Prediction of treatment responses to neoadjuvant chemotherapy in triple-negative breast cancer by analysis of immune checkpoint protein expression

“Avoiding immune destruction” has recently been established as one of the hallmarks of cancer. The programmed cell death (PD)-1/programmed cell death-ligand (PD-L) 1 pathway is an important immunosuppression mechanism that allows cancer cells to escape host immunity. The present study investigated how the expressions of these immune checkpoint proteins affected responses to neo-adjuvant chemotherapy (NAC) in breast cancer. A total of 177 patients with resectable early-stage breast cancer were treated with NAC. Estrogen receptor, progesteron receptor, human epidermal growth factor receptor 2, Ki67, PD-L1, PDL-2 and PD-1 status were assessed by immunohistochemistry. There were 37 (20.9%) patients with high PD-1 expression, 42 (23.7%) patients had high PD-L1 expression, and 52 (29.4%) patients had high PD-L2 expression. The patients with high PD-1 and PD-L1 expressions had a significantly higher rate of triple-negative breast cancer (TNBC) (p = 0.041) (p < 0.001). In TNBC, patients with high PD-1 and PD-L1 expressions had significantly higher rates of non-pCR (p = 0.003) (p < 0.001). Univariate analysis showed that PD-1 and PD-L1 expressions also significantly shortened disease free survival in TNBC (p = 0.048, HR = 3.318) (p = 0.007, HR = 8.375). However, multivariate analysis found that only PD-L1 expression was an independent prognostic factor (p = 0.041, HR = 9.479). PD-1 and PD-L1 expressions may be useful as biomarkers to predict treatment responses to NAC in breast cancer. Above all, PD-L1 expression may also be useful as biomarkers for more effective chemotherapy in TNBC.


Background
Various immunosuppressive factors from cancer cells in the tumor microenvironment inhibit host immune responses to cancer [1,2]. Several immune checkpoints exist in immune response pathways, and negative costimulatory molecules such as cytotoxic T-lymphocyteassociated protein (CTLA)-4 and PD-1 are important checkpoints in limiting self-immune responses [3,4]. Immunotherapy is effective not only in malignant melanoma and renal cell carcinoma (RCC), but anti-tumor effects have also been demonstrated in a variety of other cancers. Immune checkpoint inhibitor therapy, which turns anti-tumor T cells into effectors, has completely changed the role of cancer immunotherapy in clinical practice [5][6][7][8].
Breast cancer was not previously regarded as a tumor associated with abnormal immunity [9]. However, in a phase I trial in TNBC, the PD-1 inhibitor pembrolizumab showed anti-tumor activity, and correlations between PD-1 and PD-L1 (B7-H1) expressions and outcomes were reported [10][11][12][13]. Thus, immune checkpoint inhibitor therapy is expected to play a major role in the tailored treatment of breast cancer.
Immunohistological analysis has shown that PD-L1 expression is induced in most solid tumors, including malignant melanoma, ovarian cancer, lung cancer, RCC, and breast cancer. PD-L1 expression in cancer cells has been correlated with cancer progression, the occurrence of metastases, and survival rates [14][15][16][17]. In addition, tumor-infiltrating lymphocytes (TILs) include PD-1-positive lymphocytes, and a correlation between them and prognosis in breast cancer has been reported [11].
The effect of the tumor immune environment not only on immunotherapy effectiveness, but also on conventional anti-tumor therapy effectiveness and prognosis, has recently been demonstrated [18]. Thus, improvement of the tumor immune environment is important. In other words, the tumor immune environment plays a role in the anti-tumor effects of conventional anti-tumor drugs. Moreover, immune checkpoint proteins such as PD-1, PD-L1, and PD-L2 (B7-H2) may play an important role in improving the tumor immune environment. Given this background, the clinical significance of immune checkpoint protein expression was investigated in patients receiving NAC for breast cancer using conventional anti-cancer drugs, and whether this would be useful as a marker to predict treatment response was evaluated.
All patients received a standardized protocol of NAC consisting of four courses of FEC100 (500 mg/m 2 fluorouracil, 100 mg/m 2 epirubicin, and 500 mg/m 2 cyclophosphamide) every 3 weeks, followed by 12 courses of 80 mg/m 2 paclitaxel administered weekly [21,22]. Forty-five patients had HER2 + BC and were additionally administered weekly (2 mg/kg) or tri-weekly (6 mg/ kg) trastuzumab during paclitaxel treatment [23]. All patients underwent chemotherapy as outpatients. Therapeutic anti-tumor effects were assessed according to the response evaluation criteria in solid tumors (RECIST) criteria [24]. Pathological complete response (pCR) was defined as the complete disappearance of the invasive compartment of the lesion with or without intraductal components, including in the lymph nodes. Patients underwent mastectomy or breast-conserving surgery after NAC. All patients who underwent breast-conserving surgery were administered postoperative radiotherapy to the remnant breast. Overall survival (OS) time was the period from the initiation of NAC to the time of death from any cause. Disease-free survival (DFS) was defined as freedom from all local, loco-regional, and distant recurrences. All patients were followed up by physical examination every 3 months, US every 6 months, and CT and bone scintigraphy annually. The median followup period for the assessment of OS was 3.4 years (range 0.6-6.0 years), and for DFS it was 3.1 years (range 0.1-6.0 years). This study has been conducted in our institution, Osaka City University Graduate School of Medicine, Osaka, Japan, according to the reporting recommendations for tumor marker prognostic studies (REMARK) guidelines and a retrospectively written research, pathological evaluation, and statistical plan [25]. Asano et al. J Transl Med (2018) 16:87 Immunohistochemistry All patients underwent a core needle biopsy prior to NAC, and they underwent curative surgery involving a mastectomy or conservative surgery with axillary lymph node dissection after NAC at the Osaka City University. Immunohistochemical studies were performed as previously described on core needle biopsy specimens [26]. Tumour specimens were fixed in 10% formaldehyde solution and embedded in paraffin (FFPE), and 4-µm-thick sections were mounted onto glass slides. Slides were deparaffinized in xylene and heated for 20 min (105 °C, 0.4 kg/m 2 ) in an autoclave in Target Retrieval Solution (Dako, Carpinteria, CA, USA). Specimens were then incubated with 3% hydrogen peroxide in methanol for 15 min to block the endogenous peroxidase activity, then incubated in 10% normal goat or rabbit serum to block non-specific reactions.

Immunohistochemical scoring
Immunohistochemical scoring was performed by two pathologists specialized in mammary gland pathology, using the blind method to confirm the objectivity and reproducibility of diagnosis. The cut-off value for ER and PgR positivity was set at ≥ 1% in accordance with previous studies [27]. HER2 expression was scored according to the accepted grading system (0, no reactivity, or membranous reactivity in less than 10% of cells; 1 +, faint/ barely perceptible membranous reactivity in ≥ 10% of cells or reactivity in only part of the cell membrane; 2 +, weak to moderate complete or basolateral membranous reactivity in ≥ 10% of tumour cells; or 3 +, strong complete or basolateral membranous reactivity in ≥ 10% of tumour cells). HER2 expression was considered positive if the immunostaining score was 3 +, or in cases where the score was 2 + and included gene amplification via fluorescent in situ hybridization (FISH). For FISH analyses, each copy of the HER2 gene and its centromere 17 (CEP17) reference were counted. The interpretation followed the criteria of the ASCO/CAP guidelines for HER2 IHC classification for breast cancer: positive if the HER2/ CEP17 ratio was higher than 2.0 [28]. A Ki67-labeling index ≥ 14% was classified as positive [20].
Histopathologic analysis of TILs was evaluated on a single full-face hematoxylin and eosin (HE)-stained tumor section using criteria described [29][30][31]. To evaluate PD-1 expression, five stained areas were selected, and the number of TILs in stroma surrounding the stained cancer cells was quantitatively measured in each field under 400-times magnification (Fig. 1a). The median value of the average each field was determined, and that number was set as a cutoff value. To evaluate PD-L1 and PD-L2 expression, 3 fields of view (FOVs) in darkly stained areas were selected, and the percentage of cancer cells stained with anti-PD-L1 antibody and anti-PD-L2 antibody in each FOVs was measured under 400-times magnified microscopy (Fig. 1b, c). Based on previous reports, ≥ 10% was defined as high expression, and < 10% was defined as low expression [12,32].

Statistical analysis
Statistical analysis was performed using the SPSS version 19.0 statistical software package (IBM, Armonk, NY). The associations between PD-1, PD-L1 and PD-L2 and clinicopathological variables were evaluated using χ 2 tests. Multivariate analysis of pCR was carried out using a binary logistic regression model. The Kaplan-Meier method was used to estimate DFS and OS, and the results were compared between groups using log-rank tests. Multivariate analysis of prognostic factors was carried out using a Cox regression model. A p value < 0.05 was considered significant. Cutoff values for different biomarkers included in this study were chosen before the statistical analysis.

Discussion
Stephen Paget in 1889 proposed the "seed and soil" theory with regard to cancer metastases, and since that time, the importance of the tumor microenvironment in cancer cell proliferation has been increasingly recognized [33]. Tumor tissue is composed not only of cancer cells, but also inflammatory cells, immunocytes, vascular and lymphatic cells, fibroblasts, and fibrous tissue, and these comprise the characteristic tumor microenvironment. The tumor immune environment affects not only the effectiveness of immunotherapy, but also the prognosis and response to other treatments, such as conventional anti-tumor drugs [18]. Thus, control and improvement of the tumor immune microenvironment are important. In other words, assessment of the tumor immune environment in each individual patient can be useful in predicting treatment responses to conventional anti-cancer drugs. Therefore, the present study investigated the immune microenvironment in breast cancer patients' tumor tissues before receiving NAC and examined the correlation with treatment responses.
"Avoiding immune destruction" has recently been established as one of the hallmarks of cancer [33]. Cancer is controlled by immunological surveillance mechanisms at the stage of cancer cell growth and by immune responses to tumor antigens in actual cancer tissue [2]. In response to these immune responses, cancer cells themselves can alter their immunogenicity and induce immunosuppression mechanisms in the tumor microenvironment, thus enabling cancer cells to cleverly escape the host immune system, survive, and grow [1,34].
The PD-1/PD-L1 pathway is also an important immunosuppression mechanism that allows cancer cells to escape host immunity. Because of excessive PD-1 and PD-L1 levels in the tumor microenvironment, antibody inhibition of PD-1 and PD-L1 pathways is promising    16:87 for effectively reversing this immunosuppression in the tumor microenvironment [32]. Suppression of T cell activation by PD-1 signals is promoted in association with the interaction of PD-1 and its ligands PD-L1 and PD-L2 [4,35,36]. The present study found statistical correlations among PD-1, PD-L1, and PD-L2 expressions in all breast cancers. There might be an interaction between PD-1 and its ligand PD-L1 and PD-L2.
The present study also investigated how the expressions of these immune checkpoint proteins affected responses to NAC in breast cancer. Previous studies have shown that high, compared to low, PD-1 expression is associated with a poorer prognosis in malignant melanoma, RCC, and breast cancer [10,11,37]. Moreover, more reports about PD-L1 than about PD-1 suggest a correlation between PD-L1 expression and the degree of cancer malignancy and a poorer prognosis [12,[14][15][16]. In TNBC, PD-1 and PD-L1 expression has been reported frequently in TNBC [1,12]. In the present study, patients with high PD-1 and PD-L1 expressions had significantly higher rates of TNBC. In addition, patients with low PD-1 and PD-L1 expressions had a significantly longer DFS. In particular, low PD-1 and PD-L1 expressions in TNBC were associated with a higher pCR rate and significantly longer DFS, and low PD-L1 expression was an independent prognostic factor. These results suggest that immune escape mediated by immune checkpoints may play a role in the biological malignancy of TNBC. Among patients who received NAC, a longer DFS in patients with low PD-L1 expression suggests increased chemotherapy sensitivity. However, as limitation of this study, it is thought that pCR which has an influence on the prognosis of TNBC after NAC was included as a factor [38,39]. On the other hand, although HER2 + BC showed correlation with PD-1 and PD-L1 expression, there was no effect on prognosis.
Many anti-cancer drugs have immunosuppressive effects and are not compatible with immunotherapy, but depending on their mode of administration, immunological enhancement or reversal of immunosuppression TNBC triple-negative breast cancer, HER2 human epidermal growth factor receptor 2, BC breast cancer, pCR pathological complete response, PD-1 programmed cell death-1, PD-L programmed cell death-ligand is possible [40,41]. In the present study, a standard regimen (FEC followed by paclitaxel ± trastuzumab) was used as NAC in breast cancer. To improve immune escape on the cancer cell side, decreased sensitivity to cytotoxic T lymphocytes (CTLs) can be improved by drugs such as 5FU and paclitaxel, and so-called immunogenic cell death (ICD) of cancer cells can be induced by alkylating agents such as cyclophosphamide and anthracycline drugs [40,42]. Moreover, to improve immune escape on the host side, paclitaxel inhibition of regulatory T cells (Tregs) and 5FU inhibition of myeloid-derived suppressor cells (MDSCs) have been reported [40,42]. Such regimens are thought to enhance immune responses by these mechanisms. Furthermore, improvement of immune escape on the cancer cell side by PD-L1 inhibition and on the host side by PD-1 inhibition can enhance the anti-tumor effects of anti-cancer drugs.

Conclusions
In conclusion, PD-1 and PD-L1 expressions may be useful as biomarkers to predict treatment responses to NAC in breast cancer. Above all, PD-L1 expression may also be useful as biomarkers for more effective chemotherapy in TNBC.

Additional file
Additional file 1: Fig S1. Overall survival analysis of the correlation with outcome. Analysis of the correlation with outcome of all 177 patients, overall survival (OS) was also significantly longer in patients with low, compared to high, PD-1 and PD-L1 expressions (p = 0.048, log-rank) (p = 0.022, log-rank) (A, B). OS did not differ significantly between patients with low vs high PD-L2 expression (p = 0.615, log-rank) (C). In 61 TNBC cases, OS was also significantly longer in patients with low, compared to patients with high, PD-1 expression (p = 0.021, log-rank), but OS was not significantly different based on PD-L1 expression (p = 0.155, log-rank) (D, E). DFS and OS were also not significantly different based on PD-L2 expression (p = 0.595, log-rank) (F). In 45 HER2 + BC cases, OS was also not significantly longer in patients with low, compared to patients with high, PD-1, PD-L1 and PD-L2 expressions (p = 0.673, p = 0.620, p = 0.749, logrank, respectively) (G-I). Authors' contributions YA participated in the design of the study and drafted the manuscript. SK participated in the design of the study and manuscript editing. WG, KTakad, TM and TT helped with study data collection and manuscript preparation. KTakah, HF and ST helped with study data collection and participated in its design. MOhs helped with pathological diagnosis. KH and MOhi conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.