Alteration in the expression of MGMT and RUNX3 due to non-CpG promoter methylation and their correlation with different risk factors in esophageal cancer patients.

Promoter methylation reflects in the inactivation of different genes like O6-methylguanine-DNA methyltransferase DNA repair gene and runt-related transcription factor 3, a known tumor suppressor gene in various cancers such as esophageal cancer. The promoter methylation was evaluated for O6-methylguanine-DNA methyltransferase and runt-related transcription factor 3 in CpG, CHH, and CHG context (where H is A, T, or C) by next-generation sequencing. The methylation status was correlated with quantitative messenger RNA expression. In addition, messenger RNA expression was correlated with different risk factors like tobacco, alcohol, betel nut consumption, and smoking habit. CpG methylation of O6-methylguanine-DNA methyltransferase promoter had a positive association in the development of esophageal cancer (p < 0.05), whereas runt-related transcription factor 3 promoter methylation showed no significant association (p = 1.0) to develop esophageal cancer. However, the non-CpG methylation, CHH, and CHG were significantly correlated with O6-methylguanine-DNA methyltransferase (p < 0.05) and runt-related transcription factor 3 (p < 0.05) promoters in the development of esophageal cancer. The number of cytosine converted to thymine (C→T) in O6-methylguanine-DNA methyltransferase promoter showed a significant correlation between cases and controls (p < 0.05), but in runt-related transcription factor 3 no such significant correlation was observed. Besides, messenger RNA expression was found to be significantly correlated with promoter hypermethylation of O6-methylguanine-DNA methyltransferase and runt-related transcription factor 3 in the context of CHG and CHH (p < 0.05). The CpG hypermethylation in O6-methylguanine-DNA methyltransferase showed positive (p < 0.05) association, whereas in runt-related transcription factor 3, it showed contrasting negative association (p = 0.23) with their messenger RNA expression. Tobacco, betel nut consumption, and smoking habits were associated with altered messenger RNA expression of O6-methylguanine-DNA methyltransferase (p < 0.05) and betel nut consumption and smoking habits were associated with runt-related transcription factor 3 (p < 0.05). There was no significant association between messenger RNA expression of O6-methylguanine-DNA methyltransferase and runt-related transcription factor 3 with alcohol consumption (p = 0.32 and p = 0.15). In conclusion, our results suggest that an aberrant messenger RNA expression may be the outcome of CpG, CHG, and CHH methylation in O6-methylguanine-DNA methyltransferase, whereas outcome of CHG and CHH methylation in runt-related transcription factor 3 promoters along with risk factors such as consumption of tobacco, betel nut, and smoking habits in esophageal cancer from Northeast India.

Expression of Interleukin-8, Interleukin-12 and Interleukin-13 in Esophageal Squamous Cell Carcinoma: Biomarker Potentiality and Prognostic Significance.

PURPOSE: Interleukin-8 (IL8), Interleukin-12 (IL12) and Interleukin-13 (IL13) are cytokines that play regulatory role in cancer pathogenesis. We analysed their expression profile to evaluate as molecular biomarkers of esophageal squamous cell carcinoma (ESCC) and their association with different parameters and patient survival. METHODS: Expression analysis was performed by Real time quantitative polymerase chain reaction and receiver operating characteristic (ROC) curve analysis was done. The expression profiles were associated with different clinicopathological and dietary factors. Survival and hazard analysis were also performed. RESULTS: IL8 expression showed upregulation in tissue (p = 0.000) and blood samples (p = 0.481), IL12 expression showed downregulation in tissue samples (p = 0.064) and upregulation in blood samples (p = 0.689) and IL13 expression showed upregulation in tissue (p = 0.000) and blood samples (p = 0.006). IL13 expression in tissue showed the highest area under the curve (AUC) value (0.773) for ESCC diagnosis, followed by IL8 expression in tissue (0.704) and IL13 expression in blood (0.643). This study also reveals the correlation of studied cytokines in tissue and blood level. Different clinicopathological and dietary factors showed significant association (p < 0.05) with IL8, IL12 and IL13 expression and with survival of ESCC patients. IL8 expression in blood and IL12 expression in tissue and blood showed significant association (p < 0.05) with patient survival. CONCLUSION: Altered expression of IL8, IL12 and IL13 may be associated with ESCC progression. Overexpression of IL8 and IL13 in tissue samples may be potential biomarkers for ESCC screening. Additionally, both survival and hazard analysis data indicate the effects of different parameters on the prognosis of ESCC patients.

Altered expression of TGF-ß1 and TGF-ßR2 in tissue samples compared to blood is associated with food habits and survival in esophageal squamous cell carcinoma.

In the transforming growth factor ß (TGF-ß) signaling pathway, TGF-ß1 and TGF-ß receptor 2 (TGF-ßR2) are essential regulatory components which play an important role in different type of cancer. Expressions of TGF-ß1 and TGF-ßR2 were done by real-time qPCR in both biopsy and blood samples collected from esophageal squamous cell carcinoma (ESCC) patients (n = 76). The expression profiles were correlated with different lifestyle factors and clinicopathological parameters. Kaplan-Meier survival analysis and Cox regression analysis were performed to estimate survival and hazard outcomes of different parameters. TGF-ß1 showed upregulation in 91% tissue samples (2.84 ± 1.34*) and 55% blood samples (2.43 ± 1.24*) whereas expression of TGF-ßR2 showed downregulation in 89% tissue samples (0.27 ± 0.23*) and 75% blood samples (0.30 ± 0.26*). Among all the parameters, TGF-ß1 expression is significant with histopathology grade, consumption of betel nut and smoked food whereas TGF-ßR2 expression is significant only with dysphagia grade in both blood and tissue samples and while analyzing both male and female patients separately. Consuming alcohol and hot food, difference in tumor stage and metastasis were found to have statistically significant (P < 0.05) impact on survival and mortality of male patients while consuming hot food, tobacco, metastasis and TGF-ßR2 expression in tissue level were found to associate with survival and mortality of female patients. Expression of both TGF-ß1 and TGF-ßR2 in tissue samples may be prospective biomarkers for screening of ESCC among the Northeast population. Survival outcomes and hazard analysis supports the importance of some clinicopathological and lifestyle factors on ESCC development, whereas expression study depicts association of change in expression of the studied genes in ESCC patients. *Mean fold change.

IL-10 Polymorphisms and Haplotypes Predict Susceptibility to Hepatocellular Carcinoma Occurrence in Patients with Hepatitis C Virus Infection from Northeast India.

Chronic hepatitis C virus (HCV) infection leads to variable outcomes, ranging from prolonged slow hepatic damage leading to cirrhosis, and hepatocellular carcinoma (HCC). Polymorphism in cytokines IL-10 and IL-12 that impact the immune response to HCV infection may play a role in determining this outcome. This study was aimed to determine if polymorphisms in IL-10 and IL-12B contribute to HCV susceptibility and the risk of developing HCC in patients from Northeast India. IL-10 - 1082, -819, -592 polymorphisms and IL-12B -1188 polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism in a total of 266 HCV-infected patients and 100 age- and sex-matched controls. In the HCV-infected subjects, 110 patients had chronic hepatitis C (CHC), 96 with liver cirrhosis, and 60 with HCC. Serum levels of IL-10 were also measured and correlated with disease severity. Haplotype analysis for IL-10 polymorphisms was carried out. Statistical data were analyzed using SPSS ver. 22.0. The frequency of IL-10 - 592 AA genotype/A allele was significantly higher in HCC patients than in CHC patients. The intermediate IL-10-producing ACC haplotype was significantly more frequent in HCC and cirrhotic patients than in CHC patients. No significant association was found for IL-10 - 819, -592 and IL-12B -1188 polymorphisms with the susceptibility to HCV infection or occurrence of HCC in HCV-infected patients. IL-10 - 592 CA polymorphism and IL-10 ACC haplotype are significant biomarkers of HCC in HCV-infected patients from Northeast India. Higher serum levels of IL-10 were also linked to higher disease severity.

Role of VDR, GC, and CYP2R1 Polymorphisms in the Development of Hepatocellular Carcinoma in Hepatitis C Virus-Infected Patients.

Aims: This study was designed to determine if vitamin D receptor (VDR), carrier globulin/binding protein (GC), and cytochrome P-450 family 2, subfamily R, polypeptide 1 (CYP2R1) gene polymorphisms are risk factors in the development of hepatocellular carcinoma (HCC) in hepatitis C virus (HCV)-infected patients from Northeast India. Materials and Methods: A total of 351 HCV-infected patients were enrolled of which 167 were diagnosed with chronic hepatitis C (CHC), 124 with liver cirrhosis (LC), and 60 with HCC together with 102 age- and sex-matched healthy controls. VDR (BsmI, ApaI, and TaqI), GC (rs4588, rs7051), and CYP2R1 (rs10741657) gene polymorphisms were genotyped for all subjects. Statistical data were analyzed using SPSS ver. 22.0. Results: The frequency of the ApaI CC genotype, ApaI C allele, and bAt haplotype of the VDR gene was significantly higher in HCC and LC patients than controls. After adjusting for other covariates (age, gender, platelet count, AST, ALT, serum albumin, and viral load) logistic regression analysis showed that the ApaI CC genotype and bAt haplotype were independent predictors of HCC development. No significant associations was found for the GC and CYP2R1 polymorphisms examined with the occurrence of HCC. Conclusions: The presence of the VDR ApaI CC genotype and bAt haplotype appear to be important indicators in the development of HCC among HCV-infected patients. Larger studies are needed to further clarify and establish this potential causal relationship.

Survey of alcohol-related cirrhosis at a tertiary care center in North East India.

BACKGROUND: Alcohol use is increasing in North East India and is important to estimate the influence of these changes in the epidemiology of alcohol related cirrhosis. METHODS: Among 1000 consecutive patients of cirrhosis, diagnosed by a combination of clinical, radiological and/or histopathological features, etiology was established by history of significant alcohol abuse, determining viral and autoimmune markers and by metabolic screening. Patients not confirmed to be cirrhotic were excluded from the study. All cases were studied to determine clinical features, complications, disease prognosis, and mortality. Alcoholic cirrhotics were then compared with nonalcohol etiology. RESULTS: 72.2 % alcoholic cirrhosis were compared with 27.8 % patients of nonalcohol etiology and alcoholic cirrhotics were younger (45 + 9.4 years vs. 47.9 + 12.5 years), predominantly males (M/F ratio 37:1 vs. 1.8:1) with significantly high incidence of jaundice (38.5 % vs. 30.5 %), night blindness (14.4 % vs. 3.6 %), ascites (76.3 % vs. 69.1 %), upper gastrointestinal bleed (46.4 % vs. 34.5 %), and hepatic encephalopathy (24.1 % vs. 10.4 %). Biochemical parameters that were significantly higher in alcoholics were mean bilirubin (4.7 + 8.7 vs. 3.1 + 4.7 mg/dL), AST/ALT ratio (2.03 vs. 1.4), gamma-glutaryl transaminase levels (209.7 + 37.9 vs. 93.9 + 14 IU/mL), and serum ammonia (75.1 + 55.7 vs. 52.1 + 45.4 mg/dL). Mean model for end-stage liver disease, scores, and Child C disease was significantly higher in alcoholics (18.6 + 7.7 vs. 15.6 + 6.4) and (54.1 % vs. 37 %), respectively, representing advanced disease at presentation. Mortality within 1 month was significantly higher among alcoholic cirrhosis (9.8 % vs. 3.2 %). CONCLUSION: Thus, alcoholic cirrhosis is of major concern in North East India as majority patients are in most productive age group and presented with advanced disease. Short-term mortality was high among alcoholic cirrhotics. Proper education and legislation are essential to mitigate the consequences of this disease.

Polymorphisms in Heat Shock Proteins A1B and A1L (HOM) as Risk Factors for Oesophageal Carcinoma in Northeast India.

BACKGROUND: To investigate polymorphisms in heat shock proteins A1B and A1L (HOM) and associated risk of oesophageal carcinoma in Northeast India. MATERIALS AND METHODS: The study includes oesophageal cancer (ECA) patients attending general outpatient department (OPD) and endoscopic unit of Gauhati Medical College. Patients were diagnosed based on endoscopic and histopathological findings. Genomic DNA was typed for HSPA1B1267 and HSPA1L2437 SNPs using the polymerase chain reaction with restriction fragment length polymorphisms. RESULTS: A total of 78 cases and 100 age-sex matched healthy controls were included in the study with a male: female ratio of 5:3 and a mean age of 61.4±8.5 years. Clinico-pathological evaluation showed 84% had squamous cell carcinoma and 16% were adenocarcinoma. Dysphagia grades 4 (43.5%) and 5 (37.1%) were observed by endoscopic and hispathological evaluation. The frequency of genomic variation of A1B from wild type A/A to heterozygous A/G and mutant G/G showed a positive association [chi sq=19.9, p= <0.05] and the allelic frequency also showed a significant correlation [chi sq=10.3, with cases vs. controls, OR=0.32, p≤0.05]. The genomic variation of A1L from wild T/T to heterozygous T/C and mutant C/C were found positively associated [chi sq= 7.02, p<0.05] with development of ECA. While analyzing the allelic frequency, there was no significant association [chi sq= 3.19, OR=0.49, p=0.07]. Among all the risk factors, betel quid [OR =9.79, Chi square= 35.0, p<0.05], tobacco [OR = 2.95, chi square=10.6, p<0.05], smoking [OR=3.23, chi square=10.1, p<0.05] demonstrated significant differences between consumers vs. non consumers regarding EC development. Alcohol did not show any significant association [OR= 1.34, chi square=0.69, p=0.4] independently. CONCLUSIONS: It can be concluded that the present study provides marked evidence that polymorphisms of HSP70 A1B and HSP70 A1L genes are associated with the development of ECA in a population in Northeast India, A1B having a stronger influence. Betel quid consumption was found to be a highly significant risk factor, followed by smoking and tobacco chewing. Although alcohol was not a potent risk factor independently, alcohol consumption along with tobacco, smoking and betel nut was found to contribute to development of ECA.