Abstract P5-04-07: Multispectral imaging allows visualization and quantification of multiple immunologic cell types in breast tumor tissues
Authors: Mittendorf Elizabeth A , Wang Chichung , Yau Peng , Roman Kristin , Wu Yun , Alatrash Gheath , Hoyt Clifford 
Published: May 2015

Abstract
Background: Multiple studies have identified tumor infiltrating lymphocytes (TIL) in breast cancer and shown them to have prognostic and predictive significance. TIL are comprised of CD8+ and CD4+ T cells, regulatory T cells (Treg) and B cells. To date, the majority of studies have identified TIL using H&E staining. Studies utilizing IHC to stain for TIL generally have been limited to evaluating a single immune cell phenotype such as CD8+ T cells, while studies evaluating multiple immune cell types have generated single cell suspensions from tumors for analysis by flow cytometry to determine relative percentages of specific immune cell populations. Although these studies have confirmed that immune infiltrates in tumors are heterogeneous, they have not addressed the spatial relationship between tumor and immune cells, which could be critical to anti-tumor immune effects. This study was undertaken to demonstrate the feasibility of multispectral imaging and multiplexed immunofluorescence to visualize and quantify specific immune infiltrates in the tumor and surrounding stroma on single FFPE tissue sections.
Methods: Single FFPE slides from 9 HER2+ breast cancer patients receiving neoadjuvant chemotherapy were stained with primary antibodies targeting cytokeratin, CD8, CD4, FoxP3 (Treg), CD20 (B-cells) and PD-L1 (T cell inhibitory molecule). Tyramide signal amplification was used to improve signal and specificity, as well as to reduce background. The Vectra multispectral imaging instrument was used for image acquisition and InForm software was used to quantitate the density (number of cells per square millimeter) of specific cell types in the tumor and in the surrounding stroma.
Results: Multispectral imaging successfully captured and quantified multiple immune cell types in all tissues (images to be shown at time of presentation). CD8+ and CD4+ T cell densities as well as PD-L1 expression in the tumor and surrounding stroma are shown.
Summary of immune cell infiltrate Within Tumor Within Stroma SampleCD8 densityCD4 densityPDL1 H-scoreCD8 densityCD4 densityPDL1 H-score1050631301207342523059189238312260022111331104305083833106345020808206131751011891591110700417754621858511431739186812198591617448342570196
For patients not achieving a pathologic complete response (pCR), the density of both the CD8 (p=.03) and CD4 (p=.05) infiltrates in the stroma were significantly greater than in the tumor. For patients achieving a pCR, there was no significant difference in the densities of stromal and intratumoral CD8 (p=.11) or CD4 (p=.75) infiltrates suggesting that T cell infiltration into the tumor from the stroma is critical.
Conclusion: Multispectral imaging allows different immune cell phenotypes to be visualized and quantified simultaneously in the same tissue section enabling further study of the relationships and distribution of these cells within the tumor and tumor microenvironment, and their spatial distribution and proximity to the tumor cells. This technology will enable improved understanding of the immune infiltrate in breast tumors thereby facilitating the rational design and use of immunotherapeutic agents in combination with standard systemic therapies.
Citation Format: Elizabeth A Mittendorf, Chichung Wang, Peng Yau, Kristin Roman, Yun Wu, Gheath Alatrash, Clifford Hoyt. Multispectral imaging allows visualization and quantification of multiple immunologic cell types in breast tumor tissues [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 P5-04-07.