CAPTIVATE is a multi-center translational and observational trial, that aims to investigate the impact of the gut microbiome on treatment outcomes in women with untreated, stage I-III Triple Negative undergoing neoadjuvant treatment with and without immune checkpoint inhibitors. As part of the trial, stool samples, core tumors biopsies and research bloods samples for the analysis will be collected at various points of the study.

The current standard of care for patients with early disease triple-negative breast cancer is neoadjuvant chemotherapy. The addition of the immune checkpoint inhibitor (ICI) to this the NACT treatment regimen has recently been shown to substantially improve the outcome of this patient population. Several translational studies have suggested the potential influence of the gut microbiome in modulating the efficacy of ICI-based therapies. In addition to the influence on response to ICI, several studies also suggest a strong association between the gut microbiome and side effects with ICI therapy.

The aim of this prospective longitudinal study is to investigate the complex interactions between the gut microbiome, the tumour biology of early-stage triple negative breast cancer, the efficacy and tolerability of neoadjuvant chemotherapy (NACT) followed by immune checkpoint inhibitors, including the possible impact on the cognitive function and the long-term outcome. A specific focus will be to study the association of the gut microbiome and the risk of breast cancer recurrence in the high-risk group of patients with residual disease after NACT. The study will furthermore assess dynamic changes in both the gut microbiome and the tumour biology, and their association with long-term outcome. Furthermore, the association of nutritional input and the gut microbiome will be studied. Results of this trial might be the basis for future interventional studies that might stratify treatment modalities based on the gut microbiome or attempt to modulate treatment outcomes through targeting the gut microbiome.

At least 200 patients undergoing neoadjuvant chemotherapy plus immunotherapy and at least 100 patients undergoing neoadjuvant chemotherapy alone will be recruited into the study.

The study poses very little risk for research participants; blood samples and faecal samples are routinely performed in hospitals.

Informed consent will be obtained prior to the participants undergoing procedures that are specifically for the purposes of the study and our outside standard, routine care at participating sites. Ample time will be given for consideration by the patient before taking part. Written informed consent will only be obtained from those who the Investigator feels assured have understood the implications of participation in the study.

Inclusion Criteria:

• Willing and able to provide written informed consent prior to study entry
• Female ≥ 18 years of age
• Histologically confirmed operable primary breast cancer with a tumor size of ≥1 cm
• Triple-negative disease:
• defined as tumours with <10% of tumours cells positive for ER and PR1 on IHC staining or an IHC score (Allred) < 3
• HER2-negative tumours defined as 0, 1+ or 2+ intensity on IHC and no evidence of amplification of the HER2 gene on ISH. defined as tumours with
• Patient planned to undergo neoadjuvant chemotherapy (as per institutional standard) with/without immunotherapy
• Representative formalin-fixed paraffin embedded (FFPE) breast tumours samples with an associated pathology report that are determined to be available and sufficient for central testing OR tumours accessible for biopsy.
• Ability to comply with the protocol, including but not limited to, completion of the patient-reported outcomes questionnaires Exclusion Criteria: Patients meeting any of the following exclusion criteria are not be enrolled in the study.
• Previous systemic or local treatment for the new primary breast cancer currently under investigation (including surgery, radiotherapy, cytotoxic and endocrine treatments)
• Received therapeutic oral or intravenous antibiotics within 14 days prior to randomization
• Known distant metastases

A. Patients with triple-negative early stage breast cancer who will be treated with neoadjuvant chemotherapy.

B. Patients with triple-negative early stage breast cancer who will be treated with neoadjuvant chemotherapy followed by immunotherapy (immune checkpoint inhibitors).