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


Here, we evaluate tumor infiltrating lymphocyte composition and PD-L1 expression in breast DCIS. Our cohort is primarily pure DCIS of nuclear grade 2–3, and our data reveal several characteristics of this cohort. First, tumor infiltrating lymphocytes are present within all cases of DCIS examined. A trend toward different patterns of tumor infiltrating lymphocyte distribution and CD8/Foxp3 ratios are seen in ER relative to ER+ DCIS and younger women relative to older women with DCIS. Second, while DCIS carcinoma cells do not express cell surface PD-L1 in this study, the majority of DCIS-associated tumor infiltrating lymphocytes are PD-L1+. The three DCIS cases with high PD-L1 expression on tumor infiltrating lymphocytes (>50% PD-L1+ tumor infiltrating lymphocytes) are triple negative and high nuclear grade. In three cases where DCIS was associated with concurrent infiltrating ductal carcinoma, no cell surface PD-L1 expression was seen on the carcinoma cells of either the in situ or invasive component. Although the conclusions are limited by the small number of cases with concurrent invasion, this finding is in agreement with our previous study showing concordance of carcinoma cell PD-L1 status by frank infiltrating ductal carcinoma and its associated DCIS.[24] Third, PD-L1 expression by tumor infiltrating lymphocytes is associated with a younger patient age. Fourth, PD-L1 expression by tumor infiltrating lymphocytes is associated with higher overall levels of tumor infiltrating lymphocytes within DCIS. Our findings suggest an active immune response within breast DCIS and support tumor infiltrating lymphocyte expression of PD-L1 as a marker of downregulation of the body's immune response within DCIS. The presence of PD-L1+ tumor infiltrating lymphocytes with in situ breast carcinoma suggests that investigation of immune-based therapies may be warranted even in pre-invasive disease.

Most studies of the immunobiology of breast carcinomas have focused on frankly invasive primary breast carcinomas, with little attention to immune infiltrates associated with in situ lesions. Studies in mouse models of breast cancer have identified robust CD8+ T-cell responses associated with hyperplastic, pre-neoplastic lesions.[28] Genomic analyses of DCIS and invasive breast cancers reveal activation of interleukin signaling pathway profiles, extracellular matrix pathways, and cell–cell adhesion pathways; activation of these pathways is absent or low in adjacent benign breast tissues. Furthermore, genomic profiling of DCIS shows patterns consistent with cytotoxic T-cell signaling, expression of chemokines known to recruit T cells, IFN-α signaling, and NOX4 activity, an oxygen-sensing NADPH oxidase related to the production of reactive oxygen species by granulocytes.[14] One study reported clusters of tumor infiltrating lymphocytes adjacent to involved ducts or in the interductal stroma, often associated with plump endothelial-lined vessels suggestive of high endothelial venule-like vasculature.[29] DC-based vaccination of DCIS patients also suggest that distinct DCIS phenotypes may have different immunogenicity at baseline, as vaccination produced more complete responses in ER DCIS than in ER+ DCIS.[30,31]

Our findings suggest that an active adaptive immune response may exist within DCIS. Nuclear grade 2–3 DCIS is associated with tumor infiltrating lymphocyte phenotypes similar to those seen in invasive breast carcinomas. This includes higher levels of tumor infiltrating lymphocytes in ER tumors,[11] higher levels of FoxP3+ Tregs in ER tumors,[13] strong expression of PD-L1 in ER tumors and triple-negative breast carcinomas,[11] and tumor infiltrating lymphocyte PD-L1 expression when higher overall levels of tumor infiltrating lymphocytes are present.[21] The two cases of DCIS that eventually recurred had large numbers of tumor infiltrating lymphocytes, but high levels of FoxP3+ Treg. Although the conclusions are limited by the small number of recurrences in this series, our data are consistent with prior reports that increased numbers of FoxP3+ Tregs in DCIS are associated with increased recurrence risk.[10] These data together suggest that even very early immune responses to breast DCIS may predict future disease behavior.

Our work also demonstrates that while DCIS tumor cells lack PD-L1 expression, the majority of tumor infiltrating lymphocytes associated with DCIS do express PD-L1. We have previously shown that the majority of tumor infiltrating lymphocytes associated with infiltrating ductal carcinoma also express PD-L1,[24] but that 21% primary infiltrating ductal carcinoma have PD-L1+ carcinoma cells. This dichotomy suggests there may be differences between the character of the immune response to in situ and invasive disease. Little is known about the evolution of the immune response and immunologic alterations within the tumor microenvironment along the continuum from in situ to invasive disease. It is not clear whether DCIS eventually develops expression of PD-L1 and gives rise to PD-L1+ invasive carcinoma, or if, alternatively, the presence of an invasive PD-L1+ tumor modulates the immune response within DCIS, leading to expression of PD-L1 by the DCIS tumor cells. If the infiltrating component does not generate a robust enough immune response, then insufficient IFN-γ may be generated to upregulate PD-L1 on the surface of DCIS cells. Studies are needed to elucidate the mechanisms behind the lack of PD-L1 expression by pure DCIS and the expression of PD-L1 by invasive tumors.

In addition, there is limited data on the spatial distribution of the immune response within either DCIS or invasive ductal carcinoma. In other tumor types, PD-L1 expression is often seen expressed primarily at the 'leading edge' of the tumor or at the tumor-stromal interface.[19] This supports the possibility that interactions between invasive tumors and the stromal/immune environment drive PD-L1 expression on tumor cells, which could then modulate the immune milieu of associated in situ lesions. In studies of melanoma, IFN-γ and other cytokines were shown to colocalize with tumor cells in PD-L1+ tumors, but not in PD-L1 tumors.[19,32] Such spatial distribution of inflammatory mediators could also contribute to differences in PD-L1 expression in DCIS and invasive breast carcinoma. Indeed, pure DCIS lesions are architecturally distinct from most invasive carcinomas because, while some DCIS are mass-forming lesions that spatially have a 'leading edge,' the majority of DCIS spread diffusely, segmentally, or irregularly through the breast ducts and lack a distinct 'leading edge.'

The expression of immune checkpoint molecules in DCIS identifies them as potential targets for DCIS therapy and secondary breast cancer prevention. DCIS can be responsive to immune-based therapy,[30,31,33] and PD-L1 antagonists are active in triple-negative breast carcinomas.[34,35] Although PD-L1 is not expressed on the surface of DCIS tumor cells in pure in situ lesions, the expression of PD-L1 by DCIS tumor infiltrating lymphocytes suggests that targeting PD-L1 is worth investigating as a treatment strategy for high-risk DCIS. ER and HER-2+ DCIS and DCIS in young women have the greatest numbers of tumor infiltrating lymphocytes. These patients may be ideal for testing immune-based breast cancer prevention strategies, and this may be particularly true for young women who have a higher DCIS CD8/FoxP3 ratio.

Additionally, we examine tumor infiltrating lymphocyte subsets and PD-L1 expression in DCIS classified into subtypes by ER, PR, and HER-2 status. Our study is limited by the small sample size and lack of nuclear grade 1 cases. In addition, the tissue microarray methodology limits analysis of the geography of tumor infiltrating lymphocytes and PD-L1 expression, including the 'leading edge' of tumors known to be immunologically important in invasive carcinomas.[36] However, 2–5 cores per tumor were taken to mitigate sampling limitations. Moreover, DCIS also tends to be more diffuse or segmental than invasive breast cancers, which tend to be mass forming, potentially limiting the importance of the 'leading edge' in purely in situ lesions. Finally, the tissue microarray methodology also limits our ability to detect lymphoid aggregates, which have been shown to be important in invasive breast carcinomas.[2]

In conclusion, we demonstrate differential patterns of tumor infiltrating lymphocyte infiltrates in distinct phenotypes of DCIS. Differences are seen relative to ER expression and patient age. Additional studies are needed to further characterize the association of immune parameters with the presence of concurrent infiltrating ductal carcinoma and eventual recurrence in DCIS. We previously reported differential patterns of tumor infiltrating lymphocytes across matched primary and metastatic breast carcinomas.[23] Our findings in breast DCIS are similar to patterns of tumor infiltrating lymphocytes in primary breast carcinomas, and distinct from patterns of tumor infiltrating lymphocytes in metastatic breast carcinomas. The presence of tumor infiltrating lymphocytes in DCIS, combined with the expression of PD-L1 on tumor infiltrating lymphocytes within the DCIS microenvironment, suggests the potential for immune-based therapies to treat DCIS. Further characterization of the DCIS immune microenvironment may yield additional targets for immune-based therapy and prevention of recurrence, and may help elucidate the role of the immune response in the evolution of breast cancer from in situ to invasive and ultimately to recurrent or metastatic disease.