Overview

Background: Patients with breast cancer (BC) seem to have a higher prevalence of thyroid autoimmunity (TA), and this association has a solid pathogenic rationale: 1) I have discovered that BC tissue expresses thyroid peroxidase (TPO), a major thyroid autoantigen which exists in different isoforms by alternative splicing; 2) the immune system has a key role in both autoimmunity and cancer; 3) immunity and gut microbiota are deeply connected. Serum anti-TPO autoantibodies (TPOAb), the hallmark of TA, are highly prevalent among BC patients and they are associated with a better BC prognosis is retrospective studies. The magnitude of the relationship between BC and TA needs to be defined in longitudinal studies, unbiased by concurrent anti-cancer treatments.

Hypothesis: Patients with both BC and TA have a better BC prognosis, since they express intra-tumoral breast-specific TPO isoforms that are related with TPO-specific immune responses, and microbiota patterns, that contrast neoplastic progression, not present in patients affected with only BC and not associated TA.

Aims: 1) To verify the prevalence and prognostic impact of TA in the first unbiased longitudinal cohort of BC patients, recruited before anti-cancer treatments; 2) To investigate breast-specific TPO isoforms expression in BC tissue; 3) to characterise TPO-specific tissue-resident immune responses in paired human samples derived from BC and thyroid-related tissues/organs; 4) to explore the gut microbiota associated with BC and TA.

Experimental design: Monocentric 5-year longitudinal study. BC patients and non-cancer controls will be sampled from paired blood, stool, tumoral-breast samples (BC only), and neck lymph nodes (LNs). LNs contain thyroid-draining lymphocytes and are easily accessible by ultrasound-guided fine-needle-aspiration, a well-tolerated technique which I routinely operate for research purposes. Methods used to address aims: 1) Serum thyroid function and TPOAb measurement to assess TA; 2) RT-PCR amplification and product sequencing of tumor-derived RNA in BC patients, to identify TPO isoforms expression; 3) Flow-cytometry immunophenotyping of TPO-specific lymphocytes; 4) Metagenomics, metabolomics and metaproteomics for microbiota analysis.

Expected results: BC patients have a higher prevalence of TA than non-cancer controls (Aim 1A). Compared with BC-only patients, those affected with both BC and TA have a better 5-year prognosis (Aim 1B), express breast-specific TPO isoforms (Aim 2), have increased tumor-infiltrating and neck-LN-resident TPO-specific lymphocytes with anti-neoplastic activity, i.e. T follicular helper cells (Tfh) (Aim 3), and show peculiar microbiota signatures (Aim 4).

Impact on cancer: This study will clarify the prognostic role of TA in BC patients by combining paired clinical data, intra-tumoral antigen-expression and related lymphocytic infiltration, and microbiota patterns, generating novel clinical and therapeutic potential applications. Serum thyroid function and TPOAb, as well as intra-tumoral TPO isoforms, may be assessed as routine BC markers. Precision immunotherapy for BC may target TPO isoforms; the benefits of improved tumor control would likely overweight the downside of thyroid adverse effects, easily manageable with levothyroxine replacement treatment. The immune cell subsets identified in this study may suggest other immunotherapies for BC. Finally, future clinical trials of probiotics, antibiotics, or fecal material transplant, may be designed to target the microbiota signatures of BC patients with and without TA.

Eligibility

Inclusion Criteria:

The study will include patients undergoing mammography (either for screening or for a suspect nodule) who meet all of the following criteria:

• signed informed consent
• age 18-80 years
• female sex

Based on the mammography results and the outcome of any biopsy performed due to the presence of breast nodule(s), patients will subsequently be categorized into cases (diagnosed with breast cancer) and controls (not diagnosed with breast cancer).

Exclusion Criteria:

Subjects who do not meet the mentioned inclusion criteria, or who meet at least one of the following exclusion criteria, will be excluded from recruitment:

• Pregnancy, within 6 months postpartum, and/or active breastfeeding
• Prior organ, bone marrow, or hematopoietic stem cell transplantation
• Prior splenectomy
• Therapy with biological agents, immunosuppressants, or immunomodulators
• History of breast cancer