Combined Association of Tumoral PD-L1 Expression and Pretreatment Presence of Epstein-Barr Virus DNA With Risk Stratification and Prognosis of Patients With Nasopharyngeal Carcinoma

Abstract

Background:

Little is known about whether the combination of tumor programmed death-ligand 1 (PD-L1) expression and pretreatment EBV DNA status can help stratify nasopharyngeal carcinoma (NPC) patients by risk of metastasis or predict prognosis.

Methods:

PD-L1 expression was assessed using immunohistochemical staining of 78 non-keratinizing NPC patients with clinical data. Survival outcomes and independent prognostic factors were identified.

Results:

Seventy-eight patients were included, high PD-L1 expression was observed in 25 of 43 patients (58%) with metastasis, while it was observed only in 7 of 35 patients (20%) without metastasis. Multivariate analyses showed that progression-free survival (PFS) was independently predicted by tumoral PD-L1 expression and pretreatment EBV DNA status. When combining, 93.75% patients with high PD-L1 and EBV infection developed distant metastasis, and those patients were associated with worse PFS.

Conclusions:

Both PD-L1 expression and pretreatment EBV DNA are closely related to metastasis and prognosis of NPC patients. Their combination can facilitate risk stratification and prognosis prediction, which may improve disease treatment and management.

Introduction

Nasopharyngeal carcinoma (NPC), can be distinguished from other head and neck cancers based on its clinical presentation, pathological features, and epidemiology, as well as its close association with the Epstein-Barr virus (EBV) (1). Cases of NPC have been observed worldwide, and the reported number of new cases and the number of deaths in both males and females of NPC in 2020 were 133,354 and 80,008 worldwide. The age standardized incidence and mortality rates were both remarkedly higher in Southeastern Asia than in any other region (2–4). The main treatment modality for early-stage NPC is radiotherapy (5, 6). In patients with locally advanced NPC, treatment using a combination of radiotherapy and chemotherapy has increased the 5-year local control rate to 90% (7, 8). However, about 10-20% patients can experience recurrence or distant metastasis (9–12). The treatment options for recurrence and metastatic patients are limited, and the efficacy also needs further improvement (13). Thus, identifying effective markers for stratifying NPC patients by risk of metastasis may aid in their timely treatment and management.

NPC is a virus-driven malignancy (14, 15) that is characterized by EBV infection and the presence of immune cell infiltration around the cancer nests (16). Pretreatment and postradiotherapy plasma EBV DNA levels correlate with disease outcomes and long-term survival of patients with advanced NPC (17). Although pretreatment plasma EBV DNA has been suggested as a reliable biomarker for the diagnosis, risk stratification, and prognosis of NPC patients (17, 18), studies differ in the cut-off values that they apply (17, 19) and methods to quantify EBV DNA in plasma have not been standardized (19, 20). In addition, a randomized controlled trial analyzed plasma EBV DNA in NPC patients after chemoradiation to identify high-risk patients for adjuvant chemotherapy, results showed that adjuvant chemotherapy with cisplatin and gemcitabine did not improve relapse-free survival (21). This study indicated that the EBV DNA levels alone were unable to identify high-risk patients for adjuvant chemotherapy (21). There is an urgent need to identify efficient markers to help with risk stratification and development of treatment options for NPC patients.

Programmed death-ligand 1 (PD-L1), also known as B7-H1 or CD274, is a surface glycoprotein that induces T cell anergy or apoptosis by binding to its receptor, programmed cell death protein-1 (PD-1), on T lymphocytes (22, 23). PD-L1 is widely expressed on antigen-presenting and other immune cells, and is upregulated in tumor cells in a broad range of tumors, including NPC (24–28). The expression of PD-L1 is correlated with tumor grade or prognosis in several types of carcinomas (22, 23, 29), and patients with EBV-associated malignancies have high levels of PD-L1 (26, 30). PD-L1 has recently been reported as a prognostic indicator in NPC patients, but these findings remain controversial (31–34). Victor H. F. Lee and colleagues reported that patients with higher PD-L1 expression on tumors had longer locoregional failure-free survival and marginally longer PFS (31). Another study also demonstrated that overexpression of PD-L1 on tumor cells was associated with superior OS (34). However, several studies have shown that PD-L1 expression predicts poor prognosis for NPC patients. Yajuan Zhou et al. found that patients with a higher PD-L1 H-score could have a greater risk of death (33). A meta-analysis study including 1,315 patients revealed that PD-L1 overexpression was associated with a poor OS (32). These studies with inconsistent results indicated that PD-L1 alone may not be a perfect prognostic indicator of NPC.

EBV associated malignancies had distinct regulation mechanisms on the expression of PD-L1. In NPC, PD-L1 expression was higher in EBV-positive NPC cell lines than in EBV-negative cell lines, and that PD-L1 expression can be enhanced by the exogenous and endogenous induction of the latent membrane protein 1 (LMP1) and IFN-gamma pathways (35). EBV-encoded circular RNA also upregulated the PD-L1 expression in NPC by retinoic acid-inducible gene I (36). Therefore, a combination of PD-L1 expression and EBV infection status may be effective at identifying NPC patients at higher risk of metastasis or poor survival.

In this study, we examined the tumoral PD-L1 expression and pretreatment plasma EBV DNA status in a cohort of patients with non-keratinizing NPC to assess the combined effect of these two factors on risk stratification and prognosis prediction.

Materials and Methods

Patients

We included patients with a histologically confirmed diagnosis of non-keratinizing NPC and who received intensity-modulated radiation therapy at West China Hospital, Sichuan University (Chengdu, China) between January 2008 and August 2016. Biopsy specimens, a complete clinical history and follow-up data for at least three years were available for each included patient. We excluded patients with other malignant diseases, severe comorbidities, and those with Eastern Cooperative Oncology Group (ECOG) scores ≥ 2. This study was approved by the Institutional Review Board of West China Hospital of Sichuan University, and the need for written informed consent was waived by the Institutional Review Board.

Data Collection

Medical records were reviewed for demographic information, including age, sex, smoking status, stage of disease, quantitative reverse transcription polymerase chain reaction (RT-PCR) data for plasma EBV DNA load at diagnosis, and survival outcomes. Disease stages were determined based on the American Joint Committee on Cancer tumor-node-metastasis staging (AJCC, 7th edition) (37). Progression-free survival (PFS) was defined as the time from diagnosis until progression, while overall survival (OS) was defined as the time from diagnosis until death due to any cause or the last follow-up.

Quantitative RT-PCR

Peripheral venous blood (3mL) was obtained at the time of diagnosis (before treatment) and centrifuged at 1600 g for 15 min in tubes containing EDTA. Plasma EBV DNA was isolated and analyzed using a commercial kit (Sansure Biotech, Hunan, China) based on a quantitative RT-PCR assay (20, 38) that targets the BamHI-W fragment of the EBV genome. Any detectable amplification was treated as a positive result.

Immunohistochemistry of PD-L1

Formalin-fixed, paraffin-embedded NPC tissues biopsied at diagnosis were retrieved from the pathology department. The sections were dewaxed, rehydrated through a graded alcohol series, placed in 95°C ethylene diamine tetra-acetic acid buffer (pH 8) for 40 min to retrieve antigens, then incubated in 3% H₂O₂ for 15 min at room temperature. The sections were stained overnight at 4°C with anti-PD-L1 primary antibody (clone SP142, ZSGB-Bio, Beijing, China), followed by incubation with secondary antibody for 45 min at 37°C and immunoreactions were visualized using the ChemMate EnVision+ detection kit (Peroxidase/Dab, Rabbit/Mouse, K5007, Dako, Glostrup, Denmark). Separate full slides containing NPC tissue of known PD-L1 status were used as positive and negative controls for PD-L1 staining. Tissue images were taken using a phase contrast microscope.

PD-L1 expression was semi-quantified in terms of the proportion of total cells that stained positive as well as the intensity of the positive stain by two experienced pathologists who were blinded to the clinical data. The optimal cut-off point for PD-L1 expression based on the percentage of positive tumor cells was determined based on the area under the receiver operating characteristic curve (ROC). Using the ROC value, the optimal cut-off point for tumoral PD-L1 expression was calculated as 45%. PD-L1 expression was dichotomized into two groups (high and low), when staining was observed on ≥ 45% of the tumor cells, it was classified as high expression.

Statistical Analysis

Correlations between metastasis and PD-L1 expression or EBV status were assessed using the chi-squared test. Patients were stratified into four groups based on their tumor PD-L1 expression levels and pretreatment EBV infection status: (A) patients with low PD-L1 expression and no EBV infection, (B) patients with low PD-L1 expression and EBV infection, (C) patients with high PD-L1 expression and no EBV infection, or (D) patients with high PD-L1 expression and EBV infection. Factors affecting PFS and OS were assessed using the Kaplan-Meier method and univariate log-rank tests. Multivariate Cox regression was used to identify independent predictors of survival. Differences associated with two-sided p < 0.05 were considered significant. All statistical analyses were performed using SPSS 25.0 (Chicago, IL).

Results

Clinical Characteristics of Patients

In this study, we recruited a total of 78 patients with non-keratinizing NPC (63 men) with a median age of 44 and range of 22-65 years old. Follow-up data for at least 5 years were available for 94% of the patients; the median follow-up time was 60.4 months (range, 9.4-110.8 months). Of all patients, 43 (55%) had metastatic NPC (Table 1). The expression of PD-L1 was not significantly different between males and females or between smokers and non-smokers. In addition, the percentage of high PD-L1 expression was also not significantly different among different T stages and disease stages (Table 1).

PD-L1 was detected in 66 patients (85%), primarily in the cytoplasm and membrane of tumor cells in a heterogeneous manner. Expression levels varied substantially across patients (Figure 1).

Figure 1

High PD-L1 Expression and Positive EBV Status Are Associated With Metastasis and Worse Prognosis

High PD-L1 expression was observed in 25 of 43 patients (58%) with metastatic NPC, while it was observed only in 7 of 35 patients (20%) without metastasis (p = 0.001, Figure 2A). This suggests that patients with high tumoral PD-L1 expression levels are more likely to develop metastatic NPC. In addition, Kaplan-Meier analysis showed that low PD-L1 expression was associated with better PFS (p = 0.003; Figure 3A) but not OS (p = 0.658; Figure 4A).

Figure 2

Figure 3

Figure 4

Similarly, serum EBV DNA was detectable at diagnosis in 28 of 41 patients (68%) with metastatic NPC, significantly higher than the frequency of 14 of 34 patients (41%) without metastasis (p = 0.022, Figure 2B). Undetectable EBV DNA at diagnosis was associated with significantly better PFS (p = 0.014, Figure 3B), but not OS (p = 0.160, Figure 4B).

Tumor PD-L1 Expression Levels and EBV Status Are Independent Predictors of PFS

PFS showed a significant association with N stage, disease stage, EBV status, and PD-L1 expression (Table 2). Based on the univariate analysis, we found that high PD-L1 expression was associated with higher risk of disease progression (HR 2.66, 95%CI 1.36-5.19, p = 0.004), as was EBV positivity (HR 2.39, 95%CI 1.16-4.90, p = 0.014; Table 2).

Multivariate analysis identified three independent predictors of PFS in our cohort: N stage, high PD-L1 expression (HR 2.85, 95%CI 1.35-6.04, p = 0.006), and EBV-positive status (HR 2.90, 95%CI 1.41-5.97, p = 0.004) (Table 3). None of the clinical parameters showed a significant association with OS.

Association Between EBV Status and PD-L1 Expression in NPC Patients

Previous studies have reported that EBV infection could upregulate PD-L1 expression in NPC cell lines (35). Thus, we analyzed the association between EBV status and PD-L1 expression in all the patients. In the 33 patients with negative EBV status, 44% of patients had high PD-L1expression, and in the 42 patients with positive EBV status, the high PD-L1 expression rate was 50%. We did not observe a significant association between PD-L1 expression and EBV status by the chi-square test in our cohort (p=0.813, Table 1).

Combination of EBV Status and PD-L1 Expression Levels Can Stratify Patients by Risk of Metastasis and Prognosis

In this study, we stratified the patients into four groups based on their EBV status and tumoral PD-L1 expression levels. When we compared the metastasis rates among these four groups, we found that patients with high PD-L1 expression levels and positive EBV status had the highest incidence of metastasis (93.75%), while the patients with low PD-L1 expression levels and negative EBV status had the lowest incidence of metastasis (26.3%). Patients with high PD-L1 expression and positive EBV status had the worst PFS among all four groups (p = 0.001, Figures 3C, D). The was no significant difference in overall survival of four groups (p=0.320, Figures 4C, D).

Discussion

NPC is an EBV-associated malignancy with high metastatic potential (39), and metastasis is the most frequent reason for treatment failure (40). Therefore, it is important to identify a reliable and efficient marker to predict metastasis risk. In this study, we examined the joint role played by tumoral PD-L1 expression levels and EBV status in predicting metastasis risk and prognosis of NPC patients. Our findings reveal that patients exhibiting high PD-L1 expression and EBV-positive status at NPC diagnosis are more likely to experience metastasis and shorter PFS.

Several studies have reported correlations between plasma EBV DNA levels and the prognosis and treatment outcomes of NPC patients (17, 18). However, the stratifying power of EBV DNA levels on their own may be limited (21). Better results may be obtained by using a combination of two or more biomarkers, as shown for the combinations of tumor volume and EBV status, pretreatment EBV DNA levels and clinicopathological variables, or pretreatment EBV DNA levels and cervical node necrosis (41–43). However, none of these studies stratified patients based on treatment modality. With the current advances in immunotherapy, it is worth considering the addition of immunotherapy to chemotherapy, especially for patients at high risk of metastasis (44).

PD-L1, which is expressed on immune, antigen-presenting, and tumor cells, plays an important role in T cell tolerance and immune escape. This might be the reason why patients with higher PD-L1 expression had shorter PFS and correlates with metastasis in this cohort. The mechanisms of PD-L1 upregulation induced by EBV infection are complex. In NPC, PD-L1 expression was reported to be regulated by EBV-induced LMP1 and IFN-gamma pathways (35). Recently, Ge and colleagues found that EBV-encoded circular RNA CircBART2.2 upregulated the expression of PD-L1 in NPC and further inhibited the T-cell function (36). In other malignancies with positive EBV infection, IFN-gamma mediated signaling pathways, EBV microRNA, somatic gene mutations, and epigenetic modifications were also reported to regulate PD-L1 expression (45, 46). Therefore, PD-L1 may be an effective target for immunotherapy as well as a useful biomarker for predicting metastasis and survival. Many studies have investigated the role of PD-L1 expression and EBV in the prognosis and risk stratification of NPC, as well as other EBV-associated malignancies, but most of them did not combine tumor PD-L1 expression with EBV-DNA to further stratify NPC patients (30, 33, 47–51) Hu and colleagues stratified NPC patients into three groups based on the combination of EBV DNA load and PD-L1 expression on tumor infiltrating lymphocytes (TILs). The results indicated that a high log EBV-DNA load with low TIL PD-L1 was a poor prognostic factor for PFS (52).

The results of present study indicate that tumoral PD-L1 expression and pretreatment plasma EBV status act as independent prognostic indicators, and both of them are associated with metastasis in NPC patients. Our results show that patients with high tumoral PD-L1 expression levels and detectable EBV DNA have the highest incidence of metastasis and worst PFS, indicating that the combination of these two biomarkers can help identify high-risk patients. For those patients with higher risk for metastasis and worse survival stratified by the combination of PD-L1 and EBV status, more intensive anti-tumor treatment may necessary to reduce the incidence of metastasis and recurrence, further to improve patients’ survival. Adding anti-PD-L1/PD-1 immunotherapy to conventional treatment regimens for these high-risk patients may further prolong their PFS. Several PD-1 blocking antibodies have shown promising results in the treatment of recurrent and metastatic NPC failed on previous treatment (53–56). Recently, results of two-phase 3 clinical trials showed that anti-PD-1 antibodies plus gemcitabine-cisplatin chemotherapy as first-line treatment in advanced NPC patients had superior PFS compared to chemotherapy alone, and this regimen could be a new standard of care for metastatic or recurrent NPC (57, 58). This progress might help to improve the treatment efficacy of patients with high risk stratified by PD-L1 expression and positive EBV status.

To our knowledge, this is the first study to examine the combination of tumoral PD-L1 expression and pretreatment plasma EBV DNA status as biomarkers for risk stratification and prognosis in NPC. The EBV DNA status is routinely tested during the management of NPC, and PD-L1 expression can be evaluated using low-cost immunohistochemistry. Using these two indices together may provide good risk stratification without the need for complex nomograms. Although our study was able to identify biomarkers that can be used for risk stratification in NPC patients, our findings were limited by the small sample size of the cohort and by heterogeneity in PD-L1 expression within the same patient. Future studies should investigate these associations further and validate the relationship between metastasis and PD-L1 expression and EBV status using larger samples.

This study revealed that pretreatment plasma EBV status and tumoral PD-L1 expression levels are closely related to prognosis of NPC patients. Using the two biomarkers together may facilitate risk stratification and timely, effective treatment.

Funding

This study was supported by grants from the Department of Science and Technology of Sichuan Province (Grant No. 2020YFS0276), the Health Department of Sichuan Province (130087) and the Wu Jieping Medical Foundation (320.6750.13317).

Publisher’s Note

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References

• 1

  NakanishiYWakisakaNKondoSEndoKSugimotoHHatanoMet al. Progression of Understanding for the Role of Epstein-Barr Virus and Management of Nasopharyngeal Carcinoma. Cancer Metastasis Rev (2017) 36(3):435–47. doi: 10.1007/s10555-017-9693-x

  • CrossRef
  • Google Scholar
  • View reference in article

• 2

  TangLLChenWQXueWQHeYQZhengRSZengYXet al. Global Trends in Incidence and Mortality of Nasopharyngeal Carcinoma. Cancer Lett (2016) 374(1):22–30. doi: 10.1016/j.canlet.2016.01.040

  • CrossRef
  • Google Scholar
  • View reference in article

• 3

  World Health Organization.(2021). Available at: https://gco.iarc.fr/today/data/factsheets/cancers/4-Nasopharynx-fact-sheet.pdf [Accessed November 20, 2021].

  • Google Scholar
  • View reference in article

• 4

  SungHFerlayJSiegelRLLaversanneMSoerjomataramIJemalAet al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin (2021) 71(3):209–49. doi: 10.3322/caac.21660

  • CrossRef
  • Google Scholar
  • View reference in article

• 5

  SprattDELeeN. Current and Emerging Treatment Options for Nasopharyngeal Carcinoma. OncoTargets Ther (2012) 5:297–308. doi: 10.2147/OTT.S28032

  • CrossRef
  • Google Scholar
  • View reference in article

• 6

  LeeAWNgWTPanJJChiangCLPohSSChoiHCet al. International Guideline on Dose Prioritization and Acceptance Criteria in Radiation Therapy Planning for Nasopharyngeal Carcinoma. Int J Radiat Oncol Biol Phys (2019) 105(3):567–80. doi: 10.1016/j.ijrobp.2019.06.2540

  • CrossRef
  • Google Scholar
  • View reference in article

• 7

  LinSPanJHanLGuoQHuCZongJet al. Update Report of Nasopharyngeal Carcinoma Treated With Reduced-Volume Intensity-Modulated Radiation Therapy and Hypothesis of the Optimal Margin. Radiother Oncol (2014) 110(3):385–9. doi: 10.1016/j.radonc.2014.01.011

  • CrossRef
  • Google Scholar
  • View reference in article

• 8

  XiaoWWHuangSMHanFWuSXLuLXLinCGet al. Local Control, Survival, and Late Toxicities of Locally Advanced Nasopharyngeal Carcinoma Treated by Simultaneous Modulated Accelerated Radiotherapy Combined With Cisplatin Concurrent Chemotherapy: Long-Term Results of a Phase 2 Study. Cancer (2011) 117(9):1874–83. doi: 10.1002/cncr.25754

  • CrossRef
  • Google Scholar
  • View reference in article

• 9

  ZongJLinSLinJTangLChenBZhangMet al. Impact of Intensity-Modulated Radiotherapy on Nasopharyngeal Carcinoma: Validation of the 7th Edition AJCC Staging System. Oral Oncol (2015) 51(3):254–9. doi: 10.1016/j.oraloncology.2014.10.012

  • CrossRef
  • Google Scholar
  • View reference in article

• 10

  LeeNXiaPQuiveyJMSultanemKPoonIAkazawaCet al. Intensity-Modulated Radiotherapy in the Treatment of Nasopharyngeal Carcinoma: An Update of the UCSF Experience. Int J Radiat Oncol Biol Phys (2002) 53(1):12–22. doi: 10.1016/S0360-3016(02)02724-4

  • CrossRef
  • Google Scholar

• 11

  KamMKTeoPMChauRMCheungKYChoiPHKwanWHet al. Treatment of Nasopharyngeal Carcinoma With Intensity-Modulated Radiotherapy: The Hong Kong Experience. Int J Radiat Oncol Biol Phys (2004) 60(5):1440–50. doi: 10.1016/j.ijrobp.2004.05.022

  • CrossRef
  • Google Scholar

• 12

  BaumlJMCohenRBAggarwalC. Immunotherapy for Head and Neck Cancer: Latest Developments and Clinical Potential. Ther Adv Med Oncol (2016) 8(3):168–75. doi: 10.1177/1758834016631529

  • CrossRef
  • Google Scholar

• 13

  PrawiraAOostingSFChenTWDelos SantosKASalujaRWangLet al. Systemic Therapies for Recurrent or Metastatic Nasopharyngeal Carcinoma: A Systematic Review. Br J Cancer (2017) 117(12):1743–52. doi: 10.1038/bjc.2017.357

  • CrossRef
  • Google Scholar
  • View reference in article

• 14

  YoungLSRickinsonAB. Epstein-Barr Virus: 40 Years on. Nat Rev Cancer (2004) 4(10):757–68. doi: 10.1038/nrc1452

  • CrossRef
  • Google Scholar
  • View reference in article

• 15

  ChenCJHsuWLYangHILeeMHChenHCChienYCet al. Epidemiology of Virus Infection and Human Cancer. Recent Results Cancer Res (2014) 193:11–32. doi: 10.1007/978-3-642-38965-8_2

  • CrossRef
  • Google Scholar
  • View reference in article

• 16

  LinXGudgeonNHHuiEPJiaHQunXTaylorGSet al. CD4 and CD8 T Cell Responses to Tumour-Associated Epstein-Barr Virus Antigens in Nasopharyngeal Carcinoma Patients. Cancer Immunol Immunother: CII (2008) 57(7):963–75. doi: 10.1007/s00262-007-0427-8

  • CrossRef
  • Google Scholar
  • View reference in article

• 17

  LinJCWangWYChenKYWeiYHLiangWMJanJSet al. Quantification of Plasma Epstein-Barr Virus DNA in Patients With Advanced Nasopharyngeal Carcinoma. N Engl J Med (2004) 350(24):2461–70. doi: 10.1056/NEJMoa032260

  • CrossRef
  • Google Scholar

• 18

  ChanATLoYMZeeBChanLYMaBBLeungSFet al. Plasma Epstein-Barr Virus DNA and Residual Disease After Radiotherapy for Undifferentiated Nasopharyngeal Carcinoma. J Natl Cancer Inst (2002) 94(21):1614–9. doi: 10.1093/jnci/94.21.1614

  • CrossRef
  • Google Scholar

• 19

  LertbutsayanukulCKannarunimitDNetsawangBKitpanitSChakkabatCHansasutaPet al. Optimal Plasma Pretreatment EBV DNA Cut-Off Point for Nasopharyngeal Cancer Patients Treated With Intensity Modulated Radiation Therapy. Japanese J Clin Oncol (2018) 48(5):467–75. doi: 10.1093/jjco/hyy027

  • CrossRef
  • Google Scholar
  • View reference in article

• 20

  LeQTZhangQCaoHChengAJPinskyBAHongRLet al. An International Collaboration to Harmonize the Quantitative Plasma Epstein-Barr Virus DNA Assay for Future Biomarker-Guided Trials in Nasopharyngeal Carcinoma. Clin Cancer Res (2013) 19(8):2208–15. doi: 10.1158/1078-0432.CCR-12-3702

  • CrossRef
  • Google Scholar
  • View reference in article

• 21

  ChanATCHuiEPNganRKCTungSYChengACKNgWTet al. Analysis of Plasma Epstein-Barr Virus DNA in Nasopharyngeal Cancer After Chemoradiation to Identify High-Risk Patients for Adjuvant Chemotherapy: A Randomized Controlled Trial. J Clin Oncol (2018) 36:JCO2018777847. doi: 10.1200/JCO.2018.77.7847

  • CrossRef
  • Google Scholar
  • View reference in article

• 22

  GhebehHMohammedSAl-OmairAQattanALeheCAl-QudaihiGet al. The B7-H1 (PD-L1) T Lymphocyte-Inhibitory Molecule Is Expressed in Breast Cancer Patients With Infiltrating Ductal Carcinoma: Correlation With Important High-Risk Prognostic Factors. Neoplasia (2006) 8(3):190–8. doi: 10.1593/neo.05733

  • CrossRef
  • Google Scholar
  • View reference in article

• 23

  ThompsonRHGillettMDChevilleJCLohseCMDongHWebsterWSet al. Costimulatory Molecule B7-H1 in Primary and Metastatic Clear Cell Renal Cell Carcinoma. Cancer (2005) 104(10):2084–91. doi: 10.1002/cncr.21470

  • CrossRef
  • Google Scholar
  • View reference in article

• 24

  StraubMDrecollEPfarrNWeichertWLangerRHapfelmeierAet al. CD274/PD-L1 Gene Amplification and PD-L1 Protein Expression Are Common Events in Squamous Cell Carcinoma of the Oral Cavity. Oncotarget (2016) 7(11):12024–34. doi: 10.18632/oncotarget.7593

  • CrossRef
  • Google Scholar
  • View reference in article

• 25

  KimJWEderJP. Prospects for Targeting PD-1 and PD-L1 in Various Tumor Types. Oncol (Williston Park) (2014) 28 Suppl 3:15–28.

  • Google Scholar
  • View reference in article

• 26

  ChenBJChapuyBOuyangJSunHHRoemerMGXuMLet al. PD-L1 Expression Is Characteristic of a Subset of Aggressive B-Cell Lymphomas and Virus-Associated Malignancies. Clin Cancer Res (2013) 19(13):3462–73. doi: 10.1158/1078-0432.CCR-13-0855

  • CrossRef
  • Google Scholar
  • View reference in article

• 27

  GandiniSMassiDMandalaM. PD-L1 Expression in Cancer Patients Receiving Anti PD-1/PD-L1 Antibodies: A Systematic Review and Meta-Analysis. Crit Rev Oncology/hematol (2016) 100:88–98. doi: 10.1016/j.critrevonc.2016.02.001

  • CrossRef
  • Google Scholar

• 28

  KonishiJYamazakiKAzumaMKinoshitaIDosaka-AkitaHNishimuraM. B7-H1 Expression on Non-Small Cell Lung Cancer Cells and Its Relationship With Tumor-Infiltrating Lymphocytes and Their PD-1 Expression. Clin Cancer Res (2004) 10(15):5094–100. doi: 10.1158/1078-0432.CCR-04-0428

  • CrossRef
  • Google Scholar

• 29

  MuCYHuangJAChenYChenCZhangXG. High Expression of PD-L1 in Lung Cancer may Contribute to Poor Prognosis and Tumor Cells Immune Escape Through Suppressing Tumor Infiltrating Dendritic Cells Maturation. Med Oncol (2011) 28(3):682–8. doi: 10.1007/s12032-010-9515-2

  • CrossRef
  • Google Scholar

• 30

  LiuYJTsangNMHsuehCYehCJUengSHWangTHet al. Low PD-L1 Expression Strongly Correlates With Local Recurrence in Epstein-Barr Virus-Positive Nasopharyngeal Carcinoma After Radiation-Based Therapy. Cancers (2018) 10(10):374. doi: 10.3390/cancers10100374

  • CrossRef
  • Google Scholar

• 31

  LeeVHLoAWLeungCYShekWHKwongDLLamKOet al. Correlation of PD-L1 Expression of Tumor Cells With Survival Outcomes After Radical Intensity-Modulated Radiation Therapy for Non-Metastatic Nasopharyngeal Carcinoma. PloS One (2016) 11(6):e0157969. doi: 10.1371/journal.pone.0157969

  • CrossRef
  • Google Scholar

• 32

  LiuXShanCSongYDuJ. Prognostic Value of Programmed Cell Death Ligand-1 Expression in Nasopharyngeal Carcinoma: A Meta-Analysis of 1,315 Patients. Front Oncol (2019) 9:1111. doi: 10.3389/fonc.2019.01111

  • CrossRef
  • Google Scholar

• 33

  ZhouYShiDMiaoJWuHChenJZhouXet al. PD-L1 Predicts Poor Prognosis for Nasopharyngeal Carcinoma Irrespective of PD-1 and EBV-DNA Load. Sci Rep (2017) 7:43627. doi: 10.1038/srep43627

  • CrossRef
  • Google Scholar

• 34

  ZhuQCaiMYChenCLHuHLinHXLiMet al. Tumor Cells PD-L1 Expression as a Favorable Prognosis Factor in Nasopharyngeal Carcinoma Patients With Pre-Existing Intratumor-Infiltrating Lymphocytes. Oncoimmunology (2017) 6(5):e1312240. doi: 10.1080/2162402X.2017.1312240

  • CrossRef
  • Google Scholar

• 35

  FangWZhangJHongSZhanJChenNQinTet al. EBV-Driven LMP1 and IFN-Gamma Up-Regulate PD-L1 in Nasopharyngeal Carcinoma: Implications for Oncotargeted Therapy. Oncotarget (2014) 5(23):12189–202. doi: 10.18632/oncotarget.2608

  • CrossRef
  • Google Scholar

• 36

  GeJWangJXiongFJiangXZhuKWangYet al. Epstein-Barr Virus-Encoded Circular RNA CircBART2.2 Promotes Immune Escape of Nasopharyngeal Carcinoma by Regulating PD-L1. Cancer Res (2021) 81(19):5074–88. doi: 10.1158/0008-5472.CAN-20-4321

  • CrossRef
  • Google Scholar

• 37

  EdgeSBByrdDRComptonCCFritzAGGreeneFLTrottiAet al. AJCC Cancer Staging Manual Seventh Edition. New York, NY: Springer International Publishing (2010).

  • Google Scholar

• 38

  XieXQLuoYMaXLLiSSLiuLZhangHet al. Clinical Significance of Circulating Tumor Cells and Their Expression of Cyclooxygenase-2 in Patients With Nasopharyngeal Carcinoma. Eur Rev Med Pharmacol Sci (2019) 23(16):6951–61. doi: 10.26355/eurrev_201908_18735

  • CrossRef
  • Google Scholar

• 39

  ChanAT. Nasopharyngeal Carcinoma. Ann Oncol (2010) 21 Suppl 7:vii308–12. doi: 10.1093/annonc/mdq277

  • CrossRef
  • Google Scholar

• 40

  ZhangLChenQYLiuHTangLQMaiHQ. Emerging Treatment Options for Nasopharyngeal Carcinoma. Drug design Dev Ther (2013) 7:37–52. doi: 10.2147/DDDT.S30753

  • CrossRef
  • Google Scholar

• 41

  ChenQYGuoSYTangLQLuTYChenBLZhongQYet al. Combination of Tumor Volume and Epstein-Barr Virus DNA Improved Prognostic Stratification of Stage II Nasopharyngeal Carcinoma in the Intensity Modulated Radiotherapy Era: A Large-Scale Cohort Study. Cancer Res Treat (2018) 50(3):861–71. doi: 10.4143/crt.2017.237

  • CrossRef
  • Google Scholar

• 42

  TangLQLiCFLiJChenWHChenQYYuanLXet al. Establishment and Validation of Prognostic Nomograms for Endemic Nasopharyngeal Carcinoma. J Natl Cancer Inst (2016) 108(1):djv291. doi: 10.1093/jnci/djv291

  • CrossRef
  • Google Scholar

• 43

  DuYYLuoDHSunXSTangLQMaiHQChenQYet al. Combining Pretreatment Plasma Epstein-Barr Virus DNA Level and Cervical Node Necrosis Improves Prognostic Stratification in Patients With Nasopharyngeal Carcinoma: A Cohort Study. Cancer Med (2019) 8(16):6841–52. doi: 10.1002/cam4.2481

  • CrossRef
  • Google Scholar

• 44

  LvJWLiJYLuoLNWangZXChenYP. Comparative Safety and Efficacy of Anti-PD-1 Monotherapy, Chemotherapy Alone, and Their Combination Therapy in Advanced Nasopharyngeal Carcinoma: Findings From Recent Advances in Landmark Trials. J Immunother Cancer (2019) 7(1):159. doi: 10.1186/s40425-019-0636-7

  • CrossRef
  • Google Scholar

• 45

  NakanoHSaitoMNakajimaSSaitoKNakayamaYKaseKet al. PD-L1 Overexpression in EBV-Positive Gastric Cancer Is Caused by Unique Genomic or Epigenomic Mechanisms. Sci Rep (2021) 11(1):1982. doi: 10.1038/s41598-021-81667-w

  • CrossRef
  • Google Scholar

• 46

  LiXZhangW. Expression of PD-L1 in EBV-Associated Malignancies. Int Immunopharmacol (2021) 95:107553. doi: 10.1016/j.intimp.2021.107553

  • CrossRef
  • Google Scholar

• 47

  AhmedMMGebrielMGMoradEASaberIMElwanASalahMet al. Expression of Immune Checkpoint Regulators, Cytotoxic T-Lymphocyte Antigen-4, and Programmed Death-Ligand 1 in Epstein-Barr Virus-Associated Nasopharyngeal Carcinoma. Appl Immunohistochem Mol Morphol (2021) 29(6):401–8. doi: 10.1097/PAI.0000000000000903

  • CrossRef
  • Google Scholar

• 48

  WangYQZhangYJiangWChenYPXuSYLiuNet al. Development and Validation of an Immune Checkpoint-Based Signature to Predict Prognosis in Nasopharyngeal Carcinoma Using Computational Pathology Analysis. J Immunother Cancer (2019) 7(1):298. doi: 10.1186/s40425-019-0752-4

  • CrossRef
  • Google Scholar

• 49

  PaydasSBagirESeydaogluGErcolakVErginM. Programmed Death-1 (PD-1), Programmed Death-Ligand 1 (PD-L1), and EBV-Encoded RNA (EBER) Expression in Hodgkin Lymphoma. Ann Hematol (2015) 94(9):1545–52. doi: 10.1007/s00277-015-2403-2

  • CrossRef
  • Google Scholar

• 50

  LiZLaiYSunLZhangXLiuRFengGet al. PD-L1 Expression Is Associated With Massive Lymphocyte Infiltration and Histology in Gastric Cancer. Hum Pathol (2016) 55:182–9. doi: 10.1016/j.humpath.2016.05.012

  • CrossRef
  • Google Scholar

• 51

  FangWHongSChenNHeXZhanJQinTet al. PD-L1 Is Remarkably Over-Expressed in EBV-Associated Pulmonary Lymphoepithelioma-Like Carcinoma and Related to Poor Disease-Free Survival. Oncotarget (2015) 6(32):33019–32. doi: 10.18632/oncotarget.5028

  • CrossRef
  • Google Scholar

• 52

  HuBSunMWangZZhengYCaiWShiHHet al. Prognostic Value of Programmed Cell Death-Ligand 1 Expression in Tumor-Infiltrating Lymphocytes and Viral Load in Peripheral Blood Mononuclear Cells for Epstein-Barr Virus-Positive Nasopharyngeal Carcinoma. Clin Chem (2020) 66(9):1219–27. doi: 10.1093/clinchem/hvaa170

  • CrossRef
  • Google Scholar

• 53

  HsuCLeeSHEjadiSEvenCCohenRBLe TourneauCet al. Safety and Antitumor Activity of Pembrolizumab in Patients With Programmed Death-Ligand 1-Positive Nasopharyngeal Carcinoma: Results of the KEYNOTE-028 Study. J Clin Oncol (2017) 35(36):4050–6. doi: 10.1200/JCO.2017.73.3675

  • CrossRef
  • Google Scholar

• 54

  MaBBYLimWTGohBCHuiEPLoKWPettingerAet al. Antitumor Activity of Nivolumab in Recurrent and Metastatic Nasopharyngeal Carcinoma: An International, Multicenter Study of the Mayo Clinic Phase 2 Consortium (NCI-9742). J Clin Oncol (2018) 36(14):1412–8. doi: 10.1200/JCO.2017.77.0388

  • CrossRef
  • Google Scholar

• 55

  FangWYangYMaYHongSLinLHeXet al. Camrelizumab (SHR-1210) Alone or in Combination With Gemcitabine Plus Cisplatin for Nasopharyngeal Carcinoma: Results From Two Single-Arm, Phase 1 Trials. Lancet Oncol (2018) 19(10):1338–50. doi: 10.1016/S1470-2045(18)30495-9

  • CrossRef
  • Google Scholar

• 56

  WangFHWeiXLFengJLiQXuNHuXCet al. Efficacy, Safety, and Correlative Biomarkers of Toripalimab in Previously Treated Recurrent or Metastatic Nasopharyngeal Carcinoma: A Phase II Clinical Trial (POLARIS-02). J Clin Oncol (2021) 39(7):704–12. doi: 10.1200/JCO.20.02712

  • CrossRef
  • Google Scholar

• 57

  MaiHQChenQYChenDHuCYangKWenJet al. Toripalimab or Placebo Plus Chemotherapy as First-Line Treatment in Advanced Nasopharyngeal Carcinoma: A Multicenter Randomized Phase 3 Trial. Nat Med (2021) 27(9):1536–43. doi: 10.1038/s41591-021-01444-0

  • CrossRef
  • Google Scholar

• 58

  YangYQuSLiJHuCXuMLiWet al. Camrelizumab Versus Placebo in Combination With Gemcitabine and Cisplatin as First-Line Treatment for Recurrent or Metastatic Nasopharyngeal Carcinoma (CAPTAIN-1st): A Multicentre, Randomised, Double-Blind, Phase 3 Trial. Lancet Oncol (2021) 22(8):1162–74. doi: 10.1016/S1470-2045(21)00302-8

  • CrossRef
  • Google Scholar