Abstract 575: PD-L1 positive tumor-infiltrating lymphocytes and mutational load in breast cancer
Authors: Sobral-Leite Marcelo , Vijver Koen Van de , Michaut Magali , Horlings Hugo M. , Severson Tesa M. , Schouten Philip C. , Linden Rianne van der , Kersten Kelly , Mulligan Anna Marie , Weerasooriya Nayana , Sanders Joyce , Cimino-Mathews Ashley , Peters Dennis , Hooijer Gerrit K. , Hooijberg Erik , Broeks Annegien , Bernards Rene , Linn Sabine , Andrulis Irene L. , Vijver Marc J. van de , Wessels Lodewyk F. , Kok Marleen , Visser Karin E. de , Schmidt Marjanka K. 
Published: July 2017

Abstract
Background: PD-1 blockade has emerged as an effective treatment for a subset of cancer patients. Studies have shown that PD-L1 expression is associated with likelihood of response to PD-1 blockade. In order to select the right breast cancer patient for immunotherapy, characterization of the immune landscape of breast tumors is required. Therefore, we assessed PD-L1 expression and tumor-infiltrating lymphocytes (TILs) in different breast tumor subtypes and the link with prognosis. We also sequenced a panel of genes to assess the mutational load in triple negative tumors (TNBC) and investigate the association with PD-L1 positive TILs.
Material and methods: We analyzed 438 tumor samples from breast cancer patients of all ages treated between 1986 and 2007 with surgery, with or without adjuvant therapy. PD-L1 was stained using whole slide specimens (E1L3N® antibody) after methodological validation. Pathologists quantified TILs based on International TILs Working Group recommendations and scored PD-L1 based on the percentage of positive (tumor and/or immune) cells; as negative if 0%, positive if ≥1%, and high if &gt;50%. Mutational load was assessed based on DNA kinome sequencing. Associations were measured by Cox/logistic regression model, including pathological variables. Multiplex imaging of 20 immune-infiltrated areas from four ER negative tumors were performed using the Vectra® system based on immunofluorescence staining panel of: CD4, CD68, CD8, FOXP3 and PD-L1.
Results: PD-L1 expression and TILs were higher in ductal (compared with lobular), high grade and estrogen receptor (ER)-negative tumors (p&lt;0.001). TILs (density ≥5%) were significantly associated with worse distant metastasis-free survival (DMFS) only in ER-positive tumors (n=204): HR=2.72; 95%CI: 1.07-6.94. PD-L1 positivity (≥1%) followed the same trend: HR=1.66; 95%CI: 0.87-3.15. However, in ER-negative tumors (n=171), high PD-L1 expression (&gt;50%) was significantly associated with better DMFS: HR=0.51; 95%CI: 0.27-0.98. TNBC with high PD-L1 expression of TILs (&gt;50%) showed an association with increased mutation load (p=0.019) and a trend for better DMFS (HR=0.41; 95%CI: 0.16-1.04) compared with tumors lacking TILs. Further characterization of PD-L1 positivity in the immune-infiltrated cells was conducted by a multiplex imaging analysis. Preliminary results indicated that PD-L1 is expressed in CD68+, CD4+, FOXP3+ and CD8+ immune-cells.
Conclusion: Our findings suggest that PD-L1 positive TILs are associated with worse prognosis in ER-positive breast cancer and with better outcome in ER-negative group. In TNBC, high mutational load correlates with high PD-L1 positive TILs.
Citation Format: Marcelo Sobral-Leite, Koen Van de Vijver, Magali Michaut, Hugo M. Horlings, Tesa M. Severson, Philip C. Schouten, Rianne van der Linden, Kelly Kersten, Anna Marie Mulligan, Nayana Weerasooriya, Joyce Sanders, Ashley Cimino-Mathews, Dennis Peters, Gerrit K. Hooijer, Erik Hooijberg, Annegien Broeks, Rene Bernards, Sabine Linn, Irene L. Andrulis, Marc J. van de Vijver, Lodewyk F. Wessels, Marleen Kok, Karin E. de Visser, Marjanka K. Schmidt. PD-L1 positive tumor-infiltrating lymphocytes and mutational load in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 575. doi:10.1158/1538-7445.AM2017-575