GSTM1 and GSTT1 genes analysis in head and neck cancer patients

Abstracts

OBJECTIVE: To establish the clinical and demographic profile and identify risk factors among patients with head and neck cancer and relate them to the polymorphism of GSTT1 and GSTM1. METHODS: One hundred patients with head and neck cancer and 100 control group individuals without history of neoplasm were analyzed. . The molecular analysis were made by multiplex polymerase chain reaction. For statistical analysis, data were tabulated and compared by the Fisher’s exact test, the Chi-square test and multiple logistic regression were also used. RESULTS: There was prevalence of smokers (OR = 5.32, CI 95% CI = 2.04-13.86 p = 0.0006), alcohol drinkers (OR = 5.04, CI 95% = 2.19-11.59 p = 0.0001) in head and neck cancer patients . The GSTT1 null genotype was found in 47% of the patient and 41% of the control group (OR = 0.67; CI 95%= 0.34-1.35; p = 0.2648). Likewise , the GSTM1 null genotype was found in 66% of the patient and 75% of the control group (OR = 2.25; CI 95%= 1.05 - 4.84; p = 0.0368). The combined GSTT1 and GSTM1 gene null genotype shown association between GSTM1*0/GSTT1 and occurrence of head and neck carcinoma (OR = 7.64; CI 95%= 1.72-34.04; p = 0.0076). Analysis of clinical-pathological features showed association between GSTT1 null genotype and larynx, the inverse relation between this genotype and pharynx. CONCLUSION: In our study it was possible to establish association between GSTM1 null genotypes and head and neck cancer.

Head and neck; Neoplasms; Tobacco; Glutathione transferase; Alcohol consumption

OBJETIVO: Estabelecer o perfil clínico e demográfico bem como identificar os fatores de risco entre os pacientes com câncer de cabeça e pescoço e relacioná-los ao polimorfismo dos genes GSTT1 e GSTM1. MÉTODOS: Foram estudados 100 pacientes com carcinoma espinocelular de cabeça e pescoço e 100 indivíduos sem história de neoplasia. A análise molecular foi realizada pela técnica de PCR multiplex. Para a análise estatística, os dados foram tabulados e comparados pelo teste exato de Fisher. Foi também utilizado o teste do Qui quadrado e de regressão logística múltipla. RESULTADOS: Há predomínio de indivíduos tabagistas (OR= 5,32; IC95%= 2,04-13,86; p=0,0006), etilistas (OR= 5,04; IC95%= 2,19-11,59; p= 0,0001) em pacientes com neoplasia de cabeça e pescoço. Foi identificado genótipo nulo do gene GSTT1 em 47% dos pacientes e 41% dos controles (OR= 0,67; IC 95%= 0,34-1,35; p= 0,2648). Da mesma forma, identificou-se o genótipo nulo do gene GSTM1 em 66% dos pacientes e 75% dos controles (OR= 2,25; IC95%= 1,05-4,84; p= 0,0368). A análise dos genótipos combinados demonstrou associação entre GSTM1*0/GSTT1 e a ocorrência de carcinoma de cabeça e pescoço (OR= 7,64; IC 95%= 1,72-34,04; p= 0,0076). A análise dos parâmetros clínicos mostrou que é possível identificar associação entre genótipo GSTT1 nulo e neoplasia na laringe, o inverso ocorrendo com este genótipo e a faringe. CONCLUSÃO: Em nosso estudo foi possível estabelecer a associação entre a nulidade do genótipo GSTM1 e dos genótipos combinados GSTT1/GSTM1 *0 ([ ] / [-] a ocorrência de câncer de cabeça e pescoço.

Neoplasias de cabeça e pescoço; Glutationa transferase; Tabaco; Bebidas alcoólicas

ORIGINAL ARTICLE

GSTM1 and GSTT1 genes analysis in head and neck cancer patients

Cássia Veridiana Dourado Leme^I; Luis Sérgio Raposo^II; Mariangela Torreglosa Ruiz^III; Joice Matos Biselli^IV; Ana Lívia Silva Galbiatti^V; José Victor Maniglia^VI; Érika Cristina Pavarino-Bertelli^VII; Eny Maria Goloni-BertolloVIII, ^*

^IGraduanda em Medicina - Faculdade de Medicina de São José do Rio Preto – FAMERP, São José do Rio Preto, SP

^IIMestre em Ciências da Saúde e professor do departamento de Otorrinolaringologia e Cirurgia de Cabeça e Pescoço pela Faculdade de Medicina de São José do Rio Preto – FAMERP, São José do Rio Preto, SP

^IIIDoutora em Ciências da Saúde pela Faculdade de Medicina de São José do Rio Preto – FAMERP, São José do Rio Preto, SP

^IVMestre e doutoranda em Ciências da Saúde pela Faculdade de Medicina de São José do Rio Preto – FAMERP, São José do Rio Preto, SP

^VGraduada em Ciências Biológicas e mestranda em Ciências da Saúde pela Faculdade de Medicina de São José do Rio Preto – FAMERP, São José do Rio Preto, SP

^VIProfessor livre-docente em Otorrinolaringologia e Cirurgia de Cabeça e Pescoço pela Faculdade de Medicina de São José do Rio Preto – FAMERP, São José do Rio Preto, SP

^VIIProfessora livre-docente em Genética Humana e médica pela Faculdade de Medicina de São José do Rio Preto – FAMERP, São José do Rio Preto, SP

^VIIILivre Docente em Genética Humana e Médica - Professora do Departamento de Biologia Molecular da Faculdade de Medicina de São José do Rio Preto – FAMERP, São José do Rio Preto, SP

ABSTRACT

OBJECTIVE: To establish the clinical and demographic profile and identify risk factors among patients with head and neck cancer, relating them to the polymorphism of GSTT1 and GSTM1.

METHODS: One hundred patients with head and neck cancer and 100 control group individuals without history of neoplasm were analyzed. The molecular analysis was made by multiplex polymerase chain reaction. For statistical analysis, data were tabulated and compared by the Fisher's exact test, the Chi-square test and multiple logistic regression were also used.

RESULTS: There was prevalence of smokers (OR = 5.32, CI 95% CI = 2.04-13.86 p = 0.0006), alcohol drinkers (OR = 5.04, CI 95% = 2.19-11.59 p = 0.0001) in head and neck cancer patients. The GSTT1 null genotype was found in 47% of the patient and 41% of the control group (OR = 0.67; CI 95%= 0.34-1.35; p = 0.2648). Likewise, the GSTM1 null genotype was found in 66% of the patient and 75% of the control group (OR = 2.25; CI 95%= 1.05 - 4.84; p = 0.0368). The combined GSTT1 and GSTM1 gene null genotype shown association between GSTM1*0/GSTT1 and occurrence of head and neck carcinoma (OR = 7.64; CI 95%= 1.72-34.04; p = 0.0076). Analysis of clinical-pathological features showed association between GSTT1 null genotype and larynx, the inverse relation between this genotype and pharynx.

CONCLUSION: In our study it was possible to establish an association between the nullity of GSTM1 genotype and that of combined GSTT1/GSTM1*O ([]/[-] genotypes and head and neck cancer.

Key words: Head and neck neoplasias. Glutathione transferase. Tobacco. Alcoholic drinks.

INTRODUCTION

Head and neck cancer (HNC) is a term that designates malignant neoplasias occurring in upper aerodigestive tract, that is, deriving from the oral cavity epithelium, pharynx, larynx, whose incidence of occurrence correspond, respectively, to 40%, 15% and 25%.^1-3. The most common histological type is the squamous cells or spinocellular carcinoma occurring in 90% of the patients.^3,4 This type of cancer is the fifth more common type in the world and is associated with low life expectancy rates and high morbidity rates when diagnosed in advanced stages.³

In Brazil, according to the Instituto Nacional do Câncer (National Institute of Cancer - INCA), the estimation for oral cavity cancer for the year 2010 is 13,250 new cases for male sex and 4,880 in women, in a total amount of 18,130 new cases.⁵.

Various factors are related with the development of head and neck cancer, but smoking and alcohol drinking are considered independent risk factors.^2,3,6,7 When associated, they have a synergistic effect, increasing the risk of developing cancer in this anatomical region in even 15 times.^3,8,9 There is a higher incidence among male individuals, but an increase in its occurrence among women is evidenced due to the increase in alcohol and tobacco consumption.³

Tobacco is constituted by carcinogenic agents, with N-nitrosamines and aromatic amines, which, interacting with genetic material, may result in the formation of DNA adducts, which favor cellular mutations and reactive hyperplasia in the mucosa of upper aerodigestive tract. Alcohol is not genotoxic, but it might act in synergy with tobacco, suppressing the removal of nitrosamines in inhibiting the various isoforms of the cytochrome P450 superfamily (CYPs), enzymes involved in the cellular detoxification process, increasing adducts formation.^9-11.

Two groups of enzymes are involved in the process of biometabolization of chemical compounds of the tobacco and alcohol: the enzymes of oxidative metabolism (Phase I) and conjugating enzymes (Phase II). Phase I oxidative enzymes, mainly the families pertaining to the cytochrome P-450 superfamily (CYPs), convert many compounds into highly reactive metabolites. On the other hand, Phase II enzymes act deactivating Phase I products, making metabolites hydrophilic and liable to excretion as a result of their conjugation with the endogenous substrate (glutathione, sulphate, glucose, acetate) by means of the action of glutathione S-transferases (GSTs), UDP-glucuronyl transferases and N-acetyltransferases.(NATs) ^12-17.

The glutathione S-transferases (GSTs) are an important family of enzymes involved in the biosynthesis and metabolism of many substances, including detoxification of exogenous chemical carcinogens, such as aromatic polycyclic hydrocarbons present in the tobacco. They comprise four classes of genes (α, μ, π, and θ) and each class, on their turn, include various genes.¹⁸ Polymorphisms in genes that codify the GSTM1 and GSTT1 may alter their expression or function and result in activation or detoxification of carcinogenic compounds.^12-16.

The GSTM1 gene (1p13.1chromosome) is polymorphic and 20% to 50% of the individuals did not express the enzyme due to a genic deletion in homozygosis (GSTM1*0) ¹⁹; this frequency is higher in Caucasoids and Asians than in Africans.²⁰ The GSTT1 gene (22q11.2 chromosome) is equally polymorphic, presenting null genotype by deletion (GSTT1*0) and consequent complete loss of enzymatic activity. The frequency of individuals who do not express the enzyme is higher in Asians (60%) and Africans (40%) than in Caucasoids (20%).^{21, 22}

The polymorphisms of the GSTM1 and GSTT1 genes involved may result in differences in the enzymatic activity, possibly favoring mechanisms that increase the susceptibility to cancer. Studies relating these polymorphisms of deletion with the occurrence of head and neck carcinoma diverge between themselves: some demonstrate the association of these neoplasias with the null genotype of GSTM1,^{7, 21, 23-25} while others do not.^26-30 The same occurs with the null genotype of GSTT1, showing an association with the disease^7,23,25 or not.^26,27,30-32 This way, the development of head and neck cancer is the result of the interaction between environmental factors and genetic heritage.^24,33

Therefore, the objectives of this work were to establish the clinical, sociodemographic profile and identify risk factors (smoking and alcohol drinking) of head and neck cancer patients.

METHODS

This study was approved by the Ethics Committee of the teaching institution SISNEP-0976.0.140.000-05. Participants were included after the obtention of the Informed Consent Form and submitted to a standardized questionnaire, information is maintained under secrecy.

In this case-control study individuals who did not present a family history of cancer and did not possess a personal background of cancer were evaluated. Thus, 100 patients were evaluated in out-patient follow up in the Otorhinolaryngology and Head and Neck Surgery service in a university hospital in the North-West of the São Paulo State, who received the histopathological diagnosis of head and neck spinocellular carcinoma, regardless of clinical staging. All the samples of peripheral blood were collected before the beginning of the treatment. The control group includes 100 individuals from other services offered by the same university hospital.

Varieties analyzed were age, gender, and exposition to risk factors (smoking and alcohol). Individuals who consumed more than 100 cigarettes (commercial or handmade) for the whole life were classified as smokers, and the patients who drank more than four alcoholic drinks (distilled or fermented) per week during six months or more, according to Ahrendt et al., 2000.³⁴

Clinical data referring to the primary site of the tumor and clinical staging were classified according to the Union Internationale Contrele Cancer (UICC), 2002 and the American Joint Commitee for Cancer (AJCC), 1997.³⁵

The genomic DNA was obtained through peripheral blood and the amplification by PCR performed, according to Miller et al.³⁶ The product of PCR was submitted to electrophoresis in 1.5% agarose gel, colored with ethidium bromide, in which GSTT1 is observed as a fragment of 480 base pairs (bp), GSTM1 with 219 bp and the CYP1A1 with 312 pb. A sequence of the exon 7 of the CYP1A1 gene was used as internal control of amplification.

Demographic data and those pertaining to genotypic distribution of the polymorphisms were tabled and compared by the Fisher's exact test and the Chi square test. The model of logistic regression was used to determine the effect of the dependent variables in the head and neck cancer and to group the clinical-pathological characteristics as dependent variables. The results presented in odds ratio (OR) and 95% confidence interval (IC - 95%). The level of significance was established in 5% (p=0.05).

RESULTS

Data pertaining to the sociodemographic profile (gender and identity) and exposition to risk factors (smoking and alcohol drinking habits) were analyzed. In this study male individuals were dominant in the group of patients, with mean age of 58.46 years; the control group is represented by 68% of male individuals and mean age of 55.32 years. Age median is equal to 56 years (Table 1).

The two main habits associated to HNC, smoking and alcohol drinking, were both expressively more frequent in the group of individuals with neoplasia (Table 1). The frequency of genetic polymorphisms are equally presented in Table 1.

Data of the multivariate analysis model (Table 1) show that smoking [OR: 5.32; IC 95% (2.04-13.86); p=0.0006], and drinking [OR: 5.04; IC 95% (2.19-11.59); p=0.0001] and the null genotype GSTM1 [GSTM1*0] [OR: 2.25; IC 95% (1.05-4.84); p=0.0368] are predicting factors for head and neck cancer.

Table 2 presents the results of the combined genotypes analysis, in which GSTT1/GSTM1*0 ([ ] / [-]) presents an increase for the risk of head and neck cancer [OR= 7.64; IC 95% (1.72-34.04); p= 0.0076].

In relation to the localization of the neoplasia, primary anatomical sites occurred with the frequency of 35% in the oral cavity, 26% in the pharynx, 36% localized in the larynx, and in 3% it was possible to identify the primary site of the tumor.

The analysis of the clinical parameters and polymorphisms showed association between the GSTT1 null genotype with its occurrence in the larynx [OR= 5.33; IC 95% (1.99-14.36); p=0.0009] and inverse relation in the pharynx [OR= 0.32; IC 95% (0.12-0.89); p=0.0282]. For the other anatomic sites studied, besides the tumor extension (parameter T), lymphonodal commitment (parameter N) and occurrence of metastases (parameter M), no associations could be identified.

DISCUSSION

This study confirms the fact that the development of head and neck cancer is related to some habits, as smoking and alcohol drinking. Epidemiological studies show that the exposition to tobacco and alcohol are the strongest risk factors for the disease.^18.37

In this study we have evidenced an increased frequency of the GSTM1 null genotype in patients with head and neck cancer, when compared to a group of individuals with no history of neoplasia. Results of meta-analysis demonstrate the association of the GSTM1 null genotype and the increase of risk for the development of head and neck cancer.^38-41

In a multivariate logistic analysis, we have evidenced that the GSTT1/GSTM1*0 ([ ]/ [-]) combined genotype provides a greater susceptibility for the development of spinocellular carcinoma of head and neck, similar results to those found by other authors. ^7,23,42,43

Various studies could not correlate the null GSTT1 genotype with the head and neck cancer,^26,27,43 which was a result found in our study. Similarly, there was no association between this type of neoplasia and the null combined genotypes null GSTT1 and null GSTM1, as observed by Singh et al.⁷ On the other hand, a Chinese study has shown a correlation between the double null genotype GSTT1 and GSTM1 with nasopharynx cancer among men.⁴⁴

With respect to the relation between genes deletion and specific anatomic sites, Duarte et al.²⁵ found an association between the GSTM1 null genotype and the increase in risk of development of leukoplasia in the oral cavity. On the other hand, in our study we have identified a relation between the occurrence of larynx carcinoma with GSTT1 null genotype and, on the contrary, a protective effect of this genotype in the pharynx. These data are due to embryological, histological, and molecular differences between anatomic regions, resulting from then the peculiar biologic behavior of this type of tumor in the various anatomic localizations. ^45,46

Histopathological parameters with tumor extension, invasion of lymphonodes and metastases were evaluated, but they remain controversial in the literature, because there is a relation in some studies, such as Mathias et al.⁴⁷ who revealed the association between GSTT1 null genotype with the occurrence of T3/T4 tumors and the absence of lymphonodes; while Losi-Guembarovski et al.³² did not find a statistically significant association, which was what occurred in our study.

CONCLUSION

In our study we were able to establish, by means of multivariate logistic analysis, the association of male gender, advanced age, smoking and alcohol habits with the occurrence of head and neck cancer, as well as the GSTM1 genotype's nullity; there was a higher occurrence in oral cavity and association between the occurrence of this tumor in the larynx with the null genotype.

Financial support: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Centro Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

No conflicts of interest declared concerning the publication of this article.

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Artigo recebido: 07/07/09

Aceito para publicação: 22/02/10

Research conducted at the Faculdade de Medicina de São José do Rio Preto - FAMERP, São José do Rio Preto, SP, Brazil

*

Correspondence: Av. Brigadeiro Faria Lima, 5416 - Vila São José São José do Rio Preto - SP, Brazil CEP: 15090-000

Publication Dates

• Publication in this collection
  05 Aug 2010
• Date of issue
  2010

History

• Received
  07 July 2009
• Accepted
  22 Feb 2010