Background. Hormone receptor (HR)+ breast cancer (BC) causes the majority of BC-related deaths in the US (Siegel, Miller et al. 2020). Standard treatment includes surgery, endocrine therapy +/- chemotherapy or radiation therapy (RT), depending on surgical procedure and risk assessment). However, approximately 50% of patients with HR+ do not experience disease eradication after standard-of-care therapy, ultimately relapse and succumb to the disease. It has been postulated that such cases of relapse reflect situations in which treatment is unable to elicit a strong immune response that would enable long-term disease control. Objective and procedures. To obtain insights into the immunological alterations accompanying disease relapse in HR+ BC exposed to RT, we harnessed an endogenous model of BC driven in immunocompetent mice by the implantation of a slow-release medroxyprogesterone acetate (MPA, M) pellet and oral administration of the carcinogen DMBA (D). This model recapitulates multiple key aspects of human luminal B BC, including a relatively ´cold´ microenvironment in basal conditions and hence limited sensitivity to PD-1 blockage (Buque, Bloy et al. 2020). To identify immunological alterations associated with disease relapse after RT, we undertook an in-depth characterization of the tumor (by DNAseq and RNAseq) and systemic (by flow cytometry on the splenic compartment) microenvironment of C57BL/6 female mice bearing tumors that recovered normal growth after either 21 Gy in a single fraction, or 3 consecutive doses of 10 Gy each (10 Gy X 3, total dose 30 Gy), which we previously demonstrated to mediate differential local control of the disease, with the latter approach being more efficient. Results. We observed a trend for reduction in splenic macrophage activation and abundance of B cells, T cells and NKT cells amongst tumors relapsing after single-dose vs fractionated RT, alongside an increased abundance of immunosuppressive TREG cells, elevated markers of exhaustion in both CD4+ and CD8+ cells and limited responsiveness to ex vivo stimulation in terms of TNFalpha secretion. Moreover, mice with M/D-driven tumors relapsing after single-dose RT exhibited a more pronounced secretion of IL17 by splenic CD8+ cells, which has been previously associated with immunosuppression in the BC microenvironment (Chabab, Barjon et al. 2020). As compared to control tumor-naïve mice, the splenocytes of mice with M/D-driven tumors relapsing after fractionated RT were sub-efficient at responding to ex vivo stimulation with cytokine and effector molecules, potentially linked to disease progression. DNAseq and RNAseq data are being analyzed and results will be available shortly. Impact: Identifying immunological correlates of relapse after RT can direct strategies to overcome resistance in HR+ BC patients, the majority of BC patients. If successful, this can inform therapeutic approaches to enable superior therapeutic responses in patients with HR+ BC, hence significantly reducing BC-related deaths.

Citation Format: Norma Bloy, Aitziber Buque, Giulia Petroni, Takahiro Yamazaki, Ai Sato, Bhavneet Bhinder, Olivier Elemento, Silvia C. Formenti, Lorenzo Galluzzi. Immunological characterization of mouse HR+ mammary tumors relapsing after radiation therapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-036.