Role of Tumor Microbiota in the Response to Neoadjuvant Therapy in Patients Affected by Triple Negative Breast Cancer

Overview

Triple-negative breast cancer (TNBC) is an aggressive subtype of cancer characterized by substantial heterogeneity in treatment response. Despite significant advances in therapeutic strategies, it remains difficult to accurately predict which patients will derive the greatest benefit from treatment. For this reason, identifying novel factors that may contribute to understanding and predicting treatment response became a conisiderable research priority. In recent years, increasing evidence has highlighted the potential role of microorganisms present in the intestine (intestinal microbiota) and within tumor tissue (intratumoral microbiota) in modulating immune system activity and influencing the efficacy of anticancer treatments. At the same time, the composition and the functional state of immune cells within the tumor, known as the tumor immune microenvironment (TIME), represent important determinant in the host's ability to build a response against the disease. In addition, small particles released by cells, known as extracellular vescicles (EVs), circulate in the blood and carry biological information reflecting the characteristics of their cells of origin. EVs analyisis may therefore provide a minimally invasive tool to monitor the disease and predict response to treatment through blood sampling. This study aims to further investigate the role of intratumoral microbiota and tumor microenvironment in patients with TNBC. A better understanding of the interactions of these components with fecal microbiota and extracellular vescicles may contribute to the identification of new predictive biomarkers and, in the future, support the development of more personalized and effective therapeutic strategies.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the negligible expression of estrogen, progesterone, and HER2 receptors. Compared with other breast cancer subtypes, TNBC has historically had more limited therapeutic options and is associated with a heterogeneous clinical course and a variable response to treatment. Evidence shows that the introduction of combined chemotherapy and immunotherapy in the neoadjuvant treatment setting has improved the rate of pathological complete response (pCR) in patients with TNBC. However, not all patients benefit equally from treatment, and a substantial proportion of patients do not achieve pCR. Identifying biological factors that contribute to this variability is therefore important to improve patient stratification and to recognize predictive biomarkers for treatment response. Increasing evidence indicates that the microbiota may play an important role in regulating the response to anticancer treatment, by modulating the activity of the host immune system. In several tumor types, such as melanoma, non-small cells lung carcinoma (NSCLC) and colorectal carcinoma (CRC), composition and functional activity of the intestinal microbiota have been associated with the efficacy of chemotherapy and immune checkpoint-based treatments. Specifically, the intestinal microbiota can influence systemic immune activity through interactions with immune cells, microbial metabolites and inflammatory pathways. Additionally, the tumor itself also contains a distinct microbial community. Tumor-associated microorganisms may interact directly with tumor cells and influence inflammatory signaling, antigen presentation, immune cell recruitment, and the functional state of immune cells. These processes can contribute to the composition and activity of the tumor immune microenvironment (TIME), which is an important determinant of tumor progression and response to systemic therapy. Hence, alterations in the composition of the microbiota may contribute to either an immune-permissive or an immunosuppressive tumor environment, influencing the ability of the tumor to respond to neoadjuvant therapy. Consequently, the relationship between the intestinal microbiota, intratumoral microbiota and TIME emerges as a promising area of investigation in TNBC. Preliminary studies conducted through collaboration between Istituti Clinici Maugeri and the Istituto Nazionale dei Tumori (INT) in patients with HER2-positive breast cancer have shown that the intestinal microbiota has an impact on the efficacy of trastuzumab and may distinguish patients who respond to treatment from those who do not achieve pCR. In addition, studies conducted by the INT group have shown that specific bacteria can be detected within breast tumor tissue, independently of the breast cancer subtype. Specifically, the presence of Staphylococcus spp. within the tumor microenvironment (TME) was associated with immunosuppressive features, including increased infiltration of regulatory T-cells (Tregs) and a macrophage population shifted toward the immunosuppressive M2 state. These findings support the hypothesis that the intratumoral microbiota may contribute to shape the local immune environment and may, therefore, influence the response to treatment. The present study is based on the hypothesis that the composition of the intestinal and intratumoral microbiota might be associated with the characteristics of the TIME and with the response to neoadjuvant therapy in patients with TNBC. Accordingly, the study will investigate these components within the same prospective cohort, allowing the relationships between microbiota composition, TIME, and therapeutic response to be explored. This study will also include the investigation of circulating extracellular vescicles (EVs). EVs are membrane-enclose particles released by different cell types, including tumor cells and immune cells. EVs carry molecular cargo which includes proteins, nucleic acids, lipids, and other biologically active molecules, that potentially reflect the biological features and state of the cells from which they originate, including information related to the tumor and its surrounding microenvironment. Consequently, circulating EVs have gained significant attention as potential biomarkers in breast cancer. EVs analysis may therefore provide a minimally-invasive source of biological information that complements the analysis of tumor tissue. Previous work conducted by the ICS Maugeri group has characterized circulating EVs in patients with breast cancer and healthy individuals, developing ultrasensitive assays for the quantification of tumor-associated markers expressed on EVs subpopulations detected in plasma. In the present study, circulating EVs will be investigated as potential predictors of response to neoadjuvant therapy. The analysis of EV-associated markers may also provide additional information on tumor and immune-related processes that could be associated with treatment response. The primary objective of the study is to characterize the composition of the tumor microbiota and the features of the TIME in patients with TNBC undergoing neoadjuvant therapy. Secondary objectives include the investigation of the relationship between the intestinal and intratumoral microbiota, the evaluation of their association with response to neoadjuvant therapy, and the investigation of circulating EVs as surrogate biomarkers of the TIME. Overall, the study aims to provide a more comprehensive characterization of the biological factors associated with treatment response in TNBC, and, in the longer term, a better understanding of the interactions between the microbiota and the TIME could also help identify biological pathways that may represent potential therapeutic strategies.