Gastrointestinal Microbiome and Response to Immunotherapy in Metastatic Malignant Melanoma

February 23, 2026 updated by: Institute of Oncology Ljubljana

Prognostic and Predictive Value of Gastrointestinal Microbiome in Metastatic Melanoma Treated With Immunotherapy

The aim of this prospective clinical study is to evaluate the prognostic and predictive significance of the gastrointestinal microbiome in patients with metastatic malignant melanoma treated with first-line immunotherapy using immune checkpoint inhibitors (PD-1 inhibitors and CTLA-4 inhibitors). Although immunotherapy has significantly improved survival outcomes, treatment response remains unpredictable and a substantial proportion of patients develop immune-related adverse events, pseudoprogression, or hyperprogression.

The gastrointestinal microbiome is an important regulator of immune homeostasis and may influence systemic immune response. This study investigates whether specific microbiome composition is associated with objective treatment response assessed according to iRECIST criteria, progression-free survival (PFS), and the occurrence of immune-related adverse events.

Patients treated at the Institute of Oncology Ljubljana between March 2022 and March 2024 were enrolled. In addition to standard-of-care immunotherapy, participants underwent protocol-defined collection of stool and peripheral blood samples at predefined time points for microbiome and immune profiling analyses.

Study Overview

Status

Status

Indicates the current recruitment status or the expanded access status.
Active, not recruiting

Conditions

Conditions

The disease, disorder, syndrome, illness, or injury that is being studied. On ClinicalTrials.gov, conditions may also include other health-related issues, such as lifespan, quality of life, and health risks.

Intervention / Treatment

Intervention / Treatment

A process or action that is the focus of a clinical study. Interventions include drugs, medical devices, procedures, vaccines, and other products that are either investigational or already available. Interventions can also include noninvasive approaches, such as education or modifying diet and exercise.

Detailed Description

This prospective, non-randomized clinical study evaluates the prognostic and predictive value of the gastrointestinal microbiome in patients with metastatic malignant melanoma treated with immune checkpoint inhibitors (PD-1 inhibitors and CTLA-4 inhibitors) in the first-line setting. Although immunotherapy has improved survival in metastatic melanoma, clinical outcomes remain heterogeneous and a substantial proportion of patients experience immune-related adverse events, pseudoprogression, or hyperprogression. Identification of biomarkers that predict response and toxicity remains clinically important.

The gastrointestinal tract contains a high concentration of microbes and lymphoid tissue, and the immune system and microbiome exist in close interaction. The gastrointestinal microbiome influences systemic immune response through cytokine production and regulation of T-cell activity. Previous studies suggest that microbiome composition and diversity may be associated with overall survival (OS), progression-free survival (PFS), and treatment response in patients receiving immune checkpoint inhibitors. Antibiotic-induced dysbiosis has been associated with reduced efficacy of immunotherapy, and fecal microbiota transplantation has shown potential in overcoming resistance to PD-1 inhibitors.

The primary objective of this study is to determine whether the predominant composition of the gastrointestinal microbiome is associated with objective response to PD-1 and CTLA-4 inhibitors in patients with metastatic malignant melanoma, assessed according to iRECIST criteria. The primary hypothesis is that a specific microbiome profile is associated with treatment response and progression-free survival (PFS).

Secondary objectives include evaluation of the association between microbiome composition and immune-related adverse events, disease progression during treatment, and peripheral blood immune cell populations (CD3+, CD4+, CD8+, CD4/CD8 ratio, and macrophages).

A total of 132 patients treated with first-line immune checkpoint inhibitors at the Institute of Oncology Ljubljana between March 2022 and March 2024 were enrolled. Clinical data were collected in anonymized form and include sex, age, date of diagnosis, performance status at treatment initiation, laboratory parameters (including LDH and S100), melanoma localization, BRAF status, treatment duration, radiologic response, and immune-related adverse events.

In addition to standard-of-care treatment, participants underwent study-specific biological sample collection according to a predefined schedule. Stool samples were collected prior to treatment initiation, at week 12 (+2 weeks) and week 28 (+2 weeks), at suspected progression/hyperprogression, and at the occurrence of immune-related adverse events. If participants received antibiotic therapy, stool collection was postponed and performed three weeks after completion of antibiotics. Samples were analyzed at the Biotechnical Faculty, University of Ljubljana using amplicon sequencing of 16S rRNA with Illumina 16S/ITS Nextera two-step PCR and MiSeq 2×300 technology. Bioinformatic and statistical analyses were performed using the UPARSE pipeline, Silva NR SSU and LTP SSU databases, Mothur software package, and the R package Phyloseq.

Peripheral venous blood samples were collected up to four weeks before treatment initiation, at week 12 (+2 weeks) and week 28 (+2 weeks), at suspected progression/hyperprogression, and at the occurrence of immune-related adverse events. Additional blood samples were analyzed at the Department of Cytology, Institute of Oncology Ljubljana using flow cytometry to determine immune cell populations, including CD3+, CD4+, CD8+, CD4/CD8 ratio, and macrophages. Samples were analyzed using FACSCanto 10 and FACSDiva software.

Radiologic evaluation was performed using PET/CT or CT triplet (head, chest, abdomen) within four weeks prior to treatment initiation and at week 12 (+2 weeks) and week 28 (+2 weeks). Additional imaging was performed at suspected progression or based on clinical judgement. Treatment response was evaluated according to iRECIST criteria and categorized as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). Pseudoprogression was defined as temporary radiologic progression without clinical deterioration followed by subsequent reduction of tumor burden.

Study Type

Study Type

Describes the nature of a clinical study. Study types include interventional studies (also called clinical trials), observational studies (including patient registries), and expanded access.
Interventional

Enrollment (Estimated)

Enrollment

The number of participants in a clinical study. The "anticipated" enrollment is the target number of participants that the researchers need for the study.
150

Phase

Phase

The stage of a clinical trial studying a drug or biological product, based on definitions developed by the U.S. Food and Drug Administration (FDA). The phase is based on the study's objective, the number of participants, and other characteristics. There are five phases: Early Phase 1 (formerly listed as Phase 0), Phase 1, Phase 2, Phase 3, and Phase 4. Not Applicable is used to describe trials without FDA-defined phases, including trials of devices or behavioral interventions.
  • Not Applicable

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Locations

  • Slovenia
      • Ljubljana, Slovenia, 1000
        • Institute of Oncology Ljubljana

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Eligibility Criteria

The key requirements that people who want to participate in a clinical study must meet or the characteristics they must have. Eligibility criteria consist of both inclusion criteria (which are required for a person to participate in the study) and exclusion criteria (which prevent a person from participating). Types of eligibility criteria include whether a study accepts healthy volunteers, has age or age group requirements, or is limited by sex.

Ages Eligible for Study

  • Adult
  • Older Adult

Accepts Healthy Volunteers

No

Description

Inclusion Criteria

  • Age ≥ 18 years
  • Cytologically or histologically confirmed malignant melanoma
  • Unresectable stage IIID or stage IV disease according to AJCC (8th edition, 2018)
  • ECOG performance status 0-2
  • Indication for first-line systemic immunotherapy (nivolumab, combination ipilimumab/nivolumab, or pembrolizumab)
  • CT or PET/CT imaging performed within 4 weeks prior to first administration of immunotherapy
  • Written informed consent obtained prior to study participation

Exclusion Criteria

  • Previous systemic therapy for melanoma
  • ECOG performance status 3-4
  • Contraindications to immunotherapy (e.g., known immunodeficiency, active immunosuppressive treatment, or active autoimmune disease requiring systemic therapy)
  • Presence of another active malignancy, except adequately treated basal cell carcinoma, squamous cell carcinoma of the skin, or other solid tumors with no evidence of recurrence for ≥3 years

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

  • Primary Purpose: Treatment
  • Allocation: Non-Randomized
  • Interventional Model: Parallel Assignment
  • Masking: None (Open Label)

Arms and Interventions

Participant Group / Arm

Participant Group / Arm

A group or subgroup of participants in a clinical trial that receives a specific intervention/treatment, or no intervention, according to the trial's protocol.
Intervention / Treatment

Intervention / Treatment

A process or action that is the focus of a clinical study. Interventions include drugs, medical devices, procedures, vaccines, and other products that are either investigational or already available. Interventions can also include noninvasive approaches, such as education or modifying diet and exercise.
Other: PD-1 inhibitor therapy
Patients with metastatic malignant melanoma treated in the first line with PD-1 inhibitors (pembrolizumab or nivolumab).
Drug: PD-1 and CTLA-4 inhibitors
Combination immunotherapy ipilimumab/nivolumab
Other: Combination PD-1 and CTLA-4 inhibitor therapy
Patients with metastatic malignant melanoma treated in the first line with combined immunotherapy with PD-1 and CTLA-4 inhibitors (ipilimumab/nivolumab).
Drug: PD-1 inhibitors
Pembrolizumab, nivolumab

What is the study measuring?

Primary Outcome Measures

Primary Outcome Measures

In a clinical study's protocol, the planned outcome measure that is the most important for evaluating the effect of an intervention/treatment. Most clinical studies have one primary outcome measure, but some have more than one.
Outcome Measure
Measure Description
Time Frame
Objective Treatment Response According to irRECIST
Time Frame: Baseline (within 4 weeks before treatment initiation) and up to 28 weeks (+2 weeks) after treatment initiation
Treatment response evaluated radiologically using PET/CT or CT triplet and assessed according to irRECIST criteria (CR, PR, SD, PD).
Baseline (within 4 weeks before treatment initiation) and up to 28 weeks (+2 weeks) after treatment initiation

Secondary Outcome Measures

Secondary Outcome Measures

In a clinical study's protocol, a planned outcome measure that is not as important as the primary outcome measure for evaluating the effect of an intervention but is still of interest. Most clinical studies have more than one secondary outcome measure.
Outcome Measure
Measure Description
Time Frame
Progression-Free Survival (PFS)
Time Frame: From treatment initiation up to 24 months
Time from treatment initiation to disease progression (according to irRECIST) or death, whichever occurs first.
From treatment initiation up to 24 months
Immune-Related Adverse Events
Time Frame: From treatment initiation up to 24 months
Occurrence of immune-related adverse events (e.g., colitis and other immune-related adverse events) during immune checkpoint inhibitor therapy.
From treatment initiation up to 24 months

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

Sponsor

The organization or person who initiates the study and who has authority and control over the study.
Institute of Oncology Ljubljana

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Actual)

Study Start

The actual date on which the first participant was enrolled in a clinical study. The "anticipated" study start date is the date that the researchers think will be the study start date.
March 1, 2022

Primary Completion (Actual)

Primary Completion

The date on which the last participant in a clinical study was examined or received an intervention to collect final data for the primary outcome measure. Whether the clinical study ended according to the protocol or was terminated does not affect this date. For clinical studies with more than one primary outcome measure with different completion dates, this term refers to the date on which data collection is completed for all the primary outcome measures. The "anticipated" primary completion date is the date that the researchers think will be the primary completion date for the study.
March 31, 2024

Study Completion (Estimated)

Study Completion

The date on which the last participant in a clinical study was examined or received an intervention/treatment to collect final data for the primary outcome measures, secondary outcome measures, and adverse events (that is, the last participant's last visit). The "anticipated" study completion date is the date that the researchers think will be the study completion date.
March 31, 2026

Study Registration Dates

First Submitted

First Submitted

The date on which the study sponsor or investigator first submitted a study record to ClinicalTrials.gov. There is typically a delay of a few days between the first submitted date and the record's availability on ClinicalTrials.gov (the first posted date).
February 5, 2026

First Submitted That Met QC Criteria

First Submitted That Met QC Criteria

The date on which the study sponsor or investigator first submits a study record that is consistent with National Library of Medicine (NLM) quality control (QC) review criteria. The sponsor or investigator may need to revise and submit a study record one or more times before NLM's QC review criteria are met. It is the responsibility of the sponsor or investigator to ensure that the study record is consistent with the NLM QC review criteria.
February 17, 2026

First Posted (Actual)

First Posted

The date on which the study record was first available on ClinicalTrials.gov after National Library of Medicine (NLM) quality control (QC) review has concluded. There is typically a delay of a few days between the date the study sponsor or investigator submitted the study record and the first posted date.
February 23, 2026

Study Record Updates

Last Update Posted (Actual)

Last Update Posted

The most recent date on which changes to a study record were made available on ClinicalTrials.gov. There may be a delay between when the changes were submitted to ClinicalTrials.gov by the study's sponsor or investigator (the last update submitted date) and the last update posted date.
February 25, 2026

Last Update Submitted That Met QC Criteria

Last Update Submitted That Met QC Criteria

The most recent date on which the study sponsor or investigator submitted changes to a study record that are consistent with National Library of Medicine (NLM) quality control (QC) review criteria. It is the responsibility of the sponsor or investigator to ensure that the study record is consistent with the NLM QC review criteria.
February 23, 2026

Last Verified

Last Verified

The most recent date on which the study sponsor or investigator confirmed the information about a clinical study on ClinicalTrials.gov as accurate and current. If a study with a recruitment status of recruiting; not yet recruiting; or active, not recruiting has not been confirmed within the past 2 years, the study's recruitment status is shown as unknown.
February 1, 2026

More Information

Terms related to this study

Keywords

Additional Relevant MeSH Terms

Other Study ID Numbers

Other Study ID Numbers

Identifiers or ID numbers other than the NCT number that are assigned to a clinical study by the study's sponsor, funders, or others. These numbers may include unique identifiers from other trial registries and National Institutes of Health grant numbers.
  • OI-MEL-MICROBIOME-2022
  • ERIDSPRA-0028/2022 (Other Identifier: Clinical Research Unit, Institute of Oncology Ljubljana)

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

NO

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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