br Conflict of interest statements br Acknowledgements br In

Conflict of interest statements
Acknowledgements
Introduction Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide and is the major cause for cancer morbidity and mortality in developing countries. Age standardized incidence rate in India is 12.6 per 100 000 population [1]. The risk factors for OSCC include environmental, high risk strains of human papilloma virus and genetic factors [2]. Genetic risk factors include single nucleotide polymorphisms (SNP) that increase the mutation rate in oral epithelial cells. Most of the OSCC associated SNPs occur in genes involved in pathways of carcinogen metabolism, DNA repair, ochratoxin a control, extra-cellular matrix integrity, immunity and inflammation [3], [4]. Metabolism of carcinogens occurs through bioactivation and detoxification process. Genetic polymorphisms that impair the activity of detoxifying enzymes can contribute to carcinogenesis. Some of the detoxifying enzymes with established role in carcinogenesis are glutathione-S-transferases (GSTs), microsomal epoxide hydrolase (mEH), and uridine 5′-diphosphate (UDP)-glucuronosyltransferase (UGTs) [5]. GSTs are phase II detoxifying enzymes involved in cellular protection from xenobiotics (including carcinogens) and oxidative stress. GSTs are classified into eight classes as α (alpha), μ (mu), κ (kappa), ω (omega), π (pi), σ (sigma), θ (theta), and ζ (zeta) and are expressed in various tissues like liver, kidney, lung, stomach, ovaries, colon, pancreas, prostate and spleen [6], [7]. Of the eight groups, GST pi is the predominant form expressed in the oral mucosa which is often found to be overexpressed in oral cancers [8]. c.341C > T SNP in GSTP1 gene (rs1138272) is a functional polymorphism that affects the enzymatic activity of GST pi [9]. In light of the importance of GST pi in the protection of oral mucosa against carcinogens, it is possible that functional polymorphism that affects its activity can contribute towards susceptibility to oral carcinogenesis. We hypothesized that c.341C > T SNP in GSTP1 gene may increase the susceptibility to oral cancer by lowering the protection of oral mucosa against exogenous carcinogens. To the best of our knowledge there is no information on the association between GSTP1 c.341C > T polymorphism with OSCC and therefore the present work was undertaken. The objectives of this study were (i) to determine the association of GSTP1 c.341C > T polymorphism with the risk of OSCC and (ii) to determine the influence of GSTP1 c.341C > T polymorphism on GST pi expression in the tumor.
Materials and methods
Statistical analysis Sample size and power of the study was calculated using OpenEpi web tool using 95% confidence interval [13]. Normal approximation with continuity correction was considered for power calculation. Statistical analysis was carried using the web based calculation available at www.OpenEpi.com (updated 2013/04/06). Allele and genotype frequencies of the two groups were compared using relevant contingency tables. Difference between the groups was determined by calculating P – value from chi-square test (Fisher’s exact test). The study population was tested for conformity to Hardy-Weinberg equilibrium using the web program by Rodriguez and co-workers [14]. P-values <0.05 were considered as statistically significant. Post-hoc power of the study was calculated using the online calculator ‘Online Sample Size Estimator’ (www.osse.bii.a-star.edu.sg).
Results The demographic profile of the patients and the histopathological profile of the tumor samples are given in Table 1. Age of patients ranged from 21 to 80 years with a mean age of 54.2 ± 12.03 years. Most of the patients were females (78%) and were from lower socio-economic strata. All the patients in the study group had addiction to one or more carcinogenic substance like betel nut, chewable tobacco, gutkha (consists of betel leaf, tobacco, areca nut slaked lime, spices, and catechu packed in tins or pouches), smoking or alcohol. The mean duration of the addiction was 30 ± 12 years. All the patients were habituated to chewing betel nut and tobacco while 5.6% of the patients were habituated to tobacco, gutkha chewing and alcohol followed by smoking (7.8%). None of the tumour samples were positive for HPV. Thus, tobacco chewing and not HPV infection was the major risk factor in the patient group. The main complaints during initial presentations were burning sensation in oral cavity (88%), oral ulcer (54%) and trismus (14%). Mean duration of symptoms was 10 ± 4 months. Buccal mucosa was the most commonly involved site (63%); the other sites of lesion were lower alveolus, anterior 2/3rd of tongue, retromolar trigone (RMT) and floor of mouth. 10 patients in the study had locally advanced disease involving buccal mucosa and lower alveolus and it was difficult to identify the epicenter of the disease. They have been termed as lower gingivobuccal sulcus (GBS) cancers. This type of cancers are common in Indian subcontinent. Majority of the patients showed stage IVa tumor (69%); 22% showed stage III and another 9% showed stage II. Squamous cell carcinoma was found to be well differentiated in 73%, moderately differentiated in 24% and poorly differentiated in 2% of the patients. Type 2 diabetes mellitus was the major co-morbidity seen among the patients (∼11%).