Abstract 1669: Characterization of B-cells infiltrating human breast cancer and their presence in peritumoral tertiary lymphoid structures
Authors: Garaud Soizic , Buisseret Laurence , Gu Chunyan , Migliori Edoardo , Lodewyckx Jean-Nicolas , Duvillier Hugues , Craciun Ligia , Larsimont Denis , Willard-Gallo Karen 
Published: May 2015

Abstract
Recent advances in tumor immunology show an important link exists between tumor infiltrating lymphocytes (TIL) and patient outcome. In human breast cancer (BC) more extensive lymphocyte infiltration is associated with a better prognosis and can also predict responses to pre-operative chemotherapy. Tertiary lymphoid structures (TLS) detected at the tumor periphery have been correlated with a good prognosis in lung and colorectal cancer. Our recent work was the first to demonstrate that TLS are present in BC, located adjacent to the tumor bed in extensively infiltrated tumors. TLS organization is similar to a lymph node, with a CD3+ T-cell zone adjacent to a CD20+ B-cell follicle where germinal centers containing CD23+ follicular dendritic cells, Bcl6+ TFH cells and Ki67+ cells are found. Our laboratory recently discovered that TLS-associated CD4+ Tfh cells are present in extensively infiltrated tumors and showed they signal a good prognosis. This important presence of Tfh-containing TLS adjacent to the tumor bed suggests that B-cells may also play a role in generating effective anti-tumor immune responses. We therefore undertook to fully characterize the B-cells infiltrating human BC. Flow cytometric analysis of fresh breast tumor homogenates detected an increase in CD45+ leukocytes in all BC subtypes compared with normal and non-tumor non-adjacent (NANT) breast tissue irrespective of lymphocyte infiltration levels. The infiltrate was dominated by CD3+ T-cells (65-86% of CD45+) in normal and malignant tissues. In contrast, B-cells were elevated in tumors compared to normal and NANT tissue, particularly in extensively infiltrated high proliferative BC subtypes. This increase was paralleled by a relative decrease in CD8+ T-cells. CD45/CD19/CD38/IgD labeling revealed that approximately 50% of the infiltrating B-cells are memory cells in contrast to normal tissues, which contain less than 15%. Centroblasts and centrocytes, which are follicular B cells, were significantly increased in extensively infiltrated tumors in association with Tfh cells. We assessed immunoglobulin isotypes in primary supernatants from breast tissue homogenates, detecting decreased IgA and increased IgG and IgM per mm3 in the tumor. Patient sera, particularly from high proliferative subtypes, had elevated IgG4 compared with low proliferative subtypes and healthy donors. Immunofluorescent analysis of embedded tissues show that TLS contain a marginal zone region(CD20+CD27+IgD-), a follicular mantle zone (CD20+IgD+) and a germinal center zone (CD20+Ki67+CD35+CD21+PD-1+). Tumor-associated TLS are surrounded by T-cells (CD4+ and CD8+). These data reveal that when B-cells are present in breast tumors they are principally associated with organized TLS and may therefore play an important functional role in comprehensive anti-tumor immune responses, an aspect currently under further investigation.
Citation Format: Soizic Garaud, Laurence Buisseret, Chunyan Gu, Edoardo Migliori, Jean-Nicolas Lodewyckx, Hugues Duvillier, Ligia Craciun, Denis Larsimont, Karen Willard-Gallo. Characterization of B-cells infiltrating human breast cancer and their presence in peritumoral tertiary lymphoid structures. [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 1669. doi:10.1158/1538-7445.AM2014-1669