Abstract 3137: Towards analysis of the immune microenvironment in ductal carcinoma in situ
Authors: Almekinders Mathilde M. , Visser Lindy L. , Thijssen Bram , Kristel Petra , Linden Rianne van der , Broeks Annegien , Hooijberg Erik , Visser Karin de , Lips Esther H. , Wesseling Jelle 
Published: August 2018

Abstract
Introduction
Since the introduction of population-based mammographic screening, the incidence of ductal carcinoma in situ (DCIS) increased manifold. DCIS lesions are non-obligate precursors to invasive breast cancer, because only a minority of DCIS patients later develops invasive breast cancer. DCIS patients are treated intensively with surgery, frequently supplemented by radiotherapy and/or endocrine treatment. However, treatment of DCIS lesions did not result in a decreased incidence of advanced stages of breast cancer, suggesting overdiagnosis and hence overtreatment exists. Because the immune microenvironment plays an important role in cancer progression, we performed a pilot study to assess the amount, composition and spatial distribution of immune cells aiming at the identification of biomarkers that distinguish aggressive from indolent DCIS.
Methods
A representative series of 32 paraffin-embedded DCIS lesions was studied with multispectral immunohistochemical imaging, providing simultaneous detection and quantification of CD20+ B-cells, CD8+ T-cells, CD4+ T-cells, CD4+Foxp3+ regulatory T-cells, CD68+ macrophages and pankeratin. Cellular density of immune cell subsets per tissue compartment and spatial distribution was analyzed by Inform software, SPSS and R. The number of CD4+FoxP3+ T-cells within 30µm of a CD8+ T-cell was assessed and expressed in a CD4+FoxP3+ T-cell per CD8+ T-cell ratio. Immune cell density and composition were correlated to grade and immunohistochemical ER, Her2 and p53 status.
Results
Multispectral immunohistochemical quantification showed a range of 30 to 2100 lymphocytes/mm2 in the stroma of DCIS lesions. High grade positively correlated with higher number of stromal lymphocytes/mm2 (p&lt;0.01). Negative ER status, positive Her2 status and aberrantly expressed p53 was significantly associated with higher number of stromal lymphocytes/mm2, CD8+ T-cells/mm2, CD4+FoxP3+ regulatory T-cells/mm2 and CD20+ B-cells/mm2 (p&lt;0.05). Within the DCIS-epithelium, the number of CD4+FoxP3+ regulatory T-cells positively correlated with negative ER-status (p=0.02) and positive Her2 status (p=0.03). The spatial distribution of the number of CD4+Foxp3+ T-cells within 30 μm of a CD8+ T-cell (expressed in a Treg per CD8+T-cell ratio) varied from 0 to 0.23 in the stromal compartment and from 0 to 0.60 in the DCIS compartment.
Conclusions
Within the immune microenvironment, CD20+ B-cells, CD8+ T-cells, CD4+ T-cells, CD4+Foxp3+ regulatory T-cells and CD68+ macrophages were successfully and simultaneously detected. Stromal lymphocyte density and CD8+ T-cell, CD4+ T-cell, CD4+FoxP3+ regulatory T-cell and CD20+ B-cell density positively correlated with negative ER status, positive Her2 status and aberrant expression of p53. The next step will be to analyze this multiplex panel in our nationwide DCIS cohort (1989-2005, median follow-up 12.0 years) for correlation with clinical outcome.
Citation Format: Mathilde M. Almekinders, Lindy L. Visser, Bram Thijssen, Petra Kristel, Rianne van der Linden, Annegien Broeks, Erik Hooijberg, Karin de Visser, Esther H. Lips, Jelle Wesseling. Towards analysis of the immune microenvironment in ductal carcinoma in situ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3137.