Immune checkpoint blockade (ICB) is currently approved for patients with triple-negative breast cancer (TNBC), whereas responses to ICB are also observed in a small subgroup of Estrogen Receptor (ER)-positive breast cancer. The cut-off for ER-positivity (≥1%) is based on likelihood of endocrine treatment response, but ER-positive breast cancer represents a very heterogeneous group. This raises the question whether selection based on ER-negativity should be revisited to select patients for ICB treatment in the context of clinical trials. Stromal tumor-infiltrating lymphocytes (sTILs) and other immune parameters are higher in TNBC compared to ER-positive breast cancer, but it is unknown whether lower ER levels are associated with more inflamed tumor microenvironments (TME). We collected a consecutive series of primary tumors from 173 HER2-negative breast cancer patients, enriched for tumors with ER expression between 1 and 99% and found levels of stromal TILs, CD8 + T cells, and PD-L1 positivity in breast tumors with ER 1–9% and ER 10–50% to be comparable to tumors with ER 0%. Expression of immune-related gene signatures in tumors with ER 1–9% and ER 10–50% was comparable to ER 0%, and higher than in tumors with ER 51–99% and ER 100%. Our results suggest that the immune landscape of ER low tumors (1–9%) and ER intermediate tumors (10–50%) mimic that of primary TNBC.

566 Background: Immune checkpoint blockade (ICB) is currently only approved for patients with triple-negative breast cancer (TNBC). However, the cut-off used for ER expression (<1% and in some countries <10%) has been developed as a biomarker for endocrine treatment response and not for selection for likelihood of response to ICB. While stromal tumor-infiltrating lymphocytes (sTILs) and PD-L1 expression are higher in TNBC compared to ER-positive tumors, the distribution of these and other key immune parameters in tumors with very low (1-9%), low (10-50%), intermediate (51-99%) and high (100%) ER levels is unknown. Methods: We collected a consecutive series of treatment-naïve tumor blocks of ER+/HER2- breast tumors diagnosed between 2010 and 2019. All available tumor blocks were used from the groups with ER expression between 1-9% and 10-50%. For the other groups, we randomly selected tumor blocks aiming for similar group sizes. This resulted in the following subgroups: ER 0% (n=46), 1-9% (n=17), 10-50% (n=22), 51-99% (n=37) and 100% (n=51). sTILs were scored using H&E slides. Immunohistochemistry was performed for CD8 and PD-L1 (22C3, scored on immune cells, cut-off ≥1%). Gene expression analysis was performed using the NanoString nCounter Breast Cancer 360 panel. Results: We found the highest levels of sTILs and stromal CD8+ cells in tumors with ER0% with comparable levels in tumors with ER1-9% and ER10-50% (Table). The proportion of PD-L1 positive tumors was 86% in tumors with ER0%, 81% in tumors with ER1-9%, 76% in tumors with ER 10-50% and 59% and 50% in tumors with ER51-99% and 100% respectively. As expected, a higher differentiation grade correlated with lower levels of ER expression. Differential gene expression demonstrated that expression of immune-related signatures, such as IDO1, antigen presenting machinery, CD8+ T cells and IFNγ, was comparable in tumors with ER1-50% as compared to ER0%, but statistically significantly higher as compared to tumors with ER100%. Conclusions: Our data suggest that breast tumors with low levels of ER expression (1-9%, 10-50%) comprise a separate entity within ER-positive breast cancer regarding their immune landscape. Here we show that not only tumors with very low ER levels (1-9%) mimic TNBC in terms of immune landscape but also that tumors with low ER levels (10-50%) might be more likely to respond to ICB than tumors with high levels of ER expression. [Table: see text]