Patient Characteristics
From February 2016 to September 2017, 154 UC patients were assessed for eligibility, and ultimately 15 subjects were recruited and randomized. Of these, 7 individuals were randomly assigned to the FMT and 8 to the placebo arm. Three subjects (1 in the FMT and 2 in the placebo group) did not meet endoscopic criteria for inclusion (Mayo score ≥1) and were excluded from the study (Fig. 1). The remaining 12 subjects (6 in each group) received at least one dose of study treatment. While all 6 subjects allocated to the FMT arm completed all treatments and follow-up assessments, 1 patient in the placebo group dropped out at 6 weeks due to worsening disease. The two study groups were exhibited comparable baseline demographic and clinical characteristics (Table 1).
Table 1. Baseline Subject Demographics
|
|
Group
|
Variable
|
|
Active
|
Placebo
|
N
|
|
6
|
6
|
Age
|
Mean(SD)
|
41 (15)
|
52 (15)
|
Sex
|
#(%) Male
|
4 (67%)
|
3 (50%)
|
Duration UC
|
Mean(SD) yrs
|
8.9 (9.1)
|
9.8 (10.6)
|
BMI*
|
Mean(SD)
|
24 (3)
|
29 (4)
|
Treatment with Biologic
|
#(%) yes
|
2 (33%)
|
1 (17%)
|
Fecal calprotectin
|
Mean(SD)
|
573 (659)
|
408 (277)
|
Total Mayo score
|
Mean(SD)
|
6.3 (2.0)
|
6.7 (1.2)
|
Mayo Symptom subscore
|
Mean(SD)
|
4.8 (1.5)
|
4.3 (1.0)
|
Mayo Endoscopic subscore
|
Mean(SD)
|
1.5 (0.8)
|
2.3 (0.5)
|
Endoscopic UCEIS^ score
|
Mean(SD)
|
7.0 (1.8)
|
8.5 (1.8)
|
Histologic Severity Score**
|
Mean(SD)
|
3.4 (1.2)
|
4.3 (2.0)
|
IBDQ^^ total score
|
Mean(SD)
|
142.8 (16.8)
|
120.2 (25.1)
|
IBDQ bowel system subscore
|
Mean(SD)
|
4.2 (0.7)
|
4.3 (0.9)
|
Baseline clinical characteristics of subjects randomized to both the active FMT and placebo study arms. *Body Mass Index ^Ulcerative Colitis Endoscopic Index of Severity *Geboes Score ^^Inflammatory Bowel Questionnaire
Safety Evaluation
Among study subjects that received at least one dose of active or placebo therapy, adverse events possibly or probably related to FMT were few (4 total) and were equally distributed between groups (2/6 vs 2/6; p = 1.00) (Table 2). The only serious adverse event was a worsening of disease activity, which occurred in one subject from each group. Both of these subjects required escalation of therapy (prednisone taper) during the treatment period (at 6 and 4 weeks following initial FMT, respectively). Mild adverse events included nausea (36 hours prior to colonoscopic delivery of placebo material) and fever (24 hours following FMT). Notably, this febrile patient also reported fever 24 hours prior to the initial FMT procedure, making causality uncertain. No infectious complications occurred.
Frozen FMT capsules were distributed to subjects in 4-week allotments (28 pills). cFMT capsules were maintained in home freezers for a total of 84 doses per subject. Medication adherence logs revealed strong adherence with <1% of missed doses across both arms (9/1,008); however, many subjects expressed frustration regarding the strict study guidelines imposed to ensure capsule temperature stability, particularly travel restrictions.
Table 2. Adverse Events by Treatment Assignment
Adverse events
|
FMT (n = 6)
|
Placebo (n = 6)
|
p value
|
AE possibly or probably related to FMT or sham FMT, n (%)
|
2/6 (33)
|
2/6 (33)
|
1.0
|
AE Type and severity, n (%)
|
|
|
nausea, mild
|
0
|
1 (50)
|
1.0
|
fever, mild
|
1 (50)
|
0
|
1.0
|
worsening disease, severe
|
1 (50)
|
1 (50)
|
1.0
|
Adverse events by treatment group that were possibly or probably related to FMT.
Comparisons were made by Fisher’s exact test.
Clinical and Histologic Outcomes
At baseline, no significant differences in histologic or endoscopic scoring were detected between the two groups (Table 1). At 12-week follow-up, the mean endoscopic UCEIS score decreased from 6.6 ± 2.0 to 6.2 ± 2.3 in the FMT group and increased from 7.4 ± 2.6 to 7.6 ± 1.8 in the placebo group. The mean histologic Geboes score decreased from 3.4 ± 1.2 to 2.3 ± 2.4 in FMT-treated subjects, and from 4.3 ± 2.0 to 4.1 ± 2.0 in the placebo group. While this study was not powered to predict a clinical response, values for clinical and physiologic markers of disease activity are presented for informative purposes (Table 3). Cumulatively, these observations enabled identification of 3 FMT subjects who responded favorably to treatment (Subjects E, W, F). All showed consistent evidence of mucosal and systemic healing. No such individuals were identified in the placebo group. These 3 potential ‘responders’ exhibited decreases in their average total Mayo score by 3.7 ± 3.1 points, endoscopic UCEIS scores by 1.3 ± 0.6 points, histologic Geboes scores by 2.1+ 0.9 points, and average fecal calprotectin levels by 212.6 ± 64 mcg/g. Total IBDQ scores increased by an average of 51.6 ± 38 points. None of these subjects required escalation or changes in their IBD-related pharmaceutical regimens during the course of the trial. Alternatively, the remaining FMT subjects (n = 3) had inconsistent histologic and endoscopic changes, compounded by a less impressive increase in their total IBDQ score (average increase 33 ± 26 points) and all required prednisone tapers prior to the end of the trial. Changes in fecal calprotectin are unfortunately difficult to assess in this group of ‘non-responders’ due to a missing data point, and one subject (N) being above the limit of detection at both timepoints (Table 3). Photomicrographs of biopsy samples obtained before and after treatment are shown in Figure 2. Placebo subjects exhibited inconsistent changes over time, with no clear improvements in symptomatology or clinical evidence of disease. Four of six (66%) placebo subjects required escalation or adjustments in their pharmacologic treatment regimens: one (subject I) at week 2 (this subject dropped out of the trial at this point) and the other 3 study subjects at the end of the treatment period (weeks 12 and 13).
Table 3. Changes in clinical, endoscopic, and histologic evidence of disease by subject
|
Study Code
|
Age
|
Sex
|
Extent of Disease
|
Duration of Disease (yrs)
|
Maintenance Therapy
|
BMI
|
Change in Total Mayo Score
|
Change in Endoscopic UCEIS Score
|
Change in Endoscopic Mayo Score
|
Change in Histologic Score
|
Change in Fecal Calprotectin (mcg/g)
|
Change in Total IBDQ Score
|
FMT
|
E
|
46
|
F
|
pan-colitis
|
5.5
|
Mesalamine
|
20.9
|
-7
|
-2
|
0
|
-3
|
-285
|
92
|
W
|
35
|
M
|
pan-colitis
|
7.5
|
Vedolizumab
|
27.8
|
-3
|
-1
|
0
|
-1.3
|
-189
|
47
|
F
|
20
|
M
|
pan-colitis
|
3.8
|
Mesalamine
|
25
|
-1
|
-1
|
0
|
-2
|
-164
|
16
|
A
|
65
|
F
|
L-Sided
|
26.2
|
Mesalamine
|
20.9
|
3
|
0
|
1
|
-1.2
|
? => 375
|
8
|
N
|
44
|
M
|
pan-colitis
|
0.2
|
Sulfasalazine
|
25.6
|
1
|
-3
|
0
|
0.9
|
>1000 => >1000
|
59
|
P
|
38
|
M
|
pan-colitis
|
10.2
|
Mercaptopurine
|
25.2
|
-3
|
2
|
-1
|
0
|
-42
|
32
|
Placebo
|
B
|
68
|
M
|
pan-colitis
|
4.4
|
Mesalamine
|
28.8
|
-2
|
-1
|
0
|
0.8
|
-132
|
21
|
G
|
58
|
M
|
L-Sided
|
27.8
|
Mesalamine
|
26.9
|
1
|
0
|
0
|
-2
|
4
|
32
|
Y
|
65
|
M
|
L-Sided
|
0.4
|
Mesalamine
|
36.15
|
0
|
0
|
0
|
0
|
74
|
4
|
V
|
47
|
F
|
pan-colitis
|
8.8
|
Adalimumab
|
29.2
|
-1
|
0
|
0
|
0
|
27
|
35
|
T
|
31
|
F
|
pan-colitis
|
0.8
|
Mesalamine
|
29.1
|
0
|
-1
|
0
|
0
|
-137
|
32
|
I
|
40
|
F
|
pan-colitis
|
16.3
|
Mesalamine
|
25
|
DROPPED OUT DUE TO WORSENING DISEASE ACTIVITY
|
^Ulcerative Colitis Endoscopic Index of Severity *Geboes Score; IBDQ, Inflammatory Bowel Disease Questionnaire; L-Sides, left-sided disease; BMI, Body Mass Index; yrs, years; wks, weeks
A retrospective analysis to identify baseline differences between FMT subjects who responded favorably relative to non-responders revealed greater mucosal damage at the time of FMT induction in those who responded poorly, including increased total Mayo score (7.0 + 2.0 vs. 5.7 ± 2.1), endoscopic Mayo score (2.0 ± 1.0 vs. 1.0 ± 0.0), UCEIS score, (8.3 ± 1.5 vs. 5.7 ± 2.1), histologic Geboes score (4.3 ± 0.1 vs. 2.5 ± 1.0), and fecal calprotectin levels (929 ± 1144 vs. 335 ± 50 mcg/g) (Table 4).
Table 4. Baseline Clinical Characteristics by FMT Response
|
Responders n=3 (Mean ± SD)
|
Non-Responders n=3 (Mean ± SD)
|
Clinical Variables
|
|
Age (years)
|
34 (13)
|
49 (14)
|
Sex (# (%) male)
|
2 (67%)
|
2 (67%)
|
BMI
|
24.6 (3.5)
|
23.9 (2.6)
|
Number current UC meds
|
1.0 (0.0)
|
1.7 (0.6)
|
Number prior UC meds
|
3.0 (2.6)
|
2.7 (1.5)
|
Total Mayo score
|
5.7 (2.1)
|
7.0 (2.0)
|
Endo Mayo score
|
1.0 (0.0)
|
2.0 (1.0)
|
UCEIS score
|
5.7 (0.6)
|
8.3 (1.5)
|
Calprotectin
|
335 (50)
|
929 (1144)
|
IBDQ total score
|
137 (12)
|
149 (21)
|
Histologic score
|
2.5 (1.0)
|
4.3 (0.1)
|
Duration of disease (years)
|
5.6 (1.9)
|
12.2 (13.1)
|
Longitudinal Phenotyping of Peripheral Blood T-cells
Baseline T cell populations of interest were first compared between UC subjects and healthy controls. The frequency of total lymphocytes obtained following PBMC separation, as well as the CD4:CD8 ratio, were similar between groups. T regulatory cell frequencies were also similar (mean of 3.12% ± 0.41 in UC patients vs. a mean of 3.42% ± 0.54 in controls) with comparable proportions positive for IL-17A and IL-10. No T regulatory cells were IFN. The frequency of mucosal-associated invariant T (MAIT) cells was decreased in UC patients (0.62% ± 0.15 vs. 1.67% ± 0.46) and IL-17A positivity occurred almost exclusively in UC-derived MAIT cell populations (3.42 ± 1.27 vs. 0.1759 ± 0.09). Alternatively, IFN secretion was increased in MAIT cells from healthy controls (46.97 ± 7.15 vs. 24.16 ± 6.01), (Fig. 3).
T cell populations were examined before FMT, and then at weeks 4, 8, and 12 during the cFMT maintenance period. By week 4, the frequency of total PBMC lymphocytes and CD4:CD8 T cell ratios increased in all FMT subjects with poor clinical response, while variable changes were observed in the remaining FMT and placebo subjects. One notable exception was subject F who showed a prominent decrease in CD4:CD8 ratio through week 8, after which a reverse dynamic to baseline occurred. Longitudinal frequencies of T regulatory and MAIT cell populations remained relatively constant across groups. By week 4, IL-17A+ MAIT and IFN+ MAIT cells decreased in FMT subjects with positive clinical responses, remained suppressed through week 8, and then returned to baseline by week 12. IL-17A + MAIT cells remained suppressed in subject E. The number of subjects is insufficient to evaluate the statistical significance of these observed changes.
Intestinal Microbiota Analysis by 16S Sequencing
Relative abundances
Across all time points, stool samples were dominated at the phylum level by Firmicutes, and Bacteroidetes, which accounted for 88.90% of all sequence reads. Bacteria present at lower proportions included Proteobacteria, and Actinobacteria, accounting for 6.9% and 4.0% of total reads, respectively. At the genus level, samples were dominated by Clostridiales and Bacteroidales, with a lower proportion of Burkholderiales, Bifidobacteriales, Selenomonadlaes, Enterobacteriales, Lactobacillales observed at various time points. A strong antibiotic effect was observed following a 7-day course of Metronidazole and Ciprofloxacin in all subjects. Changes included a decrease in gram negative and anaerobic bacteria of the Firmicutes and Bacteroidetes phyla and an increase in gram positive Actinobacteria, including from the genus Bifidobacteriales, and Lactobaccilales Fig. 4. This effect was associated with a decrease in alpha (Shannon) diversity and increased divergence (Jensen-Shannon) from baseline. While these changes were mitigated by the cessation of antimicrobials, neither group returned to their own baseline by 18-week follow-up (Figs 5 and 6).
Alpha and Beta Diversity
No difference in alpha or beta diversity was observed between treatment groups at baseline (Fig. 5). FMT did not increase alpha (Shannon) diversity in recipients but did lead to community-level changes in the gut microbiota creating measurable similarity (beta diversity, Jensen-Shannon divergence index) between FMT subjects and their donor. This convergence, which we termed ‘Donor Divergence Index’, remained statistically significant through 8 weeks of dosing (p < 0.01) and although losing significance (p= 0.16), could still be detected at week 20, > 8 weeks following cessation of oral cFMT therapy (Fig. 6).