Fecal Microbiota Transplantation for Grade IV Steroid Refractory GI-GvHD: Interim Results a Non-randomized, Open-label, Phase 1 Clinical Study

Background: Gastrointestinal (GI) tract graft- vs -host disease (GvHD) is a major cause of post-allo-HCT morbidity and mortality. Patients with steroid-refractory GI-GvHD face a poor prognosis and limited therapeutic options. Here, we report an interim analysis on the safety and efficacy of fecal microbiota transplantation (FMT) in treating steroid-refractory GI-GvHD. Methods: We did a non-randomized, open-label, phase 1 clinical study on patients with grade IV steroid-refractory GI-GvHD. FMT efficacy was evaluated using indexes of abdominal pain, diarrhea and bloody purulent stool at 14 and 21 days after the diagnosis of steroid-refractory GI-GvHD. The primary outcomes referred to clinical complete remission or partial remission. Secondary outcomes referred to EFS (event free survival) and OS (overall survival) at Day 90 and the end of the research. Safety was evaluated according to adverse events during FMT and the whole follow-up period. The study was registered with ClinicalTrials.gov as #NCT03148743. Results: A total of 56 patients with steroid-refractory GI-GvHD were enrolled. Of them, 24 patients with grade IV steroid-refractory GI-GvHD were assigned to FMT and 18 to the control group. The characteristics of the two group patients at baseline were similar. At Day 14 after FMT, 13 (54.2%) patients in FMT group and none (0%) of 18 control group achieved clinical remission (p<0.05), while 20(83%) patients in FMT group and 7(39%) in control group showed effective response (clinical remission+partial remission) (RR 7.86, 95% CI 1.88–32.9; p=0.005). At Day 21, the clinical remission was significantly greater in FMT group than in control group (14 (58.3%) of 24 vs 3(16%) of 18; RR 6.0, 95% CI 1.22–29.45; p=0.027). Within a follow up of 90 days, the FMT group showed better OS (HR, 7.0; 95% CI, 1.53-32.08; p=0.012). At the end of the research, the median survival time was >600 days in FMT group and 107 days in control group (HR, 4.73; 95% CI, 1.58-14.14; p=0.005). Both the EFS (HR, 0.24; 95% CI, 0.06-0.95; p=0.055) and OS (HR, 5.97; 95% CI, 1.52-23.43; p=0.01) kept increasing during the follow-up in FMT group. Overall, the mortality rate was lower in FMT group (HR, 5.97; 95% CI, 1.52-23.43; p=0.01). No difference was observed in the occurrence of other side effects, such as hemorrhagic cystitis, infection of bacteria & fungi, CMV&EBV, septicemia, TMA, cardiac events, thrombocytopenia and epilepsy. Conclusions: The diversity of intestinal microbiota can be affected by allo-HSCT. FMT is effective and safe in treating grade IV steroid refractory GI–GvHD.


Background
As a treatment for most acute leukemia, the hematopoietic stem cell transplantation (HSCT) can offer a potential cure but may have many complications that include infections, multi-organ failure, and graft-versus-host disease (GvHD) [1][2][3]. GvHD, especially gut acute GvHD (GI-aGvHD), is a major cause of post-allo-HCT morbidity and mortality [3,4].
Conventionally, glucocorticoids are used in the first-line therapy for GI-GvHD. Unfortunately, almost half of the patients do not respond efficiently [3,4]. The survival of patients developing GI-GvHD treated with standard steroid is in a range of 5-30% [4][5][6][7]. By far, few second-line treatments have been established and are urgently needed [7].
Human gut microbiota is associated with many chronic disease which is composed of more than 100 trillion microbes [8]. The influence of intestinal microbiota on immune responses, including post-allo-HCT, has been increasingly recognized [9][10][11], and became one of the main targets for the treatment of acute GvHD [3,7]. Under normal physiological condition, the diversity of gut microbiota in human GI tract is associated with intestinal inflammation and immune responses [3,12] that may affect the outcome of GvHD treatment. As the intestinal microbial diversity collapses after allo-HSCT [13,14], the abnormal gut microbiota may damage GI mucosa, and consequently influencing the immune response [3].
The fecal microbiota transplant (FMT), a procedure that use the clinical method of infusing a fecal suspension from a healthy donor into recipient's GI tract, can quickly restore the recipient's intestinal microbiota and repair the intestinal mucosal barrier, that may resolve the inflammatory response and readjust the immune system [15,16]. As a novel therapeutic method, FMT has been proven to be effective for recurrent clostridium difficile infection [17,18].
Our pilot study and other available research suggest that FMT can serve as a therapeutic option in treating steroid-refractory GI-GvHD [3,13,14,19].
Here, we assess the safety and efficacy of the FMT in a phase I study involving patients with steroid-refractory gastrointestinal tract GvHD.

Patient Characteristics
A total of 56 patients with steroid-refractory GI-GvHD were enrolled.
Clostridium difficile infection was not observed among all patients, and were not responders to methylprednisolone (mPSL) at ≥ 2mg/kg per day.
Immunosuppressant as the secondary-line therapy was given to all the patients. Eight patients were excluded, including four reluctant to participate in the study and four failing to meet inclusion criteria (one for primary disease recurrence, two for combined TMA, one for combined CMV before FMT). Of 27 patients in FMT group, the data of three patients with < grade IV GI-GvHD were not selected for statistical analysis. Of 21 patients in the control group, the data of three patients (one with missed follow-up and two with < grade IV GI-GvHD) were not used for statistical analysis (Fig.1).
The patients' information were shown in Table 1 and supplement table1. For 24 FMT patients, the median age was 29 years (range 13 to 55). The male/female ratio was 16/8. The median stool volume was 865 mL/day (range 360 to 2,100 mL/day). The median stool frequency was 6 times/day (range 3 to 21 times/day). The median abdominal pain score was 3 (range 1 to 4) ( Table 1 and Supplement Table1). For the 18

Clinical Outcomes
With Cox regression model, we first declared that immunosuppressant did not affect the outcomes of the two groups (Supplement Table1).
At Day 14(day after steroid-refractory GI-GvHD was diagnosed), 13  At 28 th day, the rate of clinical remission and effective response were higher in FMT group than control group(Supplement Table2).

Safety of FMT
Only one patient experienced thrombocytopenia after FMT and one patient developed cardiac event at day 3 after FMT. Although we were not sure whether there was a possible association between these events and FMT, it cannot be completely exclude. No other severe adverse events were observed in FMT group during seven days of follow-up after FMT. Other common adverse events include incomplete ileus in one patient，fever in one patient, vomiting and low fever in two patients, and grade-3 rash in two patients, no special treatment only symptomatic treatment need for these patients.
In overall results, the mortality rate was low in FMT group (

Fecal Microbiota and Immunity functions Analysis
Given the severity and emergency of steroid-refractory grade IV GI-GvHD, only 10 patients got fecal samples at baseline and weeks 1 after FMT.
Available fecal samples were used for microbiota analyses (N=10). Compared with that of the donors, the diversity of fecal microbiota in fecal samples of the patients was decreased (Fig.4A). Proteobacteria increased while firmicutes decreased at phylum level in the microbiota of the patients (Fig.4B). After Week 1, the microbiota composition was reconstructed in FMT patients, showing a trend back to normal (Fig.4CD). The bacterial diversity improved at Week 1 after FMT in half patients (5/10) (Supplement Table1). Similar to the results of our prior study, the ratio of firmicutes to proteobacteria was restored (7/10), proteobacteria decreased(9/10), and firmicutes increased (6/10) after FMT( Figure S1). Bacteroidetes increased (7/10) in the fecal microbiota of patients with steroid-refractory GI-GvHD (Supplement Table1) after FMT.

Discussion
Gut-GvHD related complications, especially steroid-refractory GI-GvHD, appeared to be one of cause of post-transplantation death [3,4], and the disruption of gut microbiota was linked to GvHD and transplant-related mortality [20,21].
The FMT may re-structured the gut microbiota of a patient which may consequently reinforce the patient's immune system [18]. The FMT had been proven to be very effective for the treatment of recurrent Clostridium difficile infection [17] and other human diseases (inflammatory bowel disease) [3,15,16]. Some case reports and our pilot study suggested that FMT may serve as a therapeutic option for steroid-refractory GI-GvHD [3,13,14,19].  (Table 3). Some papers reported that FMT transmitted drug-resistant E. coli, leading to patient death [22]. No similar events were observed in our study, which may be attributed to our strict FMT criteria.

Conclusion
Although up to date no reports from any phase I clinical trial of FMT treated GI GvHD were found, our study was also limited in some aspects. It was conducted at a single institution, and thus, our findings may not be applied directly to patients at other institutions [20]. Another, given the severity and emergency of steroid-refractory grade IV GI-GvHD, the trial was not randomized and double blind controlled. And also for this, only partial patients got all samples for gut microbiome analysis, we couldn't get the gut microbiome dynamics in all patients, and immunosuppressant was given and its effect could not be completely ruled out in this research. Next, not all patients showed similar responses to FMT, and we did not get enough data to compare responding and non-responding patients, which may be explained by the pathological complexity of steroid-refractory GI-GvHD.
In summary, FMT may be effective and safe in treating GI-GvHD, which should be verified with more studies.

Study design and participants
We undertook an open-label, non-randomized, phase 1 clinical study at the Research (CIBMTR) criteria were used to assess the grades of GI GvHD [3,23,24]. Criteria for diagnosing steroid-refractory gut GvHD had been described previously [3]. We excluded the patients with uncontrollable infection, irreversible organ failure, and other abnormal conditions that might interfere with the evaluation(Supplement Protocol).

Procedures
The fecal materials were handled in sealed, fully automatic machines GenFMTer (Nanjing, China). The fecal microbiota collected from four healthy donors (two females aged 23 years, and two males aged 20 years) were conserved in -80℃ with glycerine(Supplement protocol). As these patients couldn't tolerate gastroscopy or enteroscopy, forty to fifty ml of frozen fecal microbiota were suspended in 150-200 ml of warm normal saline and delivered into the intestine of the recipients through a nasojejunal tube or gastric tube after steroid-refractory GI-GvHD was diagnosed [3]. If not got improvement, FMT would be repeated in the following week.

Outcomes
The primary outcomes were described with clinical remission or partial remission at Day 14th, Day 21th and Day 28th after steroid-refractory GI-GvHD was diagnosed.
Secondary outcomes were: EFS and OS at Day 90 after steroid-refractory GI-GvHD was diagnosed; EFS and OS after steroid-refractory GI-GvHD was recorded till the end of November1 2018 FMT efficacy were evaluated according to the severity of symptoms such as abdominal pain, diarrhea (frequency and volume), and bloody purulent stool within 14 and 21 days after FMT was accomplished. For abdominal pain score, 0.5 was given to occasional pain, 1 to mild pain, 2 to moderate pain, 3 to severe pain without intervention, and 4 to severe pain with intervention.
Clinical remission was defined as a condition in which diarrhea and intestinal spasms and/or bleeding disappeared, or stool volume decreased by ≥500mL on average within 3 days. Clinical improvement was defined as a condition in which the stool volume decreased by <500mL, or the abdominal pain value and bleeding relieved. The period during follow-up after first FMT with no progress of GI-GvHD, no death, no GvHD involvement in other organs, no new infection with CMV and EBV were defined as event free survival time (EFS) [3].
OS (overall survival) referred to the period from when steroid-refractory GI-GvHD was diagnosed to November1 2018. All deaths, including relapse related or other causes in these period, were included in the statistics.
For each patient, the safety was evaluated according to adverse events (including death or drop-out) during FMT and follow-up time. and R software to analyze the 16S sequences data. Composition of fecal bacteria was analyzed at phylum level. Moreover, the Shannon diversity index was used to depict the diversity of microbiota(Supplement Methods) [3,25].

Statistical Analysis
SPSS16.0 (SPSS, Inc., Chicago, IL, USA) for statistical analyses was used to construct actuarial rate curves and to calculate log-rank hazard ratios (HRs) and significance determinations, Fisher's exact tests, and risk determinations.
Cochran's and mantel-haenszel statistical methods examined the differences between the groups. The survival package from "R" statistical software (Vienna, Austria) was used for permutation tests. For the latter, variables were determined for each day of the patient's hospitalization and HRs, confidence intervals, and significance determinations were calculated using Cox proportional hazards models with time varying covariates.

Ethics approval and consent to participate
Protocols and other trial related procedures were approved by the Institutional Review Board of the Hospital. All the patients signed written informed consent.

Availability of data and material
Please contact author for data requests

Competing interests
The authors declare that they have no competing interests.

Funding
This study was supported in part by grants from of National Key R&D Program