Abstract 1105: A prospective study of lymphocytes infiltrating the breast cancer microenvironment
Authors: Buisseret Laurence , Garaud Soizic , Duvillier Hugues , Naveaux Celine , Duquenne Sebastien , Wind Alexandre de , Sotiriou Christos , Willard-Gallo Karen 
Published: May 2015

Abstract
Recent studies in breast cancer (BC) show that strong links exist between the host immune response and clinical outcome. Immune cells have been associated with both pro-tumor and anti-tumor activities with the balance between key players likely determining the ultimate clinical result. In an effort to understand how protective immune responses are generated we performed a prospective analysis of tumor infiltrating lymphocytes (TIL) in patients with primary BC. TIL present in fresh tissue homogenates (GentleMACS with no enzymatic digestion) from untreated primary breast tumors (n=120) were immunophenotyped using 10-color flow cytometry. TIL were compared for density and cellular composition to non-adjacent non-tumor breast tissue from the same patient (NANT, n=116) as well as to normal breast tissue from mammary reductions (n=28). TIL organization and distribution in tumors was analyzed using immunohistochemistry or immunofluorescence (confocal microscopy) on paraffin sections from patients in the series. Analysis of fresh tissues revealed that the density (absolute numbers of leukocytes) in normal tissue and NANT are remarkably similar (median: 4.4-vs-5.7 CD45+ cells/mm3 of tissue, respectively) and could be used to establish a cutoff for TIL negative tumors. Using NANT as an internal control, 64.5% tumors were TIL negative (minimally infiltrated). Among total leukocytes, tumors have higher frequencies of CD19+ B cells and CD4+ T cells compared with normal tissue. Furthermore, the number of CD4+ are higher than CD8+ in TIL positive tumors, which is an inversion of the CD4/CD8 ratio in TIL negative tumors (the latter ratio is similar between TIL negative tumors and NANT). In all of the breast tissues (normal, NANT and tumor) the CD4+ and CD8+ T cells are predominantly CD45RO+ memory cells, with a significant proportion of these TIL expressing the immune checkpoint receptor PD-1. In contrast, higher numbers of naïve relative to memory B cells were generally detected in both normal and tumor tissues but the balance between these B cell populations was more heterogeneous than for T cells. The distribution pattern of TILs (CD4+, CD8+ and B cells) in infiltrated tumors was both widely dispersed in some regions of the tumor bed and minimally infiltrated in other areas. Lymphocyte aggregates were principally concentrated in the stromal and peritumoral regions, with these areas being a preferential environment for B cells. Closer examination of the lymphoid aggregates revealed they were tertiary lymphoid structures containing a T cell zone predominantly composed of CD4+ T cells and a B cell follicle with an active germinal center. These data suggest that establishment of organized immune structures adjacent to the tumor bed is an effective mechanism for generating anti-tumor memory T and B cell responses.
Citation Format: Laurence Buisseret, Soizic Garaud, Hugues Duvillier, Celine Naveaux, Sebastien Duquenne, Alexandre de Wind, Christos Sotiriou, Karen Willard-Gallo. A prospective study of lymphocytes infiltrating the breast cancer microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1105. doi:10.1158/1538-7445.AM2014-1105