INTRODUCTION
============

Esophageal cancer is the 8^th^ most common tumor and the 6^th^ cause of tumor associated death in the world \[[@r1]\].  The 5-year survival rate of this disorder is only 15--25%, owing to the aggressive nature of the disease and resistance to therapy.  Approximately 50% of esophageal cancer patients worldwide occur in China.  Patients typically present with progressive dysphagia when diagnosed \[[@r2]\].  Esophageal cancer can arise in the upper (\<10% of cases), middle (\>50%), or lower (\>20%) segment of the esophagus \[[@r2], [@r3]\].  Patients in high-risk areas in China therefore undergo routine endoscopic screening, which is useful for early diagnosis and effective treatment of esophageal cancer. 

Surgical or endoscopic resection and chemoradiotherapy are the standard treatments for esophageal cancer, although the therapeutic strategy varies according to stage \[[@r4], [@r5]\].  Many patients who present with resectable esophageal cancer ultimately develop local recurrence and metastasis \[[@r6]\].  These patients are primarily managed with chemotherapy (e.g. fluoropyrimidine/platinum) \[[@r7]\].  Additional chemotherapy methods include capecitabine, S-1, infusional 5-fluorouracil (5-FU) and other 5-FU pro-drugs, and oxaliplatin or cisplatin \[[@r7]\].  The response rates to 5-FU in combination with cisplatin were reported to be 30--40% and 40--50% for esophageal adenocarcinoma and esophageal squamous cell carcinoma (ESCC), respectively \[[@r6]\].  In total, improvements of chemotherapy were disappointing over the last 3 decades, and current efforts should pay attention to targeted therapies and immunotherapy.  Several new drugs such as ramucirumab, capecitabine, oxaliplatin, and everolimus have also demonstrated therapeutic efficacy in esophageal cancer \[[@r6], [@r8]--[@r12]\].  However, some patients have developed resistance to these therapies, and the underlying mechanisms were complex and poorly understood.  Drug resistance may in part be mediated by the long non-coding RNA CCAT1 \[[@r13]\].  Additionally, microRNA-10b and CDKN3 have been shown to contribute to cisplatin resistance \[[@r14], [@r15]\]. 

Esophageal cancer contains 2 primary pathological subtypes: adenocarcinoma and ESCC.  Similar genetic and environmental risk factors for esophageal adenocarcinoma and ESCC have been identified including gender, race, obesity, nutrition, smoking, and alcohol consumption \[[@r1]\].  Interestingly, polymorphisms in the *programmed death-1* (*PD-1*) gene (located in chromosome 2q37.3) have been associated with esophageal cancer risk and prognosis \[[@r16]--[@r18]\]. 

PD-1, a 50--55 kDa type I transmembrane glycoprotein, was first isolated from activated T cells \[[@r19], [@r20]\].  It consists of a transmembrane domain, extracellular domain, and intracellular region.  It is reported to express on the surface of some immune cells including activated monocytes, T cells, natural killer cells, B cells, and NK T cells \[[@r21], [@r22]\].  PD-1 negatively regulated the regulatory and effector T cells.  It plays a critical role in suppressing the immune response to promote self-tolerance \[[@r23]\].  However, by suppressing the immune response, it can contribute to cancer progression.  PD-1 gene expression was associated with T cell activation-induced apoptosis in murine T cell hybridomas \[[@r24]\].  Additionally, persistent PD-1 expression in tumor-infiltrating lymphocytes was related with poor prognosis and cancer recurrence \[[@r25]\].  Two FDA-approved monoclonal antibodies targeting PD-1 including pembrolizumab and nivolumab have demonstrated efficacy for several cancer treatment \[[@r26], [@r27]\].  Anti-PD-1 therapy promoted regression of advanced tumors and improved survival, particularly among subsets of patients with solid tumors, and demonstrated durable effects and tolerable toxicities \[[@r27]\].  Biomarkers including density of tumor infiltrating lymphocyte, mismatch-repair deficiency, PD-L1 expression, and tumor mutational burden, predicted treatment effect of anti-PD-1 therapy \[[@r26]\]. 

*PD-1* gene polymorphisms were associated with the risk of various cancers \[[@r1], [@r28]--[@r30]\].  Beyond foregoing disorders, PD-1 is overexpressed in different cancers, including esophageal cancer.  For example, the rs2227981, rs2227982, and rs3608432 polymorphisms were related to lung adenocarcinoma risk and prognosis \[[@r31]\].  However, no associations were observed in basal cell carcinoma \[[@r32]\].  The *PD-1*.5 C/T polymorphism increased the risk of cervical \[[@r33]\], lung \[[@r34]\], gastric \[[@r35]\], colon \[[@r36]\], thyroid cancers \[[@r37]\].  The rs2227982 C\>T polymorphism associated with gastric cardia adenocarcinoma risk \[[@r38]\].  Interestingly, it was reduced the risk of breast cancer \[[@r39]\] and increased the risk of ovarian cancer \[[@r40]\].  Hua et al.  mentioned that *PD-1* gene polymorphisms may regulate the breast cancer susceptibility and prognosis in Chinese individuals \[[@r41]\], while inconsistent findings were obtained in the study by Haghshenas et al.  \[[@r42]\].  Thus, several meta-analyses were conducted to solve these conflicting findings \[[@r43]--[@r45]\].  Data summarized that *PD-1* rs11568821 and rs2227981 polymorphisms decreased the overall cancer risk, and *PD-1* rs7421861 polymorphism was associated with an increased risk of overall cancer \[[@r43]\].  No significant association between some SNPs (rs2227982, rs10204525, rs36084323, and rs2890658 polymorphisms) and overall cancer risk was obtained \[[@r43]\].  To be honest, these loci of *PD-1* gene might be potential biomarkers for predicting susceptibility to cancers and therapeutic markers for cancer treatment. 

Considering the vital role of *PD-1* gene polymorphisms in cancers, we designed this hospital-based case-control study containing 814 esophageal cancer patients and 961 healthy controls.  The aims of this case-control study were to investigate whether three *PD-1* gene polymorphisms (rs10204525, rs36084323, and rs7421861) were related with esophageal cancer risk and clinical features in Chinese subjects. 

RESULTS
=======

Population characteristics
--------------------------

We performed a case-control study consisting of 814 esophageal cancer patients and 961 age- and gender-matched controls.  The baseline characteristics of these patients including TNM stage, pathological grade, and distant metastases are shown in [Table 1](#t1){ref-type="table"}.  The mean ages of the controls and cases were 60.91 and 60.66years, respectively.  No differences were observed in smoking or alcohol between the two groups.  The majority of the patients (85%) were diagnosed with ESCC. 

###### Patient demographics and risk factors in esophageal cancer. 

  ----------------------------- ------------------ --------------------- ---------
  **Characteristics**           **Case (N=814)**   **Control (N=961)**   ***P***
  Age                           60.66 (36-82)      60.91 (38-85)         0.495
  Sex                                                                    0.440
   Male                         577(70.9%)         665(69.2%)            
   Female                       237(29.1%)         296(30.8%)            
  Smoking                                                                0.771
   YES                          430(52.8%)         501(52.1%)            
   NO                           384(47.2%)         460(47.9%)            
  Alcohol                                                                0.358
   YES                          470 (57.7%)        534(55.6%)            
   NO                           344(42.3%)         427(44.4%)            
  TNM stage                                                              
   I+II                         424(52.1%)                               
   III+IV                       390(47.9%)                               
  Pathological grading                                                   
   Well differentiation         320(39.3%)                               
   Moderately differentiation   378(46.4%)                               
   Poorly differentiation       116(14.3%)                               
  Histology                                                              
   Squamous cell carcinoma      782(96.1%)                               
   Others                       32(3.9%)                                 
  Distant metastasis                                                     
   M0                           723(88.8%)                               
   M1                           91(11.2%)                                
  ----------------------------- ------------------ --------------------- ---------

TNM stage = Tumor node metastasis stage

PD-1 polymorphisms increase the risk of esophageal cancer
---------------------------------------------------------

We evaluated the associations between three polymorphisms in *PD-1* (rs10204525, rs7421861, and rs36084323) and the risk of esophageal cancer.  The distributions of the genotypes of the *PD-1* variants among the case and control populations are presented in [Table 2](#t2){ref-type="table"} and [Supplementary Figure 1](#SD1){ref-type="supplementary-material"}.  The GG genotype of rs10204525 polymorphism increased the risk of esophageal cancer compared to the more common AA genotype (GG vs. AA: adjusted odds ratio \[OR\] = 1.65, 95% confidence interval \[CI\] = 1.12--2.45; *P* = 0.012).  This association was significant under recessive, dominant, and allelic models.  The TT genotype of rs7421861 was related with a 1.45-fold higher risk of esophageal cancer compared to the CC genotype (TT vs. CC: OR = 1.45, 95% CI = 1.06--1.99); *P* = 0.022).  We did not observe an association between rs36084323 polymorphism and esophageal cancer risk. 

###### Genotype frequencies of PD-1 gene polymorphisms in cases and controls. 

  ---------------- -------------- -------------------- ----------------------- --------------------- --------------- ---------------- --------------------- ------------------
  **Models**       **Genotype**   **Case (n, %)^a^**   **Control (n, %)^a^**   **OR (95% CI)**       ***P*-value**   **P (FDR)^b^**   **OR (95% CI)^c^**    ***P*-value^c^**
  **rs10204525**                                                                                                                                            
  Co-dominant      AA             420(51.7%)           551(57.4%)              1.00(reference)                                                              
  Heterozygote     AG             329(40.5%)           359(37.4%)              1.20(0.99-1.46)       0.066                            1.20(0.99-1.46)       0.070
  Homozygote       GG             63(7.8%)             50(5.2%)                **1.65(1.12-2.45)**   **0.012**                        **1.67(1.13-2.48)**   **0.010**
  Dominant         AA             420(51.7%)           551(57.4%)              1.00(reference)                                                              
                   GG+AG          392(48.3%)           409(42.6%)              **1.26(1.04-1.52)**   **0.017**                        **1.26(1.04-1.52)**   **0.017**
  Recessive        AG+AA          749(92.2%)           910(94.8%)              1.00(reference)                                                              
                   GG             63(7.8%)             50(5.2%)                **1.53(1.04-2.25)**   **0.030**                        **1.55(1.06,2.28)**   **0.025**
  Allele           A              1169(72.0%)          1461(76.1%)             1.00(reference)                                                              
                   G              455(28.0%)           459(23.9%)              **1.24(1.07-1.44)**   **0.005**       **0.015**                              
  **rs36084323**                                                                                                                                            
  Co-dominant      GG             673(82.8%)           761(79.2%)              1.00(reference)                                                              
  Heterozygote     GA             132(16.2%)           188(19.6%)              0.79(0.62-1.02)       0.066                            0.79(0.62-1.01)       0.064
  Homozygote       AA             8(1.0%)              12(1.2%)                0.75(0.31-1.86)       0.539                            0.75(0.30-1.85)       0.530
  Dominant         GG             673(82.8%)           761(79.2%)              1.00(reference)                                                              
                   AA+GA          140(17.2%)           200(20.8%)              0.79(0.62-1.01)       0.056                            0.79(0.62-1.00)       0.054
  Recessive        GA+GG          805(99.0%)           949(98.8%)              1.00(reference)                                                              
                   AA             8(1.0%)              12(1.2%)                0.79(0.32-1.93)       0.600                            0.78(0.32,1.92)       0.589
  Allele           G              1478(90.9%)          1710(89.0%)             1.00(reference)                                                              
                   A              148(9.1%)            212(11.0%)              0.81(0.65-1.01)       0.058           0.058                                  
  **rs7421861**                                                                                                                                             
  Co-dominant      CC             343(42.2%)           457(47.6%)              1.00(reference)       \-                               \-                    \-
  Heterozygote     CT             370(45.5%)           411(42.8%)              1.19(0.98-1.46)       0.072                            1.20(0.98-1.46)       0.074
  Homozygote       TT             100(12.3%)           92(9.6%)                **1.45(1.06-1.99)**   **0.022**                        **1.44(1.05,1.97)**   **0.024**
  Dominant         CC             343(42.2%)           457(47.6%)              1.00(reference)       \-                               \-                    \-
                   TT+CT          470(57.8%)           503(52.4%)              **1.25(1.03-1.50)**   **0.023**                        **1.24(1.03,1.50)**   **0.024**
  Recessive        CT+CC          713(87.7%)           868(90.4%)              1.00(reference)       \-                               \-                    \-
                   TT             100(12.3%)           92(9.6%)                1.32(0.98-1.79)       0.067                            1.32(0.97,1.78)       0.074
  Allele           C              1056(64.9%)          1325(69.0%)             1.00(reference)       \-                               \-                    \-
                   T              570(35.1%)           595(31.0%)              **1.20(1.04-1.38)**   **0.010**       **0.015**                              
  ---------------- -------------- -------------------- ----------------------- --------------------- --------------- ---------------- --------------------- ------------------

^a^The genotyping was successful in 812 cases and 960 controls for rs10204525; The genotyping was successful in 813 cases and 961 controls for rs36084323; The genotyping was successful in 813 cases and 960 controls for rs7421861. 

Bold values are statistically significant (P \<0.05). 

^b^P (FDR) values were calculated with for false discovery rate (FDR) and P \< 0.05/3 was considered significant. 

^c^Adjust for sex, age, smoking and drinking. 

We next stratified patients by age, gender, pathological subtype, alcohol consumption, and smoking.  The genotype numbers among different subgroups are summarized in [Supplementary Figure 2](#SD1){ref-type="supplementary-material"}.  The association between rs10204525 polymorphism and the esophageal cancer risk was stronger among men, those who smoked or consumed alcohol, and those ≥ 60 years old ([Table 3](#t3){ref-type="table"}).  The rs7421861 polymorphism demonstrated a significant association with esophageal cancer risk among men and among smokers ([Table 3](#t3){ref-type="table"}).  The rs36084323 polymorphism was only related to esophageal cancer risk among men.  Finally, rs10204525 and rs7421861 polymorphisms increased the risk of ESCC ([Supplementary Tables 1](#SD2){ref-type="supplementary-material"} and [2](#SD2){ref-type="supplementary-material"}). 

###### Stratified analyses between PD-1 gene polymorphisms and the risk of esophageal cancer. 

  ---------------- ------------------------------ ------------------------ ---------------------- ---------------------------- ----------------------------- ---------------------------- ----------------------------
  **Variable**     **Genotypes (case/control)**   **Heterozygous model**   **Homozygous model**   **Recessive model**          **Dominant model**                                         
                   **Wild**                       **Heterozygote**         **Homozygous**                                                                                                 
  **rs10204525**   **AA**                         **AG**                   **GG**                 **AG vs. AA**                **GG vs. AA**                 **GG vs. AA+AG**             **GG+AG vs. AA**
  Sex                                                                                                                                                                                     
   Male            299/388                        236/244                  41/33                  1.26(0.99-1.59); 0.057       1.61(0.99-2.61); 0.052        1.47(0.92-2.35); 0.112       **1.30(1.04-1.63); 0.023**
   Female          121/163                        93/115                   22/17                  1.09(0.76-1.56); 0.642       1.74(0.89-3.43); 0.107        1.68(0.87-3.25); 0.122       1.17(0.83-1.65); 0.361
  Smoking                                                                                                                                                                                 
   Yes             222/290                        168/185                  38/26                  1.19(0.90-1.56); 0.219       **1.91(1.13-3.24); 0.017**    **1.78(1.06-2.98); 0.029**   1.28(0.98-1.65); 0.066
   No              198/261                        161/174                  25/24                  1.22(0.92-1.62); 0.169       1.37(0.76-2.48); 0.292        1.26(0.71-2.25); 0.429       1.14(0.94-1.63); 0.124
  Alcohol                                                                                                                                                                                 
   Yes             240/301                        191/209                  38/24                  1.15(0.88,1.49); 0.303       **1.99(1.16,3.40); 0.013**    **1.87(1.11,3.17); 0.020**   1.23(0.96,1.58); 0.100
   No              180/250                        138/150                  25/26                  1.29(0.95,1.73); 0.110       1.34(0.75,2.39); 0.330        1.21(0.69,2.14); 0.512       1.29(0.97,1.71); 0.085
  Age (years)                                                                                                                                                                             
   \<60            207/238                        134/158                  22/21                  0.98(0.73,1.31); 0.868       1.21(0.64,2.26); 0.559        1.22(0.66,2.25); 0.531       1.00(0.75,1.33); 0.989
   ≥60             213/313                        195/201                  41/29                  **1.43(1.10,1.85); 0.008**   **2.08(1.25,3.45); 0.005**    **1.78(1.09,2.92); 0.022**   **1.51(1.17,1.94); 0.001**
  **rs36084323**   **GG**                         **GA**                   **AA**                 **GA vs. GG**                **AA vs. GG**                 **AA vs. GG+GA**             **AA+GA vs. GG**
  Sex                                                                                                                                                                                     
   Male            484/520                        87/135                   6/10                   **0.69(0.52-0.93); 0.015**   0.65(0.23-1.79); 0.399        0.69(0.25-1.91); 0.472       **0.69(0.52-0.92); 0.011**
   Female          189/241                        45/53                    2/2                    1.08(0.70-1.68); 0.724       1.28(0.78-9.14); 0.809        1.26(0.18-8.99); 0.820       1.09(0.71-1.68); 0.697
  Smoking                                                                                                                                                                                 
   Yes             355/395                        71/101                   4/5                    0.78(0.56-1.09); 0.152       0.89(0.24-3.34); 0.863        0.93(0.25-3.49); 0.916       0.79(0.57-1.09); 0.154
   No              318/366                        61/87                    4/7                    0.81(0.56-1.16); 0.243       0.66(0.19-2.27); 0.507        0.68(0.20-2.35); 0.546       0.80(0.56-1.13); 0.201
  Alcohol                                                                                                                                                                                 
   Yes             388/417                        75/111                   6/6                    0.73(0.53,1.00); 0.053       1.08(0.34,3.36); 0.901        1.14(0.37,3.56); 0.821       0.86(0.59,1.24); 0.416
   No              285/344                        57/77                    2/6                    0.89(0.61,1.30); 0.558       0.40(0.08,2.01); 0.268        0.41(0.08,2.05); 0.278       0.74(0.54,1.02); 0.307
  Age (years)                                                                                                                                                                             
   \<60            300/322                        58/86                    5/10                   0.72(0.50,1.05); 0.086       0.54(0.18,1.59); 0.261        0.57(0.19,1.68); 0.309       0.70(0.49,1.00); 0.053
   ≥60             173/439                        74/102                   3/2                    0.85(0.61,1.19); 0.348       **1.77(0.29,10.62); 0.535**   1.81(0.30,10.90); 0.515      0.87(0.63,1.21); 0.407
  **rs7421861**    **CC**                         **CT**                   **TT**                 **CT vs. CC**                **TT vs. CC**                 **TT vs. CC+CT**             **TT+CT vs. CC**
  Sex                                                                                                                                                                                     
   Male            233/318                        272/283                  71/63                  **1.31(1.04-1.66); 0.025**   **1.54(1.05-2.25); 0.026**    1.34(0.94-1.92); 0.109       **1.35(1.08-1.70); 0.009**
   Female          110/139                        98/128                   29/29                  0.97(0.67-1.39); 0.858       1.26(0.71-2.24); 0.423        1.28(0.74-2.22); 0.370       1.02(0.73-1.44); 0.900
  Smoking                                                                                                                                                                                 
   Yes             174/240                        195/221                  61/39                  1.22(0.93-1.60); 0.160       **2.16(1.38-3.37); 0.001**    **1.95(1.28-2.99); 0.002**   **1.36(1.05-1.76); 0.021**
   No              169/217                        175/190                  39/53                  1.18(0.89-1.58); 0.253       0.95(0.60-1.50); 0.809        0.87(0.56-1.35); 0.535       1.13(0.86-1.49); 0.377
  Alcohol                                                                                                                                                                                 
   Yes             191/261                        222/225                  44/45                  0.73(0.53,1.00); 0.053       1.08(0.34,3.36); 0.901        1.14(0.37,3.56); 0.821       0.86(0.59,1.24); 0.416
   No              152/196                        148/186                  56/47143               0.89(0.61,1.30); 0.558       0.40(0.08,2.01); 0.268        0.41(0.08,2.05); 0.278       0.74(0.54,1.02); 0.307
  Age (years)                                                                                                                                                                             
   \<60            143/205                        173/170                  47/42                  0.72(0.50,1.05); 0.086       0.54(0.18,1.59); 0.261        0.57(0.19,1.68); 0.309       0.70(0.49,1.00); 0.053
   ≥60             200/252                        197/241                  53/50                  0.85(0.61,1.19); 0.348       1.77(0.29,10.62); 0.535       1.81(0.30,10.90); 0.515      0.87(0.63,1.21); 0.407
  ---------------- ------------------------------ ------------------------ ---------------------- ---------------------------- ----------------------------- ---------------------------- ----------------------------

Bold values are statistically significant (*P* \<0.05). 

Cross-over analysis
-------------------

We next analyzed the joint effects of the *PD-1* polymorphisms and either smoking or alcohol consumption on esophageal cancer risk ([Table 4](#t4){ref-type="table"}).  The GG genotype of rs10204525 did not confer an increased risk to esophageal cancer.  Additionally, smoking had no association with the risk of esophageal cancer.  However, smokers with the GG genotype of rs10204525 polymorphism showed an increased risk of esophageal cancer compared to non-smokers with the AA genotype (OR = 1.93, 95% CI = 1.13--3.28; *P* = 0.014).  These data indicate that there is a strong interaction between the GG genotype of rs10204525 and smoking.  The TT genotype of rs7421861 was also not associated with an increased risk of esophageal cancer.  However, smokers with the TT genotype of rs7421861 had a significantly increased risk of esophageal cancer.  No interaction between rs36084323 and either smoking or alcohol consumption was observed. 

###### Genetic (G) and environmental (E) factors 2\*4 fork analysis. 

  ---------------- ---------- ---------- ------------- ---------------------------- ----------------------------
  **G^a^**         **E^b^**   **Case**   **Control**   **OR (95%CI); P value**      **Reflecting information**
  **rs10204525**                                                                    
   GG vs. AA       Smoking                                                          
    +              \+         38         26            **1.93(1.13,3.28); 0.014**   G, E combined effect
    +              \-         25         24            1.37(0.76,2.48); 0.291       G alone effect
    -              \+         222        290           1.01(0.78,1.30); 0.944       E alone effect
    -              \-         198        261           1.00 (reference)             Common control
   AG vs. AA       Smoking                                                          
    +              \+         168        185           1.20(0.91,1.58); 0.206       G, E combined effect
    +              \-         161        174           1.22(0.92,1.62); 0.169       G alone effect
    -              \+         222        290           1.01(0.78,1.30); 0.944       E alone effect
    -              \-         198        261           1.00 (reference)             Common control
   GG vs. AA       Drinking                                                         
    +              \+         38         50            1.06(0.66,1.68); 0.819       G, E combined effect
    +              \-         25         24            1.45(0.80,2.62); 0.220       G alone effect
    -              \+         240        301           1.11(0.86,1.43); 0.434       E alone effect
    -              \-         180        250           1.00 (reference)             Common control
   AG vs. AA       Drinking                                                         
    +              \+         191        209           1.27(0.97,1.67); 0.088       G, E combined effect
    +              \-         138        150           1.28(0.95,1.73); 0.109       G alone effect
    -              \+         240        301           1.11(0.86,1.43); 0.434       E alone effect
    -              \-         180        250           1.00 (reference)             Common control
  **rs36084323**                                                                    
   AA vs. GG       Smoking                                                          
    +              \+         4          5             0.99(0.27,3.73); 0.993       G, E combined effect
    +              \-         4          7             0.71(0.21,2.45); 0.585       G alone effect
    -              \+         355        366           1.21(0.98,1.48); 0.079       E alone effect
    -              \-         318        395           1.00 (reference)             Common control
   GA vs. GG       Smoking                                                          
    +              \+         71         101           0.87(0.62,1.22); 0.431       G, E combined effect
    +              \-         61         87            0.87(0.61,1.25); 0.450       G alone effect
    -              \+         355        366           1.21(0.98,1.48); 0.079       E alone effect
    -              \-         318        395           1.00 (reference)             Common control
   AA vs. GG       Drinking                                                         
    +              \+         6          6             1.21(0.39,3.78); 0.747       G, E combined effect
    +              \-         2          6             0.40(0.08,2.01); 0.251       G alone effect
    -              \+         388        417           1.12(0.91,1.38); 0.277       E alone effect
    -              \-         285        344           1.00 (reference)             Common control
   GA vs. GG       Drinking                                                         
    +              \+         75         111           0.82(0.59,1.14); 0.229       G, E combined effect
    +              \-         57         77            0.89(0.61,1.30); 0.558       G alone effect
    -              \+         388        417           1.12(0.91,1.38); 0.277       E alone effect
    -              \-         285        344           1.00 (reference)             Common control
  **Rs7421861**                                                                     
   TT vs. CC       Smoking                                                          
    +              \+         61         39            **2.01(1.28,3.15); 0.002**   G, E combined effect
    +              \-         39         53            0.95(0.60,1.50); 0.809       G alone effect
    -              \+         174        240           0.93(0.70,1.23); 0.617       E alone effect
    -              \-         169        217           1.00 (reference)             Common control
   CT vs. CC       Smoking                                                          
    +              \+         195        221           1.13(0.86,1.50); 0.379       G, E combined effect
    +              \-         175        190           1.18(0.89,1.58); 0.252       G alone effect
    -              \+         174        240           0.93(0.70,1.23); 0.617       E alone effect
    -              \-         169        217           1.00 (reference)             Common control
   TT vs. CC       Drinking                                                         
    +              \+         56         47            1.54(0.99,2.39); 0.056       G, E combined effect
    +              \-         44         45            1.26(0.79,2.01); 0.330       G alone effect
    -              \+         191        261           0.94(0.71,1.25); 0.687       E alone effect
    -              \-         152        196           1.00 (reference)             Common control
   CT vs. CC       Drinking                                                         
    +              \+         222        225           1.27(0.96,1.69); 0.093       G, E combined effect
    +              \-         148        186           1.03(0.76,1.39); 0.868       G alone effect
    -              \+         191        261           0.94(0.71,1.25); 0.687       E alone effect
    -              \-         152        196           1.00 (reference)             Common control
  ---------------- ---------- ---------- ------------- ---------------------------- ----------------------------

^a^G (+): PD-1 gene rs10204525/rs36084323/rs7421861 variants (Heterozygous or homozygous); G (-): wild type

^b^E(+): smoking/non-smoking; E(-): non-smoking/non-drinking

*PD-1* gene polymorphisms correlate with the clinical features of esophageal cancer patients
--------------------------------------------------------------------------------------------

We next investigated the relationship between *PD-1* gene polymorphisms and the clinical characteristics of esophageal cancer patients ([Table 5](#t5){ref-type="table"}).  The GG genotype of rs10204525 polymorphism increased the risk of distant metastasis (OR = 2.21, 95% CI = 1.16--4.23; *P* = 0.014) and higher TNM stage (OR = 1.81, 95% CI = 1.05--3.12; *P* = 0.032).  The AG genotype of rs10204525 was related to an increased risk of ESCC (OR = 1.61, 95% CI = 1.05--2.46; *P* = 0.029).  Finally, the TT genotype of rs7421861was associated with higher TNM stage. 

###### The associations between PD-1 gene polymorphisms and clinical characteristics of esophageal cancer. 

  ----------------------------- ---------------------------- ---------------------------- ---------------------------- ----------------------------
  **Characteristics**           **Genotype distributions**                                                             
  **rs10204525**                **AA**                       **AG**                       **GG**                       **AG+GG**
  Pathological grading                                                                                                 
   MD/WD                        212//156                     139/135                      26/28                        165/163
   OR (95%CI); *P*-value        1.0 (reference)              0.76(0.55-1.04); 0.083       0.68(0.39-1.21); 0.190       0.75(0.55-1.01); 0.054
  Pathological grading                                                                                                 
   PD/WD                        52/156                       55/135                       9/28                         64/163
   OR (95%CI); *P*-value        1.0 (reference)              1.22(0.78-1.91); 0.375       0.96(0.43-2.18); 0.930       1.18(0.77-1.81); 0.452
  Distant metastasis                                                                                                   
   M1/M0                        52/368                       60/269                       15/48                        75/317
   OR (95%CI); *P*-value        1.0 (reference)              **1.58(1.06-2.36); 0.026**   **2.21(1.16-4.23); 0.014**   **1.67(1.14-2.46); 0.008**
  Tumor node metastasis stage                                                                                          
   T3+T4 / T1+T2                206/214                      153/176                      40/23                        193/199
   OR (95%CI); *P*-value        1.0 (reference)              0.90(0.68-1.21); 0.489       **1.81(1.05-3.12); 0.032**   1.01(0.77-1.33); 0.958
  Histology                                                                                                            
   Squamous/Not Squamous        349/71                       292/37                       49/14                        341/51
   OR (95%CI); *P*-value        1.0 (reference)              **1.61(1.05-2.46); 0.029**   0.71(0.37-1.36); 0.301       1.36(0.92-2.01); 0.121
  **rs36084323**                **GG**                       **GA**                       **AA**                       **GA+AA**
  Pathological grading                                                                                                 
   MD/WD                        321/268                      60/49                        5/3                          65/52
   OR (95%CI); *P*-value        1.0 (reference)              1.02(0.68-1.54); 0.916       1.39(0.33-5.88); 0.652       1.04(0.70-1.56); 0.834
  Pathological grading                                                                                                 
   PD/WD                        93/268                       23/49                        ½                            13/52
   OR (95%CI); *P*-value        1.0 (reference)              1.35(0.78-2.34); 0.279       1.44(0.13-16.08); 0.765      0.72(0.38-1.38); 0.323
  Distant metastasis                                                                                                   
   M1/M0                        80/593                       10/122                       2/6                          10/130
   OR (95%CI); *P*-value        1.0 (reference)              0.61(0.31-1.21); 0.151       2.47(0.49-12.45); 0.257      0.57(0.29-1.13); 0.104
  Tumor node metastasis stage                                                                                          
   T3+T4 / T1+T2                320/353                      67/65                        4/4                          71/69
   OR (95%CI); *P*-value        1.0 (reference)              1.14(0.78-1.65); 0.500       1.10(0.27-4.45); 0.890       1.14(0.79-1.63); 0.495
  Histology                                                                                                            
   Squamous/Not Squamous        569/104                      115/17                       7/1                          122/18
   OR (95%CI); *P*-value        1.0 (reference)              1.24(0.71-2.14); 0.449       1.28(0.16-10.51); 0.818      1.24(0.72-2.12); 0.434
  **rs7421861**                 **CC**                       **CT**                       **TT**                       **CT+TT**
  Pathological grading                                                                                                 
   MD/WD                        157/136                      175/149                      46/34                        221/183
   OR (95%CI); *P*-value        1.0 (reference)              1.02(0.74-1.40); 0.915       1.17(0.71-1.93); 0.533       1.05(0.77-1.42); 0.770
  Pathological grading                                                                                                 
   PD/WD                        50/136                       46/149                       20/34                        66/183
   OR (95%CI); *P*-value        1.0 (reference)              0.84(0.53-1.33); 0.459       1.60(0.84-3.04); 0.148       0.98(0.64-1.51); 0.930
  Distant metastasis                                                                                                   
   M1/M0                        44/299                       41/329                       6/94                         47/423
   OR (95%CI); *P*-value        1.0 (reference)              0.85(0.54-1.33); 0.472       0.43(0.18-1.05); 0.058       0.76(0.49-1.17); 0.207
  Tumor node metastasis stage                                                                                          
   T3+T4 / T1+T2                149/194                      185/185                      56/44                        241/229
   OR (95%CI); *P*-value        1.0 (reference)              1.30(0.97-1.75); 0.079       **1.66(1.06-2.60); 0.027**   **1.37(1.04-1.81); 0.027**
  Histology                                                                                                            
   Squamous/Not Squamous        296/47                       314/56                       81/19                        395/75
   OR (95%CI); *P*-value        1.0 (reference)              0.89(0.59-1.35); 0.587       0.68(0.38-1.22); 0.191       0.84(0.56-1.24); 0.374
  ----------------------------- ---------------------------- ---------------------------- ---------------------------- ----------------------------

Bold values are statistically significant (*P* \<0.05).  PD = Poorly differentiation, MD= Moderately differentiation, WD= Well differentiation

*PD-1* polymorphisms are related with PD-1 levels and prognosis among esophageal cancer patients
------------------------------------------------------------------------------------------------

The PD-1 expression levels were measured by qRT-PCR and ELISA in 150 esophageal cancer patients with different genotypes of rs10204525 and rs7421861 polymorphisms.  Results showed PD-1 expression levels are significantly higher in GG genotype versus AA genotype carriers (*P* = 0.013; [Figure 1](#f1){ref-type="fig"}).  Similar findings were obtained for the rs7421861 polymorphism (*P* = 0.024).  Similarly, increased PD-1 plasma levels are shown by Elisa ([Figure 2](#f2){ref-type="fig"}).  Moreover, higher PD-1 expressions showed worse overall survival among esophageal cancer patients ([Figure 3](#f3){ref-type="fig"}).  Thus, the mutant genotype of rs10204525 or rs7421861 polymorphism might contribute to worse survival of esophageal cancer patients by increasing the PD-1 expression. 

![** Relative PD-1 mRNA expression among patients in each genotype group.** (** A**) rs10204525; (**B**) rs7421861.](aging-12-102845-g001){#f1}

![** Plasma PD-1 levels among patients in each genotype group.** (** A**) rs10204525; (**B**) rs7421861.](aging-12-102845-g002){#f2}

![** Kaplan-Meier survival curves demonstrating the association between PD-1 expression and overall survival among esophageal cancer patients.**](aging-12-102845-g003){#f3}

DISCUSSION
==========

In this study, we investigated the relationship between *PD-1* gene variants and the risk of esophageal cancer and found *PD-1* gene rs7421861 and rs10204525 polymorphisms increased the risk of esophageal cancer in Chinese individuals.  The combination of smoking and these genotypes showed a significantly higher risk for esophageal cancer.  In addition, genotypes of rs10204525 and rs7421861 polymorphisms were shown to be associated with increased PD-1 gene and protein levels.  Furthermore, higher PD-1 expressions were correlated with worse survival of esophageal cancer patients. 

Increased expression of programmed death-ligand 1 (PD-L1) has frequently been observed in the brain, gastrointestinal tract, lung, liver, colorectum, kidney, pancreas, ovary, bladder, and esophagus cancers \[[@r46]--[@r49]\].  Monoclonal antibodies targeting PD-1 block the interaction with PD-L1 and have demonstrated efficacy in various malignancies \[[@r50]\].  PD-L1 expression was associated with clinical features in esophageal cancer and kidney clear cell carcinoma \[[@r51], [@r52]\].  Overexpression of PD-1 was also observed in hepatocellular carcinoma and adjacent tissue, and was correlated with the rs10204525 polymorphism in PD-1 \[[@r53]\]. 

The relationship between *PD-1* gene loci and cancer risk has been extensively studied, but the conclusions are inconsistent.  As reported, *PD-1* gene rs2227982 C\>T polymorphism other than rs10204525 A\>G or rs7421861 T\>C polymorphism was associated with gastric cardia adenocarcinoma \[[@r38]\].  PD-1 was highly expressed on liver cancer tissues and adjacent tissues and the PD-1 level was remarkably associated with *PD-1* gene rs10204525 polymorphism \[[@r53]\].  Yeo et al.  showed PD-L1 expression was unregulated in lung squamous cell carcinoma \[[@r54]\].  Moreover, the *PD-L1* 8923 A/C polymorphism \[[@r55]\] and *PD-1*.5 C/T polymorphism \[[@r34]\] are risky factors of non-small cell lung cancer (NSCLC).  Another study observed a significant link between the *PD-1* gene rs2227982 polymorphism and breast cancer risk in northwest Chinese women \[[@r39]\].  A meta-analysis concluded the *PD-1* gene rs36084323 polymorphism decreased cancer risk among Asians \[[@r44]\]. 

The AA genotype of rs10204525 in *PD-1* gene was previously associated with an increased risk of esophageal cancer and proposed to be a predictive biomarker for ESCC \[[@r17]\].  The rs36084323 T\>C polymorphism previously reduced the risk of esophagogastric junction adenocarcinoma (EGJA) while the rs7421861 polymorphism increased the risk of EGJA in Chinese subjects \[[@r18]\].  In addition, the *PD-1* T~rs10204525~G~rs2227982~C~rs36084323~A~rs7421861~ haplotype significantly associated with a decreased risk of EGJA \[[@r18]\].  The rs10204525 polymorphism was not related to ESCC risk in the full cohort.  However, a stratified analysis demonstrated that it was associated with a reduced risk of ESCC among male and younger patients \[[@r16]\].  Here we found *PD-1* rs10204525 and rs7421861 polymorphisms, but not rs36084323, increased the risk for esophageal cancer, which are obviously inconsistent with the above studies.  The conflicting findings may be attributed to some causes, such as clinical heterogeneity.  The pathological types were different among these studies, as we studied several pathological types, rather than single cancer types such as EGJA \[[@r18]\] and ESCC \[[@r16]\].  Secondly, the sample sizes of these studies were diverse.  Thirdly, living environment and diets may also affect the results. 

Furthermore, stratified analyses of age, smoking, sex, and alcohol status were conducted.  The rs10204525, rs36084323, rs7421861 polymorphisms increased the risk of esophageal cancer among men in our study.  Subgroup analysis revealed that rs10204525 polymorphism elevated the risk of esophageal cancer among patients who consumed alcohol and among patients ≥ 60 years old.  Cross-over analysis indicated that smoking in combination with either the rs10204525 or rs7421861 polymorphism significantly contributed to an increased risk of esophageal cancer. 

Next, we explored the associations between *PD-1* gene polymorphisms and clinical features of esophageal cancer.  It was found that rs7421861 and rs10204525 polymorphisms were associated with distant metastasis, and that rs7421861 was also associated with higher TNM stage.  However, rs36084323 polymorphism was not associated with esophageal cancer metastasis.  Interestingly, both the rs10204525 and rs7421861 polymorphisms were associated with higher PD-1 gene and plasma levels in esophageal cancer patients.  Furthermore, Kaplan-Meier survival curves showed higher PD-1 gene expression contributed to worse survival of esophageal cancer patients.  These results are in line with those of previous studies, which demonstrated associations between PD-L1 polymorphisms and poor prognosis \[[@r54]\] and survival \[[@r56]\] among lung cancer patients.  Thus, we assumed *PD-1* gene variants increased the PD-1 gene levels, thereby contributing to esophageal cancer metastasis and worse survival. 

This present study did have some potential limitations.  First, the limited sample size of this study could not exactly uncover the relationship of the *PD-1* gene rs7421861, rs10204525, rs36084323 polymorphisms with esophageal cancer susceptibility.  Second, the cases and controls were selected only from Chinese population.  Different diet culture, living environment, habits and customs may also contribute to the development of this disorder and selection bias to the whole ethnic groups can still not be ignored.  Third, further functional analyses were necessary to uncover how the *PD-1* gene polymorphisms affect esophageal cancer.  Fourth, we could not perform related experiments to explore the underlying mechanisms by which the *PD-1* variants conferred an increased risk to esophageal cancer.  Moreover, only three variants of *PD-1* gene were explored.  Last, potential gene-gene or gene-environment interactions were not obtained. 

In summary, the rs7421861 and rs10204525 polymorphisms in *PD-1* gene increase the risk of esophageal cancer in a Chinese Han population.  These polymorphisms could be potential diagnostic and therapeutic biomarkers.  However, the conclusions still need further validation by more studies with large sample sizes in other ethnicities. 

MATERIALS AND METHODS
=====================

Subjects
--------

814 patients with newly diagnosed esophageal cancer and 961 cancer-free controls were enrolled from the Affiliated Huai'an No.1 People's Hospital of Nanjing Medical University.  All patients were ≥ 18 years old with no history of other cancers.  Patients with history of esophageal disease, a second primary tumor, or tumor of unknown origin were excluded.  The controls consisted of individuals who received a comprehensive health examination and had no related history of cancer or autoimmune disease. 

Patient demographics and other clinical data were collected using a written questionnaire.  Smokers were regarded as smoking more than one cigarette per day for at least 1 year.  Drinkers were defined as consumption of alcoholic beverages more than once a week for ≥ 1 year.  The Institutional Review Board of Huai'an No.1 People's Hospital approved this study.  Written informed consent was got from all participants.  Patient confidentiality was maintained according to the Helsinki declaration. 

DNA extraction and genotyping
-----------------------------

Peripheral blood (2 mL) was collected from each patient following surgery.  Patients were enrolled in the study if post-operative pathological analysis confirmed a diagnosed of esophageal cancer.  Using a Puregene DNA Purification Kit, Genomic DNA was obtained from peripheral blood (Gentra, Minneapolis, MN, USA).  DNA concentration and purity were analyzed by absorbance and gel electrophoresis, respectively.  Genotyping was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) on a MassARRAY system (Sequenom, San Diego, CA, USA).  Approximately 5% of the samples were randomly used for repeat assays and a 100% concordance rate was observed. 

Quantitative RT-PCR
-------------------

Total RNA was isolated from peripheral venous blood using the Trizol reagent (Invitrogen, Carlsbad, CA, USA).  RNA was reverse-transcribed into cDNA using the SuperScript^TM^ II Reverse Transcriptase (Invitrogen).  Relative PD-1 expression was quantified by real-time PCR using TaqMan^®^ assays.  Beta-actin was used as an internal reference.  The forward and reverse primers were as follows: 5\'-GCACGAGGGACAATAGGA-3\', 5\'-GAC AATGGTGGCATACT C-3\' (PD-1); 5\'-AGGTCGGTGT GAACGGATTTG-3\', 5\'-TGTAGACCATGTAGTTG AGGTCA-3\' (GAPDH).  Relative gene expression of PD-1 was calculated by the 2^-△△CT^ method. 

ELISA
-----

PD-1 levels in plasma were evaluated using a human PD-1 ELISA kit (Sino Biological, Beijing, China).  The absorbance was assessed by use of a Tecan Infinite F50 Absorbance Microplate Reader (Tecan, Männedorf, Switzerland).  Plasma PD-1 levels were quantified using a standard curve. 

Kaplan-Meier survival analysis
------------------------------

The prognostic value of PD-1 mRNA expression in ESCC was assessed using OncoLnc ([http://www.  oncolnc.org](http://www.oncolnc.org)), which contains survival data for 144 patients derived from The Cancer Genome Atlas dataset.  Hazard ratios and 95% CIs were obtained using a Cox proportional-hazards model. 

Statistical analysis
--------------------

All statistical analyses were performed using SPSS 13.0 (IBM, Armonk, NY, USA).  Graphs were generated using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).  The observed genotype frequencies in controls were calculated for Hardy-Weinberg equilibrium test using goodness-of-fit chi-square tests \[[@r57]\].  Categorical variables were assessed using χ^2^ tests and displayed as frequencies (percentages).  Continuous variables in a normal distribution were evaluated using independent samples t-tests or one-way analysis of variance and expressed as the mean ± standard deviation.  Logistic regression assuming allelic, dominant, recessive, and co-dominant models was performed to analyze the associations between the polymorphisms and disease risk. * P* values \< 0.05 were considered significant \[[@r58], [@r59]\]. 

Supplementary Material
======================

**AUTHOR CONTRIBUTIONS:** Conceived of and designed the study, BZ; Designed the methodology, BZ and CC; Validated the results, JQZ; Performed the analysis, CC and JQZ; Contributed analytical resources, JQZ; Collected the data, CC; Wrote the manuscript, BZ; Edited the manuscript, CC. 

**CONFLICTS OF INTEREST:** The authors declare that they have no conflicts of interest.