Feasibility of antibiotic-assisted fecal microbiota transplantation with immunotherapy for esophageal and gastric cancer.

1.
Background
Advances in immune checkpoint inhibitor (ICI) therapy have improved the prognosis of various types of cancer. Although the prognosis of unresectable advanced or recurrent esophageal or gastric cancer has also improved, the median overall survival (OS) for patients who received first-line therapy with fluoropyrimidine and platinum chemotherapy plus anti-programmed cell death protein 1 (PD-1) antibodies or ICIs and have disease progression is estimated to be 13–17 months. Therefore, the development of more effective treatment options remains an unmet need [1–4]. In recent years, the intestinal microbiota has been recognized to regulate the host’s immune environment and may enhance systemic immunity and antitumor immunity. An imbalance in the intestinal microbiota is called dysbiosis, and a link to carcinogenesis has been suggested [5,6]. This is supported by reports showing that the intestinal microbiota profiles of cancer patients differ from those of healthy individuals [7,8]. Based on these findings, fecal microbiota transplantation (FMT) has been explored as a strategy for modulating the intestinal microbiota and thereby potentiating the effects of ICIs. FMT aims to correct dysbiosis by administering a solution of intestinal microbiota prepared from stool collected from donors into the intestines of recipient. In a study of patients with metastatic malignant melanoma, the response rate improved when FMT from responders was administered to non-responders following administration of antibiotics [9,10]. In another study, pretreatment consisted of only intestinal cleanses with a polyethylene glycol laxative, and FMT was performed using stool collected from healthy donors [11]. Patients with malignant melanoma who had no history of anti-PD-1 antibody therapy received combination therapy consisting of a single dose of FMT from healthy donors 1 week prior to the initiation of anti-PD-1 antibody therapy (pembrolizumab or nivolumab) as first-line treatment. Subsequently, anti-PD-1 antibodies were administered continuously until disease progression or discontinuation due to toxicity, and a response rate of 65% was observed. The response rate to anti-PD-1 antibodies reported in clinical trials for malignant melanoma is around 30% [12], suggesting improvement when used in combination with FMT. Recent evidence also supports the use of FMT in ICI-resistant solid tumors. A 2024 phase Ib trial by Kim et al. [13] demonstrated that FMT from responders restored clinical benefit in patients with anti – PD-1–refractory solid cancers. This study provides additional proof-of-concept that FMT can modulate antitumor immunity and potentially overcome resistance to ICIs, supporting the rationale for A-FMT in gastrointestinal cancers. Furthermore, there are several studies in which FMT was performed in combination with or without a 2-week pretreatment with antibiotics for ulcerative colitis [14,15]. The results showed remission rates of 82% and 58% with and without antibiotic pretreatment, respectively, indicating that the remission rate was higher with pretreatment [16]. In addition, it has been reported that pretreatment with antibiotics improves the colonization rate of intestinal bacteria [17,18]. In another line of research, a correlation has been reported between cancer and intestinal microbiota abnormalities [19], and it is likely that administering antibiotics as a pretreatment for FMT in cancer patients would reset any intestinal microbiota abnormalities and promote the colonization of more favorable intestinal bacteria. Although esophageal and gastric cancers differ in etiology, molecular pathways, and histologic characteristics, emerging evidence indicates that intestinal microbiota – mediated modulation of systemic antitumor immunity may influence response to ICIs across malignancies. Considering these differences, the present study evaluates each cancer type independently in separate cohorts, while investigating the shared immunological rationale supporting A-FMT as a potential strategy to enhance ICI responsiveness. Given the body of evidence suggesting that FMT may increase the efficacy of ICI by altering the intestinal microbiota, this study aims to evaluate the safety and efficacy of FMT combined with antibiotics as pretreatment (A-FMT) in patients with esophageal or gastric cancer scheduled to receive treatment ICI-containing regimens.

2.
Objective
To evaluate the safety and efficacy of A-FMT in patients with esophageal or gastric cancer scheduled to receive ICI-containing regimens.

3.
Study design
This is a phase I-II clinical trial designed to assess the safety and preliminary efficacy of A-FMT in combination with ICIs for patients with esophageal or gastric cancer. In the study treatment, A-FMT, a single dose of intestinal microbiota solution prepared from the stool of healthy donors will be transplanted after administration of antibiotics for 1 week (at least 5 days). The intestinal microbiota solution will be prepared by Juntendo University and Metagen Therapeutics, Inc., transported to the National Cancer Center Hospital, and administered to patients (Figure 1).

3.1.
Administration of antibiotics
After obtaining consent for the trial, screening tests will be conducted to confirm the inclusion and exclusion criteria. If the criteria are met, registration will proceed. After registration, three types of antibiotics (amoxicillin, fosfomycin, and metronidazole) will be orally administered for 1 week (at least 5 days).
The combination of amoxicillin, fosfomycin, and metronidazole was selected based on their complementary antimicrobial spectra. Amoxicillin provides broad activity against Gram-positive and some Gram-negative bacteria; fosfomycin offers additional coverage against Gram-negative organisms, including intracellular species; and metronidazole targets obligate anaerobes, which constitute a major proportion of the intestinal microbiota. Using these agents in combination is expected to reduce the overall bacterial load and transiently disrupt the existing microbial community, thereby creating an intestinal environment that may enhance donor microbiota engraftment, as suggested in previous studies of antibiotic-assisted FMT.
Amoxicillin: Administer amoxicillin hydrate 1500 mg/day orally in three divided doses.

Fosfomycin: Administer fosfomycin calcium hydrate 3000 mg/day orally in three divided doses.

Metronidazole: Administer metronidazole 750 mg/day orally in three divided doses.

3.2.
Protocol treatment
At least 2 days after the completion of antibiotics, 200 mL of intestinal microbiota solution manufactured at Juntendo University will be administered once via total colonoscopy. An ICI-containing regimen will be started on the day after FMT.
This study has two parts: a safety part and an expansion part. The safety of A-FMT will be evaluated in the safety confirmation part using two different patient cohorts. If the predefined safety criteria are met, the study will proceed to the expansion part, where additional safety data will be collected and the effectiveness of the treatment will be explored.
Cohort 1: Chemotherapy* + nivolumab or pembrolizumab.
*Esophageal squamous cell carcinoma (ESCC): fluorouracil + cisplatin.
*Gastric or gastroesophageal junction adenocarcinoma: SOX, CAPOX, or FOLFOX.
SOX:  tegafur/gimeracil/oteracil  potassium + oxaliplatin; CAPOX: capecitabine + oxaliplatin; FOLFOX: fluorouracil + oxaliplatin + levofolinate calcium.
Cohort 2 (ESCC only): nivolumab + ipilimumab.
In the expansion part, both the efficacy and safety of A-FMT will be evaluated. The following patient cohorts are planned at the time of this study design.
Esophageal cancer cohort 1: first-line treatment with fluorouracil + cisplatin + nivolumab or pembrolizumab.
Esophageal cancer cohort 2: first-line treatment with nivolumab + ipilimumab.
Gastric cancer cohort: first-line treatment with SOX/CAPOX/FOLFOX + nivolumab.

4.
Methods
4.1.
Eligible patients
The inclusion and exclusion criteria for patients are shown in Table 1.

4.2.
Eligible donors
The inclusion and exclusion criteria for donors are shown in Table 2. Obtaining consent from donors, confirming eligibility, stool collection, and stool storage will be conducted as part of a collaborative research project between Juntendo University and Metagen Therapeutics, Inc. entitled “Basic Research on Microbiome Bank for Manufacturing and Management of Standardized Safe FMT Suspension” (UMIN000049692).

4.3.
Endpoints
Primary endpoint: Incidence of dose-limiting toxicity (DLT).
Secondary endpoints: Response rate, disease control rate, progression-free survival, overall survival, and incidence of adverse events.

4.4.
Sample size calculation and statistical analysis
In the safety confirmation part, the safety of A-FMT will be evaluated in patients with esophageal or gastric cancer who are scheduled to receive ICI-containing regimens. In the safety confirmation part, 3 to 6 patients are planned to be enrolled in each cohort, giving a total of 6 to 12 patients.
In the expansion part, the study will enroll patients with esophageal or gastric cancer who are scheduled to receive ICI-containing regimens. In addition to collecting additional safety information, the expansion part will evaluate efficacy based on the response rate. The number of patients in the expansion phase was determined based on futility analysis, with planned enrollment of 10 patients per cohort. The trial design aims to control the probability of early termination (p0) for ineffectiveness when the null hypothesis is true, using the first stage of Simon’s two-stage design. The secondary endpoint for efficacy evaluation was the response rate. Given that the objective response rate (ORR) for first-line treatment is approximately 50% for 5-FU + cisplatin + nivolumab or pembrolizumab, and 35% for nivolumab + ipilimumab in esophageal cancer and 55% for SOX/CAPOX/FOLFOX + nivolumab in gastric cancer, we defined the threshold ORRs as 45%, 30%, and 50%, respectively, with ORRs below these values regarded as ineffective. Under the null hypotheses of ORR ≤45%, ≤ 30%, and ≤50%, the study would be terminated for futility if the number of responders is ≤ 5 of 10, ≤4 of 10, or ≤ 6 of 11, respectively. Based on these criteria, p0 values under the null hypothesis were calculated as 0.74, 0.85, and 0.73, respectively. Therefore, thresholds for early termination due to futility were set at 5/10, 4/10, and 6/11 responders. If the number of responders reaches ≥6/10, ≥5/10, or ≥7/11, consideration will be given to proceeding to the next phase. Based on the above, the planned enrollment for each cohort is set at 10 patients (with the required effective patient number also set to 10).

4.5.
Evaluation
4.5.1.
Evaluation of DLT
The safety evaluation period for DLT is defined as the period from day 1 (the day of FMT administration) to day 28. DLT is defined as follows, with adverse events graded according to the Common Terminology Criteria for Adverse Events v5.0.
Grade 3 or 4 non-hematologic toxicity (excluding nausea, vomiting, and diarrhea in the absence of appropriate prophylactic interventions) for which a causal relationship with FMT cannot be ruled out.

A delay of 14 days or more in the initiation of chemotherapy (either the first dose or the second cycle) due to adverse events for which a causal relationship with FMT cannot be ruled out.

4.5.2.
Safety evaluation
Evaluate items 1) to 5) below prior to antibiotic administration initiation and from 7 days before to the day of initiation of each course of the regimen including ICI. During protocol treatment, evaluate items 6) to 9) every 4 weeks. Collect blood samples for FACS analysis (PBMC) and omics analysis (serum) at 8 weeks ±1 week. Additionally, collect stool samples (11) after antibiotic administration but before FMT, at 4 weeks ±1 week, and at 8 weeks ±1 week (3 stool collection tubes). Specifically, within 14 days prior to registration, at 4 weeks ±1 week, or at 8 weeks ±1 week, additionally collect stool in a dedicated tube for inflammatory markers (1 stool collection tube).
Regarding tissue examination: 12) Tumor/non-tumor tissue biopsies will be performed within 14 days prior to registration and at 8 weeks ±1 week post-registration; however, the pre-registration examination is not mandatory. 13) Intestinal epithelial biopsies will be performed, whenever possible, after antibiotic administration but before FMT, and at 4 weeks ±1 week post-treatment.

4.5.3.
Evaluation items

PS (ECOG), Weight.

Peripheral Blood Count: White Blood Cell Count, Neutrophil Count (ANC: Band + Segmented), Lymphocyte Count, Hemoglobin, Platelet Count.

Blood Biochemistry: Albumin, Total Bilirubin, AST, ALT, Creatinine, Sodium, Potassium, ALP, LDH, Calcium, CRP, FBS (Fasting Blood Sugar), Direct Bilirubin, Uric Acid, Total Protein, BUN, Cl.

Subjective and Objective Findings (Described per CTCAE v5.0-JCOG).
General/Adverse Reactions and Administration Site Conditions: Fever, Fatigue.

Immune System Disorders: Allergic Reaction.

Skin and Subcutaneous Tissue Disorders: Urticaria, Maculopapular Rash, Palmar-Plantar Erythrodysesthesia Syndrome, Hyperpigmentation.

Blood and Lymphatic System Disorders: Anemia.

Eye disorders: Lacrimation.

Musculoskeletal and connective tissue disorders: Arthralgia, Arthritis.

Gastrointestinal disorders: Diarrhea, Colitis, Nausea, Vomiting, Stomatitis.

Metabolism and nutrition disorders: Anorexia.

Endocrine disorders: Hypopituitarism, adrenal insufficiency, hyperthyroidism, hypothyroidism.

Nervous system disorders: Olfactory neuropathy, neuralgia, dysgeusia, meningitis, encephalopathy, myasthenia gravis, peripheral sensory neuropathy, peripheral motor neuropathy, Guillain-Barré syndrome.

Cardiac disorders: Myocarditis, pericarditis, pericardial effusion.

Respiratory, thoracic, and mediastinal disorders: Pneumonitis.

Infections and infestations: Bronchial infection, pulmonary infection, urinary tract infection, infectious enterocolitis, sepsis.

Immune system disorders: Autoimmune disorders※.

※ Autoimmune disorders (immune-related adverse events): Rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid syndrome, polymyositis, dermatomyositis, scleroderma, Sjögren’s syndrome, IgG4-related disease, vasculitis syndromes, mixed connective tissue disease, autoimmune hepatitis, primary biliary cholangitis, ulcerative colitis, Crohn’s disease, autoimmune pancreatitis, idiopathic thrombocytopenic purpura, adrenal insufficiency, type 1 diabetes, pemphigus, psoriasis vulgaris, pemphigoid, vitiligo, sarcoidosis syndrome, Wegener’s syndrome (granulomatosis with polyangiitis, hypophysitis, uveitis), etc.
(5) Concomitant Medications/Therapies (Only drugs/therapies used for adverse events where a causal relationship with antibiotics and FMT cannot be ruled out will be collected via eCRF).

(6) Endocrine Tests: TSH, free T3, free T4, ACTH, cortisol.

(7) Diabetes Test: HbA1c.

(8) Blood Biochemistry: KL-6.

(9) Tumor markers: Esophageal cancer CEA, SCC Gastric cancer CEA, CA19-9.

(10) Blood samples.

(11) Stool samples.

(12) Tumor and non-tumor tissue biopsies.

(13) Intestinal epithelial biopsies.

4.5.4.
Efficacy evaluation parameters
Tumor assessments will be performed based on CT scans every 8 weeks until disease progression or discontinuation of the protocol treatment. After 24 weeks, evaluations will be conducted every 12 weeks.

4.6.
Protocol treatment discontinuation and completion criteria
Protocol treatment will continue unless discontinuation criteria are met; therefore, there is no protocol treatment completion.
4.6.1.
Criteria for protocol treatment discontinuation
All protocol treatment will be discontinued if any of the following criteria are met:
The treating physician determines that continuing protocol treatment is not beneficial from an efficacy perspective.

The treating physician determines that continuing protocol treatment is not beneficial from a safety perspective.

The patient requests discontinuation of protocol treatment for reasons related to adverse events.

The patient requests discontinuation of protocol treatment for reasons unrelated to adverse events.

The patient dies during protocol treatment.

Protocol treatment is effective, and curative surgery/radiation therapy is performed.

Protocol treatment is discontinued for reasons other than 1) through 6).

4.7.
Prohibited concurrent therapies and supportive care

Anticancer drugs other than those specified in the protocol.

Surgery and radiation therapy for cancers intended for curative treatment.

*Radiation therapy for symptom palliation is permitted.

4.8.
Date management
This study will create case report forms to collect the data necessary for this research. The primary source materials for the clinical research used in this trial refer to all records used for diagnosis and treatment of enrolled patients, including medical records, test results, diagnostic imaging, pathology reports, efficacy assessment imaging, and consent documents.
Specimens and information related to registered patients for this trial shall be stored in accordance with Article 53 of the Enforcement Regulations of the Clinical Research Act (Ministry of Health, Labour and Welfare Ordinance No. 17 of 2018). The retention period for records related to this trial at participating institutions, as well as the retention period for source documents, shall be 10 years from the date the clinical research concludes.
4.8.1.
Definition of the analyzed population
In this study, the Full Analysis Set (FAS), the per-protocol set, and the safety analysis set are defined as follows. Patients deemed ineligible after registration are excluded from all analyses.
4.8.1.1.
All registered cases
The “All Registered Cases” set comprises all registered patients excluding duplicate or erroneous registrations.

4.8.1.2.
Full analysis set (FAS)
FAS is defined as the population of all registered cases from which patients who received at least one dose of either protocol treatment and who were confirmed to have a major protocol violation relative to the “Patient Eligibility Criteria” are excluded.

4.8.1.3.
Per-protocol set (PPS)
PPS is the population derived from the largest FAS by excluding cases that did not complete any of the protocol treatments, cases for which outcome data were not obtained, and cases that received prohibited or restricted concomitant treatments.

4.8.1.4.
Safety analysis set (SAS)
SAS comprises all enrolled patients who received at least one dose of any protocol treatment drug.

4.8.2.
Follow-up survey
Until the completion of the main trial, a follow-up survey will be conducted simultaneously for subjects who discontinued protocol treatment, approximately every 48 weeks starting from the registration date of the first case. The survey will include: confirmation of survival, presence or absence of tumor enlargement, receipt of subsequent treatment for esophageal cancer or gastric cancer, date of treatment initiation, and details of treatment. Confirmation of survival may be conducted via telephone or letter.

4.9.
Translational research
In accompanying translational research, we will collect fecal samples, blood samples, tumor tissue biopsies, and non-tumor intestinal epithelial biopsies from participants who have provided informed consent, and we will conduct comprehensive analyses (Figures 2 and 3). Flow cytometry will be performed on tumor biopsy samples and peripheral blood mononuclear cells to characterize immune cell populations including T cells (CD4+, CD8+, Tregs), natural killer cells, mucosal-associated invariant T cells, and myeloid-derived suppressor cells. Immunohistochemical staining will also be conducted on tumor biopsy samples to assess changes in the tumor immune microenvironment induced by FMT. Additionally, we will perform multi-omics analyses – including metabolomics, proteomics, transcriptomics, epigenomics, and genomics – as well as bacterial quantification using biopsy specimens, blood specimens, and stool specimens in order to identify prognostic biomarkers and predictive factors of treatment efficacy. Through these analyses, we aim to surrogate biomarkers and identify factors contributing to the efficacy of the protocol treatment. Finally, we will examine the relationship between A-FMT therapy and the composition, dynamics, therapeutic impact, safety, and clinical relevance of the intestinal microbiota, together with detailed characterization of the tumor microenvironment. Above all, the antibiotic-mediated clearance of baseline microbiota, the engraftment of donor-derived bacteria, and other relevant microbiome features will be evaluated using the results from shotgun metagenomic analyses. Because the translational analyses are exploratory, specific bioinformatic pipelines and statistical methods will be determined based on the properties of the collected datasets.

4.10.
Data safety and monitoring
Samples and information pertaining to registered patients for this trial shall be stored in accordance with Article 53 of the Enforcement Regulations of the Clinical Research Act (Ministry of Health, Labour and Welfare Ordinance No. 17 of 2018). Data monitoring will be conducted through site monitoring and central monitoring based on data entered from case report forms collected by the data center. Site monitoring will be conducted by staff of the Planning and Management Office of the National Cancer Center Hospital Clinical Research Support Division, including on-site visits to verify source documents, and monitoring results will be summarized. Central monitoring will be conducted once a year in principle and will be outsourced to a contract research organization. The Safety and Efficacy Evaluation Committee shall be established as an organization independent of the principal investigator. This will enable objective data monitoring, thereby reducing bias in trial results and improving the reliability of the trial, as well as ensuring the safety of the participants.
4.10.1.
Research termination and suspension
When terminating the research itself, the Principal Investigator shall promptly report the details of the termination and its reasons to the Accredited Clinical Research Review Committee and the Minister of Health, Labor and Welfare. Appropriate measures shall be taken for the subjects of this trial. If necessary, the Principal Investigator shall seek the opinion of the Accredited Clinical Research Review Committee regarding the timing and method of research completion associated with measures for the subjects. Furthermore, even after submitting a termination report, disease reports, periodic reports, etc., shall continue to be submitted until the clinical research is completed.
Termination refers to ending the research earlier than planned for any of the following reasons. Suspension refers to temporarily halting case registration when any of the following reasons are suspected, etc.
When it is determined that the objectives of this trial have been achieved.

When it is determined that the likelihood of achieving the trial’s objectives is extremely low.

When information obtained during the trial indicates a safety concern.

When information from sources other than the trial indicates a safety concern.

When information from sources other than the trial negates the trial’s significance.

When delays in case registration or other factors make completion of the trial difficult.

4.10.2.
Ethics
Prior to patient registration, the principal investigator or co-investigators must provide the patient with an informed consent document approved by the National Cancer Center Hospital Institutional Review Board and explain the following details verbally. All participating researchers will conduct the trial in accordance with the “Declaration of Helsinki” (https://www.wma.net/policies-post/wma-declaration-of-helsinki/) and Japan’s “Clinical Research Act” (Act No. 16 of 2017; http://www.mhlw.go.jp/stf/seisakunitsuite/bunya/0000163417.html) and the “Clinical Research Act Enforcement Regulations” (Ministry of Health, Labour and Welfare Ordinance No. 17 of 2018). The clinical trial registration number for this trial in the Japan Registry of Clinical Trials is jRCTs031240170.

4.10.3.
Procedure for modifying protocol content
When modifying protocol content, the changes must be submitted to the certified clinical research review committee and reported to the Minister of Health, Labour and Welfare. Furthermore, if the protocol or informed consent document is amended or revised, the principal investigator must promptly distribute the latest version of the protocol or informed consent document to all personnel involved in the clinical research.

4.10.4.
Consent
After explaining the trial, allowing sufficient time for consideration, and confirming the patient fully understands the trial details, obtain the patient’s personal consent to participate. If the patient personally consents to participate in the trial, obtain the patient’s signature on the consent form. The principal investigator or co-investigator must confirm that the consent form includes the name of the physician who provided the explanation, the date of explanation, the name of the patient who received the explanation and consented, and the date of consent. Consent for the ancillary study is included in the main trial’s consent explanation document; written consent for this study is obtained at the time consent is obtained for the main trial.

4.10.5.
Protection of personal information
Personal information and privacy-related information such as medical records must be strictly protected and handled with care under the principle of respecting individual dignity. We implement thorough management measures in accordance with the Personal Information Protection Act and strive to protect privacy. For patient identification and verification purposes, only the minimum necessary information to identify individuals will be used: medical record number, date of birth, initials, and registration number. That is, personally identifiable information beyond the above, such as patient names, will not be disclosed from participating medical institutions to the data center or joint research institutions.

4.10.6.
Compensation
To prepare for liability in the event of health damage occurring to research subjects during the conduct of this study, this clinical research is covered by clinical research insurance. Should unexpected health damage arise as a result of participation in this study, compensation payments, medical expenses, and medical allowances may be received from the insurance policy held by the researchers.

4.10.7.
Presentation of research findings
Primary publication papers will be submitted to English-language journals.
Presentations at conferences and publications (review articles) for the purpose of introducing the study, excluding analysis results of the study’s endpoints, as well as presentations at conferences and publications of patient background distribution and safety data after registration closure, will be conducted.

5.
Conclusion
This phase I-II clinical trial will evaluate the safety and efficacy of A-FMT in combination with ICIs in patients with unresectable advanced or recurrent esophageal or gastric cancer. By resetting the intestinal microbiome with antibiotics followed by FMT from healthy donors, the aim is to overcome immune resistance and enhance the therapeutic response to ICIs. In parallel, comprehensive translational research will be conducted to identify predictive biomarkers and gain insights into the tumor microenvironment and host immunity.
Although A-FMT has potential as a novel therapeutic strategy, this study has several limitations. First, the open-label, non-randomized design may cause bias in efficacy evaluation. Second, the ideal frequency and quantity of FMT administrations have yet to be determined and are not evaluated in this study. Third, the long-term engraftment of the transplanted microbiota and its clinical implications remain unknown and require further investigation.
If proven safe and effective, A-FMT could improve treatment outcomes in gastrointestinal cancers and establish a foundation for microbiota-targeted immunotherapy in other cancer types.