Laurence Buisseret

Abstract S1-02: Lymphocytic infiltration in invasive lobular breast cancer

Background: The presence and prognostic value of tumor infiltrating lymphocytes (TILs) in invasive breast carcinoma has been demonstrated in several studies, especially in the triple-negative and HER2-positive subtypes. So far, TILs have not been investigated with sufficient detail in invasive lobular breast cancer (ILBC). Here we therefore aimed at: first, assessing the distribution of stromal TILs in ILBC; second, correlating the presence of TILs with standard clinical and pathological markers; third, exploring associations of TILs with recurrent genomic alterations; and, fourth, comparing the lymphocytic composition of ER-positive/HER2-negative lobular to ER-positive/HER2-negative ductal tumors. Material and methods: The percentage of stromal TILs was independently assessed according to Salgado et al. (Ann Oncol 2015) by three pathologists on full-face hematoxylin and eosin slides in a well-annotated retrospective series of 614 primary ILBCs previously characterized at the genomic level. The median value of TILs was used for the analyses. For the association analyses, we focused on the more homogeneous group of ER-positive/HER2-negative ILBC (555/614). Breast cancer-free interval was used as survival endpoint and the analyses were censored at 12 years of follow-up. The comparison of the lymphocytic composition (relative percentage of CD45+ TILs which are CD4+, CD8+ or CD19+) was assessed by FACS in a separate prospective cohort of 51 ER-positive/HER2-negative lobular and 112 ER-positive/HER2-negative ductal tumors. Results: The intraclass correlation coefficient between the three pathologists was 0.71 (95%CI:0.65-0.76). The median percentage of stromal TILs was 5% and the interquartile range 5-10%, with only 9% of the samples having ≥ 20%. Greater numbers of TILs were significantly associated with younger age at diagnosis, axillary lymph node involvement, high proliferative tumors as assessed by Ki67, and with the mixed non-classic ILBC subtypes. Greater numbers of TILs were associated with worse prognosis (HR=1.22; 95%CI:1.07-1.38, p=0.003) only in the unadjusted analysis, as it lost significance after adjustment for standard clinical and pathological variables. Greater numbers of TILs were observed in tumors harboring ARID1A, BRCA2, KMT2C and TP53 mutations, as well as chr3p21.31 and chr8q24.23 (PTK2) loss; whereas lower numbers were observed in tumors with ERBB3 mutations as well as chr7p and chr11q14.1 (PAK1) gains. There were no significant differences in the relative proportion of CD4+, CD8+ or CD19+ lymphocytes between ER-positive/HER2-negative lobular and ductal tumors. Conclusion: In this work, which reports to our knowledge on the largest series of ILBC ever assessed for TILs, we showed that most ILBCs were characterized by low lymphocytic infiltration. Besides the association of TILs with clinical and pathological features of ILBC patients, we found that higher TIL levels were observed in the presence of specific mutations and copy number alterations. Higher numbers of TILs were associated with worse prognosis at the univariate analysis. Finally, based on the assessed markers, we have no evidence of differential lymphocytic composition between ER-positive/HER2-negative lobular and ductal tumors. Citation Format: Desmedt C, Salgado R, Buisseret L, Zoppoli G, Fornili M, Van den Eynden G, Garaud S, Gundem G, Rothe F, Brown D, Kheddoumi N, Rouas G, Galant C, Bertucci F, Piccart M, Campbell P, Viale G, Larsimont D, Willard-Gallo K, Biganzoli E, Pruneri G, Sotiriou C. Lymphocytic infiltration in invasive lobular breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S1-02.

Abstract 1105: A prospective study of lymphocytes infiltrating the breast cancer microenvironment

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnRecent 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 naive 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.nnCitation 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