Abstract PD1-3: The significance of tumor infiltrating lymphocyte density, subset composition and organization in breast cancer
Authors: Willard-Gallo Karen , Buisseret Laurence , Garaud Soizic , Gu-Trantien Chunyan , de Wind Alexandre , Duquenne Sébastien , Larsimont Denis , Sotiriou Christos , Piccart Martine 
Published: May 2015

The clinical relevance of tumor infiltrating lymphocytes (TIL) in breast cancer (BC) has been clearly established by recent large clinical trials (presented at SABCS 2013). The relationship between protective immunity, observed in some patients, and critical features of lymphoid subset composition and organization remain unknown. Our recent work revealed that tertiary lymphoid structures (TLS), principally composed of T cells, B cells and dendritic cells, are present in the peri-tumoral regions of breast tumors and associated with a CD4+ follicular helper T (Tfh) cell presence. Through retrospective analyses, we determined that TLS were associated with good clinical outcomes in both the neo-adjuvant and adjuvant settings. To gain insight into the immune components linked with a significant TIL and TLS presence, we initiated a prospective flow cytometric study. We systematically immunophenotyped T and B cell TIL in breast tissues from tumors (n=125), non-adjacent non-tumor tissues (NANT, n=115) and normal tissue from mammary reductions (n=26) on the day of surgery. TIL organization and spatial distribution was subsequently analyzed by immunohistochemistry (IHC) and immunofluorescence (IF) on paraffin sections for a subset of patients (n=78). The fresh tissue analyses revealed that TIL density was a continuum across the 125 patients analyzed. A cutoff for TIL-positive and -negative tumors was set at 58 CD45+ TIL per mm3 of tissue based on the number of CD45+ cells present in the remarkably similar normal and NANT tissues. Applying this threshold to BC, 65% were TIL-positive with approximately one-third considered extensively infiltrated. TIL-positive tumors are characterized by an increase in CD4+ T cells and CD19+/CD20+ B cells. The median CD4/CD8 ratio was &gt;1 in TIL-positive compared to &lt;1 in TIL-negative tumors and NANT. CD4+ and CD8+ T cells were predominantly CD45RO+ memory cells, with a significant proportion expressing PD-1. Infiltrating B cells were approximately 50% memory cells in contrast to &lt;15% in normal and NANT tissues. Extensively infiltrated tumors were more frequently associated with Tfh and follicular B cells resident in TLS. IF analysis by confocal microscopy found that TLS resident cells included specific lymphocyte subsets: marginal B cells (CD20+CD27+IgD−), follicular mantle B cells (CD20+IgD+), germinal center B cells (CD20+Ki67+CD35+CD21+PD-1+) and germinal center Tfh cells (CD4+CD200+TIGIT+CXCL13+) in a B cell zone surrounded by a T cell zone containing CD4+ and CD8+ T cells. To determine whether the flow cytometry data was correlated with routine pathology, sections of the same tumors were stained by H&E and CD45 (total leukocytes) or CD3 plus CD19 (T cells + B cells; &gt;95% of CD45+ cells) IHC and scored by trained pathologists. The best correlation was observed between CD3/CD20 flow cytometry and CD3/CD19 IHC, the latter also associated with lower inter-observer variability. TIL density was positively correlated with proliferation (Ki67 & histological grade) and hormone receptor negativity while TLS were more frequent in younger women. These data suggest that organized immune responses in TLS adjacent to the tumor bed provide an effective location for generating anti-tumor memory T and B cell responses.

Citation Format: Karen Willard-Gallo, Laurence Buisseret, Soizic Garaud, Chunyan Gu-Trantien, Alexandre de Wind, Sébastien Duquenne, Denis Larsimont, Christos Sotiriou, Martine Piccart. The significance of tumor infiltrating lymphocyte density, subset composition and organization in breast cancer [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 PD1-3.