Safety and efficacy of fecal microbiota transplantation in the treatment of graft-versus-host disease

This article evaluates the efficacy and safety of FMT in the treatment of GVHD after HSCT using a systematic literature search to conduct a meta-analysis constructed of studies involving GVHD patients treated with FMT. 23 studies were included, among which 2 prospective cohort studies, 10 prospective single arm studies, 2 retrospective single arm studies, 2 case series and 7 case reports, comprise a total of 242 patients with steroid-resistant or steroid-dependent GVHD secondary to HSCT who were treated with FMT. 100 cases achieved complete responses, while 61 cases showed partial responses, and 81 cases presented no effect after FMT treatment. The estimate of clinical remission odds ratio was 5.51 (95% CI 1.49–20.35) in cohort studies, and the pooled clinical remission rate is 64% (51–77%) in prospective single arm studies and 81% (62–95%) in retrospective studies, case series and case reports. Five (2.1%) patients had FMT-related infection events, but all recovered after treatment. Other adverse effects were mild and acceptable. Microbiota diversity and composition, donor type, and other related issues were also analyzed. The data proves that FMT is a promising treatment modality of GVHD, but further validation of its safety and efficacy is still needed with prospective control studies. Clinical trial registration: Registered in https://www.crd.york.ac.uk/PROSPERO/CRD42022296288


INTRODUCTION
Hematopoietic stem cell transplantation (HSCT) is a curative procedure for a variety of hematological malignancies, immune deficiencies and other diseases. One of the major complications of allogeneic HSCT (allo-HSCT), acute graft-versus-host disease (aGVHD), is significantly associated with the poor clinical outcome owing to its high morbidity and mortality [1,2]. Amongst all allogeneic hematopoietic cell transplant patients, 30-50% develop aGVHD (grade I-IV) and 14% experience severe aGVHD (grade III-IV) [3,4]. Among unrelated donor allo-HSCT, mortality related to GVHD accounts for 11-16% of deaths for adults and 1-7% for children under 18 [5]. The long-term death rate of steroid-resistant GVHD is approaching 90% [6]. Since GVHD remains a major cause of mortality in patients undergoing HSCT, prevention and treatment of GVHD is of great significance.
The human gastrointestinal (GI) tract contains over 1000 bacterial species, 100-fold more genes than the human genome [7], and gut microbiota population plays an important role in human health [8][9][10]. The human gut is also rich in immune cells, making it the largest immune organ in the human body (gutassociated lymphoid tissue-GALT) and, consequently, one of the major target organs of the aGVHD. As a result, the GI tract is involved in virtually all fatal cases of aGVHD [11]. Increasing evidence points out that the gut microbiota plays a critical role in the modulation of immune response. Post-HSCT patients, especially those who later develop aGVHD, usually present with dysbiosis and a reduction of stool bacterial diversity [12,13], with an enrichment of pathobionts (e.g., enterococci), reduction in some specific bacteria as Blautia spp., as well as other members of the Clostridia class [14]. Decreased abundance of commensal bacteria belonging to the Blautia genus is associated with higher aGVHD incidence and poor overall survival (OS) [15]. Patients with a reduction in stool bacterial diversity are more likely to undergo GVHD and GVHD-related mortality [16][17][18][19][20][21]. Concomitantly, GVHD exacerbates dysbiosis, disrupting gut barrier, diminishing the antimicrobial response against pathogenic bacteria, ultimately increasing the risk of gut microbiota translocation into the bloodstream [17,22,23]. It was shown recently, that lower gut microbiota diversity correlates with the incidence of treatmentrelated mortality, GVHD and reduces OS [24].
Regarding that steroid-refractoriness leads to the fatal outcomes of GI aGVHD and due to increasing role of microbiota pathogenesis, strategies targeting microbiota offer a promising avenue for GVHD prevention and treatment. Fecal microbiota transplantation (FMT), an innovative strategy to modify gut microbiota, has been drawing worldwide attention in recent years. FMT refers to the transfer of fecal microbiota from healthy donors into the GI tract of patients through oral capsule, nasogastric/duodenal tube, colonoscopy, or enema etc., to restore gut microbiota homeostasis in the recipients [25]. FMT is strongly recommended for the treatment of recurrent Clostridioides (formerly Clostridium) difficile infection [26], while recent evidence suggest that FMT could be a promising intervention for other conditions, including inflammatory bowel disease and metabolic syndrome [27].
Studies have shown that FMT could improve the clinical outcome of patients undergoing HSCT [28]. It was suggested that FMT reduced the incidence of adverse events after HSCT [29] and decolonized GI tract from antibiotic-resistant bacteria [30][31][32]. Consequently, Chinese Society of Hematology approved FMT as one of the possible treatments for steroid-resistant aGVHD in their consensus published in 2020 [4]. In general, first description of FMT to treat aGVHD showed very encouraging results, but whether it could be widely adopted in clinical practice remains unknown. The aim of this systematic review and meta-analysis is to evaluate the efficacy and safety of FMT in the treatment of GVHD after allo-HSCT.

Literature search strategy
A systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [33]. The search was conducted in duplicate in PubMed, EMBASE, Web of Science and the Cochrane Library. Search restrictions including human studies only, published from the construction of the databases to May 1st of 2022 in English only. Citations of the reviews and meta-analyses were also checked to identify possible additional studies. The detailed search strategy is outlined in Supplementary Table 1.

Inclusion and exclusion criteria
Inclusion criteria. (1) Randomized controlled trials, cohort studies, casecontrol studies, case series and case reports; (2) published in English; (3) with the target population suffering from acute or chronic GVHD after allo-HSCT; (4) undergoing FMT; (5) reporting any of the following: the response rates, remission and adverse events.

Exclusion criteria.
(1) Reviews, animal experiments, basic experiments and repeatedly published literatures; (2) Lack of information to be extracted.
Data extraction. Data extraction from eligible articles was performed by two investigators independently using Endnote 2.0 software for deduplication of the literature. The following clinical information was extracted from each study: the first author, year of publication, type of disease, number of patients, route of administration, donor identity, follow-up days, outcomes, and adverse events.
Quality assessment. Quality assessment was carried out by two authors independently and disagreements were resolved by the discussion, with the senior author (GY) involvement. The qualities of cohort studies were evaluated using Newcastle-Ottawa Scale (NOS) [34] on the following criteria: representativeness of the GVHD after HSCT cohort, ascertainment of FMT, demonstration that outcome of interest was not present at the start of the study, assessment of the outcome, the length (at least 3 months) and adequacy of follow-up. IHE Quality Appraisal Checklist for Case Series Studies [35] was used to assess the quality of case series and studies without control groups. Studies with 12 or more yes responses were considered to be of acceptable quality.
Statistical analysis. Studies with and without control groups were pooled in meta-analysis using the Review Manager 5.3 software and R 4.0.3 respectively [36,37]. The rates of clinical response were estimated using a random effects model with 95% confidence interval (CI). Mantel-Haenszal method was used in cohort studies and the R package 'metaprop' was adopted in analyzing single arm studies. The presence of heterogeneity was assessed using Q statistic (Chi-square test) and the I 2 statistic was used to assess the degree of heterogeneity between the trials. Funnel plots were employed to measure the potential publication bias. Sensitivity analyses were conducted to identify which study had the most impact on I 2 and P for heterogeneity.

Risk of bias
Methodological quality of all studies is shown in Supplementary Tables 2-5. Two prospective studies with control group reached 8 points each, by NOS. Other publications (except case reports) obtained 12 or more yes responses by IHE Quality Appraisal Checklist for Case Series Studies. Also, the conference report presented by Malard et al. [48] describing one Phase 2 clinical trial and one compassionate use program (Early Access Program) passed the quality assessment after our comprehensive investigation of their other related articles [48,[61][62][63].

Patients characteristics and FMT outcomes
Among all 23 studies included in the meta-analysis, a total of 242 patients with GVHD after allo-HSCT were treated with FMT (Table 1). Most had grade III or IV aGVHD, while 26 patients served as controls in 2 studies. Among all patients who received FMT, 161 (66.5%) responded, with 100 complete responses (CR), 61 partial responses (PR) and 81 no response (NR). In the study of Goloshchapov et al. [60], among the 8 patients in the control group (receiving normal saline or oral placebo capsule), 1 had CR, 4 had PR and 3 had NR and died, while in the 19 patients of FMT group, 9 reached CR, 9 reached PR and 1 patient died. In the second clinical study with control group [39], 23 and 18 patients were assigned to the FMT and the control group (without FMT) respectively. Twenty-eight days after FMT treatment, 20 patients (87.0%) in the FMT group and 11 patients (61.1%) in the control group showed an effective response. Next-generation FMT product "MaaT013", a standardized, pooled donor, high-richness microbiota biotherapeutic, was reported by Malard et al. [48] to treat patients with intestinal aGVHD in 2020. MaaT013 was investigated in a Phase 2 clinical trial (HERACLES, NCT03359980) and is being administered in an early access program (EAP) in France [48]. During EAP, 52 patients with steroid-dependent or steroid-resistant, Grade II-IV, GI aGVHD were treated with MaaT013, and 30 out of 52 patients (58%) showed objective responses of which 17 patients (32.7%) had CR, 9 patients (17.3%) had very good PR, and 4 patients (7.7%) showed PR [48]. OS at the 12-month follow-up was 38%, with 59% in responders and 7% in non-responders [48].

FMT dosage, form, donors and route of administration
The route of administration of FMT were heterogenous, with 34 recipients obtaining FMT through oral capsules, 9 receiving fresh or frozen fecal microbiota solution through colonoscopy, 89 through nasoduodenal tube or/and gastroduodenoscopy and 76 using "MaaT013" through nasogastric tube or enema. The response rate of upper gut (nasoduodenal tube or gastroduodenoscopy) was better (79.22%) while other three routes were similar (Table 2). However, Goeser et al. [54] mentioned that based on data of refractory CDI, delivery of FMT by capsules shows less discomfort, higher safety but comparable efficacy compared to invasive methods.
A total of 21 (8.7%) recipients had a relative as the fecal donor, while others received fecal samples from unrelated donors.
Concerning the times of FMTs performed per patient, most cases ranged from 1 to 3, however maximum reached up to 10. The volume of fecal solutions transplanted varied, with the most common 200-250 mL of fecal suspension per dose, the concentrations are not the same, leading to no comparability among studies.

Meta-analysis
The meta-analysis outcomes of two prospective studies with control groups are shown in Fig. 2, and the results of prospective single arm studies are shown in Fig. 3, meanwhile, Fig. 4 shows the result after combining the retrospective studies, case series and case reports. The funnel plots ( Supplementary Figs. 1, 2) show moderate publication bias. The estimate of the objective response odds ratio (OR) was 5.51 (95% CI 1. 49-20.35) in the two-arm studies, with low heterogeneity (Chi 2 = 0.40, df = 1, P = 0.53; I 2 = 0%). The pooled clinical remission (complete and partial response) rate of GVHD after FMT was 64% (51-77%) in the 10 prospective single arm studies, and 81% (62-95%) in retrospective studies (2 studies), case series (2 studies) and case reports (7 studies). A sensitivity analysis was performed to investigate the impact of each study on the response rate. The forest plots of prospective and retrospective studies show that for every study, there was no significant difference compared with the original results ( Supplementary Figs. 3, 4).

Adverse events
Adverse events reported including infections, nausea, vomiting, abdominal pain, diarrhea, constipation, abdominal distension, fever, anorexia, general malaise, as well as sore throat and GI bleeding due to invasive procedures. Infections occurred with the higher incidence as compared to FMT performed to treat C. difficile infections [64]. Even though most of the infections reported in the GVHD patients were considered as not related to FMT, there had been 5 recipients undergone severe infections related to FMT, with 2 cases of sepsis, 1 septic shock, 1 norovirus infection and 1 adenovirus infection. All 5 patients recovered by prompt treatment. A total of 90 deaths had been reported but none relates to FMT.

DISCUSSION
The first study applying FMT to the treatment of patients with GVHD was conducted by Kakihana et al. [49] in 2016. A total of 4 patients with steroid-resistant or steroid-dependent gut aGVHD were included, resulting in 3 CR and 1 PR, with no severe adverse event attributed to FMT, proving FMT as a potential treatment option for aGVHD. According to our meta-analysis, the overall remission (complete and partial remission) rate of GVHD after FMT was 64% (51-77%) in the 10 prospective single arm studies, and the OR was 5.51 (95% CI 1. 49-20.35) in the 2 two-arm studies. As for the retrospective studies (2 studies), case series (2 studies) and case reports (7 studies), the pooled overall remission rate was 81% (62-95%).
As patients routinely receive systemic immunosuppressive therapies for at least 6 months after HSCT, most FMT recipients being treated for aGVHD are immunosuppressed, leading to an increased risk of gut microbiota translocation and infection. Therefore, the safety of FMT in treating GVHD had to be evaluated. In our study, only 5 patients went through serious FMT-related  infections, which were stated in the Adverse events part before. Other severe infections were not related to FMT. Other adverse events, including nausea, vomiting, abdominal pain, diarrhea, abdominal distension, fever, were mild and transient. Factors affecting the efficacy and safety of FMT include donor selection, material preparation, route of administration, dosage, frequency of administration, and the status of gut preparation [65][66][67][68]. The gut barrier might have been severely damaged during late periods of GVHD, which increases the risk of gut microbiota translocation after FMT [58]. Therefore, performing FMT earlier might increase safety and efficacy of the procedure [58]. It was shown that the donor selection, relative or unrelated      one, has no impact on FMT efficacy and safety, and stool banks even provides more thorough safety procedures [69,70]. Potentially, an 'ideal' microbiota donor should be the same as the hematopoietic stem cell donor, which could result in a faster and easier development of immunological tolerance within the recipient's immune system, potentially reducing the occurrence of GVHD. It should be confirmed that the donor does not have dysbiosis, which, however, would complicate the process of donor screening.
The fecal microbiota could be instilled into the recipient's GI tract through the upper gut, mid-gut or lower gut, including oral capsule, nasogastric/duodenal tube, gastroscopy, GI stoma, colonoscopy and enema [71]. In our study, using nasoduodenal, nasojejunal or nasogastric tube to transplant fresh or frozen fecal microbiota seems more effective, but there is a clear overrepresentation of this route. No significant difference on the efficacy was shown among other routes of microbiota transplantation or whether the sample is frozen or not. Previous reports on remission rates for different transplant routes are controversial. Quraishi et al. [72] and Zhao et al. [73] found lower GI deliveries of FMT were more effective than upper GI but Fehily et al. [74] got the opposite result. Most metaanalyses recently show no difference between fresh and frozen FMT [72][73][74][75][76][77]. The FMT delivery method must also be chosen upon the willingness and health condition of the recipients, as well as the technical conditions and experiences of the treating person.
Recent studies have shown that multiple gut microbiota infusions could be more beneficial [7]. Most patients included in this study received multiple FMTs, with GVHD partially or completely remitted. A case study in our center showed that combining FMT with ruxolitinib, the best documented second-line therapy applied in SR-aGvHD [78,79], could potentiate the response and result in sustained remissions [58]. The initial experience suggested the synergistic effect of FMT with other immunomodulatory drugs and show potentially better results.
The mechanism of FMT on treating GVHD remains unclear. One possible mechanism is that FMT leads to positive alterations in dominant gut microbiota species forcing new immune interactions between the microbiota and the lymphatic system. Beneficial bacteria such as Bacteroides, Lactobacillus, Bifidobacterium, and Faecalibacterium were observed dominant after FMT mitigating GVHD, meanwhile bacteria strongly correlated with GVHD (e.g., Enterococci, Proteobacteriaceae) remained dominant at the recurrence of aGVHD [49], suggesting that increasing the number of beneficial bacteria, inhibiting the proliferation of pathogenic bacteria, enriching the diversity of intestinal flora is most important [24]. Healthy gut microbiota may provide regeneration signals for damaged intestinal epithelium and repair intestinal mucosa via production of metabolites such as butyrate, etc [43]. They can also produce metabolites with anti-inflammatory effects, such as short-chain fatty acids, resulting in an increase on the expression of IL-10 and IL-17 through inhibiting histone deacetylase and NF-κB signaling pathway, thus alleviating inflammation and the symptoms of GVHD [22,80]. FMT may lead to an antiinflammatory change in the immune environment. An increase in regulatory T cells and type 3 innate lymphoid cells, along with a drop of relative numbers of CD8 + T cells and Th17 cells was observed [44,49], indicating that FMT may shift the systemic allogeneic immune response to an anti-inflammatory state while maintaining the immune tolerance to symbiotic bacteria. With further studies, the mechanisms of FMT on treating diseases related to GI tract are to be clarified.
There are some limitations of our work. Firstly, this review is lack of studies with highest-level quality, more large-scale randomized controlled trials (RCTs), cohort studies and case-control studies are needed. Secondly, all studies in the review are small sample sized (≤52 participants). Furthermore, heterogeneity among studies regarding patients' baseline characteristics, concomitant treatment, volume and routes of FMT forced us to do mostly pooled analyses and descriptive statistics. Last, the lack of some critical information, including potential cooperation or antagonism between FMT and other treatment, also leads to insufficient assessment of the efficacy and safety of FMT.
In this systematic review, we can draw a conclusion that FMT has positive therapeutic effect on treating GVHD after allo-HSCT. FMT could be currently considered as a safe and promising treatment modality of GVHD. The time of transplantation, route, frequency and dose of FMT need further exploration and standardization. The mechanism of FMT acting in GVHD is related to an increase of the gut microbiota's diversity, but details remain unclear and need to be further clarified. There are a few ongoing trials studying the effectiveness of FMT in the treatment of aGVHD (NCT04269850, NCT03819803, NCT03812705, NCT04285424, NCT03359980) whose results should bring us more reliable data.

DATA AVAILABILITY
All processed data are available.  Fig. 4 Forest plot of the combination of retrospective studies (2), case series (2) and case reports (7) describing the effect of fecal microbiota transplantation to treat graft-versus-host disease. CI confidence interval.