Abstract PS4-10-11: Pre and Post Treatment Microbiota Profiling in Archival Tumour Tissue fromEarly-Stage Triple-Negative Breast Cancer

Abstract Background Emerging evidence highlights the role of the microbiota in breast cancer prognosis and treatment response, with tissue-resident bacteria potentially influencing the tumour microenvironment and immune modulation. Formalin-fixed paraffin-embedded (FFPE) tissue represents a vast, clinically annotated resource in pathology archives, offering a unique opportunity for retrospective microbiome studies. However, its suitability remains uncertain due to DNA fragmentation, low bacterial biomass, and background contamination. We accessed the feasibility of using FFPE breast tumour tissue for bacterial DNA analysis in early-stage triple-negative breast cancer (TNBC) patients receiving neoadjuvant chemotherapy. Methods Breast tumour specimens from 23 patients enrolled in an observational biobank protocol (NCT01840293) were obtained. A total of 38 samples were analysed: 18 baseline biopsies and 20 surgical resections. Paired baseline and surgical specimens were available for 70% (16/23) of patients. Median age at diagnosis was 42 years. All patients received standard-of-care neoadjuvant chemotherapy before primary surgery. 61% (14/23) of patients achieved a pathological complete response (pCR). DNA was extracted using a protocol optimised for FFPE tissue and assessed via Qubit fluorometry. Bacterial DNA was amplified using polymerase chain reaction (PCR) with primers targeting the 16S rRNA V1-V2 and V3-V4 regions. Quantification was performed using qPCR targeting the V6 region of the 16S rRNA gene, with a synthetic gene block as standard. Human DNA was quantified by qPCR targeting the RNase P gene, with human genomic DNA as standard. Water was used as a no-template control on each qPCR plate, alongside block-matched tissue-free FFPE shaves to monitor background contamination. Purified bacterial DNA was used as a positive control to validate PCR assays. Laboratory procedures were designed to minimise contamination, though archival FFPE specimens were not originally prepared for microbial analysis, limiting control over all sources of background DNA. Results Resection samples yielded average DNA concentrations of 85.75 µg/µl; biopsies averaged 95 µg/µl, consistent with FFPE expectations. Tissue-free negative controls yielded ∼0.1 µg/µl, confirming minimal background DNA from the paraffin matrix. Despite adequate DNA yield, PCR targeting bacterial genes produced no visible amplicons. Positive controls yielded clear bands on agarose gel electrophoresis. qPCR detected similar levels of bacterial DNA in tumour samples and matched tissue-free negative controls, suggesting minimal genuine bacterial DNA and potential background amplification from paraffin contamination. Template-free negative controls showed no amplification. RNase P detection by qPCR confirmed amplifiable human DNA in tumour samples (resection and biopsy), with no amplification in negative controls. Conclusion Archived specimens collected without consideration for microbiota analysis pose challenges for microbial profiling. Despite careful optimisation and contamination-aware laboratory procedures, bacterial DNA signals in tumour samples were indistinguishable from tissue-free controls, likely due to contamination introduced during non-sterile sample collection and embedding. These findings highlight FFPE limitations for tumour microbiota analysis and have informed the refinement of our prospective protocol (NCT06709651), including a shift toward fresh-frozen sampling. While FFPE archives for microbial research would be transformative, background DNA from initial handling remains a major barrier. Funding Supported by the Breast Cancer Research Foundation, Health Research Board, Science Foundation Ireland and Breakthrough Cancer Research (Precision Oncology Ireland). Citation Format: E. da Silva Morais, E. Lynch, M. A. Hennessy, C. P. Devoy, G. Walsh, E. Crowley, C. Girleanu, J. Barry, J. Fay, L. Young, M. Tangney, R. Connolly. Pre and Post Treatment Microbiota Profiling in Archival Tumour Tissue fromEarly-Stage Triple-Negative Breast Cancer abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-10-11.