Effect of Primary Systemic Therapy on PD-1, PD-L1, and PD-L2 mRNA Expression in Advanced Breast Cancer

Objective: The association between PD-1, PD-L1, and PD-L2 expression and prognosis has been extensively studied in various cancers but remained controversial in breast cancer. Besides, little is known about the prognostic value of PD-1, PD-L1, and PD-L2 upregulation or downregulation following systemic therapy (chemotherapy and hormonal therapy) in breast cancer. Therefore, we aim to investigate the change of PD-1, PD-L1, and PD-L2 expression in mRNA level after primary systemic therapy in breast cancer patients and its clinical implications. Methods: Expression of PD-1, PD-L1, and PD-L2 mRNA were measured before-after chemotherapy and hormonal therapy with real-time PCR in 80 advanced breast cancer patients. The correlation between alteration of PD-1, PD-L1, and PD-L2 expression and clinicopathological characteristics as well as overall survival was also statistically analyzed. Results: Chemotherapy and hormonal therapy altered PD-1, PD-L1, and PD-L2 expression in breast cancer with most patients have an increase expression. As much as 57.1%, 62.9% and 60% patients have an increase PD-1, PD-L1, and PD-L2 expression after chemotherapy, while 60%, 60%, and 64% patients have an increase PD-1, PD-L1, and PD-L2 expression after hormonal therapy. Alteration of PD-1, PD-L1, and PD-L2 expression was not correlated with all clinicopathological characteristics. Increase in PD-1, PD-L1, and PD-L2 expression was significantly associated with better OS (p=0.031, p=0.019, and p=0.019 for PD-1, PD-L1, and PD-L2, respectively), which remained significant in multivariate analysis including age, stage, primary systemic therapy, histology grade, subtype and primary tumor histology (HR PD-1 0.5 (95% CI 0.28-0.88) p=0.031; HR PD-L1 0.43 (95% CI 0.24-0.8) p=0.019; HR PD-L2 (95% CI 0.24-0.87) p=0.019). Conclusion: Expression of PD-1, PD-L1, and PD-L2 in breast cancer patients is mostly enhanced after chemotherapy and hormonal therapy, and the enhancement is associated with good OS. This result revealed the potential of measuring PD-1, PD-L1, and PD-L2 mRNA expression in predicting clinical outcome.


Introduction
Positive PD-L1 expressions correlated with good response to immune checkpoint inhibitor therapy (anti-PD-1/PD-L1). Currently, measuring PD-L1 expression using immunohistochemistry (IHC) has been used to determine type of patients who respond immune checkpoint inhibitor therapy. However, the use of PD-L1 IHC has several limitations such as different cut offs, different scoring systems, variable detection antibodies, and processing variability (Bertucci et al., 2015;Patel and Kurzrock, 2015). This could affect the result which leads to conflicting data in several studies (Schalper et al., 2014). Therefore, the use of alternative method such as real-time PCR to assess PD-L1 expression may help to overcome such limitations. It has been shown that there is positive correlation between PD-L1 protein and mRNA expression, which indicated potential of measuring PD-L1 mRNA expression to assess response to anti-PD-1/PD-L1 therapy (Kim et al., 2018).
Chemotherapy and hormonal therapy are type of systemic therapy that majorly used to treat breast cancer patients in advanced stages. The idea of combining immune checkpoint inhibitor therapy with chemotherapy or hormonal therapy has been proposed to enhance the response rate and duration and improve survival (Esteva et al., 2019;Hühn et al., 2019;Luo and Fu, 2016;Page et al., 2019). Several studies reported that some chemotherapy agents could change PD-L1 expression (Luo and Fu, 2016). On the other hand, the study of hormone therapy effect to PD-1, PD-L1, or PD-L2 expression is very rare. To investigate the potential benefit of combination therapy between hormonal therapy/chemotherapy and immunotherapy, it is crucial to understand the impact of chemotherapy or hormonal therapy to PD-L1 expression as well as PD-1 and PD-L2 expression. Besides, little is known about the prognostic value of PD-1, PD-L1, and PD-L2 upregulation or downregulation following systemic therapy in breast cancer. Therefore, in this study, we aimed to assess PD-1, PD-L1, PD-L2 mRNA expression before-after primary systemic therapy (chemotherapy and hormonal therapy) using real-time PCR and investigate the association with clinicopathological features and overall survival of advanced breast cancer patients.

Patients' samples collection and therapy given
This study was a retrospective study conducted from 2011 to 2017 (n=80) at Dharmais National Cancer Center Hospital, Indonesia. Patients' tumor tissues of advanced breast cancer (stages 3B and 4) patients were taken before and after primary systemic therapy (chemotherapy and hormonal therapy). Tissue samples were divided into two pieces, one piece for histological examination and another piece was directly put in cryotubes containing 1 mL of RNAlater then stored at -80 o C to keep RNA integrity and quality. Criteria for stage 3B and 4 was determined based on American Joint Committee on Cancer 7 th edition guideline (American Cancer Society, 2010).
From 80 samples, 35 patients were received primary chemotherapy and 45 patients received primary hormonal therapy. All patients were given systemic therapy before the patient undergone surgery. Hormonal therapy group received Aromatase Inhibitor, Tamoxifen or GNRH-analogue during 6 months of treatment. The chemotherapy group received FAC (5-Fluorouracil, Adriamycin, and Cyclophosphamide) which were given for 6 cycles.
Patients who have a mastectomy before primary systemic therapy, pregnant, and refuse to participate were excluded. The patient followed up was done continuously to obtain data of death, censored patients, and patients with new symptoms. All patients agreed to be involved in this study after signing informed consent. This study was approved by Ethical Committee at Dharmais Hospital-National Cancer Center, Indonesia (Number of Ethical Clearance: 9/KEPK/II/2019 and 10/KEPK/ II/2019).

Extraction of total RNA and cDNA synthesis
Isolation of total RNA from tissue samples was done using RNA Tissue Mini Kit (Qiagen) according to manual instruction book provided by the kit. The total RNA was then measured for its concentration and purity using Nanodrop spectrophotometer. Maximum 2,000 ng of RNA was reverse transcribed to cDNA using High Capacity cDNA synthesis kit (Applied Biosystem). The process of cDNA synthesis was conducted based on standard procedure from the kit. Generated cDNA from all samples was then diluted to 100 ng to be used in real time PCR.

Statistical analysis
Statistical analysis was performed using IBM SPSS 21. Statistical comparisons between PD-1, PD-L1, and PD-L2 and clinic pathological were assessed by the Chi-Square test (χ 2 test). Correlation between fold change value of PD-1, PD-L1 and PD-L2 were assessed by Spearman Correlation. Analysis between effect therapy and fold change of PD-1, PD-L1 and PD-L2 were assessed by Independent T-test. Overall Survival (OS) were estimated using the Kaplan-Meier method. Cox proportional hazards model was used to estimate the prognostic factor of PD1, PD-L1, and PD-L2 on overall survival. All analyses were hypothesis-driven by P < 0.05 was considered statistically significant.

Relationship between PD-1, PD-L1 and PD-L2 expression
The relationship between expression changes of PD-1 and PD-L1, PD-1 and PD-L2 and PD-L1 and PD-L2 showed a significant positive correlation with a very strong close relationship (R-value: 0.762, 0.746, and 0.834 for PD-1, PD-L1, and PD-L2 respectively). We showed that samples with increased PD-1 expression also have increased PD-L1 and PD-L2 expression (Figure 2).

Association between PD1, PDL1, and PDL2 expression after therapy and clinicopathology
We found that the alteration of PD-1, PD-L1 and PD-L2 expression was not associated with age, primary tumor histology, histology grade, ER status, PR status, Her2 status, Ki67 status and molecular subtype. However, increased PD-1, PD-L1, and PD-L2 expression were found more on breast cancer patients with higher ages (>40   Table 3. Overall Survival (OS) by PD-1, PDL-1, PDL-2 Expression and Multivariate Analysis years), ductal histology, higher grade, positive estrogen receptor (ER) status, negative HER2 status, and positive progesterone receptor (PR) status ( Table 2).

Association of PD-1, PD-L1, and PD-L2 expression with survival in breast cancer
Further analysis was undertaken to explore the potential association of PD-1, PD-L1, and PD-L2 with patient prognosis and survival. Kaplan-Meier survival analysis indicated that increased PD-1, PD-L1, and PD-L2 expression after primary systemic therapy were associated with statistically significant better overall survival (Figure 3). Increased PD-1 expression was associated with longer OS than decreased PD-1 expression in advanced breast cancer (HR=0.55, 95% CI 0.32 -0.94; p=0.031). Increased PD-L1 expression was associated with longer OS than decreased PD-L1 expression (HR=0.52, 95% CI 0.30 -0.90; p=0.019). Increased PD-L2 expression was also associated with longer OS than decreased PD-L2 expression in advanced breast cancer (HR=0.52, 95% CI 0.29 -0.89; p=0.019). Our data identified significant association between better overall survival and increased PD-1, PDL-1 and PDL-2 expression that was confirmed by multivariate analysis including prior age, stage, primary systemic therapy, histology grade, subtype and primary tumor histology (Table 3).

Discussion
In the present study, we have analyzed PD-1, PD-L1, and PD-L2 mRNA expression in breast cancer tissue from advanced stages patients. Our study found that expression of PD-1, PD-L1, and PD-L2 in breast cancer patients is mostly increased after chemotherapy with 57.1%, 62.9% and 60% patients have an increase in PD-1, PD-L1, and PD-L2 expression, respectively (Table 2). While 42.9%, 37.1%, and 40% breast cancer patients have their PD-1, PD-L1, and PD-L2 expression decreased after chemotherapy. It has been explained that chemotherapy can alter the expression of PD-1, PD-L1, and PD-L2 expression in several cancer types. However, the change depends on chemotherapeutic agents and cell line that were used in the experiment (Chacon et al., 2016;Ghebeh et al., 2010;Peng et al., 2015;Zhang et al., 2008).
Some chemotherapy agents that have been reported to increase PD-1, PD-L1, or PD-L2 expression are paclitaxcel, etoposide, gemcitabine, decitabine, and cisplatin (Luo and Fu, 2016). Etoposide and paclitaxel induced PD-L1 expression in breast cancer cell line leading to the activation of co-inhibitory signals (Zhang et al., 2008). Carboplatin-paclitaxel treatment also induced PD-L1 expression in ovarian cancer cell lines (Peng et al., 2015). Both PD-L1 and PD-1 expression in leukemia cells were upregulated after decitabine treatment (Yang et al., 2013). Cisplatin could increase the expression of PD-L1 in hepatoma H22 cells when the concentration is less than IC 50 (Qin et al., 2010). Gemcitabine or paclitaxel was also enhanced PD-L1 expression in human pancreatic cell lines both in protein and mRNA level (Doi et al., 2017). Nonetheless, some chemotherapy drugs could downregulate PD-1, PD-L1, or PD-L2 expression. Oxiliplatin inhibit PD-L2 expression thus limiting immunosuppression by tumor cells and dentritic cells (Lesterhuis et al., 2011). Treatment with panobinostat suppresses PD-1 expression in lymphoma (Oki et al., 2014). Research by Sheng et al. (Sheng et al., 2016) revealed the downregulation of PD-L1 expression in tumor cells of NSCLC patients after treatment with neoadjuvant chemotherapy (paclitaxel, pemetrex, and TKI). After chemotherapy, positive PD-L1 expression changed from 75% to 37.5% (Sheng et al., 2016).
In this study, 5-FAC (5-Fluorouracil, Adriamycin, and Cyclophosphamide) was used in the treatment of breast cancer patients. It has been reported that 5-Fluorouracil induce PD-L1 surface expression on breast cancer cell lines (Zhang et al., 2008). Doxorubicine (adriamycin) is reported to upregulate PD-L1 nuclear expression, although downregulate its surface expression in tumor. Thus, these previous finding supported our results that after 5-FAC treatment, PD-1, PD-L1, and PD-L2 expression in most breast cancer patients are increased. Meanwhile, the effect of cyclophosphamide on PD-1, PD-L1, or PD-L2 expression hasn't been known. Moreover, it has been proposed that combination between 5-Fluorouracil, Adriamycin, or Cyclophosphamide with anti PD-1/ PD-L1 might give positive impact on cancer patients (Bailly et al., 2020).
The exact mechanism on how chemotherapy work on tumor microenvironment and affect PD-1, PD-L1, and PD-L2 expression is still not clear. However, some studies reported that some chemotherapeutic agents involved in several biological pathway. Chemotherapeutic agents through interferon (IFN)-γ-independent and IFN-γdependent may upregulate PD-L1 expression by activating different signal such as JAK/STAT3, PI3K/AKT, RAS/ RAF, or release several immune suppression cytokine (Luo and Fu, 2016). In breast cancer, signaling through key proliferative pathways, like PI3K/ AKT and MEK/ ERK is known to induce PD-L1 expression (Crane et al., 2009;Hasan et al., 2011).
Similar with chemotherapy, the PD-1, PD-L1, and PD-L2 mRNA expression are mostly increased after patients underwent hormonal therapy. Percentages of increased PD-1, PD-L1, and PD-L2 expression after hormonal therapy are 60%, 60%, and 64% respectively ( Table 2). It has been reported that some hormonal therapy could induce PD-L1 expression in several cancer. Expression of PD-L1 is increased in MCF7 cells (breast cancer cell line) after treatment with estrogen receptor (ER) antagonist. Treatment with tamoxifen is also increased PD-L1 expression in mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT) breast cancer mice models (Hühn et al., 2019). It also has been shown that aromatase inhibitor (AI) therapy might increase the expression of both PD-L1 and chemokine receptor CCR7 in tumors (Turnbull et al., 2020;West et al., 2018). In a prostate cancer trial, enzalutamide plus pembrolizumab was associated with increased PD-L1 expression in tumor and dendritic cells, and increased PD-1-positive in circulating T-cells (Bishop et al., 2015;Graff et al., 2016).
It is not clear that how hormone therapy affects PD-1, PD-L1, and PD-L2 expression. Nonetheless, hormone therapy is known to change hormone level such as estrogen. It has been shown that alteration of estrogen level might alter PD-L1 expression. A study by Huhn et al., (2019) showed that estrogen deprivation could upregulate PD-L1 expression and triggers a wide inflammatory transcriptional program in ER+ breast cancer which includes secretion of cytokine such as as IL-6 and IFNγ that trigger the activation of the JAK/STAT pathway and TNFα that activate NF-kB signaling. Another studies showed that addition of 17β-estradiol (E2) could induce PD-1 and PD-L1 expression suggesting that E2 signaling might be involved in PD-1/ PD-L1 pathway (Rothenberger et al., 2018). In this study, aromatase inhibitor (AI), tamoxifen, and GnRH analogue are given to breast cancer patients. It has been reported that AI and tamoxifen could induce PD-L1 expression (Hühn et al., 2019;Turnbull et al., 2020;West et al., 2018). Meanwhile, the effect of GnRH analogs on PD-L1 expression is still unknown. However, the administration of GnRH analogs is known to decrease the concentration of circulating estrogen in premenopausal women (Huerta-Reyes et al., 2019). Moreover, it has been shown that reduction of estrogen (estrogen deprivation) could increase PD-L1 expression (Hühn et al., 2019). This may explain how GnRH analogs induce PD-L1 expression.
Interestingly the alteration of PD-1, PD-L1, and PD-L2 expression was associated each other. Mostly, samples with increased PD-1 expression also increased in PD-L1 and PD-L2 expression (Figure 2). Another study also revealed that there is positive correlation between the PD-1 and PD-L1 mRNA expression levels in blood samples of ITP patients (Zhong et al., 2016). Yearley et al., (2017) showed positive correlation between PD-L1 and PD-L2 protein expression in breast cancer patients. This finding suggested that chemotherapy and hormone therapy might affect the same pathway involved in the alteration of PD-1, PD-L1, and PD-L2 expression.
We also found that alteration of PD-1, PD-L1, and PD-L2 expression are associated with survival whereas increased PD-1, PD-L1, and PD-L2 were significantly associated with good OS, while decreased PD-1, PD-L1, and PD-L2 is associated with worse OS (Figure 3). This finding is linear with previous reports which showed that high PD-1, PD-L1, and PD-L2 expression is significantly associated with good survival. Various studies have reported that PD-1, PD-L1, and PD-L2 expression were associated with longer recurrence-free survival, longer disease-specific survival, longer OS, DFS, and PFS in breast cancer (Ali et al., 2015;Baptista et al., 2015;Sabatier et al., 2015.;Schalper et al., 2014;Uhercik et al., 2017;Yearley et al., 2017). The relationship between survival and PD-L1 expression in breast cancer might indicate the presence of strong antitumor immune response mediated by TILs which leading to PD-L1 upregulation. Therefore, previous results are supported our finding. In addition, higher PD-L1 and PD-L2 mRNA expression were associated with better OS to atezolizumab (anti PD-L1) in melanoma, RCC, NSCLC, and metastatic urothelial (Schmid et al., 2016;Yearley et al., 2017).
To the best of our knowledge, this study is the first that analyzed the relationship between alteration of PD-1, PD-L1, and PD-L2 mRNA expression after primary systemic therapy with survival rate. We have shown that PD-1, PD-L1, and PD-L2 expression in most samples are increased after chemo and hormonal therapy and the enhancement is associated with good survival. Since PD-L1 expression was used to assess response to PD-1/PD-L1 checkpoint inhibitor therapy, this finding indicated reassessment of PD-L1 expression after chemotherapy or hormonal therapy should be performed. Besides, because of high PD-L1 expression include expression in mRNA level is associated with good clinical outcome of anti-PD-1/PD-L1 therapy (Patel and Kurzrock, 2015;Schmid et al., 2016), we could suggest that PD-1/ PD-L1 checkpoint inhibitor therapy might improve outcome of breast cancer patients who have an increased PD-L1 expression after completion of chemotherapy or hormonal therapy.
It has been shown that the combination of chemotherapy/ hormone therapy and immunotherapy might provide effective and durable anti-tumor immune response and facilitate the clearance of the residual breast cancer cells, and reducing the percentage of patients that progress into metastatic disease (Hühn et al., 2019;Luo and Fu, 2016). Therefore, our finding may support the idea of combining chemo or hormone therapy with anti PD-1/ PD-L1. This finding also revealed that PD-1, PD-L1, and PD-L2 mRNA expression potentially could be used to predict clinical outcome of breast cancer patients. However, the limitation of this study is mRNA expression is not the same with protein expression due to post transcriptional modifications. Thus, further study to compare PD-L1 mRNA expression with PD-L1 protein expression using IHC as gold standard is needed to confirm this finding.
In conclusion, Expression of PD-1, PD-L1, and PD-L2 majorly increased after primary systemic therapy. Increase in PD-1, PD-L1, and PD-L2 expression after therapy was significantly associated with good OS. Strong positive correlation between PD-1, PD-L1, and PD-L2 alteration after systemic therapy suggested chemotherapy or hormonal therapy may affect the same pathway to alter PD-1, PD-L1, and PD-L2 expression. Our finding implied reassessment of PD-L1 expression and the potential benefit of anti-PD-1/PD-L1 therapy after completion of systemic therapy. This finding also revealed the potential to measure PD-1, PD-L1, and PD-L2 mRNA expression to predict clinical outcome of advanced stages breast cancer patients. However, subsequent study by comparing mRNA expression to PD-L1 IHC is needed to confirm the result.

Author Contribution Statement
R.K and M.A.A designed the study and took the lead in writing the manuscript. M.A.A carried the experiment and performed the measurement. F.S contributed to sample preparation. Y.P performed statistical analysis; All authors discussed the results and contributed to the final manuscript.