Gastrointestinal Microbiome and Response to Immunotherapy in Metastatic Malignant Melanoma

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: Active, not recruiting
• Conditions: Metastatic Malignant Melanoma
• Intervention / Treatment: Drug: PD-1 and CTLA-4 inhibitors; Drug: PD-1 inhibitors

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: Interventional
• Enrollment (Estimated): 150
• Phase: Not Applicable

Contacts and Locations

Study Locations

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

Participation Criteria

Eligibility Criteria

• 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

How is the study designed?

Design Details

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

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
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

Outcome Measure	Measure Description	Time Frame
Objective Treatment Response According to irRECIST	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

Outcome Measure	Measure Description	Time Frame
Progression-Free Survival (PFS)	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	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

• Sponsor: Institute of Oncology Ljubljana

Study record dates

Study Major Dates

• Study Start (Actual): March 1, 2022
• Primary Completion (Actual): March 31, 2024
• Study Completion (Estimated): March 31, 2026

Study Registration Dates

• First Submitted: February 5, 2026
• First Submitted That Met QC Criteria: February 17, 2026
• First Posted (Actual): February 23, 2026

Study Record Updates

• Last Update Posted (Actual): February 25, 2026
• Last Update Submitted That Met QC Criteria: February 23, 2026
• Last Verified: February 1, 2026

More Information

Terms related to this study

• Keywords: Immunotherapy; Metastatic melanoma
• Additional Relevant MeSH Terms: Neoplasms by Site; Neoplasms; Neoplasms by Histologic Type; Skin Diseases; Neuroectodermal Tumors; Neoplasms, Germ Cell and Embryonal; Neoplasms, Nerve Tissue; Neuroendocrine Tumors; Nevi and Melanomas; Skin Neoplasms; Skin and Connective Tissue Diseases; Melanoma; Antineoplastic Agents, Immunological; Antineoplastic Agents; Molecular Mechanisms of Pharmacological Action; Pharmacologic Actions; Chemical Actions and Uses; Therapeutic Uses; Immune Checkpoint Inhibitors
• Other Study ID 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