Association of cyclin D1 gene polymorphisms with risk of esophageal squamous cell carcinoma in Kashmir Valley: a high risk area.

Investigation of potential association of SNPs (G870A, rs9344; G1722C, rs678653) of cyclin D1 gene (CCND1) with susceptibility to esophageal squamous cell carcinoma (ESCC) in Kashmir valley (India). The study included 302 subjects comprising 151 ESCC cases and 151 controls. PCR-RFLP and direct sequencing were employed for genotyping. The G870A polymorphism, the individuals carrying GA + AA genotype was having 2.80-fold increased risk for development of ESCC (OR 2.8, 95% CI = 1.77-4.4; P = 0.0001) compared to GG genotype. Further a significantly higher risk was observed in individuals who consume >3 cups per day of salted tea (OR = 5.1; 95% CI = 1.6-16.7; P = 0.0016) and had smoking habits (OR = 6.3; 95% CI = 2.9-13.9; P = 0.0005). We also demonstrate for the first time in CCND1 1722 locus, the CC genotype was strongly associated with increased risk of developing ESCC (OR = 2.58; 95% CI = 1.61-4.15; P = 0.0001). In addition, the frequency of polymorphic C allele was also found to be higher in cases (OR = 1.92; 95% CI = 1.37-2.69; P = 0.0002). There appears to be an influence of CCND1 G870A/G1772C genotypes on genetic susceptibility to ESCC.

Methylation-mediated gene silencing of suppressor of cytokine signaling-1 (SOCS-1) gene in esophageal squamous cell carcinoma patients of Kashmir valley.

CONTEXT: Esophageal squamous cell carcinoma (ESCC) is a leading cause of cancer-related deaths in Jammu and Kashmir. The negative regulation of tumor suppressor gene leading to change in signaling pathway is one of the major mechanisms responsible for tumorigenic transformation. OBJECTIVE: In the present study, the role of silencing of suppressor of cytokine signaling-1 (SOCS-1) gene, a negative regulator of JAK/STAT pathway, was analyzed in ESCC. METHODS: The expression pattern of SOCS-1 gene was analyzed in esophageal tumor biopsies although normal adjacent tissues that served as controls. Reverse transcriptase polymerase chain reaction (RT-PCR), immunohistochemistry, methylation-specific PCR (MSP), and human papillomavirus (HPV) detection were performed to assess the expression pattern and promoter methylation of SOCS-1 gene including HPV status in a total of 75 surgically resected tissue specimens. RESULTS: Compared with the level of SOCS-1 expression in normal tissues, 53% (40/75) of the tumor tissues expressed either undetectable or reduced SOCS-1 expression (>50% loss of expression), which was significantly associated with advanced clinical stage or severe histopathological grade of the disease (P < 0.01). Aberrant promoter methylation of the SOCS-1 gene was found in 45% (34/75) of the esophageal tumor tissues, which was also found to be significantly associated with advanced stage of esophageal carcinoma (P < 0.01). The prevalence of HPV infection was found in 19% of tumor cases, whereas no HPV could be detected in any of the normal adjacent tissues. CONCLUSION: Transcriptional inactivation of SOCS-1 gene, primarily due to its promoter hypermethylation although HPV infection, may play an important role in esophageal carcinogenesis in Kashmir.

Transcription factor AP-1 in esophageal squamous cell carcinoma: alterations in activity and expression during human Papillomavirus infection.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a leading cause of cancer-related deaths in Jammu and Kashmir (J&K) region of India. A substantial proportion of esophageal carcinoma is associated with infection of high-risk HPV type 16 and HPV18, the oncogenic expression of which is controlled by host cell transcription factor Activator Protein-1 (AP-1). We, therefore, have investigated the role of DNA binding and expression pattern of AP-1 in esophageal cancer with or without HPV infection. METHODS: Seventy five histopathologically-confirmed esophageal cancer and an equal number of corresponding adjacent normal tissue biopsies from Kashmir were analyzed for HPV infection, DNA binding activity and expression of AP-1 family of proteins by PCR, gel shift assay and immunoblotting respectively. RESULTS: A high DNA binding activity and elevated expression of AP-1 proteins were observed in esophageal cancer, which differed between HPV positive (19%) and HPV negative (81%) carcinomas. While JunB, c-Fos and Fra-1 were the major contributors to AP-1 binding activity in HPV negative cases, Fra-1 was completely absent in HPV16 positive cancers. Comparison of AP-1 family proteins demonstrated high expression of JunD and c-Fos in HPV positive tumors, but interestingly, Fra-1 expression was extremely low or nil in these tumor tissues. CONCLUSION: Differential AP-1 binding activity and expression of its specific proteins between HPV--positive and HPV--negative cases indicate that AP-1 may play an important role during HPV-induced esophageal carcinogenesis.

Aberrant promoter methylation and reduced expression of p16 gene in esophageal squamous cell carcinoma from Kashmir valley: a high-risk area.

Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancer in Jammu and Kashmir region of India and has multi-factorial etiology involving dietary habits, genetic factors, and gene environmental interactions. Inactivation of the p16 gene expression by aberrant promoter methylation plays an important role in the progression of esophageal carcinoma. In the present investigation, we have studied the role of p16 promoter methylation in 69 histopathologically confirmed ESCC tissues and compared it with corresponding normal adjacent tissues for DNA methylation in the CpG island in the p16 promoter region by methylation-specific polymerase chain reaction (MSP) and p16 protein expression by immunoblotting. The results showed loss of p16 expression in 67% (46/69) of tumor tissues compared to only 3% in control tissues (2/69). Promoter methylation was observed in 52% (36/69) of tumor tissues and it gradually increased with the increasing severity of histological grades of the cancer (P = 0.0001). Loss of p16 expression with promoter methylation was observed in 26 of 36 cases (72%). Analysis of patients dietary habits revealed a strong association between promoter methylation and high consumption of hot salted tea (P < 0.05) which is a most favourite drink commonly consumed by Kashmiri people.

Association between copper excess, zinc deficiency, and TP53 mutations in esophageal squamous cell carcinoma from Kashmir Valley, India--a high risk area.

Trace element deficiency or excess is implicated in the development or progression in some cancers. Here we report the elevated level of copper and low level of zinc in the plasma of esophageal cancer patients in Kashmir India--a high incidence area. The average level of copper was significantly higher (P < 0.0001) for patients than for controls, with a mean concentration of 169 microg/dl and 149 microg/dl for patients and controls, respectively. The control group consisted of 55 healthy individuals matched for age, sex, and place of residence of the patients. In contrast, the average level of zinc in patients was significantly lower than in controls (P < 0.0001), with a mean concentration of 86.8 microg/dl and 96.1 microg/dl for patients and controls, respectively. The levels of both copper and zinc showed significant differences based on gender and age in patients as compared to controls. Similarly, smokers depicted a significant increase in serum copper (N = 39, P = 0.002) and a decrease in serum zinc approaching level of significance in the patient group as compared to controls. The copper and zinc levels were significantly altered in patients (N = 40) when compared to controls as a function of snuff consumption. The differences in the levels of copper and zinc showed significant association with the consumption of local salted tea up to 1,500 ml per day, but the changes were insignificant beyond that. Patients with poorly differentiated tumors (N = 7) had a higher copper concentration than those with moderately or well-differentiated tumors (P < 0.0001). To validate the general notion that imbalance in copper and zinc levels may lead to higher prevalence of TP53 mutations, we compared the 3 variables, and no association was found between copper concentration and TP53 mutation status; but patients with TP53 mutant tumor had lower zinc levels than those with no mutation. In conclusion, our results point toward a role of the trace element imbalance in the esophageal tumorigenesis in high-risk Kashmiri population exposed to a range of nitroso compounds or their precursors. Further prospective cohort studies are warranted to determine whether change in the plasma zinc and copper homeostasis may represent an independent risk factor for this malignancy as well as a possible target for preventive intervention.

Mutations in epidermal growth factor receptor gene in esophageal squamous cell carcinoma patients in kashmir- a high incidence area of India.

Activating mutations in Epidermal Growth Factor Receptor (EGFR) are common in lung adenocarcinoma of never smokers but are rare in other types of cancer. Here we have analysed mutations in exons 19 to 21 of EGFR and in exons 19 and 20 of the EGFR homolog HER2 in 54 cases of Esophageal Squamous Cell Carcinomas (ESCC) from patients recruited in Kashmir, India, a region of high incidence for this cancer. We report the detection of 3 mutations (6%) in the ATP-binding regulatory loops of the tyrosine kinase domain of EGFR (deletion 746-750, P753L, G719D). No mutation was found in HER2. This is the first report of activating EGFR mutations in ESCC, of the same type as those detected in lung adenocarcinoma of never-smokers. This suggests that a small proportion of ESCC patients in this high incidence area may benefit from treatment with EGFR tyrosine kinase inhibitors.

Combined impact of polymorphism of folate metabolism genes; glutamate carboxypeptidase, methylene tetrahydrofolate reductase and methionine synthase reductase on breast cancer susceptibility in kashmiri women.

BACKGROUND: Folate and methionine play a crucial role in DNA synthesis, repair and the epigenetic profile of cell. Hence, the alterations in the folate metabolism can lead to aberrant proliferation leading to neoplasia. Most of the studies have associated polymorphisms in methylene tetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes with reduced risk of cervical and colorectal cancer. However, the association with breast cancer is still controversial. Further, the involvement of Glutamate carboxypeptidase II (GCPII) polymorphism in cancer is not known. In the present study, we analyzed if the individual and combined effects of polymorphisms in folate pathway genes viz., MTHFR 677C > T, MTHFR 1298A > C, MTRR 66A > G and GCP II 1561 C>T, have any role in altering the susceptibility to breast cancer. METHODS: The DNA of 35 female breast cancer patients and 33 healthy individuals, in the Kashmiri population from India, were analyzed using a PCR-RFLP approach for the above mentioned polymorphisms. RESULTS: Individuals carrying the MTHFR 677CT/TT and GCPII 1561 CT genotype showed a 3.5 (95% CI: 3.1-3.7, P<0.02) and 7.7 (95% CI: 6.7-9.1, P<0.001) fold decreased risk for breast cancer than the wild types (MTHFR 677CC and GCPII 1561 CC). Subjects with MTRR 66 G-allele showed a 4.5 fold decreased risk (OR: 0.22, 95% CI: 0.20, 0.24, P<0.0005) compared to the wild type (MTRR 66A). Further, subjects with combined polymorphisms in MTHFR, GCPII and MTRR loci revealed a significant reduction of breast cancer risk. CONCLUSION: This study indicates (i) a protective role of polymorphisms in MTHFR, GCPII, MTRR against breast cancer in the study subjects, and (ii) combined effect of polymorphisms is more pronounced than single genetic polymorphism, thereby emphasizing the role of gene-gene interaction in the susceptibility to breast cancer.

The Association of Beta-catenin Gene Mutations and Human Papillomavirus in Carcinoma of Esophagus in a High-Risk Population of India.

BACKGROUND: Esophageal cancer (EC) is the sixth leading cause of death from cancer. In high-risk regions, squamous cell carcinoma is the most common type of EC, and its etiology remains poorly understood. It shows uneven geographical distribution in its occurrence, reflecting the influence of local environmental conditions, lifestyle and genetic predisposition in the development of the cancer. Kashmir, in the north of India, has been described as a high-risk area for esophageal squamous cell carcinoma (ESCC). In the present investigation an attempt was made to study the role of ß-catenin mutations and human papillomavirus in 62 ESCC patients from Kashmir. METHODS: The hot spot mutation region of ß-catenin exon 3 was evaluated in matched tumor and normal tissues using a combination of PCR-SSCP and direct sequencing. We used two different sets of consensus primers viz., GP5+ and GP6+; PGMY09 and PGMY11 in conjunction with reverse line blot assay to screen for human papillomavirus(HPV). RESULTS: None of the tumors showed the presence of commonly reported mutations in ß-catenin. In view of the fact that HPV has been linked to pathogenesis of EC, we screened all the tumor and control specimens for the presence of HPV and we didn't detect HPV in any of the matched tumor and control specimens in contrast to the positive controls we used. CONCLUSION: In conclusion our results suggest that squamous cell carcinoma of esophagus in Kashmir may arise independent of oncogenic ß-catenin mutations and HPV is unlikely to be an etiologic factor for ESCC in this region.

Studies on Association Between Copper Excess, Zinc Deficiency and TP53 Mutations in Esophageal Squamous Cell Carcinoma From Kashmir Valley, India-A High Risk Area.

Trace element deficiency or excess is implicated in the development or progression in some cancers. Here we report the elevated levels of copper and low level of zinc in the plasma of esophageal cancer patients in Kashmir India- a high incidence area. The average level of copper was significantly higher for patients than for controls (p<0.0001) with a mean concentration of 169 µg/dl and 149 µg/dl for patients and controls, respectively. In contrast, the average level of zinc in patients was significantly lower than in controls (p<0.0001) with a mean concentration of 86.8 µg/dl and 96.1 µg/dl for patients and controls, respectively. No significant difference in copper and zinc levels was observed for different age groups in controls or patients. For controls, the level of copper was not significantly different in males and females (median: 155 µg/dl for males and 144 µg/dl for females, p=0.10), but we observed a higher level of zinc in females (median: 90.5 µg/dl for males and 101 µg/dl for females, p=0.03). Copper or zinc concentrations were not significantly associated with gender, tumor site, green tea with salt (nun chai) consumption, smoking habits or snuff in cases. Patients with poorly differentiated tumors had a higher copper concentration than those with moderately or well-differentiated tumors (p<0.0001). No association was found between copper concentration and TP53 mutation status but patients with TP53 mutant tumor had lower zinc levels than those with no mutation. Our results point towards a role of the trace element imbalance in the esophageal tumorigenesis in high risk Kashmiri population exposed to a range of nitroso compounds or their precursors. Further prospective cohort studies are warranted to determine whether change in the plasma zinc and copper homeostasis may represent an independent risk factor for this malignancy as well as possible target for preventive intervention.