Abstract PS03-04: Tumor immune microenvironment modulates resistance to estrogen suppression in ER+ breast cancer
Authors: Napolitano Fabiana , Wang Yunguan , Sudhan Dhivya , Gonzalez-Ericsson Paula , Formisano Luigi , Guo Lei , Chica-Parrado M Rosario , Lin Chang-Ching , Lee Kyung-Min , Ma Hongli , Evans Nathaniel , Servetto Alberto , Mendiratta Saurabh , Barnes Spencer , Fang Yisheng , Xu Lin , Balko Justin , Mills Gordon , Labrie Marilyne , Hanker Ariella , Arteaga Carlos 
Published: May 2024

Abstract
Despite major advances in the treatment of estrogen receptor positive (ER+) breast cancer (BC), advanced disease continues to be the main cause of death from this disease. The role of the tumor immune microenvironment (TIME) in the progression of ER+ BC and its response to treatment is not completely understood. The aim of this study was to elucidate the role the TIME in the response of ER+ tumors to estrogen deprivation (ED).
We collected samples from 215 postmenopausal patients with stage I-III ER+ BC, treated with letrozole for 2-4 weeks, to induce ED. We used AQUA on pre-treatment biopsies and on-treatment surgical biopsies to assess ER, PR, HER2, and Ki67. Then, we categorized the patients’ response to ED based on the Ki67 score in on-treatment samples: ED-sensitive (ED-S) if natural log (ln) of the Ki67 score ≤1.0 or ≤2.7% Ki67+ cells vs. ED-resistant (ED-R) if ln ≥2.0 or ≥7.4% Ki67+ cells.
Firstly, we assessed TIME composition by investigating stromal tumor-infiltrating lymphocytes (sTILs) in H&E-stained FFPE of on-treatment tumor sections. ED-R tumors exhibited a significantly higher stromal TILs score (p=0.0001) relative to ED-S tumors. We next prepared tissue microarrays from 227 (on-treatment) surgical sections and subjected them to cyclic immunofluorescence (CycIF) with 38 antibodies to examine their intra-tumoral immune cell infiltration. From each tumor core, we segmented single cells and labeled them as immune, cancer, or stromal cells based on expression of a set of markers. Briefly, cells that stained strongly for CD45 (leukocyte marker), and/or CD4 (T lymphocyte marker), or CD68 (macrophage marker) were labeled immune cells. CD45/CD4/CD68-negative cells were categorized as tumor if E-cadherin and/or cytokeratin-positive, or stromal cells if -negative.
Next, we assessed cell specific spatial enrichment by quantifying the expression of immune markers in the area immediately adjacent to each tumor cell. Immune-suppressive T-reg (FOXP3+) cells were enriched in the ED-S tumors (p=0.0004), as well as PD1+ (exhausted) T cells (p=0.0004), and CD68+ cells (p &lt; 0.0001) compared to ED-R tumors. ED-R tumors exhibited higher CD20+ B cells (p &lt; 0.0001), higher CD8+ T cells (p=0.0329), in addition to higher CD45+ cells (p &lt; 0.0001), compared to ED-S tumors. RNA-sequencing of the same surgical samples showed a higher T cells cytolytic score in ED-R relative to ED-S (p=0.0058), suggesting enhanced CD8+ T cells activity, in addition to their higher infiltration. We are currently analyzing letrozole-induced changes in TIME composition using Geomix digital spatial profiler in paired pre- and on-treatment biopsies from ED-R and ED-S tumors and will be presented at the meeting.
Consistent with the CycIF findings, GSEA of hallmark gene signatures from bulk RNA-sequencing of treated tumors revealed that immune-related gene sets, such as “IFN α response”, “IFNɣ response”, and “allograft rejection” were upregulated in ED-R vs. ED-S cancers. ED-R tumors showed enrichment of CXCL9, CXCL10, and CXCL11 chemokines and their receptor, CXCR3. Publicly available datasets of patients with ER+ breast cancers showed that higher expression CXCL9 (HR 1.36; p=0.016), CXCL10 (HR 1.71; p&lt; 0.0001), and CXCL11 (HR 1.5; p=0.0016) are predictive of shorter relapse-free survival on antiestrogen therapy. We are currently investigating whether these chemokines play a causal role in resistance to ED, and if this is phenocopied by co-cultures of ER+ BC cells and CD8+ T-cells.
Conclusions
ED-resistant tumors are enriched with stromal TILs and exhibit higher immune cell intra-tumoral infiltration and CD8+T cells cytolytic activity compared to ER+ tumors sensitive to estrogen suppression. In contrast, ED-S tumors showed a more immunosuppressed milieu. The role of CXCL9, CXCL10 and CXCL11 in inducing resistance to ED warrants further investigation.
Citation Format: Fabiana Napolitano, Yunguan Wang, Dhivya Sudhan, Paula Gonzalez-Ericsson, Luigi Formisano, Lei Guo, M Rosario Chica-Parrado, Chang-Ching Lin, Kyung-Min Lee, Hongli Ma, Nathaniel Evans, Alberto Servetto, Saurabh Mendiratta, Spencer Barnes, Yisheng Fang, Lin Xu, Justin Balko, Gordon Mills, Marilyne Labrie, Ariella Hanker, Carlos Arteaga. Tumor immune microenvironment modulates resistance to estrogen suppression in ER+ breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS03-04.