Abstract PD6-02: Immunological differences between primary and metastatic breast cancer
Authors: Szekely B , Bossuyt V , Li X , Baine M , Silber A , Sanft T , Hofstatter E , Mougalian S , Baghwagar S , Neumeister V , Pelekanou V , Hatzis C , Pusztai L 
Published: February 2018

Background: Little is known about how the immune microenvironment of breast cancer evolves during disease progression. Immunological differences between primary and metastatic lesions may explain discordant results of clinical trials that showed low tumor response rates with immune checkpoint therapy in metastatic breast cancer but high rates of pathologic complete response in early stage disease. The goal of this project was to examine TIL counts, PD-L1 protein expression and immune gene mRNA expression in primary tumors (P) and metastatic lesions (M).

Methods: FFPE blocks of primary breast cancers and core needle biopsies of matching distant metastases from 54 patients (n=104 samples). TIL count was assessed on H&E slides for 39 paired tissues and is reported as % of TIL in the stroma. PD-L1 protein expression was detected with immunohistochemistry (IHC, E1L3N antibody) in 36 pairs, samples with &gt; 1% cells showing staining were considered PD-L1 positive which was determined separately for tumor and stroma. The expression of 770 immune-related genes was measured using the Nanostring PanCancer Immune Gene Panel in 31 P and 17 M, including 10 paired cases. Genes were organized into 14 immune cell type (total T, Th1, Treg, Total CD8, exhausted CD8, Cytotoxic T, B, NK, NK-CD56, Mast cell, CD45, Dendritic cell, macrophage, neutrophil) and 22 immune function metagenes. Differences in mean expression in P and M were assessed using Fisher exact and Mann-Whitney tests without adjustment for multiple comparisons due to overlap in metagene membership.

Results: Mean TIL counts (14% vs 20%, p=0.026) and stromal PD-L1 positivity by IHC (14% vs 54%, p=0.004) were significantly lower in M. PD-L1 positivity in tumor cells was similar (25% in M vs 42%, in P p=0.14). The total TIL gene expression score (2.48 vs 2.8 p=0.018) and all immune cell type metagenes, except neutrophils, had lower absolute expression levels in M. The relative abundance of neutrophils (0.035 vs -0.38, p=0.0001) and macrophages (0.62 vs 0.38 p=0.0013) increased in M. Among the 22 immune function metagenes, T, B and NK cell functions, cytotoxicity, chemokine and TNF superfamily expression, regulation and pathogen defense were significantly lower in M and none showed significantly increased expression in M. Pro-inflammatory/immune-activating cytokines of IL-6, CCL-5,-12,-19,-22 and CXCL-5,-9,-10,-11 were all significantly lower in M. The greatest drop was seen for CXCL-9 (2243 vs 422, p&lt;0.0001) and CCL-19 (2537 vs 309, p&lt;0.0001). No cytokine showed increased expression in M. Only 6 immune genes (C7, GPI, MAPK1, TAB1, TLR5, PVR) showed significantly higher (p&lt;0.05) expression in M, the greatest increase was for Complement C7 (member of the terminal complement pathway membrane attack complex; 484 vs 3499, p=0.03) and GPI (glucose 6-phosphate isomerase that induces immunoglobulin secretion; 1967 vs 3011, p=0.01).

Conclusions: Breast cancer metastases exist in an attenuated immune microenvironment. Most immune cell subtypes, immune functions, and immune-associated gene expression are lower in M compared to P, consistent with immune escape. Metastatic lesions have higher relative abundance of macrophages and neutrophils, which suggest new therapeutic opportunities.

Citation Format: Szekely B, Bossuyt V, Li X, Baine M, Silber A, Sanft T, Hofstatter E, Mougalian S, Baghwagar S, Neumeister V, Pelekanou V, Hatzis C, Pusztai L. Immunological differences between primary and metastatic breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD6-02.