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Year : 2019  |  Volume : 56  |  Issue : 1  |  Page : 24--28

Multiple logistic regression analysis predicts cancer risk among tobacco usage with glutathione S-transferase p1 genotyping in patients with head and neck cancer

Argi Anuradha1, Veerathu L Kalpana1, Natukula Kirmani2,  
1 Department of Human Genetics, Andhra University, Visakhapatnam, Andhra Pradesh, India
2 School of Biotechnology, Mahatma Gandhi National Institute of Research and Social Action, Gaganmahal Road, Domalguda, Hyderabad, Telangana, India

Correspondence Address:
Natukula Kirmani
School of Biotechnology, Mahatma Gandhi National Institute of Research and Social Action, Gaganmahal Road, Domalguda, Hyderabad, Telangana


INTRODUCTION: Numerous studies have been investigated to understand the association between glutathione S-transferase P1 (GSTP1) polymorphism and risk of head and neck cancer (HNC) but yielded contradictory results, and no studies could confirm polymorphism in GSTP1 and that tobacco usage increases the risk of HNCs. Therefore, this study aimed to understand the association of GSTP1 Ile105Val polymorphism with or without tobacco usage in carcinogenesis and clinicopathological characteristics of patients with HNC. MATERIALS AND METHODS: Binary logistic regression analysis was performed to predict HNC risk with tobacco use and GSTP1 genotyping. Five predictor variables such as gender, age, tobacco usage, familial, and GSTP1 genotypes were included in the model. RESULTS: The results of the logistic regression analysis show that the full model which considered all the five independent variables together was statistically significant, log-likelihood = −111.820, and all slopes are zero: G = 74.297, degree of freedom (DF) = 5, P = 0.000. The strongest predictor in this model is tobacco usage (odds ratio = Z = −5.16, P = 0.000). CONCLUSION: The study concludes that multiple logistic regression analysis model could predict the risk factors in case–control studies where control samples are compromised.

How to cite this article:
Anuradha A, Kalpana VL, Kirmani N. Multiple logistic regression analysis predicts cancer risk among tobacco usage with glutathione S-transferase p1 genotyping in patients with head and neck cancer.Indian J Cancer 2019;56:24-28

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Anuradha A, Kalpana VL, Kirmani N. Multiple logistic regression analysis predicts cancer risk among tobacco usage with glutathione S-transferase p1 genotyping in patients with head and neck cancer. Indian J Cancer [serial online] 2019 [cited 2020 Dec 3 ];56:24-28
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Full Text


The development of head and neck cancer (HNC) is a multifactorial process associated with a variety of risk factors, the most important being smoking tobacco, drinking alcohol, and chewing betel quid in developed countries.[1],[2] Polymorphisms in various functional genes and their encoded enzymes may alter the function by an increase or decrease in carcinogen activation/detoxification and adapt DNA repair capacity, possibly by shifting their expression and function. Genetic factors and environmental factors contributing to the development of HNCs are reported.[3],[4],[5] Recent studies have suggested that polymorphisms in genes encoding for carcinogen-metabolizing genes and DNA-repair genes[6] play critical roles in determining individual susceptibility to various cancers including HNC.[7] One of the most important systems in detoxification is the glutathione S-transferase (GST) family of enzymes.

GST gene family comprises detoxifying enzymes that catalyze the conjugation of glutathione with a wide variety of endogenous and exogenous compounds. Alterations in GST-encoded genes lead to elevated levels of enzymes, which can affect detoxification ability. Single nucleotide polymorphisms in the GST gene responsible for altered detoxification process and increases the risk of cancer with specific variant alleles of the GST gene was reported. Several SNPs exist in the GSTs which may lead to null genotypes (GSTM1 and GSTT1) or partially deficient enzyme (GSTP1) activity.[2],[8]

Among GST gene family, GSTP1 isoform is extensively studied because it catalyzes the conjugation of glutathione to toxic compounds, resulting in more water-soluble and less biologically active products that are easily excreted. GSTP1 plays a major role in DNA along with other biomolecules which protect the cell against damage or adduct formation from tobacco-related carcinogenic components. GSTP1 enzyme is present quantitatively in the oropharyngeal tissues[9] and has a role in the detoxification process of tobacco related products and in tobacco-related malignancies.

An Isoleucine to Valine substitution at codon 105 (exon 5) of GSTP1 gene has been identified; altered affinity and enzymatic activity were observed in the presence of some substrates. Studies conducted previously[10],[11],[12],[13],[14] showed a significantly higher risk with Ile/Val and/or Val/Val genotypes in HNC when compared with those with Ile/Ile genotype. Substitution of a Val allele in Ile might be associated with an increased risk of HNC, and there are no studies showing significant lower risk with Val/Val and/or Ile/Val genotypes than Ile/Ile genotype. A meta-analysis by Hashibe et al.[15] opined that Ile/Val and Val/Val genotypes increase HNC risk when compared with Ile/Ile genotype. Earlier studies[12],[16],[17],[18],[19] suggested that polymorphisms in GSTP1 genes could control the equilibrium between metabolic activation and detoxification of carcinogens and are, therefore, related to individual susceptibility to HNC. In contrast, other studies[14],[20],[21],[22],[23] did not support these findings. All these contradictory results lead to uncertainty whether GSTP1 polymorphism modifies the risk of HNC. Previous studies did not establish any association between GSTP1 gene polymorphism and tobacco usage in HNCs. Thus, the aim of this study was to evaluate the possible association between GSTP1 Ile105Val polymorphism with or without exposure to tobacco usage and risk of HNC.

 Materials and Methods

Cases and controls

This was a retrospective hospital-based case–control study. The study included 105 HNC cases confirmed by histopathological examination and 110 healthy age- and sex-matched controls. This study was approved by the Institutional Ethical Committee, and written consent form was obtained from each case and control subject before enrollment.

DNA isolation and glutathione S-transferase P1 genotyping

Extraction of DNA was carried out by the salting-out method described by Miller et al.[24] Genotyping of GSTP1 gene was performed by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) technique.[25]GSTP1 Ile 105 Val substitution was detected using PCR/RFLP approach as the substitution by guanine introduced restriction site that can be recognized by an endonuclease ALW26I (Acinetobacter lwoffii), catalog no. ER0031 (Thermo Fisher Scientific). A PCR product of 328 base pair on 1.5% agarose gel electrophoresis confirmed the presence of GSTP1 gene. PCR products were digested using restriction enzyme (ALW26I) incubated with 10 units of enzyme for 16 h at 37°C. The cleaved products were then resolved by 2% gel electrophoresis. The digested fragments were 328, 222, 106, and 105 bp for heterozygote variant alleles; 222, 106, and 105 bp for homozygous variant alleles; and 328, 106, and 105 bp for homozygous wild alleles.

Statistical analysis

Genotype frequencies of GSTP1 among cases and controls were determined for Hardy–Weinberg equilibrium using standard Chi-square tests. Crude odds ratios (ORs) and 95% confidence intervals were calculated by (For windows, MedCalc Software, Acacialaan 22, 8400 Ostend, Belgium) online statistical software for the association between genotypes and HNC risk. Adjusted OR was calculated using multiple logistic regression model using SPSS software package (version 11.0 for Windows; SPSS, Chicago, IL, USA) after adjusting for other covariates such as age, smoking, tobacco chewing, and alcohol consumption. P < 0.05 was considered statistically significant.


A total of 105 patients with confirmed diagnosis of head and neck squamous cell carcinoma (HNSCC) and 110 healthy controls were enrolled in this study. Among patients confirmed with malignancies, 67 (63.80%) were males and 38 (36.19%) were females. The mean age of all patients with HNC was 54.18 years (range 21–80 years). The mean age for males was 55.40 years and for females 52.95 years. The variation between male and female age groups was not significant. Higher numbers of patients were found in the 41–60 (58.09%) years age group. The significance of age was found to be negative [Table 1] when compared with control. Other demographics and habits were analyzed for significance with disease, and a statistical significance was found with gender. Irrespective of usage or exposure to tobacco, all males and females showed significant values. Out of all the tobacco exposure (any form of smoking or chewing) habits, reverse smoking showed significant results, whereas smoking and tobacco chewing were insignificant. Both tobacco exposure and nontobacco exposure in HNC cases were found to be statistically significant (P < 0.05) [Table 1]. The three different genotype frequencies of GSTP1 gene (Ile/Ile, Ile/Val, Val/Val) were 0.685, 0.285, and 0.028, respectively, whereas allele frequency of Ile/Ile was 0.827 and Val/Val 0.17. The study found no significance of GSTP1 gene polymorphisms in cases when compared with controls [Table 1]. The distribution of site of disease in smokers and nonsmokers of HNC cases was also studied. Statistically significant results were obtained for pharynx in relation to smoking habit when compared with larynx and oral cavity in males and females of HNC cases [Table 1]. Distribution of GSTP1 genotypes, stratified by tobacco usage in cases and controls, was evaluated. Ile/Ile (HNC cases and controls) of GSTP1 gene showed significant results in relation to tobacco smokers whereas tobacco chewers and reverse smokers were insignificant. Ile/Val (HNC cases and controls) of GSTP1 gene showed significance with tobacco chewers but was insignificant with smokers and reverse smokers. Val/Val and tobacco exposure versus nontobacco exposure groups were insignificant in this study (data not shown here).{Table 1}

From the output [Table 2], it was estimated that the coefficients for gender, tobacco usage, familial, and GSTP1 have P < 0.05, indicating that there is sufficient evidence that the coefficients are not zero using an α-level of 0.05, which concludes that these four independent variables have an impact on cancer. Whereas variables such as gender and age do not give significant result on cancer of type GSTP1. The results of the logistic regression analysis show that the full model which considered all the five independent variables together was statistically significant (P = 0.000).{Table 2}


HNC constitutes the cancers of the oral cavity, larynx, and pharynx and accounts for the sixth most frequent cancer worldwide.[26] India is one of the countries occupies the highest incidence of tobacco-related cancers as it is the second largest country having consumers and producers of tobacco in the world. In this case–control study, we did not find any association with HNC risk. Demographic and clinicopathological data showed no association between cancer risk when compared with controls because of convenience sampling in controls. It was noticed that chewers used tobacco in the form of betel quid, which consists of betel leaf and tobacco. Irrespective of tobacco usage, tobacco chewing and reverse smoking results showed significance with HNC risk. As per literature, HNSCC has been strongly associated with tobacco smoking, alcohol consumption, chewing tobacco and slaked lime, betel nut, or betel quid chewing,[27] but the risk is differentially associated with HNC subtypes.[28] Inspite of these risk factors in genotyping studies, epidemiological studies proved that factors such as poor oral health, human papillomavirus infection, polymorphisms in DNA repair genes, phase II carcinogen-metabolizing enzymes which include GSTs strongly associated in the genesis of HNSCC.[29] In the class of GST genes, it was reported that GSTP1 is more efficient and selective in detoxifying the carcinogenic epoxide of benzo(a)pyrene variant forms.[30],[31] Contrastingly, this study did not find any association with HNC risk. Of the two common polymorphisms of GSTP1, that is, in exon 5 nucleotide position at 313 adenine (A) to guanine (G) transition and at position 104 in the amino acid sequence of the protein substitution to valine (Val) from isoleucine (Ile), the previous one has a higher risk of HNSCC susceptibility. A meta-analysis by Hashibe et al. showed that the presence of at least one variant allele (Val/Val) or (Ile/Val) may increase the risk of HNSCC,[15] and several other studies showed that polymorphism in GSP1 homozygous variant (Val/Val) allele may increase the risk of HNSCC. However, the results of this study are similar to Oude Ophuis et al. and Reszka et al., where in homozygous variant of GSTP1 (Val/Val) genotypes in patients with HNSCC and in healthy controls[32],[33] no statistical differences were found for homozygous wild-type AA (Ile/Ile) and heterozygous variant (Ile/Val) or (Val/Val) genotypes, which confirmed that substitution of Ile to Val of GSTP1 gene polymorphism is not associated with altered susceptibility to HNSCC.

It is thus proved that genetic variations may not cause disease but instead may influence a person's susceptibility to environmental factors. When genotyping was correlated with tobacco usage, statistical significance was not found with HNSCC risk except Ile/Val of GSTP1 gene with tobacco chewers, but insignificant with smokers and reverse smokers. However, our results were also similar to Jourenkova-Mironova et al., McWilliams et al., and Peters et al., where there is a relationship between GSTP1 polymorphisms and smoking status in HNSCC[19],[22],[34] and the results are not consistent. In this study, there were no differences in the risk for HNC in cases who were smokers or nonsmokers with variant genotypes of GSTP1. By identifying and characterizing polymorphisms of genes involved in carcinogen activation, as well as gene–environment interactions, ultimately, the information can be used to support lifestyle modifications in genetically predisposed high-risk individuals.[21]

The control population in this study was a convenience sample, and cases and controls were not balanced for other nongenetic risk factors known to be important in HNC risk. For this reason, the study preferred a logistic regression analysis of data to control for the effect of known risk factors. For analysis, the study included gender, age, smoking, and family history, all of which have been previously associated with HNC. The results of the binary logistic regression analysis showed that the full model which considered all the five independent variables such as gender, age, smoking usage, familial history, and GSTP1 genotype together was statistically significant, degree of freedom (DF) = 5, P = 0.000, except age. This implies that the odds ratio of HNSCC indicates that tobacco usage increases HNSCC risk [Table 2].


This study did not find any association of GSTP1 genotyping alone or in combination with tobacco exposure in risk of HNSCC cases when compared with controls which might be because of convenience sampling of the control population. Thus, binary logistic regression was adopted to jointly examine the influence of gender, age, tobacco usage, familial history, and GSTP1 genotype in cases alone. The key finding is that the selected variables are considered important as it correlates with the tobacco usage with HNSCC. Hence, this study concludes that tobacco usage with GSTP1 genotype as one of the strongest predictors for HNSCC.


We would like to thank the Department of Human Genetics, Andhra University, Visakhapatnam, and School of Biotechnology, Mahatma Gandhi National Institute of Research and Social Action, Hyderabad, for laboratory facilities and King George Hospital, Visakhapatnam, for study participant selection and enrollment.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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