The Immune Microenvironment of Breast Ductal Carcinoma in Situ

Elizabeth Thompson; Janis M Taube; Hillary Elwood; Rajni Sharma; Alan Meeker; Hind Nassar Warzecha; Pedram Argani; Ashley Cimino-Mathews; Leisha A Emens


Mod Pathol. 2016;29(3):249-258. 

In This Article

Abstract and Introduction


The host immune response has a key role in breast cancer progression and response to therapy. However, relative to primary invasive breast cancers, the immune milieu of breast ductal carcinoma in situ (DCIS) is less understood. Here, we profile tumor infiltrating lymphocytes and expression of the immune checkpoint ligand programmed death ligand 1 (PD-L1) in 27 cases of DCIS with known estrogen receptor (ER), progesterone receptor, and human epidermal growth factor 2 (HER-2) expression using tissue microarrays. Twenty-four cases were pure DCIS and three had associated invasive ductal carcinoma. Tumors were stained by immunohistochemistry for PD-L1, as well as the lymphocyte markers CD3, CD4, CD8, FoxP3, and CD20. The expression of PD-L1 by DCIS carcinoma cells and tumor infiltrating lymphocytes was determined, and the average tumor infiltrating lymphocytes per high power field were manually scored. None of the DCIS cells expressed PD-L1, but 81% of DCIS lesions contained PD-L1+ tumor infiltrating lymphocytes. DCIS with moderate-diffuse tumor infiltrating lymphocytes was more likely to have PD-L1+ tumor infiltrating lymphocytes (P=0.004). Tumor infiltrating lymphocytes with high levels of PD-L1 expression (>50% cells) were seen only in triple-negative DCIS (P=0.0008), and PD-L1−tumor infiltrating lymphocytes were seen only in ER+/HER-2−DCIS (P=0.12). The presence of PD-L1+ tumor infiltrating lymphocytes was associated with a younger mean patient age (P=0.01). Further characterization of the DCIS immune microenvironment may identify useful targets for immune-based therapy and breast cancer prevention.


Data from both laboratory studies and clinical trials support an important role for the host immune response in breast cancer progression and in patient response to therapy.[1,2] Tumor infiltrating lymphocytes, both within tumor cell nests and in tumor-associated stroma, are associated with improved patient outcomes. In treatment naive triple-negative breast carcinomas, tumor infiltrating lymphocytes are an independent prognostic factor for improved survival,[3,4] decreased distant recurrence,[4,5] and increased time to metastasis.[6] The presence of tumor infiltrating lymphocytes in triple-negative breast carcinomas post-neoadjuvant therapy is also prognostic, predicting longer metastasis-free and overall survival.[3] In addition, the presence of tumor infiltrating lymphocytes predicts response to therapy, as larger numbers of tumor infiltrating lymphocytes correlate with better responses to chemotherapy in triple-negative breast carcinomas and estrogen receptor (ER)-negative breast cancer,[1,2] and to trastuzumab-based chemotherapy in human epidermal growth factor receptor-2 (HER-2)-positive breast carcinomas.[5] Multiple tumor infiltrating lymphocyte subsets, including CD8+ cytotoxic T cells, CD4+ helper T cells, and CD20+ B cells, predict patient survival across the breast carcinoma subtypes.[1,2] In contrast, high numbers of FoxP3+ regulatory T cells (Tregs) relative to CD8+ T cells predict decreased progression-free survival and overall survival[7] in breast carcinoma patients.

Gene expression profiling defined five major molecular subtypes of invasive primary breast carcinomas with unique clinicopathologic characteristics and outcomes: luminal A, luminal B, HER-2-enriched (HER-2+), basal-like, and normal breast-like.[8] These molecular subtypes have immunohistochemical correlates, where luminal A cancers are ER+ progesterone receptor (PR)+HER-2Ki67low, luminal B cancers are ER+PR+HER-2Ki67high or ER+PR+HER-2+, HER-2+ cancers are ERPRHER-2+, and basal-like carcinomas are typically ERPRHER-2 (triple-negative breast carcinoma) with cytokeratin (CK)5/6 or epidermal growth factor (EGFR) expression.[9] The luminal A phenotype has an improved prognosis relative to luminal B, HER-2+, or basal-like carcinomas.[9] These same gene expression profiles and immunohistochemical phenotypes are observed in ductal carcinoma in situ (DCIS), the only established precursor to invasive primary breast carcinomas.[10]

Although HER-2+ breast cancers and triple-negative breast carcinomas have a poor prognosis relative to other subtypes, they typically have more vigorous tumor lymphocyte infiltrates. Larger numbers of tumor infiltrating lymphocytes are seen in ER relative to ER+ carcinomas.[11] In addition, genetic correlates of the immune response predict better survival in ER and HER-2+ carcinomas, but not ER+ carcinomas.[12] Although robust immune infiltrates are associated with ER and HER-2+ carcinomas, evidence suggests functional skewing toward a pro-tumorigenic phenotype. High levels of Treg are more common in ER carcinomas,[13] and predict shorter progression-free and overall survival.[7] In addition, relative to luminal A cancers, triple-negative breast carcinomas tend to harbor tumor infiltrating lymphocytes with the T helper type 2 phenotype thought to promote tumor growth.[14]

Growing evidence supports a major role for immune checkpoint pathways, such as those mediated by interactions between programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1), in regulating antitumor responses.[1,2] PD-1 (B7-1) is upregulated following the activation of lymphocytes and belongs to the B7-CD28 family. Its primary ligands are PD-L1 and PD-L2, which are inducible by IFN-: and other inflammatory cytokines on the surface of tumor cells and tumor infiltrating T cells, B cells, macrophages, and dendritic cells.[15] The interaction of PD-1 with PD-L1/2 inhibits T-cell activation and proliferation.[16] PD-L1 may also be constitutively expressed on tumor cells as a result of oncogenic signaling or epithelial-to-mesenchymal transition.[17,18] Tumor cells may also counter an active antitumor immune response by upregulating PD-L1 expression through a process known as adaptive immune resistance.[19] PD-L1 is expressed on the tumor cells of invasive breast carcinomas and their associated tumor infiltrating lymphocytes[17,18,20] and correlates with lack of ER expression, increased numbers of tumor infiltrating lymphocytes, response to chemotherapy, and the triple-negative phenotype.[21] The role of PD-L1 expression as a prognostic factor in breast carcinoma remains unclear. In one study, PD-L1 expression was associated with improved clinical outcomes;[11] however, a separate study reported an association with negative clinical outcomes.[22]

We previously reported differential patterns of tumor infiltrating lymphocytes across matched primary and metastatic breast carcinomas,[23] and in invasive primary breast carcinomas with associated DCIS.[24] Our data suggest that tumor immunobiology evolves as tumors progress. Little is known about the immune milieu of DCIS or how the antitumor immune response evolves as breast tumors progress from in situ to invasive and then metastatic lesions. Here, we profile the composition of tumor infiltrating lymphocytes and PD-L1 expression in 27 cases of DCIS with known ER, PR, and HER-2 expression.