Gut Microbiome Effect on the Neoadjuvant Chemotherapy-induced Immunosurveillance in Triple Negative Breast Cancer

This is a prospective study of newly diagnosed TNBC patients undergoing standard of care
neoadjuvant chemotherapy and correlate gut microbiome composition and anti-tumor immune
responses with pCR.

The biopsy at diagnosis will be used as a pretreatment control for the assessment of TILs,
PD-L1 expression, immune signature profiles. Stool and peripheral blood (PB) samples will be
collected at time of consent for neoadjuvant therapy. Detailed instructions on the stool
collection will be provided to patients (please see Appendix 1). TNBC patients will be first
treated with 4 cycles of AC neoadjuvant chemotherapy (2 weeks/cycle). Prior to the initiation
of T, a tumor biopsy will be offered to the patient. Patients that are interested in having
the optional biopsy will be informed and will sign a separate consent form (please see
Appendix 2). In addition to the samples collected, patients' information regarding their
pathology and the diagnosis (and physicians' notes) will be reviewed.

Stool and PB samples will be collected. At time of surgery, after the completion of T (12
cycles, 1 week/cycle or at the discretion of the medical oncology), resected tumor and
adjacent normal, non-tumor tissue, stool and PB samples will be collected.

Pre- and post-therapy immune phenotyping/profiling will be determined in PB samples and
patient biopsies. The overall composition of the gut microbiome will also be determined in
patient stool samples.

The overview of the study is presented below:

1. Duration of T treatment is 12 weekly cycles or at the discretion of the medical
oncologist.

2. Cycle 1 refers to first dose of each treatment.

3. Tumor morphology, IHC and FISH will be performed at diagnosis of TNBC. Criteria for
newly diagnosed TNBC: <1% of ER and PR immunoreactivity and HER2— by FISH or IHC
staining 0 or 1+ and T2 mass lesion or greater.

4. For correlative studies, collection of PB will be at day 1 of cycle 1 of AC, day 1 of
cycle 1 of T and end of treatment, prior to surgery. 8x Yellow top tubes (BD Vacutainer
ACD Solution A Blood Collection tubes — 8.5ml) — 68 ml. Immunophenotying, gene
expression profiling and assessment of cytokine/chemokine production will be performed.

5. For correlative studies, Stool collection will be collected up to 48 hours prior to drug
administration on day 1 of cycle 1 and day of surgery. Sequencing of the the gut
microbiome and gene-associated pathways will be performed by 16S rRNA and shotgun
metagenomics sequencing.

6. For correlative studies, immunostaining of fixed tissue for PD-L1 expression on tumor
cells and for the in situ presence of various T cell subset markers with PD1 expression
will be performed. Isolation of RNA will be performed from formalin-fixed tissues.

7. Tumor biopsy for cycle 1, day 1 of paclitaxel is not standard care. This biopsy will be
offered and performed upon consent of patient.

8. This tissue will be provided by Pathology Department upon processing of surgical
specimen.

Inclusion Criteria:

- Histologically confirmed new diagnosis of TNBC (<1% of ER and PR immunoreactivity and
HER2— by FISH or IHC staining 0 or 1+)

- >18 years

- T2 mass lesion or greater

- Tumor amenable to percutaneous core biopsy

Exclusion Criteria:

- chronic anticoagulation therapy

- prior ipsilateral breast surgery, ipsilateral radiotherapy, hormonal therapy or
systemic chemotherapy

- prior systemic antibiotics x 6 months

- lactating

- pregnant