Abstract 2218: Prognostic relevance of immune phenotyping of the tumor immune microenvironment and its interactions with the mammary microbiota in Ethiopian breast cancer patients

Background: Breast cancer (BC) is a highly heterogeneous disease, and refining additional clinical parameters is crucial for its effective management. The tumor immune microenvironment, influenced by factors such as infiltrating intratumoral microbiota, has been shown to play a key role in cancer prognosis and treatment response. Methods: RNA was isolated from formalin-fixed paraffin-embedded (FFPE) tissue from 82 women with BC. Expression of 54 immune genes was analysed using Nanostring and differentially expressed genes (DEGs) were determined using ROSALIND®. Immune cell populations were profiled using Nanostring's cell type profiling module, while tumour infiltrating lymphocytes (TILs) were assessed via haematoxylin and eosin (H&E) staining. For microbiome profiling, DNA was isolated from 29 biopsy samples using MoBio PowerMag Soil DNA Isolation kit and sequenced with an Illumina MiSeq. In addition, the PIK3CA gene was genotyped for three hotspot mutations using qPCR. Log-rank test was performed to evaluate the prognostic significance of immune phenotypes. Results: Hierarchical clustering of the gene expression profiles revealed four discrete immune phenotypes (IP1-4) characterized by differential expression of immune-activating and immune-regulatory genes. An immunologically inert phenotype (IP2) was identified, marked by consistent downregulation of both immune suppressive and immune activating genes, alongside the lowest levels of immune cell infiltration. In contrast, IP4 exhibited an immunologically active TME, characterized by upregulation of key cytotoxic genes and the highest abundance of immune cells. IP1 and IP3 had intermediate characteristics. A significant association was found between IPs and BC subtypes: luminal A tumors predominated in IP2, while basal BC tumors were most common in IP3. No significant differences in PIK3CA mutations were observed across the IPs. Importantly, IPs with an active TME were associated with improved overall survival compared to those with an inert immune environment. Microbiota diversity and abundance did not show significant differences across the IPs. However, microbiota composition differed significantly between TIL groups. Notably, the Firmicutes and Verrucomicrobia phyla, as well as the Lactobacillaceae, Akkermansiaceae and Caulobacteraceae families, were more abundant in intermediate TIL groups. At the genus level, Delftia was significantly enriched in the high TIL group (P < 0.0001), while Akkermansia and Cutibacterium were more abundant in the intermediate TIL groups. Conclusion: Immune phenotyping of the TME identified distinct immune subgroups that are associated with prognosis and could guide treatment selection. Additionally, potential immune-microbiome interactions were observed, suggesting a role in modulating anti-tumor immunity. Citation Format: Meron Yohannes Nigussie, Zelalem Desalegn, Marcus Bauer, Kathrin Stückrath, Endale Anberbir1, Yonas Bekuretsion, Mathewos Assefa, Tariku Wakuma, Yasin Worku, Pablo S. Santos, Lesley Taylor, Adamu Adissie, Claudia Wickenhauser, Chiara Massa, Martina Vetter, Eva J. Kantelhardt, Barbara Seliger, Tamrat Abebe. Prognostic relevance of immune phenotyping of the tumor immune microenvironment and its interactions with the mammary microbiota in Ethiopian breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2218.