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

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.

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.

All samples were re-stained and IHC-based expression for estrogen receptor alpha (ERα), progesterone receptor (PR) and HER2 was determined in the central laboratory by two pathologists (RP and WB) who were blinded to the original assessments and to expression in the paired samples. Then, BCBM and the adjacent brain microenvironment were subjected to analysis of stromal tumor infiltrating lymphocytes (TILs) (CD4+, CD8+, CTLA4+), CD68+ cell infiltration, expression of PD-1, PD-L1, PD-L2, and glial fibrillary acid protein (GFAP). The staining was performed according to the manufacturers’ protocols (Table 2). The TILs and CD68+ cells were scored under a light microscope at a magnification of 400 (ocular × 10 with objective of × 40), corresponding to a total area of 1 mm² on full slides. As TILs are predominantly present in stromal parts of BCBM and are a rare occurrence in other BCBM compartments, we have consistently used the term “stromal TILs”. TILs were considered PD-1+ if cytoplasmic staining was found in at least 1 % of cells, irrespective of staining intensity. PD-1 staining was assessed in lymphoid cells, which were identified basing on morphologic features and previously performed staining for/CD4+ and CD8+, with negative results (0) or positive results (1). Due to lack of standardization criteria of PD-L expression positivity and possible intratumoral heterogeneity, PD1-L1 and PD-L2 were assessed in the whole tissue sections using the semiquantitative staining H-score, which accounts for the quantitative and qualitative features of the reactions. The intensity of staining was defined as weak (1), moderate (2), or strong (3). The intensity of the reaction was determined in a percentage of positive cells. The H-score was calculated for each biomarker by the formula:

3 × % Strong cellular staining (cytoplasmic, nuclear and/or membranous) + 2 × % Moderate staining + % Weak staining

This gave a range of 0–300. Figure 1 shows positive control staining for PD-1, PD-L1 and PD-L2.

All statistical analyses were performed using STATA software version 11. Statistical significance was defined as P < 0.05. We tested correlation between parameters of immune response in BCBM and the brain microenvironment and GFAP, PD-1, PD-L1, PD-L2, TILs (CD4+, CD8+, CTLA4+) and CD68+ cells (listed in Table 3), and assessed their prognostic relevance. This analysis included all available clinical and pathological variables (Table 1). Due to the retrospective and multicenter nature of the study we were unable to include BCBM size and accompanying cerebral edema in the analysis, or the use of steroids and diuretics. Categorical and continuous variables were compared using Pearson’s chi-squared test (c ²), Spearman’s r rank test and the Mann–Whitney U test. Overall survival (OS) was computed using the Kaplan-Meier method, starting from BCBM excision to the date of death or the last follow up. Univariate and multivariate analyses were performed using the log-rank test, Wilcoxon test, and Cox proportional hazard and logistic regression.

The study group included 84 breast cancer patients who underwent excision of BM (Table 1). Based on ERα, PR and HER2 expression, four primary tumor phenotypes were identified: hormone-receptor + and HER2– (23 cases), hormone-receptor + and HER2+ (21 cases), hormone-receptor– and HER2– (21 cases), and hormone-receptor– and HER2+ (19 cases). Of these tumors 83 % were invasive ductal carcinomas (no special type); 42 % were grade 2 and 46 % were grade 3: 50 % were ERα– and PR–, 48 % were HER2+ (IHC3+ or HER2 amplified by fluorescence in situ hybridization (FISH)). All patients underwent radical surgery for the primary tumor; 62 % received neoadjuvant chemotherapy and 42 % adjuvant chemotherapy, 32 % received adjuvant radiotherapy and 19 (50 % of the 38 HER2+ cases) received adjuvant trastuzumab. The first manifestation of progression was distant metastasis in 89 % of patients, with viscera being the most common dominant sites of metastatic disease. Forty-seven patients (56 %) developed BM as the first site of progression, 61 % of whom presented with a single brain lesion at the time of excision. The mean age at BCBM diagnosis was 53 years (range 30–81). The median length of follow up in the entire population was 61.3 months (range 8.7–209 months). The median time to BCBM occurrence from first diagnosis of breast cancer was 41.6 months (range 0.9–152.7). The most common sites of BCBM were the cerebellum and parietal lobe. After BCBM excision, 75 % of patients were administered whole brain radiotherapy, 44 % received chemotherapy and 18 % endocrine therapy. Eight HER2+ patients received trastuzumab or lapatinib. The median OS after BCBM excision was 18.3 months (range 0–99 months).

TIL (CD4+, CD8+) and macrophage/microglia (CD68+) infiltration was determined in 96 %, 98 % and 92 % of cases, respectively (Table 3). TILs were identified in both stromal and epithelial compartments of BCBM, but were generally much more abundant in the stroma (Fig. 2a, b). There was no CTLA4 expression on TILs (Fig. 2c). The median number (per mm²) of CD4+ TILs was 49 (interquartile range (IQR) 23–121), of CD8+ TILs was 69 (IQR 38–127), and of CD68+ TILs was 76 (IQR 57–104) (Table 3). CD4+ and CD8+ TILs were positively correlated (r = 0.48; P < 0.001) and both were positively correlated with CD68+ cells (r = 0.23; P = 0.043 and r = 0.27, P = 0.019, respectively) (Table 4). GFAP, a biomarker of reactive astrocytes, was expressed in 71 % of cases (Table 3). There was no correlation between GFAP expression and BCBM phenotype, TILs and CD68+ cell infiltration, or expression of PD-1 and its ligands.

PD-1 expression on TILs in BCBM was identified in 17 cases (23 %), more frequently in older patients (mean age at brain metastasis diagnosis in PD-1+ and PD-1– groups 59 and 51 years, respectively; P = 0.003), and in cases with HER2-amplified primary breast cancer. PD-1 expression was correlated positively with both TILs: CD4+ (r = 0.26; P = 0.028) and CD8+ (r = 0.33; P = 0.005; Table 4). PD-1+ patients, compared to PD-1– patients had longer OS after BCBM excision (median 27.9 months (range 0.1–88.9) vs. 13.9 months (0.0–82.6), respectively; P = 0.02) (Fig. 3a and Table 5). There was no correlation between expression of PD-1 on TILs and PD-1 ligands in BCBM (Table 4). PD-L1 and PD-L2 expression in BCBM was present in 41 (53 %) and 28 (36 %) cases, respectively, and was not related to BCBM phenotype. The mean expression for PD-L1 and PD-L2 (H-score) was 27 and 26, respectively (Table 3).

There was no impact of previous systemic adjuvant therapy including trastuzumab on the analyzed immunological parameters in the brain. In HER2+ patients, the administration of trastuzumab before BCBM development did not affect CD4+ (P = 0.77) or CD8+ TILs (P = 0.17), CD68+ infiltration (P = 0.77), or expression of PD-1 (P = 0.85), PD-L1 (P = 0.86), or PD-L2 (P = 0.80). The univariate analysis of survival included all available clinicopathologic variables (the histology, grade and expression of ER, PR and HER2 in the primary tumor, phenotype of the primary tumor and BCBM, treatments administered prior to and after BCBM, type of first progression, dominant metastatic site and location of BCBM) and all studied immune parameters (reactive astrocytes (GFAP), TILs (CD4+, CD8, microglia/macrophages (CD68+), PD-1 expression on TILs, and PD-L1 and PD-L2 expression on BCBM). The multivariate analysis included variables that were significant (P < 0.05) in the univariate analysis (Table 5). In this analysis, favorable factors for survival after BCBM excision included PD-1 expression on TILs (hazard ratio (HR) = 0.3 (0.1–0.7); P = 0.003) (Fig. 3a) and CD68+ cell infiltration (HR = 0.2 (0.1–0.5); P < 0.001) (Fig. 3b), brain radiotherapy (HR = 0.1 (0.1–0.3); P < 0.001), and endocrine therapy after the development of BCBM (HR = 0.1 (0.1–0.3) P < 0.001; Table 5). Adverse prognostic factors included a hormone receptor–/HER2+ primary tumor phenotype (HR = 2.6 (1.3–5.5); P = 0.01) and HER2 expression in BCBM (HR = 4.9 (1.3–19.2); P = 0.01).