Abstract C055: Gene Expression Profiling Identifies a Five-Gene Signature Associated with Antibiotic Exposure and Reduced Anti–PD-1 Efficacy in Metastatic Melanoma

Abstract Background: Melanoma is an immunogenic malignancy in which responses to immune checkpoint inhibitors (ICIs) are strongly shaped by both tumor-intrinsic mechanisms and host-related factors such as the gut microbiota. Growing evidence indicates that antibiotic administration before ICI therapy compromises antitumor immunity by disrupting microbial homeostasis and altering myeloid- and T-cell–mediated responses. Preclinical and clinical studies have shown that preserved microbial diversity supports effective CTLA-4 and PD-1 blockade, whereas antibiotic-induced dysbiosis can foster immunosuppressive circuits, impair antigen presentation, and ultimately reduce ICI efficacy in melanoma and other solid tumors. Building on this evidence, we investigated retrospectively how prior antibiotic exposure may influence survival outcomes and immune-related gene expression profiles (GEP) in unresectable metastatic melanoma patients treated with anti–PD-1. Methods: Eighty-seven patients with inoperable metastatic melanoma treated with anti–PD-1 monotherapy were retrospectively analyzed. Overall survival (OS) and progression-free survival (PFS) were estimated using Kaplan–Meier (KM) analyses. Median survival times were derived through inverse Kaplan–Meier methodology. GEP was performed with the NanoString IO360 panel. To identify the best genes signature the Sparse Partial Least Squares Discriminant Analysis (sPLS-DA) was applied. Results: Among the 87 patients, 18 received systemic antibiotics in the weeks preceding ICI initiation. This subgroup experienced significantly poorer outcomes, showing a median OS of 19 versus 46 months (HR = 0.62, p < 0.0001) and a median PFS of 4.8 versus 31.8 months (HR = 0.57, p < 0.0001), compared with antibiotic-naïve patients. GEP analysis revealed higher expression of TNFRSF8, a receptor involved in immune cell activation and costimulatory signaling, and HLA-DRB5, an MHC class II gene critical for antigen presentation, in antibiotic-exposed individuals. A five-gene signature associated with antibiotic treatment was identified: LILRB4, IRF5, HDC, CDK6, and BLM. This signature reflects coordinated activation of immunosuppressive pathways (LILRB4), dysregulated inflammatory transcriptional responses (IRF5), histamine-related immune modulation (HDC), and proliferative/stress-response programs (CDK6 and BLM). In multivariable analyses, antibiotic administration, the identified gene signature, and the presence of brain metastases were each independently and negatively associated with survival. Conclusions: Antibiotic exposure is associated with worse clinical outcomes in metastatic melanoma patients treated with anti–PD-1 therapy. The identified five-gene signature provides biologically meaningful insight into immune and transcriptional alterations linked to antibiotic use and may represent a valuable tool for refining risk stratification in this setting. Citation Format: Domenico Mallardo, Mario Fordellone, Lucia Festino, Miriam Paone, Vito Vanella, Assunta Esposito, Mario Claudia trojaniello, Mallardo, Maria Grazia Vitale, Francesca Sparano, Alfonso Arianna facchini, Esposito, Agostina Lagodin D'amato, Margaret Ottaviano, Alfredo Budillon, Ester Simeone, Corrado Caraco, Paolo Antonio Ascierto. Gene Expression Profiling Identifies a Five-Gene Signature Associated with Antibiotic Exposure and Reduced Anti–PD-1 Efficacy in Metastatic Melanoma [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C055.