapeutic potential in this setting.
Given the immunoregulatory potential of SCs, multiple studies have been conducted to assess the safety and efficacy of stem cell therapy (SCT) in CD. In this study, we perform a meta-analysis of feasibility and toxicity of SCT for the treatment of CD.

Methods
The study was performed following the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines [18].

Literature search
We identified relevant literature (published articles and abstracts) by performing a systematic search of three databases: PubMed, Cochrane Library CENTRAL, and Embase (initial search February 5, 2015;updated October 15, 2016). Keywords used were (all fields): ("inflammatory bowel disease" or "crohn's disease" or "crohn disease" or "enteritis") and ("stem cell*" OR "precursor cell*" OR "progenitor cell*" OR "Stromal cell*"), and any appropriate abbreviations. For PubMed, all relevant MeSH terms were used. The final queries were validated by manual review and matching results.
The conference proceeding abstracts for annual meetings of European (European Crohn's and Colitis Organisation congress) and American (Digestive Disease Week) Congresses were searched between 2002 and 2016.

Study selection
We finally performed a manual selection of studies which satisfied the following criteria: (a) observational (prospective or post hoc analysis of prospectively obtained cohort) or interventional design (randomized or non-randomized); (b) established diagnosis of CD by accepted criteria not in clinical or endoscopic remission at study outset; and (c) clear definition of efficacy and adverse events (AEs). Studies with exclusively pediatric patients (<15 years), a diagnosis of ulcerative colitis, indeterminate colitis, or an unclear diagnosis of CD and case reports were also excluded.

Data extraction and quality assessment
Eligible articles were reviewed in a blind manner by two different investigators (YQ and MYL), and the results of the primary research studies were abstracted onto specially designed data extraction forms. Disagreement in data extraction was resolved by consensus with MHC.
Assessment of the quality of randomized controlled trials (RCTs) and observational studies was performed using the Cochrane risk of bias tool [19] and Newcastle Ottawa Quality Assessment Scale (NOS) [20], respectively. For the NOS, studies scoring >7 (of 9) were considered high quality.

Outcomes assessed
The primary endpoint was clinical efficacy (e.g., clinical response or remission, fistula healing as defined by the primary study) of SCs for the treatment of patients with CD. Secondary outcomes were safety (e.g., AEs, severe AEs (SAEs)) and other measures of disease activity (endoscopic remission, clinical recurrence) of SCT for CD.

Data synthesis and analysis
We calculated incidence estimates with the variancestabilizing double arcsine transformation [21] because the inverse variance weight in fixed-effects meta-analyses is suboptimum when dealing with binary data with low incidences. Additionally, the transformed incidences are weighted very slightly towards 50%, and studies with incidences of zero can thus be included in the analysis. We used the Wilson method [22] to calculate 95% confidence intervals (CIs) around these estimates because the asymptotic method produces intervals which can extend below zero. Heterogeneity due to study variation was assessed using the χ 2 test [23,24], with a P value of <0.10 being considered as statistically significant.

Publication bias
Funnel-plot asymmetry as proposed by Egger et al. [25] was used to investigate the possibility of publication bias.
The meta-analysis was performed using the metaprop command of the meta package in R (version 3.2.0) [26] and Stata (version 12.1) with the commands metareg (for metaregression). All statistical tests were two-sided, and statistical significance was defined as a P value <0.05.

Outcomes
The initial search strategy yielded 4828 abstracts for review, of which 108 were selected for detailed review. Seventy-one studies were excluded for being non-human or safety studies or studies with exclusively pediatric patients, or for having a diagnosis of UC or indeterminate colitis or mixed irritable bowel syndrome for which the CD data could not be separated. Sixteen studies were further excluded because they were case studies or duplicate studies ( Fig. 1; Additional file 1: Table S1).

Efficacy of SCT as a systemic infusion for CD Clinical response
Eleven studies [16, 27, 29-31, 33-36, 40, 41] reported raw data on clinical response of patients with SCT. Rates of clinical response ranged from 10 to 100%. The random-effects pooled rate of clinical response was 56% (95% CI 33-76, n = 150); heterogeneity was pronounced (χ 2 = 1.88, P < 0.001; I 2 = 78.6%; Fig. 2a). Subgroup analyses were performed to assess whether the source of the stem cells, i.e., HSCT or MSCT, and the method of SC infusion had substantial effects on the efficacy of SCs as well as decreased the high heterogeneity.

Publication bias and heterogeneity
A funnel plot generated for the primary outcome suggested no publication bias (Fig. 6), as did an Egger test for fistula healing (t = −0.97, P = 0.36), but not for clinical response (t = −4.91, P = 0.003).

Discussion
Our meta-analysis suggests that SCT has good therapeutic potential with relatively low risk of AEs for patients with CD, particularly for those who have perianal disease and are treated with local therapy.
MSCs have regenerative and immunomodulatory properties which lead to reduction of inflammation and healing of affected intestinal tissue. A previous metaanalysis showed that 40.5% (95% CI 7.5-78.5) of patients  [44] compared to 23% (95% CI 7-54) in our study. Autologous and allogeneic MSC administration are both being evaluated; their unique cell surface HLA characteristics allow for therapy with unrelated donor cells without increasing the risk of rejection by the host. According to our study, the pooled rate of clinical remission was 32% (95% CI 11-62) for patients who underwent allogeneic MSCT compared to 61% (95% CI 25-88) for patients who underwent autologous SCT (Table 3). However, patients who underwent allogeneic MSCT had a relatively higher pooled rate of clinical response compared with patients who underwent autologous SCT (86 versus 31%). Whether allogeneic MSCT has a superior therapeutic efficacy for CD warrants further study.
When considering HSCT, the question remains whether its risk to benefit ratio justifies it. Observations of case reports published to date suggest that sustained clinical remission with HSCT is initially likely to result from lymph ablation by drugs used in the conditioning regimen; altered immune reconstitution may be a later effect. Temporarily disrupting the immunological memory and ceasing the chronic inflammatory burden using non-ablative HSCT has been shown to be effective in non-controlled study designs [9]. However, the recent ASTIC trial [13] failed to demonstrate a statistically significant improvement in sustained disease remission at 1-year of HSCT. According to our study, the pooled rate of clinical remission was 73% (95% CI 36-93) for patients who underwent autologous HSCT, which was higher than 23% (95% CI 7-54) for patients who received ASC. Based on these trial findings, further study of HSCT in patients with refractory CD may be warranted.
When considering fistulizing CD, the existing pharmacological treatments for complex perianal fistulas have low efficacy in inducing fistula healing (antibiotics 21-48%; thiopurines 20-40%; anti-TNFs 23% complete responders) [45]. In our study, the random-effects metaanalysis of all perianal CD studies showed that 57% (95% CI 44-69) of patients had healed fistula after local MSC injection, which is superior to all the above-mentioned treatments. The pooled OR for achieving fistula healing in patients who received SC injection versus control was 3.83 (95% CI 1.06-13.86, P = 0.04).
Whether SCT is associated with improved long-term outcomes for CD patients is unclear. Burt et al. [9] reported that the percentage of clinical relapse-free survival after autologous nonmyeloablative HSCT was 91% at 1 year, 57% at 3 years, and 19% at 5 years. The percentage of patients in remission, steroid-free, or medication-free at any post-transplantation evaluation interval more than 5 years after HSCT remained at or greater than 70, 80, and 60%, respectively. Ciccocioppo et al. [31] reported the efficacy of MSCs for refractory complex CD fistulae declined over time and fistula relapse-free survival was 50% at 2 years and 37% at 4 years. Cho et al. [38] reported that 83.3% (20/24) of patients who received MSCs maintained complete fistula closure at 2 years. In the present meta-analysis, the pooled rate of clinical recurrence was 16% (95% CI 4-34) with a follow-up >12 months. Studies assessing the long-term safety and efficacy of systemic MSC therapy for luminal CD are lacking. Further studies that investigate the impact of periodic MSC administration on long-term outcomes are necessary to establish SCT as maintenance therapy.
The safety of SCT remains a major concern that must be addressed before it can be officially approved for use for CD. During the first 12-month follow-up, viral infections were the most commonly observed complications  [46]. This is further confirmed by the ASTIC trial [13], with most importantly proven or presumed infections associated with the pancytopenia induced by the conditioning regimen. In contrast, Kniazev et al. [47] revealed no differences in the development of acute post-transfusion reactions, infectious complications, exacerbations of chronic inflammatory diseases, severe infectious complications, malignant transformation, and fatal cases in patients who received MSC therapy during 5-year follow-up. In our analysis, the pooled rate of SAEs and SAEs related to SCT were 12% (95% CI 6-23) and 8% (95% CI 3-18), respectively. When subgrouped by source of SCs, patients who received HSCs had a pooled rate of SAEs of 35% (95% CI 5-86), which is higher than with either BMSC (10%, 95% CI 2-42) or ASC (12%, 95% CI 7-18) infusion. MSCT does not require preparatory regimens involving high-dose chemotherapy and/or radiation; HSCT is thus associated with less procedure-related mortality. Overall, autologous HSCT for patients with refractory CD is feasible, but extraordinary supportive measures need to be implemented.
This meta-analysis also has limitations. First, assessment of the methodological quality determined that there were deficiencies in all the studies evaluated. Although only six RCTs [13,17,27,37,39,42] met the inclusion criteria, the majority of studies included are phase I/IIa clinical trials. Second, clinical response exhibited statistical heterogeneity, which likely reflects the variability of definitions for clinical response, study design, sources of SCs, and time to clinical or endoscopic assessment. We used a random effects model to conservatively account for the clinical and statistical heterogeneity in the pooled studies. We also performed subgroup analyses to examine differences in the overall effect estimate.

Conclusions
So far, not enough studies have so far been performed to have a clear view about the use of SCT for CD; up to now the findings are encouraging but not conclusive. SCT seems potentially effective for refractory CD and has high efficacy in inducing fistula healing. Based on subgroup analysis, systemic allogeneic BMSC transfusion had relatively higher rates of inducing clinical response, while autologous ASC local injection had relatively higher rates of inducing fistula closure. Studies are needed to standardize MSC injection in often complex fistulas with multiple tracts to determine optimal dosing and source. Systemic infusion of SCs is not yet ready for the clinic and faces multiple challenges. Toxicity will remain the most significant barrier to HSCT in patients with CD.

Additional files
Additional file 1: Table S1. Characteristics of excluded studies. (DOCX 35 kb) Additional file 2: Figure S1. Risk of bias within studies assessed by Cochrane risk of bias assessment tool. (TIF 1067 kb) Additional file 3: Table S2. Assessment of quality of observational studies using Newcastle Ottawa Quality Assessment Scale (NOS) (DOCX 14 kb)