Breast cancer is the most common malignancy in Singapore women with rising incidence across all ethnic groups (Chinese, Malays and Indians). Ductal carcinoma in situ (DCIS) is the earliest non-invasive stage of disease and has been shown to account for approximately 26% of diagnoses in all women participating in the Breast-Screen Singapore program. Despite availability of breast screening, there are still Singapore women presenting with locally advanced breast cancer. The goal of the current study was to investigate pathway and immune cell heterogeneity in low, intermediate and high nuclear grade DCIS using a newly developed method for in situ hyperplexed analysis of multiple proteins in a single FFPE tissue (MultiOmyx). FFPE samples from patients (n= 15) diagnosed with DCIS were provided by Singapore General Hospital. Patients were of Chinese origin, ranged from 50-59 years, were all post-menopausal, and included low (n=5), intermediate (n=5) and high grade (n=5) samples. All histological diagnoses were reviewed by a single pathologist. Following a single antigen retrieval step, DAPI and cytokeratin staining was conducted and imaged at 10X. Based on DAPI, cytokeratin and autofluorescence, an H&E-like image was generated for each sample. Using this image, approximately 30 regions of interest (ROI) were selected per slide/patient, including normal and DCIS regions with and without immune cells. In total, 15 biomarkers were imaged following an iterative process of IF staining and dye inactivation. These included CD4 (T-cells), CD8 (T-cells), CD20 (B-cells), CD68 (macrophages), CD10 (myoepithelial cells), CD44v6 (cancer stem cell), Her2, Her4, EGFR (ErbB family proteins), pmTOR (cell growth), SLC7A5 (Mammostrat) and epithelium and cell segmentation markers (pan-cadherin, pan-cytokeratin, S6 and Na+K+ATPase). Images were evaluated for quality and processed to generate single cell quantification data for each marker in the epithelium and stroma. Single cell data was compared with clinical and histological features (grade, DCIS/ low immune cells, DCIS/ high immune cells). Cell-based k-means clustering was then performed for biomarkers in both the epithelial cells (Her4, Her2, panCK, SLC7A5, EGFR, NaKATPase, pmTOR, and CD10) and stromal cells (CD4, CD8, CD20, CD68). Using consensus clustering, the optimum number of clusters was found to be six in both the epithelium and stroma. No distinct clusters were associated with histological grade. Normal ROIs tended to be associated with higher CD10 and EGFR, moderate CD20 and low or negative for other markers. DCIS with low immune cells tended to have higher Her4, pmTOR and moderate CD68 while DCIS with high immune cells tended to have high Her2 and moderate to high CD4, CD20 and CD68. A larger study is needed to associate the findings with outcome. This technology provides a way to elucidate mechanisms of early disease and illustrates the complex inter-play between grade, pathway activation and immune response.

Citation Format: Nicole E LaPlante, Yunxia Sui, Michael J Gerdes, Sean Dinn, Rong Zhang, Sireesha Kaanumalle, Elizabeth McDonough, Christina Lowes, Craig Allred, Fiona Ginty, Thomas Foo, Puay-Hoon Tan. Demonstration of immune cell and pathway heterogeneity in Singapore DCIS samples using novel hyperplexing method (MultiOmyx®) [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 P2-04-06.