Abstract P5-01-02: Characterization of follicular helper CD4 T cells and B cells resident in peritumoral tertiary lymphocyte structures as specific markers for an immunological grade in breast cancer
Authors: Willard-Gallo K , Garaud S , Buisseret L , Gu-Trantien C , Migliori E , Lodewyckx J-N , Naveaux C , de Wind A , Duquenne S , Larsimont D , Sotiriou C , Piccart M 
Published: August 2014

Abstract
Molecular approaches such as gene expression profiling have improved the classification of human breast cancer (BC) by identifying molecular subtypes and demonstrating that the immune infiltrate is an important prognostic or predictive factor. Our prospective study of freshly isolated CD4+ T cells infiltrating (TIL) BC discovered that along with Th1, Th2 and Th17 effector memory and Treg subpopulations the newest CD4+ Th subset, T follicular helper (Tfh) cells, was also present in the tumor. Comparison of extensive versus minimally-infiltrated tumors led to our finding that extensive immune infiltrates are distinguished by Tfh cells located in tertiary lymphoid structures (TLS) next to the tumor bed. TLS are highly organized with a CD3+ T cell zone adjacent to a CD20+ B cell follicle with germinal centers containing Bcl6+ Tfh cells, CD23+ follicular dendritic cells and Ki67+ cells. Tfh cells are known to play a critical role in generating antibody producing plasma cells and memory B cells. We demonstrated that Tfh cells are the principal cellular source of the B cell chemoattractant CXCL13 in BC. Thus, a Tfh presence paralleled the TLS incidence and together they were associated with an increased peritumoral B cell presence and a higher frequency of CD8+ T cells in the tumor bed. Our recent work shows that approximately 50% of the infiltrating B cells are memory cells in contrast to normal and non-tumor non-adjacent breast tissue (controls, less than 15%). A significant B cell presence is found in extensively infiltrated high proliferative BC subtypes with germinal center centroblasts and centrocytes consistently associated with a Tfh cell presence. We assessed immunoglobulin isotypes in supernatants of fresh breast tissue homogenates, detecting decreased IgA and increased IgG and IgM in the tumor tissue compared to controls. Patient sera, particularly from patients with high proliferative BC subtypes, had elevated IgG3 and IgG4 compared to low proliferative tumors and healthy donors. Immunofluorescent analysis found the majority of B cells infiltrating tumors have germinal center characteristics (IgD+Ki67+CD35+CD21+PD-1+) and are clustered with Tfh in the B cell follicles surrounded by a T cell zone, which together create the tumor-associated TLS. Based on their specificity for TLS, we derived a scoring system, referred to as immunological grade, to measure the extent of the lymphocytic infiltrate and the level of immune organization. This system is based on a morphological score (the density of the lymphocyte infiltrate determined by CD3/CD20 immunohistochemistry plus the number and size of peritumoral TLS assessed by two pathologists) together with a molecular score [an 8 gene qRT-PCR Tfh signature that predicts long term survival in an untreated patient cohort with &gt;10-year survival (n = 794) or pathological complete response in patients treated with preoperative chemotherapy (n = 996)]. We are testing this immunological grading system on a large retrospective series of patients to determine its value in evaluating a patient's anti-tumor immune response in pre-operative biopsies and/or the primary tumor at surgery as a new marker for use in treatment decisions.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-01-02.