PTEN polymorphisms and the risk of esophageal carcinoma and gastric cardiac carcinoma in a high incidence region of China

SUMMARY. PTEN, as a tumor suppressor gene, plays an important role in regulating cell growth, proliferation, and apoptosis. Two common polymorphisms, –9C/G and IVS4 (?/+), may alter susceptibility to the disease. To test the hypothesis that the genetic variations of PTEN play a role in the etiology of esophageal squamous cell carcinoma (ESCC) and gastric cardiac adenocarcinoma (GCA), a population‐based case‐control study was conducted in 350 ESCC patients, 257 GCA patients, and 634 healthy controls from a high‐incidence region of Hebei province, China. The PTEN polymorphisms were genotyped by polymerase chain reaction‐restriction fragment length polymorphism analysis (PCR‐RFLP). The results showed that the family history of upper gastrointestinal cancer (UGIC) significantly increased the risk of developing ESCC and GCA (the age, gender and smoking status adjusted OR = 1.73 and 1.67; 95% CI = 1.29–2.32 and 1.28–2.19, respectively). The overall distribution of the PTEN –9C/G genotype was not significantly different between cancer patients and controls. Compared with the PTEN IVS4‐/? genotype, the IVS4+/+ genotype significantly decreased the risk of ESCC and GCA development, the adjusted OR was 0.64 (95% CI = 0.44–0.94) and 0.63 (95% CI = 0.41–0.98), respectively. Stratification analysis by gender, age, smoking status and family history of UGIC showed that the PTEN IVS4?/+ genotype only reduced the risk of ESCC (adjusted OR = 0.55, 95%CI = 0.34–0.90) among subjects with family history of UGIC. While the IVS4+/+ genotype decreased the susceptibility to both ESCC and GCA (adjusted OR = 0.61 and 0.57, 95% CI = 0.37–0.98 and 0.34–0.98, respectively) among male subjects, the IVS4+/+ genotype only decreased the risk of ESCC development among subjects younger than 55 years (adjusted OR = 0.43, 95% CI = 0.21–0.85). In addition, the haplotype analysis found that the –9C/IVS4– haplotype increased the risk of developing ESCC and GCA (OR = 1.31 and 1.24, 95% CI = 1.08–1.58 and 1.001–1.53). Our results suggested that the PTEN IVS4+/+ homozygote may play a protective role in the development of ESCC and GCA, while the haplotype –9C/IVS4– might be the risk factor of the development of ESCC and GCA in the high incidence region population of Hebei province, China.     

α-Adducin gene promoter DNA methylation and the risk of essential hypertension

This study was conducted to test the association between promoter DNA methylation of α-Adducin (ADD1) gene and the risk of essential hypertension (EH). A total of 150 EH patients and 100 aged- and gender-matched controls were investigated. DNA methylation levels of five cytosine-phosphate-guanine (CpG) dinucleotides on ADD1 promoter were measured employing bisulfite pyrosequencing technology. Our results showed that females have a higher ADD1 DNA methylation than males and a significantly lower CpG1 methylation level is associated with increased risk of EH among them. As for males, a significant association between lower CpG2-5 methylation levels and increased risk of EH was shown. In addition, CpG2-5 methylation was found to be a highly significant predictor for EH among males. In females, CpG1 methylation was considered a predictor of hypertension. No significant correlations were found with biochemical measures, apart from the concentration of aspartate aminotransferase which was inversely correlated with ADD1 CpG2-5 methylation levels among female controls (r = ?0.703). These findings highlight that ADD1 methylation may have a contributing role in the pathogenesis of EH with varying implications for both genders.     

Expression of klotho and β‐catenin in esophageal squamous cell carcinoma,and their clinicopathological and prognostic significance

Esophageal carcinoma is one of the most common types of cancers in the world; the molecular mechanism underlying its tumorigenesis is still not well understood. This study was aimed at investigating the expression of klotho and β‐catenin in patients with esophageal squamous cell carcinoma (ESCC) and analyzing their association with clinicopathological variables and their effects on prognosis. The expression patterns of klotho and β‐catenin were determined by tissue microarray and immunohistochemical technique in ESCC and normal tissues, and their correlations with clinicopathological characteristics were investigated using univariate and multivariate analysis. The serum klotho levels in 40 ESCC patients and controls were measured by sandwich enzyme‐linked immunosorbent assay system (ELISA). The expression level of klotho was significantly lower in ESCC than in the adjacent noncancerous tissues (30 vs. 50%, P < 0.000), and the protein level was negative correlated with clinical staging, histological grade, lymph node metastasis, and invasion depth (P < 0.05). Whereas, the expression of β‐catenin was much higher in ESCC than their corresponding normal mucosa tissues (78.3 vs. 11.5%, P < 0.000), and the level of protein correlated only with histological grade and invasion depth (P < 0.05). Correlation analysis showed the expression level of klotho inversely correlated with that of β‐catenin (r = ?0.214, P < 0.01). Patients with klotho‐positive tumors had longer survival than those with klotho‐negative tumors (P < 0.01). Cox proportional hazards model analysis demonstrated that positive expression of klotho was an important factor indicating good prognosis (hazard ratio, 0.371; 95% confidence interval, 0.201–0.685; P < 0.01). ELISA showed that the level of serum klotho was markedly higher (461.50 ± 43.30 pg/mL) than control group (239.37 ± 20.65 pg/mL) (P < 0.001). Receiver operating characteristic analysis gave a cut‐off value of 327.031 of serum klotho with a sensitivity of 81.3% and specificity of 81.2% (P < 0.000). Our present study demonstrated for the first time that klotho might be a novel biomarker candidate for predicting progression and prognosis in patients with ESCC.     

Aberrant p16 methylation, a possible epigenetic risk factor in familial esophageal squamous cell carcinoma

Aim. Detection of methylation in the p16 gene, an inhibitor of cyclin D-dependent protein kinase, as a new tumor marker for early detection of esophageal squamous cell carcinoma (ESCC) in DNA derived from blood and serum. Method. A large family with clustering of ESCC was assessed in Khorasan province in northeastern Iran. The family had three histologically proven cases of ESCC in two consecutive generations and several other deceased cases with histories of ESCC. DNA from blood of 28 living family members in three consecutive generations, 30 sporadic ESCC cases (from serum, blood, and tumor tissues), and 30 healthy volunteers (from blood) were examined for the methylation status of p16 promoter using methylation-specific PCR (MSP). Results. Aberrant p16 promoter methylation was found in 64.3% (n=28) of ESCC family members and none (n=30) of our normal volunteers. Five of the 28 family members with esophageal cancer symptoms had negative endoscopy results for ESCC, while four of these members had p16 hypermethylation in their blood. The family members with negative endoscopy and positive p16 promoter methylation are being monitored closely for signs of ESCC development through regular check-ups and chromoendoscopies. In sporadic ESCC in northeastern Iran, 73.3% (n=30) of tumor tissue samples had p16 hypermethylation. Serum and blood samples from the same patients showed p16 hypermethylation in 26.6% and 43.3% of the samples, respectively. Conclusion. Aberrant p16 methylation may be a valuable diagnostic tool as a tumor marker for the early identification of individuals in high risk ESCC families.     

Helicobacter pylori induces promoter hypermethylation and downregulates gene expression of IRX1 transcription factor on human gastric mucosa

Background and Aim: Gene silence of IRX1 tumor suppressor by promoter CpG methylation combined with loss of heterozygosity (LOH) has been identified in human gastric cancer. This study investigated the association between methylation of IRX1 and Helicobacter pylori infection in gastric mucosa tissues and cell line. Methods: IRX1 methylation was studied by methylation specific polymerase chain reaction (MSP) and bisulfate sequencing polymerase chain reaction (BSP) methods in gastric mucosa tissues from H. pylori‐positive chronic gastritis patients or H. pylori‐negative chronic gastritis patients. Promoter activity, methylation status and gene expressing level of IRX1 were evaluated by persistent infecting H. pylori on human gastric cells GES‐1 in vitro. Electron microscopy was used to observe the effect of H. pylori infection on GES‐1 gastric mucosa cells. Results: The methylation level of IRX1 promoter in H. pylori positive chronic gastritis and H. pylori negative chronic gastritis was 55.30% ± 13.17 versus 5.20% ± 6.31, respectively (P < 0.01). H. pylori infection stimulated increased microvillus, and mucous secretion on GES‐1 cells. Infection of H. pylori induced IRX1 promoter methylation and downregulation of the promoter activity as well as gene expression significantly. Conclusions: This study firstly demonstrated that H. pylori infection contributes to IRX1 promoter methylation on gastric mucosa.     

核黄素转运蛋白C20orf54基因rs3746804位点单核苷酸多态性与食管鳞癌遗传易感性的关系

  目的 探讨核黄素转运蛋白C20orf54基因外显子3中rs3746804位点单核苷酸多态性（SNP）与食管鳞癌（ESCC）遗传易感性的关系。方法 磁珠法提取全血基因组DNA,以直接测序方法对434例包括长治、林州两地ESCC患者与554例包括长治、林州健康人群及由林州移居长治的健康移民5组人群的C20orf54基因外显子3的SNP1139C>T进行基因分型。结果 长治ESCC组与长治健康组、健康移民组在基因型CT(37.5%比51.0%、37.5%比52.0%)、CC(44.2%比34.8%、44.2%比33.0%)的分布频率上差异明显（P值均<0.05）,与林州ESCC组在基因型TT(18.3%比4.1%)、CC(44.2%比54.6%)的分布频率上有明显差异（P值均<0.05）;林州ESCC组与健康移民组在基因型TT(4.1%比15.0%)、CT(41.2%比52.0%)、CC (54.6%比33.0%)的分布频率上差异明显（P值均<0.05）,与长治健康组在基因型TT(4.1%比14.1%)、CC(54.6%比34.8%)的分布频率上差异明显（P值均<0.01）。肿瘤组（长治ESCC组+林州ESCC组）与健康组（长治健康组+林州健康组+健康移民组）在基因型CT(39.2%比48.7%)和CC (48.8%比38.2%)的分布频率上差异明显（P值均<0.01）。相对于CC基因型,CT和CT+TT基因型可以降低食管癌发生的危险性（OR=0.630,95%CI 0.481~0.826;OR=0.654,95%CI 0.507~0.844）。结论 C20orf54基因外显子3中rs3746804 位点的SNP与食管鳞癌的遗传易感性明显相关。     

Association between positive murine double minute 2 expression and clinicopathological characteristics of esophageal squamous cell carcinoma: a meta‐analysis

The correlations of murine double minute 2 (MDM2) T309G and esophageal cancer were elucidated because the association between MDM2 expression states and clinicopathological parameters of esophageal squamous cell carcinoma (ESCC) is controversial. We conducted a meta‐analysis on studies screened from PubMed, Web of Science, Embase, the Cochrane Library, and the Chinese Biomedical Literature Databases that were published before October 2014. All studies describing the association between MDM2 and ESCC were traced. Meta‐analysis was performed using the STATA software (Stata Corp., College Station, TX, USA). A total of 9 studies with 707 cases and 324 controls were included. MDM2 expression was higher in ESCC than in normal esophageal epithelium (odds ratio [OR] 10.38, 95% confidence interval [CI] 6.42–16.78, P < 0.001). High MDM2 expression was associated with early primary tumor stage (T1/T2 vs. T3/T4, OR 0.59, 95% CI 0.38–0.92, P = 0.018) and increased risk of regional lymph node metastasis (N0 vs. N1, OR 1.66, 95% CI 1.03–2.67, P = 0.039). However, no relationship was observed between MDM2 expression and the risk of distant metastasis (OR = 2.09, 95% CI 1.00–4.36, P = 0.050), and MDM2 was not significantly correlated with TP53 expression (OR 1.22, 95% CI 0.53–2.77, P = 0.643). Our analysis suggests that MDM2 acts as a potent marker of early primary tumor stage but higher risk of regional lymph node metastasis in ESCC. However, because of the limited number of studies included, the result should be further clarified by well‐designed prospective studies.     

Association of mu‐opioid receptor expression with lymph node metastasis in esophageal squamous cell carcinoma

The mu‐opioid receptor (MOR), a membrane‐bound G protein‐coupled receptor, is the main target for opioids in the nervous system. MOR1 has been found in several types of cancer cells and reported to be involved in tumor progression and metastasis. However, the expression and clinical significance of MOR1 in esophageal squamous cell carcinoma (ESCC) remain unclear. In our study, the expression of MOR1 was confirmed in ESCC cell lines (KYSE180, KYSE150, and EC109) by Western blot. MOR1 was also detected on tissue microarrays of ESCC samples in 239 cases using immunohistochemical staining. We found that MOR1 was mainly located in the cytoplasm and occasionally occurred in the membrane or nucleus of ESCC cells. Moreover, results indicated that MOR1 expression in the cytoplasm was associated with lymph node metastasis (R = 0.164, P = 0.008, Kendall's tau‐b‐test). No more associations were found between MOR1 expression status and other clinical parameters. However, no statistical significant differences were found between MOR1 expression in the cytoplasm, nucleus/membrane, and the overall survival of ESCC patients (P = 0.848; P = 0.167; P = 0.428, respectively, log‐rank test). Our results suggest that the cytoplasmic MOR1 may be a high‐risk factor for lymph node metastasis of ESCC patients. We also hypothesize that MOR1 agonists used in ESCC patients should be prudent, and opioid receptor antagonists may be novel therapeutic drugs for ESCC patients.     

Clinical effect of E‐series of prostaglandin receptor 2 and epidermal growth factor receptor signal pathways in the development of esophageal squamous cell carcinoma

Signal pathways mediated by epidermal growth factor receptor (EGFR) and E‐series of prostaglandin receptors (EPs) are closely correlated to the pathogenesis of tumor. This experiment was designed to investigate the expression and clinical significance of EP2 and EGFR in esophageal squamous cell carcinoma (ESCC). Tissue samples were collected reterospectively from 87 patients with ESCC (first diagnosed). The patients were followed up for 5 years after radical surgery. The expression of EP‐2 and EGFR were examined by tissue chip technology and immunohistochemistry methods. Clinicopathological and prognostic impact were evaluated. Overexpression of EGFR and EP‐2 was more observed in ESCC than the control group (58.6% vs. 13.9%; 52.9% vs. 4.88%, P < 0.001, respectively); which correlated with tumor infiltration depth, lymph node metastasis, and tumor‐lymph node‐metastasis staging. Both the EP‐2 and EGFR overexpression were detected in 39 specimens and exhibited the positive correlation (P < 0.001, r = 0.404). Overexpression of EP2 and EGFR exhibited significant correlation with worse 5‐year overall survival than those with negative result (17.6% vs. 27.8%, P = 0.011; 10.9% vs. 34.1%, P < 0.001, respectively). Cox proportional hazard model showed that the T‐staging, lymph node metastasis, and EGFR overexpression were the independent risk factors of the prognosis. The present study exhibited that the overexpression of EP2 and EGFR in ESCC tissues might play an important role in carcinogenesis and the progression of ESCC.     

Overexpression of cyclooxygenase‐2 is associated with chemoradiotherapy resistance and prognosis in esophageal squamous cell carcinoma patients

Our objective was to investigate whether cyclooxygenase‐2 (COX‐2) expression can predict the patient's response to chemoradiotherapy (CRT) and ensuing prognosis in esophageal squamous cell carcinoma (ESCC). The clinicopathological and follow‐up data of 112 patients with ESCC who underwent CRT from January 2001 to June 2006 were analyzed retrospectively. The immunohistochemical expression level of COX‐2 was examined for all biopsy specimens of primary tumors, and the correlation of COX‐2 expression with the patient's response to CRT and prognosis was examined. COX‐2 positive immunostaining was detected in 111 (99.1%) of the patients, including overexpression in 54 (48.2%) patients and low expression in 58 (51.8%) of the patients. The response of tumors with a low level expression of COX‐2 (70.7%, 41/58) was significantly higher than that of tumors with COX‐2 overexpression (42.6%, 23/54; P = 0.003). Patients with a low level of COX‐2 expression had a higher downstaged rate than those with a high level of COX‐2 expression (9/13 vs 2/8), but the difference was not statistically significant (P = 0.08). In the definitive CRT group (91 cases), COX‐2 overexpression was significantly associated with poor 3‐year overall survival (P = 0.028). Multivariate analysis showed that only metastatic stage (nonregional node metastasis) was an independent prognosis factor. The assessment of COX‐2 status may provide additional information to identify ESCC patients with poor chances of response to CRT and potential candidates for more individualized treatment.     

Significance of immunohistochemical over-expression of CD44v6 as an indicator of malignant potential in esophageal squamous cell carcinoma

Purpose The aim of the current study was to find out a clinicopathologic significance of CD44v6 over-expression in esophageal squamous cell carcinoma (ESCC), which has not been elucidated fully.Methods Immunohistochemical expression of CD44v6 was examined for 81 ESCCs. Correlation of CD44 over-expression with the clinicopathologic features were investigated.Results Thirty-eight ESCCs (46.9%) had over-expression of CD44v6. The proportions of the incidence of lymph node metastasis (P=0.039), lymphatic permeation (P=0.003), and blood vessel invasion (P=0.037) in ESCCs with over-expression of CD44v6 were significantly higher than those in ESCCs without over-expression of CD44v6. The stage of the tumor in ESCCs with over-expression of CD44v6 was significantly more advanced (P=0.045). Survival rates of patients with ESCC with over-expression of CD44v6 were significantly worse (P=0.0005). Moreover, CD44v6 over-expression (P=0.048) as well as blood vessel invasion (P=0.014) and stage of the tumor (P=0.010) were factors independently associated with the unfavorable prognosis of the patients with ESCC.Conclusions Over-expression of CD44v6 can be an indicator of the malignant potential of ESCC.     

Pentraxin 3 (PTX3) promoter methylation associated with PTX3 plasma levels and neutrophil to lymphocyte ratio in coronary artery disease

Background Pentraxin 3 (PTX3) is expressed in the heart under inflammatory conditions and plays an important role in atherogenesis. Patients with increased PTX3 levels may suffer from higher rates of cardiac events. Regulation of specific genes by promoter methylation is important in atherogenesis. The factors influencing PTX3 levels and the association between epigenetics and PTX3 levels have not been investigated. Methods Blood samples were collected from 64 patients admitted to the Department of Cardiology, 35 who had coronary artery disease (CAD), and 29 who were CAD-free. Plasma levels of PTX3 were measured by ELISA. PTX3 promoter methylation was evaluated via methyl-specific PCR. The severity of coronary artery lesion was evaluated by angiography. Results The level of PTX3 promoter methylation in the CAD group was 62.69% ± 20.57%, significantly lower than that of the CAD-free group, which was 72.45% ± 11.84% (P = 0.03). Lower PTX3 promoter methylation levels in the CAD group were associated with higher plasma PTX3 concentrations (r = -0.29, P = 0.02). Furthermore, lower PTX3 promoter methylation levels were associated with higher neutrophil to lymphocyte ratio (NLR) in men (r = -0.58, P = 0.002). Conclusions The present study provides new evidence that methylation of the PTX3 promoter is associated with PTX3 plasma levels and NLR in coronary artery disease. This study also shows that modification of epigenetics by chronic inflammation might be a significant molecular mechanism in the atherosclerotic processes that influence plasma PTX3 concentrations.     

Aberrant DNA methylation of G‐protein‐coupled bile acid receptor Gpbar1 predicts prognosis of acute‐on‐chronic hepatitis B liver failure

The G‐protein‐coupled bile acid receptor Gpbar1 (TGR5) has been demonstrated to be able to negatively regulate hepatic inflammatory response. In this study, we aimed to determine the methylation status of TGR5 promoter in patients with acute‐on‐chronic hepatitis B liver failure (ACHBLF) and its predictive value for prognosis. We enrolled 76 consecutive ACHBLF patients, 80 chronic hepatitis B (CHB) patients and 30 healthy controls (HCs). Methylation status of TGR5 promoter in peripheral mononuclear cell (PBMC) was detected by methylation‐specific polymerase chain reaction (MSP). The mRNA level of TGR5 was determined by quantitative real‐time polymerase chain reaction (RT‐qPCR). We found that the frequency of TGR5 promoter methylation was significantly higher in ACHBLF (35/76, 46.05%) than CHB patients (5/80, 6.25%; χ² = 32.38, P < 0.01) and HCs (1/30, 3.33%; χ² = 17.50, P < 0.01). TGR5 mRNA level was significantly lower (Z = ?9.12, P < 0.01) in participants with aberrant methylation than those without. TGR5 methylation showed a sensitivity of 46.05% (35/76), specificity of 93.75% (75/80), positive predictive value (PPV) of 87.5% (35/40) and negative predictive value (NPV) of 64.66% (75/116) in discriminating ACHBLF from CHB patients. ACHBLF patients with methylated TGR5 showed significantly poor survival than those without (P < 0.01). When used to predict 3‐month mortality of ACHBLF, TGR5 methylation [area under the receiver operating characteristic curve (AUC) = 0.75] performed significantly better than model for end‐stage liver diseases (MELD) score (AUC = 0.65; P < 0.05). Therefore, our study demonstrated that aberrant TGR5 promoter methylation occurred in ACHBLF and might be a potential prognostic marker for the disease.