Immune response in breast cancer brain metastases and their microenvironment: the role of the PD-1/PD-L axis

Duchnowska et al. Breast Cancer Research (2016) 18:43 DOI 10.1186/s13058-016-0702-8 RESEARCH ARTICLE Open Access Immune response in breast cancer brain metastases and their microenvironment: the role of the PD-1/PD-L axis Renata Duchnowska1*, Rafał Pęksa2, Barbara Radecka3, Tomasz Mandat4, Tomasz Trojanowski5, Bożena Jarosz5, Bogumiła Czartoryska-Arłukowicz6, Wojciech P. Olszewski7, Waldemar Och8, Ewa Kalinka-Warzocha9, Wojciech Kozłowski10, Anna Kowalczyk11, Sherene Loi12, Wojciech Biernat2, Jacek Jassem11 and for the Polish Brain Metastasis Consortium Abstract Background: A better understanding of immune response in breast cancer brain metastases (BCBM) may prompt new preventive and therapeutic strategies. Methods: Immunohistochemical expression of stromal tumor-infiltrating lymphocytes (TILs: CD4, CD8, CTLA4), macrophage/microglial cells (CD68), programmed cell death protein 1 receptor (PD-1), programmed cell death protein 1 receptor ligand (PD-L)1, PD-L2 and glial fibrillary acid protein was assessed in 84 BCBM and their microenvironment. Results: Median survival after BCBM excision was 18.3 months (range 0–99). Median number of CD4+, CD8+ TILs and CD68+ was 49, 69 and 76 per 1 mm2, respectively. PD-L1 and PD-L2 expression in BCBM was present in 53 % and 36 % of cases, and was not related to BCBM phenotype. PD-1 expression on TILs correlated positively with CD4+ and CD8+ TILs (r = 0.26 and 0.33), and so did CD68+ (r = 0.23 and 0.27, respectively). In the multivariate analysis, survival after BCBM excision positively correlated with PD-1 expression on TILs (hazard ratio (HR) = 0.3, P = 0.003), CD68+ infiltration (HR = 0.2, P < 0.001), brain radiotherapy (HR = 0.1, P < 0.001), endocrine therapy (HR = 0.1, P < 0.001), and negatively with hormone-receptor-negative/human epidermal growth factor receptor 2 (HER2)-positive phenotype of primary tumor (HR = 2.6, P = 0.01), HER2 expression in BCBM (HR = 4.9, P = 0.01). Conclusions: PD-L1 and PD-L2 expression is a common occurrence in BCBM, irrespective of primary tumor and BCBM phenotype. Favorable prognostic impact of PD-1 expression on TILs suggests a beneficial effect of preexisting immunity and implies a potential therapeutic role of immune checkpoint inhibitors in BCBM. Keywords: Brain metastases, Breast cancer, PD-1, PD-L, Lymphocytes Background Breast cancer has not been traditionally considered an immunogenic cancer type. However, there is an increasing body of evidence suggesting that an effective immune response may greatly impact on the clinical behavior of this malignancy. Tumor lymphocyte infiltration is associated with favorable prognosis in early triple-negative and human epidermal growth factor receptor type 2 (HER2)-positive * Correspondence: 1 Department of Oncology, Military Institute of Medicine, Szaserów St 128, 04-141 Warsaw, Poland Full list of author information is available at the end of the article breast cancer phenotypes [1–4] and may influence the response to systemic therapies [3–6]. Information on the association between the immune host response and the colonization of the brain by tumor cells is scarce. The central nervous system (CNS) has long been considered an immunologically privileged site [7]. Actually, CNS is an immune specialized site under a tight regulatory control network linking microglia, astrocytes and lymphocytes [8]. Brain metastases in preclinical and clinical models are characterized by high proliferation, apoptosis, and inflammatory response in the form of surrounding extensive © 2016 Duchnowska et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Duchnowska et al. Breast Cancer Research (2016) 18:43 reactive gliosis [9]. It is postulated that the reactive astrocytes reduce apoptosis mediated by the cytotoxic agents by sequestering calcium from the cytoplasm of tumor cells or by secreting metastasis-stimulating chemokines [10]. In the inflammatory and degenerative processes, CNS reactive glial cells actively participate in the restimulation of T cells through the secretion of some chemokines [9, 11, 12]. This increases the influx of regulatory T cell (Treg) lymphocytes, resulting in silencing of the immune response. The programmed cell death protein 1 receptor (PD-1) and its ligands, programmed cell death protein 1 receptor ligand (PD-L)1 and PD-L2, also known as B7-H1 and B7-DC, respectively, play a crucial role in the induction and maintenance of peripheral tolerance, and protect tissues from autoimmune attack [13]. The PD-1/PD-L axis is also a key getaway pathway serving in many cancers as an “immune control” [14, 15]. Several studies suggest that immune response to malignant processes in the brain may be related to the type of cancer [16–19]. Better understanding of the local immune response accompanying brain metastases (BM) may pave the way to the development of novel preventive and therapeutic strategies in breast cancer patients. This retrospective study aimed to assess the correlation between selected parameters of immune response in breast cancer brain metastases (BCBM) and their impact on overall survival. Methods Study population and data collection This study was approved by the Institutional Review Board of the coordinating center, the Military Institute of Medicine in Warsaw, Poland. The study group included breast cancer patients who underwent excision of BCBM (Table 1). The patients were diagnosed and treated between 1990 and 2014 in eight oncology centers in Poland. Demographic, clinicopathologic, and clinical follow-up data were extracted from medical records. All data were coded to secure full protection of personal information, therefore, patient consent was not sought. Pathologic analysis The starting material from each patient was an archival formalin-fixed, paraffin-embedded (FFPE) specimen obtained at surgery from the primary breast tumor and BCBM. The pathologic diagnosis was confirmed by a Board-certified pathologist (RP or WB) who reviewed FFPE tissue sections stained with hematoxylin and eosin. A representative paraffin block from each specimen was chosen for immunohistochemical analysis (IHC). In patients with more than one BCBM, only the single most representative lesion was subjected to analysis. Page 2 of 11 Immunohistochemical staining All samples were re-stained and IHC-based expression for estrogen receptor alpha (ERα), progesterone receptor (PR) and HER2 was determined in the central labor (...truncated)