Abstract PS4-37: A pro-tumorigenic mechanism of M2 tumor-associated macrophages (TAM) in triple-negative breast cancer
Authors: Korlimarla Aruna , Prabhu Jyothi , Ragulan Chantirika , Shivakumar Gnanapriya , Desai Krisha , Cheang Maggie , BS Srinath , Diwakar Ravi , Apachu Sandhya , Kumar Rekha , Sridhar TS , RajaRajan Savitha , Kaluve Rohini , Alexander Annie , Sadanandam Anguraj 
Published: February 2021

Introduction:Triple-Negative Breast Cancer (TNBC) comprises approximately 30% of all breast cancers in Indian women. Given their aggressive nature, TNBCs have high rates of systemic metastasis and mortality with only chemotherapy available for treatment. The success of immunotherapy in solid tumors has raised the hope for their utility in TNBCs as well. But only a subset of patients have a clinical response to check-point inhibitors. The cellular and molecular mechanisms that mediate the immunological response or tolerance are just beginning to be understood. Compared to ER/PR+ breast cancer, TNBC features a unique tumor microenvironment (TME) characterized by a large number of tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). The density of TILs in and of themselves do not accurately predict response to neoadjuvant chemotherapy or survival. M2 tumor-associated macrophages (M2-TAMs) have been reported to associate with solid tumors to facilitate epithelial to mesenchymal transition (EMT), tumor invasiveness, metastasis, and resistance to therapy. In this study, we have characterized presence of M2-TAM in the TNBC immune environment by examining expression of several biomarkers. Methods:Surgically excised tumor specimens from 88 Indian women with TNBC were accessed from the longitudinal observational series of SJNAHS tissue bank under IERB approved protocols and assayed on nCounter® PanCancer Immune Profiling Panel which comprised of 740 genes and characterises 14 different immune cell types. Transcriptome analysis using Non-negative Matrix factorization (NMF) based unsupervised clustering was done to arrive at stable subtypes characterized by different tumour microenvironments within TNBC. CIEBERSORT tool was used to analyse for distribution of cell types. Identification of macrophages was done by Immunohistochemistry (IHC) for CD68 and CD163 markers in TNBCs and a control group of ER+HER2-.Results:NMF analysis with an enriched immune gene signature of 111 genes, yielded 3 subtypes (ST) (37%, 27% and 36% with relative proportions of ST1, ST2 and ST3) within the TNBC. The three subtypes had distinct survival patterns with ST1 having the best prognosis with enriched TH1 gene signature and ST3 had poorest (log rank p=0.5). On application of this gene signature to TNBC groups in METABRIC and TCGA data, similar pattern emerged with a significant survival pattern of ST 3 having the poorest outcome (p=0.005). A closer examination of ST 3 revealed a signature enriched for M2 macrophages also known as TAM. Immunohistochemistry for Pan macrophage marker CD68 and an M2 specific marker CD163 between TNBC and ER/PR+ revealed difference in the two groups was significantly different (Mann Whitney p=0.03), with TNBC exhibiting 2.4 fold higher expression of CD163. CD 68, was enriched in ST2 and ST3. CD163 which is an M2 specific marker was highest in ST3 as compared to ST 1& 2 (p=0.04). This group also correlated with signal molecules secreted by macrophages containing growth factors, cytokines and chemokines, such as TGF-β, VEGF, IL-10 and CXCL and interleukins like IL4 and IL6 suggestive of TAM recruitment and polarization. It is likely that the TAM enriched subgroup is likely to be unresponsive to PD1/PDL1 inhibitors but could be targeted by novel therapeutic strategies to directly target M2-TAM. In vitro and In vivo analysis to target M2-TAM to evaluate the response to these therapeutic compounds in cell lines as well as a mouse syngeneic model is underway.Conclusions and future directions: As a cell type within the tumor microenvironment, that promotes invasion, M2-TAMs makes an ideal therapeutic target in TNBC. In addition, this sub-type lends itself to easy identification by a simple IHC assay.

Citation Format: Aruna Korlimarla, Jyothi Prabhu, Chantirika Ragulan, Gnanapriya Shivakumar, Krisha Desai, Maggie Cheang, Srinath BS, Ravi Diwakar, Sandhya Apachu, Rekha Kumar, TS Sridhar, Savitha RajaRajan, Rohini Kaluve, Annie Alexander, Anguraj Sadanandam. A pro-tumorigenic mechanism of M2 tumor-associated macrophages (TAM) in triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-37.