Dynamic evaluation of the immune infiltrate and immune function genes as predictive markers for neoadjuvant chemotherapy in hormone receptor positive, HER2 negative breast cancer.

Gene expression (GE) signatures and Tumor Infiltrating Lymphocytes (TIL) enumeration are predictive for response to neoadjuvant chemotherapy in HR- and in HER2+ breast cancer, but data are conflicting in HR+/HER2- disease. This study aimed to explore their predictive value in this subset, measured both at baseline and after short exposure to chemotherapy. Specifically, the PROMIX phase 2 trial enrolled patients with locally advanced HER2- BC to receive six cycles of epirubicin and docetaxel, plus bevacizumab during cycles 3-6. Patients underwent tumor biopsies at baseline and after cycle 2 for GE profiling and enumeration of TIL, FOXP3+ T-cells and CD163+ macrophages. An immune related gene module and the quantification of the immune infiltrate were analyzed for association with pathologic complete response (pCR), decrease in tumor size and disease-free survival (DFS). Of the 150 patients enrolled in PROMIX, 113 were HR+/HER2-. Baseline GE and immune cell enumeration data were available from 71 patients, while data after 2 cycles of chemotherapy were available from 41. At baseline, only GE was statistically significantly associated with higher pCR rates (OR 2.29, 95% CI 1.05 - 5.38, p = 0.037) and decrease in tumor size (r = 0.25, p = 0.047). In contrast, longitudinal data indicate that both GE (r = 0.54, p<0.001) and TIL abundance (p = 0.009) are stronger predictors for the reduction of tumor size, while low FOXP3+ was statistically significantly associated with an improved DFS (p = 0.027). In conclusion, GE analysis, TIL and FOXP3+ enumeration after short-term exposure to chemotherapy carry important predictive information in HR+/HER2- breast cancer at the neoadjuvant setting.

Effect of macrophages on breast cancer cell proliferation, and on expression of hormone receptors, uPAR and HER-2.

Malignant tumors, including breast cancers, are frequently infiltrated with innate immune cells and tumor-associated macrophages (TAMs) represent the major inflammatory component in stroma of many tumors. In this study, we examined the immunoreactivity of the macrophage markers CD68 and CD163 as well as the hormone receptors estrogen receptor α (ERα), progesterone receptor (PR), estrogen receptor ß1 (ERß1), human epidermal growth factor receptor 2 (HER-2), matrix metalloproteinase 9 (MMP­9), urokinase-type plasminogen activator receptor (uPAR) and the proliferations marker Ki67 in 17 breast cancer biopsies. The quantitative score for CD68+ and CD163+ strongly indicate M2 phenotype dominance in the currently investigated biopsies. We found that an increasing level of macrophages was negatively associated with ERα or PR, whereas a positive association was observed for Ki-67 or uPAR. No significant association could be seen between the level of macrophage and HER-2, ERß1 or MMP-9 expression. Effect of conditioned media (CM) generated from cultured human M1 and M2 macrophage phenotypes were investigated on the proliferation and expression of selected markers in the T47D breast cancer cell line. We found that in contrast to the in vivo situation, in particularly the CM from M1 macrophages decreased the growth and Ki67 expression in T47D, and significantly increased ERß1 mRNA levels. Moreover, in accordance to the in vivo situation the CM from the macrophages decreased the expression of ERα protein as well as ERα or PR mRNA. In conclusion our results show that macrophages alone have the capability to decrease the tumor cell expression of ERα and PR in vitro. In the tumor environment in vivo macrophages also contribute to an increase in tumor cell expression of uPAR and Ki67, suggesting that macrophages are involved in impairing the prognosis for breast cancer patients.