Abstract PD1-5: Characterizing the Tumor Immune MicroEnvironment (TIME) in high-risk ductal carcinoma in situ
Authors: Campbell Michael J , Mukhtar Rita , Obi-Okoye Ekene , Han Booyeon , Tandon Vickram , Zheng Sarah , Zhu Zelos , Endicott Max , Wicha Max , Lindstrom Linda , Au Alfred , Baehner Frederick , Gray Joe , Esserman Laura 
Published: May 2015

Abstract
Background: Ductal carcinoma in situ (DCIS) of the breast is a premalignant condition. Although DCIS is treated as an obligate precursor of invasive ductal carcinoma, the rate and latency of progression from DCIS to invasive breast cancer (IBC) in the absence of treatment are unknown. DCIS is not one condition, but rather a spectrum of disease and although DCIS itself is not a lethal condition, women with DCIS are at higher risk of developing subsequent IBC over a time period of 1-20 years depending on DCIS subtype. Features of DCIS that are associated with high risk of recurrence include large size (&gt; 5cm), high grade, comedo necrosis, palpable mass, hormone receptor (HR) negativity, and HER2 positivity. The objective of this study was to characterize the tumor immune microenvironment (TIME) of these high-risk DCIS lesions.
Methods: Forty-eight cases of high grade DCIS, enriched for large, confluent lesions and history of recurrence were age matched with 64 cases of non-high grade DCIS. IHC analyses were performed as single or two-color stains for the following antigens: CD68, CD8, CD4, CD20, HLA-DR, CD115, FoxP3, PCNA, Mac387, MRC1, ALDH, CD24, CD44, Ki-67, and HER2. HR status was determined from ER and PR staining results in pathology reports. A Nuance multispectral imaging system was used to image and spectrally unmix each stain. Protocols for automated image analysis were developed using CellProfiler software. Associations between immune cell populations and clinical parameters (tumor palpability, recurrence, HR status, HER2 status, and Van Nuys score [12-point scale: margins, age, size, grade]) were identified with non-parametric Spearman correlation tests.
Results: We found a high macrophage infiltrate associated with a high Van Nuys score, palpability, and high Ki-67. High CD115 (CSF-1 receptor) was associated with HER2+, high Ki-67, and recurrence. Mac387+ cells and FoxP3+ regulatory T cells (Treg) were associated with high Van Nuys score, comedo necrosis, high Ki-67, HR- and HER2+. Interestingly, both Mac387 and CD115 were expressed on tumor cells as well as macrophages and high CD115 staining on tumor cells was associated with recurrence. The presence of CD8+HLA-DR-negative T cells throughout a section was associated with high Van Nuys score, HR-, HER2+, and recurrence. In contrast, CD8+ T cells within the nests of tumor cells were negatively associated with Van Nuys score, palpability, and comedo necrosis. A tumor immune microenvironment score (TIME score) was developed based on the proportions of various immune cell populations. A high TIME score was significantly associated with high Van Nuys scores as well as with recurrence.
Summary: These results demonstrate that high risk DCIS features (palpability, high Van Nuys score, high proliferation, HR-, HER2+, and increased recurrence) are associated with a suppressed tumor immune micro-environment (high FoxP3+ cells, CD68+Mac387+ cells, CD8+HLA-DR-neg T cells, and upregulated CD115). These high risk lesions truly represent an opportunity to prevent cancer. Identifying these high risk lesions with the help of tumor immune microenvironment markers and manipulating the DCIS TIME via local or systemic immunotherapeutic strategies may represent an ideal preventative intervention.
Citation Format: Michael J Campbell, Rita Mukhtar, Ekene Obi-Okoye, Booyeon Han, Vickram Tandon, Sarah Zheng, Zelos Zhu, Max Endicott, Max Wicha, Linda Lindstrom, Alfred Au, Frederick Baehner, Joe Gray, Laura Esserman. Characterizing the Tumor Immune MicroEnvironment (TIME) in high-risk ductal carcinoma in situ [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD1-5.