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High expression of HLA-DQA1 predicts poor outcome in patients with esophageal squamous cell carcinoma in Northern China

Abstract Background: Our previous studies demonstrate that the major histocompatibility complex (MHC) is associated with the progression of esophageal squamous cell carcinoma (ESCC). HLA-DQA1, which belongs to the MHC Class II family, may be a potential biomarker in ESCC progression. However, the association between HLA-DQA1 and ESCC in high-incidence area of northern China has not been well characterized. The purpose of this study is to investigate the relationship of HLA-DQA1 expression with the progression and prognosis of ESCC. Methods: We analyzed the expression profiles of HLA-DQA1 in esophageal cancer (EC) samples in the TCGA database and validated HLA-DQA1 expression by immunohistochemistry, western blotting, and quantitative reverse-transcription polymerase chain reaction in matched EC and normal tissues, respectively. The correlation between HLA-DQA1 expression and clinicopathologic characteristics of ESCC was further analyzed. Result: Immunohistochemical analysis indicated that the expression level of HLA-DQA1 in ESCC tissues was significantly higher than the matched normal tissues (P < .001). HLA-DQA1 mRNA and protein expression were significantly higher in ESCC tissues compared to the matched normal tissues. Patients with family history negative or with tumor sizes >4 cm were associated with higher HLA-DQA1 expression levels. A prognostic significance of HLA-DQA1 was also found by the Log-rank method, in which high expression of HLA-DQA1 was correlated with a shorter overall survival time. The receiver operating characteristic (ROC) curve analysis yielded the area under the ROC curve value of 0.693. Univariate and multivariate analyses also suggest that high expression of HLA-DQA1 is a potential indicator for poor prognosis of ESCC. Conclusions: Our results demonstrate that HLA-DQA1 plays an important role in ESCC progression and may be a biomarker for ESCC diagnosis and prognosis, as well as a potential target for the treatment of patients with ESCC.


Introduction
Despite the current advances in the treatment strategies for esophageal cancer (EC), still, it ranks the 6th most common cause of cancer-related deaths worldwide. [1,2] The 5-year overall survival of patients with EC ranges from 15% to 25%. [3] Around 80% (328,800/400,200) of EC-related deaths occur in less developed regions, especially the rural regions around the Taihang Mountain in the junction of Henan, Hebei, and Shanxi provinces in northern China. [2,[4][5][6] In those places, esophageal squamous cell carcinoma (ESCC) is the predominant histologic type of EC. Due to the fact that ESCC is tightly associated with extensive lymphatic spread and vascular invasion, ESCC is often diagnosed at advanced stage and the survival rate of ESCC is very poor. [7] Therefore, a better understanding of the molecular mechanisms underlying ESCC progression could improve the early diagnosis, treatment strategy, and overall prognosis of this deadly disease.
Our previous studies [8][9][10] have demonstrated that the major histocompatibility complex (MHC) region of human chromosome is associated with the progression of ESCC. HLA-DQA1, which is one of the MHC Class II family members and locates on chromosome 6p21, may be a potential prognostic biomarker for ESCC. [8] A major function of MHC Class II molecules in the immune system is to present antigens derived from extracellular proteins for recognition by CD4 + T-cells. [11] Aberrant expression of HLA-II may result in insufficient immune response or autoimmunity reaction, which may result in lots of diseases including cancers. [12,13] More importantly, numerous studies have revealed that HLA-II members are involved in cancers like ESCC. [14][15][16][17] However, the association between HLA-DQA1 and ESCC in high-incidence area of northern China has not been well characterized.
In this study, we analyzed the expression profiles of HLA-DQA1 in EC samples in the database (from TCGA: The Cancer Genome Atlas), and validated the expression of HLA-DQA1 in matched EC and normal tissues by techniques including immunohistochemistry, western blotting, and reverse-transcription polymerase chain reaction (RT-PCR). The relationship between HLA-DQA1 expression level and clinicopathologic characteristics was investigated. Moreover, we assessed the link between HLA-DQA1 expression and the prognosis of ESCC. To our knowledge, no previous studies have reported HLA-DQA1 expression associated with the prognosis of ESCC.

Ethics statement
The study protocol was approved by the ethical review committee of the Third Affiliated Hospital of Xinxiang Medical University. All the assays were conducted according to Declaration of Helsinki principles. Written informed consent was obtained from all the participants.

Recruitment of patients and controls
A total of 97 cases of matched ESCC and normal tissue samples (3-7 cm away from the tumor margin) were obtained from Anyang Tumor Hospital (Henan, China) ranging from January, 2011 to February, 2012. All specimens for total RNA and protein extraction were stored at À80°C after immediate snap-frozen in liquid nitrogen. All the cases were histopathologically confirmed and there was no prior treatment to those patients. Patient demographic, clinical, and pathologic data were recorded. All the tumors were confirmed to contain >80% tumor cells by histologic examination of sequential sections. Patients were classified according to TNM staging (American Joint Committee on Cancer, 7th edition). All the personal information was concealed to ensure the privacy. The clinicopathologic characteristics of the patients with ESCC are summarized in Table 1.

Real time PCR
Total RNA of the tissues was extracted by using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Carlsbad, California), according to the manufacturers protocol. The complementary DNA was synthesized using reverse transcription kit (Tiangen, Beijing, China) and stored at À20°C. The levels of messenger RNA (mRNA) of HLA-DQA1 expression and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were determined using the ABI 7500 FAST sequence detection system (Applied Biosystems, Thermo Fisher Scientific, Inc., Carlsbad, California). GAPDH was used as a control gene for the quantitative RT-PCRs (qRT-PCRs). The primer sequences were sense/anti-sense: HLA-DQA1: All the reactions were done in triplicate using 20 mL samples containing 50 ng of complementary DNA. The cycling condition is listed as follows: 10 minutes at 95°C, 1 cycle; (95°C, 15 seconds; 60°C, 60 seconds) for 40 cycles. The data were analyzed using the ABI 7500 FAST Sequence Detection software. Each reaction was performed in triplicate and the 2 ÀDDCt method was used to calculate the expression of HLA-DQA1. [18]

Western blotting analysis
The tumor tissues were collected and lysed on ice according to the manufacture protocol. After spinning at 12,000g for 20 minutes, the concentration of protein was measured, and then the samples were denatured by boiling for 10 minutes at 100°C and loaded into sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%) gel for electrophoresis. The proteins were transferred onto polyvinylidene fluoride membrane (Millipore, Bedford, Massachusetts), which was then incubated in the blocking

Immunohistochemistry staining
Paraffin-embedded sections (5 mm) of human esophageal histologic ESCC (n = 97) and matched normal tissues (n = 97) were collected on gelatin-coated slides for HLA-DQA1 immunostaining (Abcam Group, Inc; Abcam, Cambridge, UK). The avidin-biotin-peroxidase complex method was applied as previously described. [19] In brief, after dewaxing, inactivating, endogenous peroxidase activity, and blocking cross-reactivity with preimmune serum, the sections were incubated over night at 4°C with the primary antibodies (antibody for HLA-DQA1 was diluted at 1:100). Localization of the primary antibodies was achieved by subsequent incubation of a biotinylated anti-primary antibody, an avidin-biotin complex conjugated to horseradish peroxidase, and diaminobenzidine (Vectastain elite kit, Vector Laboratories Inc., Burlingame, CA). The slides were washed 3 times with phosphate-buffered saline after incubation. As negative controls, some slides were subjected to normal serum blocking and omission of the primary antibody. Two experienced pathologists (H-JY and F-FS) were specified to read the immunostaining slides.

Statistical analysis
The SPSS 22.0 (IBM SPSS; SPSS Inc, Chicago, IL) was used for statistical analysis. In all stratification analysis, comparisons of categorical variables were made using the Pearson test. Continuous variables were compared using independent 2-sample t test.
Receiver operating characteristic (ROC) curve was plotted to determine how well the HLA-DQA1 expression level could differentiate survival time from patients with ESCC. The area under the ROC curve (AUC) >0.5 was taken to indicate reasonable biomarker performance. ROC curves optimal cutoff values were defined as the point that maximized the Youden index, defined as sensitivity À-(1-specificity). Survival analysis was conducted by Kaplan-Meier method and log-rank test. Univariate and multivariate Cox regression analyses were used to assess the prognostic value of biomarkers alone and when adjusted for clinical parameters. P < .05 was considered to be statistically significant.

Immunohistochemical analysis for HLA-DQA1 protein
Immunohistochemical analysis was used to detect HLA-DQA1 expression in 97 paired ESCC cases and matched normal tissues.
Intense nuclear or cytoplasm staining was the criterion for a "positive" reaction. We applied the criteria established by our laboratory previously to describe the patterns of positive result as follows: "scattered," in which only some isolated positive cells were identified; "papillary," where immunostain-positive cells were identified only in the esophageal epithelial papillary area; "focal," where wide clusters of positive cells were seen in some areas of the epithelia; and "diffuse," in which the sheets of positive cells were found throughout most areas of the lesions. We found that HLA-DQA1 protein was mainly expressed in ESCC tissues (81.44%, 79/97), while there was weak or no positive staining in most matched normal tissues (16.49%, 16/ 97) (Fig. 1, Table 1). Various expression pattern of HLA-DQA1 protein in ESCC tissues was observed (Fig. 1C, D). Most ESCC cases in this study showed a significantly high HLA-DQA1 protein expression when compared to the matched normal tissues (P < .001). The positive reactants of HLA-DQA1 were yellow or brown substances present primarily in the cytoplasm.
The results revealed that HLA-DQA1 expression levels were upregulated approximately 6-fold in EC (P < .001, Fig. 2A). Then, we analyzed the expression of HLA-DQA1 in ESCC cases and normal tissue samples from TCGA. As shown in Figure 2B, HLA-DQA1 expression levels were significantly upregulated in ESCC (P < .001). We also evaluated the expression levels of HLA-DQA1 in 97 paired ESCC cases and matched normal tissues by qRT-PCR. The expression of HLA-DQA1 mRNA was 8-fold higher in ESCC tissue samples, compared to which in matched normal esophageal tissue samples (P < .001, Fig. 2C). These results were confirmed by western blot (Fig. 1D). These results demonstrate that HLA-DQA1 is highly expressed in human ESCC.

Clinicopathologic characteristics and HLA-DQA1 mRNA expression level in ESCC
The clinicopathologic characteristics of the 97 ESCC cases are summarized in Table 1. To further demonstrate the clinical significance of HLA-DQA1 expression in ESCC, we used t test to examine the correlation of HLA-DQA1 expression levels and clinicopathologic factors in patients. The results demonstrated that there was an obvious positive correlation between increased HLA-DQA1 levels and family history (21.332 ± 8.753 vs 5.520 ± 2.564, P = .021) and larger tumor size (27.057 ± 13.886 vs 8.265 ± 2.645, P = .004). Patients with family history negative or with tumors sizes >4 cm were associated with higher HLA-DQA1 expression levels, whereas patients with family history positive or with tumors sizes <4 cm were associated with lower HLA-DQA1 expression levels (Fig. 2E, F). A Chi-squared test was performed to assess the clinical pathologic characteristics between the 2 groups. The results in Table 2 show that HLA-DQA1 expression was significantly associated with family history (P = .004) and tumor size (P = .029). However, there was no correlation detected in the expression of HLA-DQA1 with age (P = .586), gender (P = .836), and smoking history (P = .805) in our study (Table 2). 9.43%, while it was 27.27% in the compared groups. As it is shown in Figure 3, the overall survival is shorter in HLA-DQA1 highly expressed patients than those of low levels of HLA-DQA1 (P = .0024).

ROC curve of HLA-DQA1 expression in the prognosis of ESCC
We further analyzed ROC curve of HLA-DQA1 expression to assess the prognosis of ESCC (Fig. 4). We found that HLA-DQA1 expression level could differentiate survival time from patients with ESCC, with an AUC of 0.693 (95% confidence interval: 0.577-0.808, P = .013). And the optimal cutoff values were 1.323 (sensitivity 0.539, specificity 0.824).

HLA-DQA1 expression and the indicator for survival
The association between HLA-DQA1 expression and prognosis of patients with ESCC was evaluated by univariate analysis and multivariate analysis. As shown in Table 3, the overall survival of those patients was significantly dependent on gender (hazard ratio [HR] = 0.404, P = .016), tumor location (HR = 0.034, P = .034), and HLA-DQA1 expression levels (HR = 2.096, P = .008). Thus, these factors and smoking history were enrolled in the multivariate Cox regression analysis. Highly expressed HLA-DQA1 was demonstrated to be an independent indicator for the poor overall survival (HR = 1.958, P = .009) in ESCC, along with the gender (HR = 0.446, P = .013) and tumor location (HR = 2.886, P = .012) ( Table 3). These results indicate that HLA-DQA1 could be a useful marker for the prognosis of ESCC.

Discussion
The ESCC has long been recognized as one of the most malignant tumors worldwide, with the current median survival time <1 year. [20] With the improvement of early diagnosis and the application of new techniques, and the 5-year survival rate has increased significantly. [3] Of note, the application of endoscopic techniques, including endoscopic mucosal resection, endoscopic submucosal dissection, and radiofrequency ablation, has improved the prevention and curative treatment of early esophageal lesions. [21] Both neoadjuvant chemotherapy and chemoradio- Even through many factors were reported to be related to the progression of ESCC, no reliable prognostic markers were available in clinic. [22][23][24][25][26][27] Discovery of new prognostic marker is of urgent need for the treatment of those patients.
Over the past few decades, the roles of MHC in human disease, including cancer, have attracted much attention. Many studies have revealed that MHC has crucial roles in various cancer types by functioning as potential oncogenes or tumor suppressors. [28][29][30] In our previous studies, [8][9][10] we found that multiple genetic factors within the MHC region confer risk to ESCC from highrisk area in northern China, but the mechanism of MHC has not been studied. Further functional studies were needed to elucidate the molecular mechanisms about the MHC genes.
Based on our previous study, we have investigated HLA-DQA1 expression in ESCC samples and matched normal tissue samples. Firstly, we analyzed the expression profiles of HLA-DQA1 in EC samples in the TCGA database and validated the results in a cohort of 97 pairs of ESCC samples and normal tissue samples. The results showed that HLA-DQA1 was upregulated in ESCC tissues and correlated with poor prognosis and shorter survival rate. Specifically, patients with higher HLA-DQA1 expression levels seemed to harbor larger tumor sizes and were characterized by family history. We further determined that HLA-DQA1 expression was an independent predictor for overall survival. These results indicated that overexpressed HLA-DQA1 played an important role in the occurrence of ESCC.
Previous studies have indicated that family history, smoking, alcohol consumption, aging, and gender are risk factors for ESCC. [10,31,32] Our results demonstrated that family history (P = .004) and tumor size (P = .029) were preferentially associated with HLA-DQA1 expression. The percentage of patients expressing HLA-DQA1 is higher for negative family history than that of positive family history. The mRNA expression in this study was consistent with the protein expression in our previous study. The other factors (age, gender, drinking history, smoking history, differentiation, T classification, N classification, and location) did not significantly associated with HLA-DQA1 expression. The results further supported the potential role of HLA-DQA1 in esophageal carcinogenesis.
However, certain limitations in our study should be addressed as follow. The population of enrolled cases was relatively small, which might result in bias in the final results. Therefore, we will increase the number of samples for further investigation. Besides, the concise molecular mechanisms underlying the altered expression of HLA-DQA1 in ESCC cell lines and mouse model need to be further addressed.

Conclusion
Collectively, in this study, we showed that HLA-DQA1 was upregulated in ESCC tissues and correlated with poor prognosis in patients with ESCC. Our finding implies that HLA-DQA1 could be a potential prognostic marker in patients with ESCC.