Influence of p53 codon 72 exon 4, GSTM1, GSTT1 and GSTP1*B polymorphisms in lung cancer risk in a Brazilian population

Lung Cancer (2008) 61, 152—162

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Influence of p53 codon 72 exon 4, GSTM1, GSTT1 and GSTP1*B polymorphisms in lung cancer risk in a Brazilian population Helen N. Honma a, Eduardo M. De Capitani a, Maur´ıcio W. Perroud Jr. a, Arist´ oteles S. Barbeiro a, Ivan F.C. Toro b, Daniel B. Costa c, Carmen S.P. Lima a, Lair Zambon a,∗ a

Department of Internal Medicine, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Rua Tess´ alia Vieira de Camargo, 126, Cidade Universit´ aria ‘‘Zeferino Vaz’’, Distrito de Bar˜ ao Geraldo, Campinas, S˜ ao Paulo, Brazil b Department of Thoracic Surgery, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Rua Tess´ alia Vieira de Camargo, 126, Cidade Universit´ aria ‘‘Zeferino Vaz’’, Distrito de Bar˜ ao Geraldo, Campinas, S˜ ao Paulo, Brazil c Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Q1 330 Brookline Avenue, Rabb 430, Boston, MA 02215, United States Received 29 June 2007; received in revised form 10 December 2007; accepted 18 December 2007

KEYWORDS Carcinogenesis; GSTM1; GSTP1; GSTT1; Lung cancer;

Summary Purpose: Glutathione S-transferases (GST) modulates the effects of various cytotoxic and genotoxic agents, particularly those derived from benzo[a]pyrene, which is one of the main tobacco carcinogens. Both the mu 1 (GSTM1) and theta 1 (GSTT1) genes have a null variant allele in which the entire gene is absent. The GSTP1*B allele has an A to G transition at nucleotide 313 (codon 105) in exon 5, causing a change of isoleucine (Ile) to valine (Val), which affects the electrophile binding site of GSTP1 and results in an enzyme with reduced activity. Polymorphisms in these metabolizing enzymes may alter the response to benzo[a]pirene-induced DNA

Abbreviations: A, adenine; bp, base pairs; BPDE, benzo[a]pyrene diolepoxide; CI, confidence interval; dGMP, deoxyguanosine phosphate; DNA, deoxyribonucleic acid; dNTP, deoxynucleotides; EDTA, ethylene diamine tetracetic acid; G, guanine; GST, glutathione S-transferase; GSTM1, gene mu1 of glutathione S-transferase; GSTP1, gene pi 1 of glutathione S-transferase; GSTT1, gene theta 1 of glutathione S-transferase; HCl, acid chlorhydrique; I, isoleucine; Ile, isoleucine; KCl, potassium chloride; LC, lung cancer; MgCl2 , magnesium chloride; mM, millimole; ng, nanogram; ␮l, microliter; NSCLC, non-small cell lung cancer; OR, odds ratio; PCR, polymerase chain reaction; pmol, picomole; RFLP, restriction fragment length polymorphism; SCLC, small cell lung cancer; TNM, tumor, nodes and metastasis; U, unit; Val, valine; VV, valine/valine. ∗ Corresponding author at: Clinical Pulmonary Service, Department of Internal Medicine, Faculty of Medical Sciences, State University of Campinas, Rua Tess´ alia Vieira de Camargo, 126, Cidade Universit´ aria ‘‘Zeferino Vaz’’, Distrito de Bar˜ ao Geraldo, Campinas, CEP 13083-970 S˜ ao Paulo, Brazil. Tel.: +55 19 35217907; fax: +55 19 35217907. E-mail address: [email protected] (L. Zambon). 0169-5002/$ — see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2007.12.014

Influence of p53 codon 72 exon 4, GSTM1, GSTT1 and GSTP1*B polymorphisms Metabolism p53 codon 72 polymorphism; Polymorphism; Susceptibility; Xenobiotics

153

damage. Polymorphisms in p53 may also modulate the risk of lung cancer (LC) carcinogenesis. The aim of our study was to measure the frequency of GSTM1, GSTT1, GSTP1*B and p53 gene polymorphisms in a Brazilian population and determine the possible contribution of these genetic variations to LC risk. Patients and methods: Genomic DNA was obtained from 200 Brazilian patients with LC and 264 blood donors (control group). All samples were analyzed by PCR and PCR-RFLP to determine GSTM1, GSTT1, GSTP1*B and p53 codon 72 genotypes. Multiple logistic regressions were used to adjust for confounding factors in this case—control study. Results: No statistical significance was observed between GSTM1, GSTT1 and GSTP1*B genetic polymorphisms, either isolated or combined, with LC incidence in the studied population. However, our data showed a higher frequency of p53 codon 72 A/P plus P/P genotype in AfricanBrazilian than Caucasian-Brazilian patients with LC, and we also found a higher frequency of the P/P genotype of the p53 gene in non-smokers compared to smokers with LC. Conclusions: Genetic polymorphisms of GST and p53 codon 72 did not increase the risk of LC in Brazilian patients. The A/P plus P/P genotype of p53 codon 72 is more common in LC patients with African ethnical background and the P/P genotype more prevalent in non-smoking related LC. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Tobacco smoke is the main risk factor for lung cancer (LC) development. However, not all populations are equally susceptible to tobacco-related carcinogens [1]. Thus, the identification of genes responsible for lung carcinogenesis susceptibility may allow us to perform screening programs and chemoprevention trials in subgroups of heavy smokers. The glutathione S-transferases (GSTs), forming a family of enzymes catalyzing the detoxification of wide range of eletrophilic substrates, play a significant role in phase II biotransformation of xenobiotics. This detoxification is achieved by the conjugation of xenobiotics with glutathione, which facilitates the neutralization of their eletrophilic centre. Conjugated xenobiotics can be eliminated by urine or bile either directly or by subsequent intermediate stages of transformation in which N-acetylases and transpeptidases take their places [2]. Inter-individual variability in Glutathione S-transferase (GST) enzyme activity can also influence the susceptibility to cancers, especially in those with environmental determinants, such as LC [3,4]. Some genetic variants in GST genes, such as the GSTM1 null polymorphism, are known to abolish enzymes activity. Individuals with GSTM1 null genotype have been reported to have higher levels of polycyclic aromatic hydrocarbon —dGMP adducts in lung tissues, which can induce genetic mutations [5]. The GSTT1 gene, located on chromosome 22q11.23, has also been found to have a deletion polymorphism that results in absence of enzymatic activity [6]. GSTT1 is involved in the metabolism of smaller compounds found in tobacco smoke, such as monohalomethane and ethylene oxide [7]. GSTP1 is known to metabolize many carcinogenic compounds, among them benzo[a]pyrene diolepoxide (BPDE), which is one of the most important carcinogenic metabolites derived from tobacco smoke [8]. Given that GSTP1 is the most abundant GST isoform in the lung [9], it is anticipated to be of particular importance in the detoxification of inhaled carcinogens. The GSTP1*B allele has an A to G transition at nucleotide 313 (codon 105) in exon 5, causing

a change of isoleucine (Ile) to valine (Val), and affecting the electrophile binding site of the GSTP1 enzyme. It has been reported to result in an enzyme with reduced activity and it is possible that deficient or reduced activity of this enzyme may result in an increased susceptibility to cancer [10]. The Val variant has generally lower activity towards polycyclic aromatic hydrocarbon diol epoxides, especially BPDE, and has been shown to have a lower detoxification potential leading to a greater risk of neoplasic transformation [11]. The p53 gene is located on chromosome 17p13 and encodes a 53 kDa protein which plays a critical role in cell growth control. Variant alleles of codon 72 in exon 4 encode either arginine [Arg-CGC] or proline [Pro-CCC]. Polymorphic variants differ in biochemical and biological properties. An association between the presence of the p53 Pro72 allele and an increased risk of LC development has been suggested [12—14]. However, further validation is required and the mechanism of this possible relationship is still not well understood. Ethnic differences in the frequency of Arg72 and Pro72 alleles warrant analysis of the potential clinical relevance of this polymorphism in various populations. Other studies have also shown that the Pro72 allele confers a worse prognosis in LC patients [15,16]. The aim of our study was to estimate and compare the frequency of GSTM1, GSTT1, GSTP1*B and p53 gene polymorphisms in a Brazilian population and determine the possible contribution of these genetic variations to LC risk.

2. Subjects and methods 2.1. Study subjects The study population included 200 patients with LC (144 men and 56 women; mean age ± S.D.: 64.0 ± 9.7 years) seen at the Pulmonary unit of School Hospital at the State University of Campinas (UNICAMP) from January 2004 to December 2006. The control group consisted of 264 blood donors (160 men and 104 women) selected in the same Hospital.

154 All the procedures were carried out according to the principles of the institutional guidelines and the study was approved by the local Ethics Committee and all the patients and controls provided with written informed consent. The diagnosis of LC was performed by histological evaluation of tumour biopsies and chest computed tomography scans. Clinical data and smoking history were collected from the patient’s chart.

2.2. Genotyping analyses Genomic DNA was obtained from peripheral blood samples (12 ml) collected in EDTA tubes. The DNA was extracted using DNAzol TM Reagent (Life Technologies) proteinase K and lithium chloride method [17].

2.3. Detection of GSTM1 and GSTT1 genes deletions by multiplex-PCR Genomic DNA from peripheral blood samples of LC patients and controls were analyzed using polymerase chain reaction (multiplex-PCR). GSTM1 and GSTT1 genes were amplified in the same reaction, including the amplification of a ␤-globin gene fragment used as a control of the DNA sample [18]. Multiplex-PCR was carried out in a mixture of 10 mM Tris—HCl, pH 8.4, 3 mM MgCl2 , 50 mM KCl, 0.4 mM of each nucleoside triphosphate, 500 ng of genomic DNA and 2 U Taq polymerase (Invitrogen-Life Technologies), using the following primers: sense (5 -CTGCCCTACTTGATTGATGGG3 ) and antisense (5 -CTGGATTGTAGCAGATCATGC-3 ), sense (5 -TTCCTTACTGGTCCTCACATCTC-3 ) and antisense (5 -TCACCGGATCATGGCCAGCA-3 ), and sense  (5 -ATACAATGTATCATGCCT-CTTTGCACC-3 ) and antisense (5 -GTATTTTCCCAAGGTTTGAACTAGCTC-3 ), for the amplifications of GSTM1 [19], GSTT1 [6] and ␤-globin gene, respectively. The reaction involved 35 cycles of incubation at 95 ◦ C (1 min), 62 ◦ C (1 min), and 72 ◦ C (1 min). GST genotypes were analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide and only if the PCR signal corresponding to the ␤-globin internal gene control was evident.

2.4. Determination of the GSTP1*B genotype Each PCR reaction mixture (50 ␮l) contained 200 ng of each primer sense (5 -ACC CCA GGG CTC TAT GGG AA-3 ) and antisense (5 -TGA GGG CAC AAG AAG CCC CT-3 ), 100 ng of genomic DNA, 1.5 mM MgCl2 , 100 mM each dNTP and 1 U Taq polymerase (Invitrogen-Life Technologies). The reaction involved 30 cycles of incubation at 94 ◦ C (30 s), 55 ◦ C (30 s), and 72 ◦ C (30 s). After the confirmation of an amplified fragment of the expected size (176 bp) on an agarose gel, the PCR products were digested with 5 U of restriction enzyme BsmAI (New England Biolabs, UK) at 55 ◦ C for one hour. DNA fragments were submitted to electrophoresis through a 3.5% agarose gel and stained with ethidium bromide.

H.N. Honma et al.

2.5. p53 codon 72 BstUI polymorphism Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of the codon 72 of the p53 gene originally described by Biros et al. [20] was used to identify p53 BstUI genotypes. The two primers were 5 -CAC CCA TCT ACA GTC CCC CTT GC-3 and 5 -CTC AGG GCA ACT GAC CGT GCA AG-3 . Each PCR reaction mixture (50 ␮l) contained 20 pmol of each primer, 2 mM MgCl2 , 100 mM each dNTP, 1 U Taq polymerase (Invitrogen-Life Technologies) and 100—300 ng of genomic DNA. The reaction involved 35 cycles of incubation at 94 ◦ C (1 min), 60 ◦ C (1 min), and 72 ◦ C (1 min). After the confirmation of an amplified fragment of the expected size (318 bp) on an agarose gel, the PCR products were digested with 3 U of restriction enzyme BstUI (New England Biolabs, UK) at 60 ◦ C for 16 h. DNA fragments were submitted to electrophoresis through a 3% agarose gel and stained with ethidium bromide.

2.6. Statistical analyses The Hardy—Weinberg equilibrium was tested with the 2 statistic for the goodness-to-fit (one degree of freedom). Statistical differences between groups were calculated by the 2 or Fischer exact test. Conditional analysis was used to obtain race, age and gender-adjusted crude odds ratios (OR). All analyses were performed using the statistical package SAS System for Windows, version 8.2 of the SAS Institute Incorporation, USA.

Table 1 Age, gender, ethnic, tobacco, histology and stage characteristics of the patients and control groups Patients (n = 200)

Controls (n = 264)

p-Value

Agea <64 years ≥64 years

100 (50.0) 100 (50.0)

250 (94.7) 14 (5.3)

<0.0001b

Gender Male Female

144 (72.0) 56 (28.0)

160 (60.6) 104 (39.4)

0.0106b

Ethnic Caucasian African-Brazilian

158 (79.0) 42 (21.0)

215 (81.4) 49 (18.6)

0.5122

Tobacco Yes No

181 (90.5) 19 (9.5)

86 (32.6) 178 (67.4)

<0.0001b

Histology NSCLC SCLC

168 (84.0) 32 (16.0)

Stage (TNM) I + II III + IV

68 (34.0) 132 (66.0)

All the values given in brackets are in percentage. a Mean (range). b 2 .

Influence of p53 codon 72 exon 4, GSTM1, GSTT1 and GSTP1*B polymorphisms

3. Results DNA samples from the control population (healthy Brazilian blood donors) were in Hardy—Weinberg equilibrium for polymorphism GSTP1*B and p53 (2 = 1.05, p = 0.64; 2 = 2.15, p = 0.66; respectively) and the LC patients were also in Hardy—Weinberg equilibrium (2 = 0.030, p = 0.64; 2 = 0.144, p = 0.68; respectively). As only persons under 60 years old are allowed to donate blood in our service, LC patients showed to be older than controls (age ≥64; 50.0% versus 5.3%; p < 0.0001). Males were over-represented compare to females (72.0% versus 28.0%; p = 0.0106) in our cohort of LC. The frequency of smokers was higher in patients with LC than in controls (90.5% versus 32.6%; p < 0.0001). NSCLC was the most frequent histological alteration found (84.0%) and the most frequent stages were III plus IV (66.0%) (Table 1). The distribution of GSTM1, GSTT1, GSTP1*B and p53 polymorphisms were not significantly different between LC patients and our controls (p = 0.57, p = 0.84, p = 0.86 and p = 0.62; respectively). There were no statistically significant association between genetic polymorphisms in GSTM1, GSTT1, GSTT1*B and p53, either alone or in combination,

Table 2

155

between our patients with LC and our controls (Tables 2—4 ). Also, these polymorphisms do not seem to be associated with age, gender, stage and histology in LC cohort (Tables 5—7). However, we observed a higher frequency in genotype A/P plus P/P of the p53 gene (p = 0.010) in the African-Brazilian when compared to Caucasians with LC (Table 5). When we generated associations with other genes we also observed the following: GSTM1 present (p = 0.014) and GSTP1 Val/Val plus Ile/Val (p = 0.025) were more frequent in association with the A/P plus P/P p53 polymorphism. These associations were not observed when the patients with LC and the AfricanBrazilian control group were compared (71.4% versus 73.5%; 47.6% versus 42.9% and 50.0% versus 44.8%; respectively). We found a higher frequency of genotype P/P of the p53 gene in non-smokers compared to smokers with LC (p = 0.028) (Table 7). This difference did not reach statistical significance when we compared our non-smoker controls with our non-smoker LC group (p = 0.06), however there continued to be a strong trend towards a higher frequency of the P/P genotype of p53 in non-smoker LC patients (26.3% versus 10.7%).

The frequencies of the GSTM1, GSTT1 and p53 genotypes in 200 LC patients and 264 controls

Genotypes

Patients (n (%))

Controls (n (%))

OR* (95% CI)

p-Value

GST M1 null M1 present T1 null T1 present M1/T1 null M1/T1 present

91 (45.5) 109 (54.5) 27 (13.5) 173 (86.5) 12 (6.0) 94 (47.0)

127 (48.1) 137 (51.9) 34 (12.9) 230 (87.1) 13 (4.9) 116 (43.9)

0.69 (0.42—1.13) 1.0 (reference) 1.26 (0.61—2.61) 1.0 (reference) 0.79 (0.27—2.30) 1.0 (reference)

0.14

p53 PP AP PP plus AP AA AP plus AA PP

19 (9.5) 89 (44.5) 108 (54.0) 92 (46.0) 181 (90.5) 19 (9.5)

25 (9.5) 129 (48.9) 154 (58.3) 110 (41.7) 239 (90.6) 25 (9.5)

1.35 (0.54—3.41) 0.78 (0.47—1.32) 0.85 (0.52—1.40) 1.0 (reference) 0.99 (0.69—1.44) 1.0 (reference)

0.35 0.18 0.52

11 (5.5) 39 (19.5) 50 (25.0) 51 (25.5) 1 (0.5) 10 (5.0) 11 (5.5) 76 (38.0) 0 4 (2.0) 4 (2.0) 43 (21.5)

11 (4.2) 60 (22.7) 71 (26.9) 54 (20.5) 5 (1.9) 14 (5.3) 19 (7.2) 95 (36.0) 4 (1.5) 5 (1.9) 9 (3.4) 43 (16.3)

0.47 (0.21—1.06) 1.10 (0.28—4.40) 0.54 (0.26—1.13) 1.0 (reference) 0.77 (0.04—15.26) 1.15 (0.36—3.73) 1.07 (0.35—3.26) 1.0 (reference) 0 0.37 (0.07—2.01) 0.31 (0.06—1.56) 1.0 (reference)

0.48 0.08 0.82

GST plus p53 M1 null and PP M1 null and AP M1 null and PP plus AP M1 present and AA T1 null and PP T1 null and AP T1 null and PP plus AP T1 present and AA M1/T1 null and PP M1/T1 null and AP M1/T1 null and PP plus AP M1/T1 present and AA

0.54 0.83

1.00

0.83 0.77 0.94 0 0.25 0.44

OR* , Adjusted odds ratio by age, gender, ethnic and tobacco; PP and AP, p53 variant homozygous and heterozygous genotypes, respectively; AA, p53 wild genotype.

156 Table 3

H.N. Honma et al. The frequencies of the GSTM1, GSTT1 and GSTP1*B genotypes in 200 LC patients and 264 controls

Genotypes

Patients (n (%))

Controls (n (%))

OR* (95% CI)

p-Value

GSTs M1 null M1 present T1 null T1 present M1/T1 null M1/T1 present

91 (45.5) 109 (54.5) 27 (13.5) 173 (86.5) 12 (6.0) 19 (9.5)

127 (48.1) 137 (51.9) 34 (12.9) 230 (87.1) 13 (4.9) 25 (9.5)

0.69 (0.42—1.13) 1.00 (reference) 1.26 (0.61—2.61) 1.0 (reference) 0.79 (0.27—2.30) 1.0 (reference)

0.14

GSTP1*B Val/Val Ile/Val Val/Val plus Ile/Val Ile/Ile Ile/Ile plus Ile/Val Val/Val

25 (12.5) 93 (46.5) 118 (59.0) 82 (41.0) 175 (87.5) 25 (12.5)

30 (11.4) 129 (48.9) 159 (60.2) 105 (39.8) 234 (88.6) 30 (11.4)

1.03 (0.46—2.30) 0.93 (0.55—1.57) 0.95 (0.58—1.56) 1.0 (reference) 0.94 (0.69—1.28) 1.0 (reference)

0.87 0.75 0.83

12 (6.0) 44 (22.0) 56 (28.0) 79 (39.5) 47 (23.5) 4 (2.0) 12 (6.0) 16 (8.0) 23 (11.5) 71 (35.5) 1 (0.5) 7 (3.5) 8 (4.0) 11 (5.5) 40 (20.0)

11 (4.2) 61 (23.1) 72 (27.3) 116 (43.9) 50 (18.9) 7 (2.7) 15 (5.7) 22 (8.3) 27 (10.2) 93 (35.2) 3 (1.1) 4 (1.5) 7 (2.7) 10 (3.8) 44 (16.7)

1.15 (0.30—4.38) 0.52 (0.23—1.17) 0.63 (0.31—1.29) 0.52 (0.27—1.01) 1.0 (reference) 0.72 (0.14—3.74) 1.29 (0.40—4.18) 0.78 (0.52—1.18) 1.51 (0.52—3.67) 1.0 (reference) 0.60 (0.04—9.29) 1.02 (0.17—6.10) 0.86 (0.21—3.61) 0.76 (0.20—2.82) 1.0 (reference)

0.47 0.11 0.65 0.06

GST plus GSTP1*B M1 null and Val/Val M1 null and Ile/Val M1 null and Val/Val plus Ile/Val M1 null and Ile/Ile plus Ile/Val M1 present and Ile/Ile T1 null and Val/Val T1 null and Ile/Val T1 null and Val/Val plus Ile/Val T1 null and Ile/Ile plus Ile/Val T1 present and Ile/Ile M1/T1 null and Val/Val M1/T1 null and Ile/Val M1/T1 null and Val/Val + Ile/Val M1/T1 null and Ile/Ile + Ile/Val M1/T1 present and Ile/Ile

0.54 0.83

0.77

0.60 0.55 0.27 0.36 0.71 0.80 0.82 0.68

OR* , Adjusted odds ratio by age, gender, ethnic and tobacco; Val/Val and Ile/Val, GSTP1 variant homozygous and heterozygous genotypes, respectively; Ile/Ile, GSTP1 wild genotype.

4. Discussion In this study we have investigated if the presence of genetic polymorphisms, isolated or combined, increases LC risk, and if they are correlated with age, gender, ethnic group, stage, histopathologic diagnosis and history of tobacco exposure. We found 45.5% of homozygosis for the GSTM1 deletion in patients with LC and 48.1% in our control group. This frequency was similar to that seen in three other studies that analyzed GSTM1 polymorphisms in Brazilians [21—23]. Hosgood et al. [24] studied the presence of GSTM1, GSTT1 and GSTP1 genotypes and their influence in susceptibility to LC in an Asian population. They found that only the GSTM1 null genotype was associated with a statistically significant increased risk of LC in subjects exposed to coal smoke (p = 0.0003). Such association might be due to the habit of using coal for cooking at home, apart from tobacco smoke. Taioli et al. [25], Sorensen et al. [26], and Sreeja et al. [27] found a statistically significant increase in LC risk in

subjects with the GSTT1 null genotype, although other studies have not found the same results [28—31]. Our study was also unable to identify an association between GSTT1 and LC. We observed that 13.5% of the studied Brazilian population was homozygous for the gene deletion GSTT1 and we did not find any correlation with LC. Nelson et al. [32] reported that the null genotype of GSTT1 was present in 64% of Chinese’s, 60% of Koreans, 28% of Caucasians and in 22% of African-Americans. This null genotype is more common in the Asians than Caucasians, strengthening the idea that polymorphisms in enzymes that metabolize tobacco carcinogens have a strong ethnical link. Besides ethnic factors, environment factors should also be considered in populational studies of LC risk, since not only tobacco smoke but exposures to other fumes may affect the overall cancer risk, such as that conferred by the habit of using a wood stove for home cooking [33—36]. Miller et al. [37] in a North American population showed an association between polymorphic GSTP1 gene and LC. The patient’ single nucleotide polymorphism (SNP) frequencies were 46, 42 and 12% for genotypes Ile/Ile, Ile/Val,

Influence of p53 codon 72 exon 4, GSTM1, GSTT1 and GSTP1*B polymorphisms Table 4

157

The frequencies of the GSTP1*B and p53 genotypes in 200 LC patients and 264 controls

Genotypes

Patients (n (%))

Controls (n (%))

OR* (95% CI)

p-Value

GSTP1 Val/Val Ile/Val Val/Val plus Ile/Val Ile/Ile Ile/Ile plus Ile/Val Val/Val

25 (12.5) 93 (46.5) 118 (59.0) 82 (41.0) 175 (87.5) 25 (12.5)

30 (11.4) 129 (48.9) 159 (60.2) 105 (39.8) 234 (88.6) 30 (11.4)

1.03 (0.46—2.30) 0.93 (0.55—1.57) 0.95 (0.58—1.56) 1.0 (reference) 0.94 (0.44—2.00) 1.0 (reference)

0.87 0.75 0.83

p53 PP AP PP plus AP AA AP plus AA PP

19 (9.5) 89 (44.5) 108 (54.0) 92 (46.0) 181 (90.5) 19 (9.5)

25 (9.5) 129 (48.9) 154 (58.3) 110 (41.7) 239 (90.6) 25 (9.5)

1.35 (0.54—3.41) 0.78 (0.47—1.32) 0.85 (0.52—1.40) 1.0 (reference) 0.65 (0.27—1.57) 1.0 (reference)

0.35 0.18 0.52

0 11 (5.5) 62 (31.0) 56 (28.0) 36 (18.0) 46 (23.0) 78 (39.0) 97 (48.5) 14 (7.0)

3 (1.1) 15 (5.7) 97 (36.7) 62 (23.5) 48 (18.2) 57 (21.6) 98 (37.1) 136 (51.5) 12 (4.5)

0 1.20 (0.37—3.88) 0.85 (0.42—1.71) 1.21 (0.58—2.50) 1.0 (reference) 1.09 (0.52—2.28) 0.93 (0.32—2.67) 0.90 (0.29—2.82) 1.0 (reference)

0 0.76 0.34 0.46

GSTP1 plus p53 Val/Val and PP Val/Val and AP Ile/Va plus Val/Val and PP plus AP Ile/Val plus Val/Val and AA Ile/Ile and AA Ile/Ile and AP plus PP Ile/Ile plus Ile/Val and AA Ile/Ile plus Ile/Val and PP plus AP Val/Val and AA

0.86

0.34

0.80 0.89 0.86

OR* , Adjusted odds ratio by age, gender, ethnic and tobacco; PP and AP, p53 variant homozygous and heterozygous genotypes, respectively; AA, p53 wild genotype and Val/Val, Ile/Val, GSTP1*B variant homozygous and heterozygous genotypes, respectively; Ile/Ile, GSTP1*B wild genotype.

Val/Val, respectively. Similar frequencies were seen in our study, in which 41.0% of patients had the Ile/Ile genotype, 46.5% had the Ile/Val genotype, and 12.5% the Val/Val genotype. The above-mentioned percentages were different from those found in a population from Thailand: 63% (Ile/Ile), 35% (Ile/Val) and 2% (Val/Val) [38], but similar to those in Japanese and Thai normal controls [39,40]. Few studies reported the association of combined gene polymorphisms with LC risk. Jourenkova-Mironova et al. [41] reported an increased risk of LC associated with the combination of the GSTM1 null, GSTP1 Ile/Val plus Val/Val and GSTM3 A/A. Kihara and Noda [42] reported a potential interaction between the GSTP1 and GSTM1 genes in a Japanese population of male smokers (ages 50—69 years), in which a higher risk of LC was associated with the combination of the variant allele for GSTP1 and GSTM1 null genotype. In our current study we found no correlation of the GSTP1*B and GSTM1 null genotypes and the risk to LC in a Brazilian population. Epidemiological and functional studies of the p53 codon 72 polymorphisms suggested that Arg and Pro alleles may confer increased susceptibilities to different types of cancers, implying an interplay of different pathogenetic mechanisms by each allele [43]. The Pro allele of p53 codon 72 has been linked epidemiologically to smoking-related lung and bladder cancers

in some, but not all studies [44,45]. In our study, we have found a larger frequency of genotype P/P in nonsmokers (p = 0.028), which has been described by others [13,44]. Wang et al. [46] observed a three times higher risk of LC in light smoker women with LC and genotype P/P of p53. Lee et al. [38] in studies on squamous-cell carcinoma of the esophagus showed an effect of the P/P gene p53 genotype, regardless of the use of tobacco and alcohol. Hu et al. [47] showed that p53 tumor mutations were more common (p = 0.04) among lighter lifetime smokers (<50 pack-years) who carried the p53 codon 72 Pro allele. The p53 A/A genotype induces apoptosis with faster kinetics and suppresses transformation more efficiently than the p53 P/P genotype [48]. Many studies reported an increase of risk of LC associated with the GSTM1 null polymorphism and the P/P genotype of the p53 gene [46,50,12]. We did not find any correlation (p = 0.48) of these combined polymorphisms and LC in our study population. Besides the association of the P/P polymorphism of p53 to LC in non-smokers, we also noted a higher frequency of the A/P plus P/P genotype of p53 codon 72 in African-Brazilians with LC. Other investigators have previously reported a higher frequency of p53 polymorphisms in patients of African ethnical background with LC [49,51]. These findings indicate that polymorphisms of p53 may be important in specific subgroups of LC patients.

158

Table 5

The frequencies of the GSTM1, GSTT1 and p53 genotypes in 200 LC patients stratified by clinical pathological features

Variable

Age <64 ≥64

n

200 100 100

p-Value Gender Male Female

200 144 56

p-Value Ethnic Caucasian African-Brazilian

200 158 42

p-Value Stage I plus II III plus IV

200 68 132

p-Value Histology NSCLC SCLC

200 168 32

p-Value Tobacco Yes No

* **

Both null, n (%)

p53

GST + p53

PP plus AP, n (%)

PP plus AP plus M1 present, n (%)

PP plus AP plus T1 null, n (%)

PP plus AP plus both null, n (%)

M1 null, n (%)

T1 null, n (%)

44 (44.0) 47 (47.0)

15 (15.0) 12 (12.0)

7 (7.0) 5 (5.0)

53 (53.0) 55 (55.0)

23 (23.0) 27 (27.0)

5 (5.0) 6 (6.0)

2 (2.0) 2 (2.0)

0.670

0.534

0.761

0.776

0.933

0.7641

0.9515*

66 (45.8) 25 (44.6)

21 (14.6) 6 (10.7)

11 (7.6) 1 (1.8)

81 (56.3) 27 (48.2)

39 (27.1) 11 (19.6)

9 (6.3) 2 (3.6)

4 (2.8) 0

0.879

0.472

0.299*

0.306

0.633

0.633*

0.487*

74 (46.8) 17 (40.5)

22 (13.9) 5 (11.9)

11 (7.0) 1 (2.4)

78 (49.4) 30 (71.4)

38 (24.1) 20 (47.6)

8 (5.1) 3 (7.1)

4 (2.5) 0

0.462

0.733

0.689*

0.010

0.014**

0.08

0.07

34 (50.0) 57 (43.2)

6 (8.8) 21 (15.9)

4 (5.9) 8 (6.1)

35 (51.5) 73 (55.3)

18 (26.5) 32 (24.2)

2 (2.9) 9 (6.8)

1 (1.5) 3 (2.3)

0.359

0.194

1.000*

0.654

0.770

0.548*

0.990

77 (45.8) 14 (43.8)

20 (11.9) 7 (21.9)

9 (5.4) 3 (9.4)

93 (55.4) 15 (46.9)

44 (26.2) 6 (18.8)

8 (4.8) 3 (9.4)

3 (1.8) 1 (3.1)

0.828

0.130

0.667

0.377

0.780

0.302*

0.776*

84 (46.4) 7 (36.8)

27 (14.9) 0

12 (6.6) 0

97 (53.6) 11 (57.9)

46 (25.4) 4 (21.1)

11 (6.1) 0

4 (2.2) 0

0.425*

0.082

0.343

0.720

0.864

0.515

0.837

Fisher’s exact test; PP and AP, p53 variant homozygous and heterozygous genotypes, respectively; AA, p53 wild genotype. Genotype A/P plus P/P of p53 gene and GSTM1 present, showed higher frequency (p = 0.014).

H.N. Honma et al.

p-Value

200 181 19

GST

The frequencies of the GSTM1, GSTT1 and GSTP1 genotypes in 200 LC patients stratified by clinical pathological features

Variable

Age <64 ≥ 64

n

200 100 100

p-Value Gender Male Female

200 144 56

p-Value Ethnic Caucasian African-Brazilian

200 158 42

p-Value Stage I plus II III plus IV

200 68 132

p-Value Histology NSCLC SCLC

200 168 32

p-Value Tobacco Yes No p-Value *

200 181 19

GST Both null, n (%)

GSTP1

GST + GSTP1

VV plus IV, n (%)

M1 null plus VV plus IV, n (%)

M1 null, n (%)

T1 null, n (%)

T1 null plus VV plus IV, n (%)

Both null plus VV plus IV, n (%)

44 (44.0) 47 (47.0)

15 (15.0) 12 (12.0)

7 (7.0) 5 (5.0)

53 (53.0) 65 (65.0)

24 (24.0) 32 (32.0)

8 (8.0) 8 (8.0)

5 (5.0) 3 (3.0)

0.670

0.534

0.761

0.084

0.367

0.337

0.385*

66 (45.8) 25 (44.6)

21 (14.6) 6 (10.7)

11 (7.6) 1 (1.8)

88 (61.1) 30 (53.6)

42 (29.2) 14 (25.0)

12 (8.3) 4 (7.1)

7 (4.9) 1 (1.8)

0.879

0.472

0.299*

0.330

0.811

0.618*

0.642*

74 (46.8) 17 (40.5)

22 (13.9) 5 (11.9)

11 (7.0) 1 (2.4)

89 (56.3) 29 (69.0)

42 (26.6) 14 (33.3)

12 (7.6) 4 (9.5)

7 (4.4) 1 (2.4)

0.462

0.733

0.689*

0.136

0.214*

0.506*

0.347*

34 (50.0) 57 (43.2)

6 (8.8) 21 (15.9)

4 (5.9) 8 (6.1)

44 (64.7) 73 (55.3)

23 (33.8) 32 (24.2)

4 (5.9) 9 (6.8)

3 (4.4) 3 (2.3)

0.359

0.194

1.000*

0.239

0.533

0.392*

0.930*

77 (45.8) 14 (43.8)

20 (11.9) 7 (21.9)

9 (5.4) 3 (9.4)

97 (57.7) 21 (65.6)

47 (28.0) 9 (28.1)

12 (7.1) 4 (12.5)

7 (4.2) 1 (3.1)

0.828

0.130

0.667

0.377

0.780

0.302*

0.776*

84 (46.4) 7 (36.8)

27 (14.9) 0

12 (6.6) 0

106 (58.6) 12 (63.2)

51 (28.2) 5 (26.3)

16 (8.8) 0

8 (4.4) 0

0.425*

0.082

0.343

0.698

0.861*

0.485*

0.794*

Influence of p53 codon 72 exon 4, GSTM1, GSTT1 and GSTP1*B polymorphisms

Table 6

Fisher’s exact test; VV and IV, GSTP1*B variant homozygous and heterozygous genotypes, respectively.

159

160

Table 7

The frequencies of the GSTP1 and p53 genotypes in 200 LC patients stratified by clinical pathological features

Variable

Age <64 ≥64

n

200 100 100

200 144 56

Ethnic 200 Caucasian 158 African-Brazilian 42 p-Value Stage I plus II III plus IV p-Value Histology NSCLC SCLC

p-Value

¶

VV plus IV plus PP plus AP, n (%)

IV plus VV plus AA, n (%)

I/I plus AP plus PP, n (%)

42 (42.0) 11 (11.0)

11 (11.0) 14 (14.0)

10 (10.0) 9 (9.0)

43 (43.0) 46 (46.0)

28 (28.0) 34 (34.0)

25 (25.0) 31 (31.0)

25 (25.0) 21 (21.0)

40 (27.8) 16 (28.6)

33 (22.9) 13 (23.2)

48 (30.4) 8 (19.0)

37 (23.4) 9 (21.4)

26 (38.2) 30 (22.7)

17 (25.0) 29 (22.0)

46 (27.4) 10 (31.3)

42 (25.0) 4 (12.5)

51 (28.2) 5 (26.3)

42 (23.2) 4 (21.1)

71 (49.3) 22 (39.3)

0.906* 17 (11.8) 8 (14.3)

68 (43.0) 25 (59.5)

34 (50.0) 59 (44.7)

200 168 32

75 (44.6) 18 (56.3)

83 (45.9) 10 (52.6) 0.939

67 (46.5) 22 (39.3)

0.586* 21 (13.3) 4 (9.5)

13 (8.2) 6 (14.3)

10 (14.7) 15 (11.4)

4 (5.9) 15 (11.4)

65 (41.1) 24 (57.1)

18 (10.7) 1 (3.1)

31 (45.6) 58 (43.9)

14 (7.7) 5 (26.3) 0.0286*,¶

18 (26.5) 44 (33.3) 0.444

75 (44.6) 14 (43.8)

0.406* 23 (12.7) 2 (10.5)

41 (25.9) 21 (50.0) 0.025*,†

0.491 22 (13.1) 3 (9.4)

48 (33.3) 14 (25.0) 0.554

0.335*

0.289 200 181 19

14 (9.7) 5 (8.9)

0.340

51 (30.4) 11 (34.3) 0.461

83 (45.9) 6 (31.6)

55 (30.4) 7 (36.8) 0.961*

Fisher’s exact test; PP and AP, p53 variant homozygous and heterozygous genotypes, respectively; VV and VI, GSTP1*B variant homozygous and heterozygous genotypes, respectively. Genotype VV plus VI and genotype AP plus PP of the p53 gene, observed a higher frequency (p = 0.025). Genotype PP of gene p53, observed a higher frequency in non-smokers.

H.N. Honma et al.

* †

AP, n (%)

0.359

p-Value Tobacco Yes No

PP, n (%)

0.1916 200 68 132

GSTP1 + p53

V/V, n (%)

0.879*

p-Value

p53

I/V, n (%)

0.224*

p-Value Gender Male Female

GSTP1

Influence of p53 codon 72 exon 4, GSTM1, GSTT1 and GSTP1*B polymorphisms

5. Conclusions Our results suggest that GSTM1, GSTT1, GSTP1*B and p53 genes, isolated or combined, have no influence in the risk of LC in a Brazilian population. When comparing Caucasian and African-Brazilian patients we found a higher frequency in the A/P plus P/P genotype of the p53 gene in African-Brazilian patients. This genetic alteration could be associated with LC development in this ethnic group. We also noted a higher frequency of the P/P genotype of the p53 gene in non-smokers with LC. Stratification by ethnic group and smoking status should be considered in future studies of genetic polymorphisms and LC.

Conflict of interest The authors of the article ‘‘Influence of p53 codon 72 exon 4, GSTM1, GSTT1 and GSTP1*B polymorphisms in lung cancer risk in a Brazilian population’’, developed at the Oncology and Genetic Laboratory, Pulmonary Diseases Unit, School of Medical Sciences of the State University of Campinas (UNICAMP), S˜ ao Paulo, Brazil, declare not to have any conflict of interest whatsoever.

Acknowledgements We have been supported by governamental resources, from ˜o de Amparo e Apoio a ` Pesquisa FAPESP 04/13519-5 (Fundac ¸a no Estado de S˜ ao Paulo).

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