Background: Breast cancer (BC) evolution is influenced by tumor microenvironment. Presence of CD8+ cytotoxic T lymphocytes (TILs) has been proposed as surrogate marker of adaptive immune response, and programmed death ligand-1 (PD-L1) is a negative regulator of the tumor immune microenvironment. However, whether PD-L1 expression adversely affects breast cancer outcome is unknown (Oncotarget 2014; 6:5449). Tumor-associated macrophages (TAMs) in the tumor microenvironment may contribute to BC progression and metastagenicity. We assessed the potential correlations between PD-L1 expression, the presence of TAMs and TILs, and BC outcomes. Methods: 59 primary BCs (16 HR+, 16 HER2+, and 27 TNBC) with known clinical and pathological features and patient (pt) follow-up for a median of 3.9 years were evaluated by immunohistochemistry for expression of CD8, CD68, and PD-L1 within tumor and stroma. The average number of CD8+ cells within 10 high power fields was determined separately for invasive tumor cell nests and for stroma within each sample, and the median number of CD8+ cells within tumor vs stroma was calculated (Breast Cancer Res Treat 2011 128:703–711). Non-lymphocyte mononuclear cells in tumor and stroma were used in counting CD68+ TAMs. BCs were positive for CD8+ TILs (Cell Marque clone #C8144B) or CD68+ TAMs (Cell Marque clone #KP1) if the number of cells positive in the sample was greater than the median. PD-L1 (Dako 28-8 pharmDx) was positive if at least 1% of tumor cells expressed PD-L1. The log-rank was used to compare the survival and progression free survival between groups and Spearman's rank-order correlation tests were conducted to determine associations between CD8, CD68, and PD-L1. Results: 57% of TN, 26% of HER2+ and 13% of HR+ BCs expressed PD-L1 in tumor. TNBC pts received anthracycline/ taxane chemotherapy (62%) or taxane therapy alone (22%). HR+ and HER2+ pts received standard endocrine therapy and trastuzumab-based therapy. Stromal CD8+ TILs were associated with improved OS in the overall population (log rank p=0.026). In TNBC, PD-L1 expression was positively correlated with the presence of TILs (p=0.0002) and TAMs (p=0.0005) as well as with improved 3 year PFS and OS (log rank p=0.04 and p=0.03, respectively). Furthermore, stromal CD8+ TILs and stromal CD68+ TAMs correlated positively with each other in the TNBC group (p=0.0094). Conclusions: PD-L1 expression correlated with TILs and TAMs in TNBC and was associated with a favorable outcome. PD-L1+ TNBCs with high levels of TILs and TAMs may be primed for exceptional response to immunogenic chemotherapy alone. Whether some pts with PD-L1+ TNBCs with high TILs/TAMs will benefit additionally from an anti-PD-L1/PD-1 agent is being investigated currently. Citation Format: Mardones MA, Grosser D, Levin MK, Daoud Y, Palucka K, O'Shaughnessy J, Osborne C. PD-L1 expression in triple negative breast cancer (TNBC) is associated with improved outcomes [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-20.

BACKGROUND: Women with TNBC who do not achieve a pathologic complete response (pCR) with preoperative (preop) chemotherapy have a high risk of recurrence and death from BC. Immunotherapy is an attractive strategy as human BCs can be immunogenic, and enhancing the immune effector function may augment the cytotoxic effects of standard therapies. CLINICAL TRIAL: Following IRB-approved informed consent, 10 pts with locally advanced TNBC received preop dose-dense doxorubicin/cyclophosphamide (AC) followed by paclitaxel and carboplatin (TCb) chemotherapy, combined with antigen-loaded (TNBC antigens: Cyclin B1, WT1, and control viral antigens: CEF) autologous monocyte-derived DC vaccinations administered intratumorally and subcutaneously. DCs were generated with GM-CSF and type I interferon, loaded with antigen in the form of long peptides and activated with innate ligands (LPS and Clo75) and CD40 ligand. Vaccines were given at 4 time points prior to definitive surgery, and 3 times post-surgery, pre- and post-radiation therapy (RT). Safety was the primary study endpoint, and pCR rate in breast and axilla was a secondary endpoint. Correlative studies included assessment of immune response via ELISpot and transcriptional profiling of blood samples collected over time. RESULTS: All pts received the 4 vaccines during preop chemotherapy, and 7/10 received all 7 vaccines. At the time of definitive surgery, 4 pts achieved a pCR, 3 pts had macroscopic residual disease in the breast and axillary lymph nodes, and 3 pts had residual cancer burden scores of 1. As of June 1, 2017, all pts have been in follow-up for at least 1 year s/p completion of all vaccines, and 7/10 patients have no evidence of disease. To assess immune signatures with IFN-γ-ELISpot, PBMCs from baseline (BL) and several time points during vaccine treatment were cultured with control peptides or with peptide libraries covering vaccine antigens. Using a linear mixed model to account for repeated and missing observations we found statistically significant (α = 0.05) increases in Cyclin B1, WT1, and CEF ELISpots in at least 1 time point post-DC vaccination and in follow-up. Compared to BL, Cyclin B1 and WT1 increased at 3 day pre-RT in 8/10 and 7/10 pts, respectively. To assess transcriptional signatures, a linear mixed model was utilized to determine statistically significant differences in fold-change over time compared to the BL and healthy controls. Modular analysis of differentially expressed transcripts at BL revealed downregulation of transcripts related to the monocyte lineage in 7/10 pts. Longitudinal analysis revealed profound transcriptional changes during AC with downregulation of lymphocyte modules and upregulation of innate and inflammation modules. While the latter ones have normalized during TCb and follow-up, T cell module remained substantially downregulated throughout treatment and follow-up. CONCLUSIONS: Combination of preop chemotherapy and intratumoral and subcutaneous autologous DC vaccination is safe in locally advanced TNBC pts and is linked with profound changes in immune transcription signatures and with expansion of antigen-specific immune responses that can be detected in IFN-γ ELISpot. Citation Format: Palucka AK, Roberts LK, Zurawski SM, Tarnowski J, Turner J, Wang X, Blankenship D, Smith JL, Levin MK, Finholt JP, Burkeholder SB, Timis R, Muniz LS, Dao T, Grant M, Banchereau J, Zurawski G, Pascual V, O'Shaughnessy JA. Immune and transcriptional signatures of dendritic dell (DC) vaccination combined with chemotherapy in locally advanced, triple-negative breast cancer (TNBC) patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-01.