Abstract D063: Intratumoral macrophage analysis and characterization of HER2+ pretreatment breast tumor biopsy using multiplex immunofluorescence
Authors: Gutierrez Emmaly D , Preece Christina , Stopeck Alison , Thompson Patricia 
Published: September 2020

Abstract
Background: Latinas in the US experience overall worse breast cancer (BC) survival than non-Latinas. Higher rates of aggressive BC subtypes including estrogen receptor negative(ER-) and HER2+ in Latinas are partly suspected. High intra-tumoral immune cells including myeloid suppressive M2 type macrophages (MAC) that suppress tumor immune responses are positively associated with poor outcomes and low response to therapy. Unclear is if tumor associated immune cells differ by race/ethnicity. Objective: To develop and evaluate the performance of a multiplex immunofluorescence (IF) methods to classify tumor-associated macrophages (TAM) as M1 and M2 in HER2+ breast tumors. Methods: Two panels of three antibodies were studied in HER2+ breast cancer biopsy specimens (n=19 patients). Two myeloid panels targeting CD80, CD68, CD163, CD206 with a nuclear (DAPI) and tumor cell (cytokeratin 8/18) was investigated. Banked breast tumor tissues were deparaffinized, and stained using indirect multiplex IF. Each tissue underwent multiple staining for CD206, CD68, and CD163 with stripping. A tyramide signal amplification IF (Perkin Elmer) was used in fluorescent labeling. Whole slide analysis image analysis was performed using HALO image analysis software. Results: All biopsy tissues yielded high quality IF profiles. Analyses per patient tumor (i.e., 5 regions of high tumor cell density containing a minimum of 5000 nucleated cells) revealed a fairly homogeneous MAC profile. In contrast, significant between patient heterogeneity in intratumoral M2 MAC density and differences by ER status was observed. In this sample, Her2+ BCs contained a high intratumoral TAM content – i.e., mean 27% (range &lt;1% to 70%) of cells in tumor dense regions were positive for the pan MAC marker CD68+. Further, intratumoral CD68+ cells (all MAC) were slightly higher in abundance in ER-/HER2+ tumors at 31% compared to 20% in ER+/HER2+ tumors. Further, the relative abundance of CD68+/CD206+ cells (M2 type) in tumor rich regions showed a high degree of variability between cases with a mean 7.1% of total cells (range 0 to 48%). Notable, the relative abundance of double CD68/CD206 cells was higher in the ER+ tumors at 8.8% (range 0 to 48%) compared to 3.3% in ER- that showed a narrow range of 0 to 8%. A similar pattern was observed for the CD68/CD163 double positive M2 type that included large heterogeneity between patients and higher abundance in ER+/HER2+ tumors. In contrast, while exhibiting high heterogeneity between patients, the triple positive CD68, CD163, CD206 cells population appeared to be proportionally higher in ER-/HER2 tumors. Ongoing work includes characterization of the M1 type MAC and their relative abundance in HER2+ tumors by ER status. Conclusion: These results demonstrate the feasibility of multiplex IF to characterize TAM subsets in patient derived BC tissues. Future efforts will include relating TAM to HER2 targeted therapy response rates and examination of differences in TAM profiles in patient populations by race/ethnicity.
Citation Format: Emmaly D Gutierrez, Christina Preece, Alison Stopeck, Patricia Thompson. Intratumoral macrophage analysis and characterization of HER2+ pretreatment breast tumor biopsy using multiplex immunofluorescence [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D063.