Single-cell heterogeneity in ductal carcinoma in situ of breast

• Type of Material: Article
• Authors: Michael J. Gerdes, GE Global Research; Yesim Polar, Emory University; Yunxia Sui, GE Global Research; Alberto Santamaria Pang, GE Global Research; Nicole LaPlante, Clarient NeoGen Labs; Adrian L. Harris, University of Oxford; Puay-Hoon Tan, Singapore General Hospital; Fiona Ginty, GE Global Research; Sunil Badve, Emory University
• Language: English
• Date: 2018-03-01
• Publisher: NATURE PUBLISHING GROUP
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2018 United States & Canadian Academy of Pathology.
• Final Published Version (URL): http://dx.doi.org/10.1038/modpathol.2017.143
• Title of Journal or Parent Work: MODERN PATHOLOGY
• Volume: 31
• Issue: 3
• Start Page: 406
• End Page: 417
• Grant/Funding Information: This study is supported by the National Cancer Institute of the National Institutes of Health under Award Number R01CA194600 to S Badve and M Gerdes. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.; Funding is through NCI - R01CA194600 to S. Badve and M. Gerdes and internal funds from General Electric to F. Ginty and M. Gerdes. In addition, S. Badve is supported by 100 Voices of Hope and Joshua Edwards Endowed Chair.
• Keywords: RECOMMENDATIONS; TUMOR-INFILTRATING LYMPHOCYTES; Science & Technology; Life Sciences & Biomedicine; IMMUNOHISTOCHEMISTRY; RISK; INTRATUMOR HETEROGENEITY; LOCAL RECURRENCE; CANCER; EXPRESSION; AMERICAN SOCIETY; CLINICAL ONCOLOGY/COLLEGE; Pathology

Abstract

Heterogeneous patterns of mutations and RNA expression have been well documented in invasive cancers. However, technological challenges have limited the ability to study heterogeneity of protein expression. This is particularly true for pre-invasive lesions such as ductal carcinoma in situ of the breast. Cell-level heterogeneity in ductal carcinoma in situ was analyzed in a single 5 μm tissue section using a multiplexed immunofluorescence analysis of 11 disease-related markers (EGFR, HER2, HER4, S6, pmTOR, CD44v6, SLC7A5 and CD10, CD4, CD8 and CD20, plus pan-cytokeratin, pan-cadherin, DAPI, and Na+K+ATPase for cell segmentation). Expression was quantified at cell level using a single-cell segmentation algorithm. K-means clustering was used to determine co-expression patterns of epithelial cell markers and immune markers. We document for the first time the presence of epithelial cell heterogeneity within ducts, between ducts and between patients with ductal carcinoma in situ. There was moderate heterogeneity in a distribution of eight clusters within each duct (average Shannon index 0.76; range 0-1.61). Furthermore, within each patient, the average Shannon index across all ducts ranged from 0.33 to 1.02 (s.d. 0.09-0.38). As the distribution of clusters within ducts was uneven, the analysis of eight ducts might be sufficient to represent all the clusters ie within- and between-duct heterogeneity. The pattern of epithelial cell clustering was associated with the presence and type of immune infiltrates, indicating a complex interaction between the epithelial tumor and immune system for each patient. This analysis also provides the first evidence that simultaneous analysis of both the epithelial and immune/stromal components might be necessary to understand the complex milieu in ductal carcinoma in situ lesions.