Gustavo J. Lourenço

Polymorphisms in the 5′- and 3′-untranslated region of the VEGF gene and sporadic breast cancer risk and clinicopathologic characteristics

AbstractThe wild and the variant alleles of the C936T and G634C vascular endothelial grow factor (VEGF) polymorphisms seem to be linked to higher angiogenic phenotype than the remaining alleles and may act on breast cancer (BC) origin. We investigated the influence of the VEGF C936T and G634C polymorphisms on the occurrence and clinicopathologic characteristics of sporadic breast cancer (SBC) in 235 patients and 235 controls. Peripheral blood samples of all individuals were analysed by the polymerase chain reaction for identification of genotypes and by enzyme-linked immunosorbent assay (ELISA) for quantification of serum VEGF levels. The variant 634CC genotype isolated (16.2% versus 10.7%, P  = 0.01) and in combination with the wild 936CC genotype (10.6% versus 5.5%, P  = 0.01) were more common in patients than in controls. The carriers of the respective genotypes were under a 2.20-fold and a 3.08-fold increased risks for the disease. Additionally, the frequency of the wild 936CC genotype was higher in patients with tumours of histological grade III compared to those with tumours of I+II histological grades (84.0% versus 64.7%, P  = 0.004) and in patients with positive oestrogen receptor tumours compared to those with tumours lacking oestrogen receptor expression (84.7% versus 73.9%, P  = 0.02). Similar serum values of VEGF were seen in patients and controls with the distinct genotypes of the VEGF. The data suggest that the VEGF wild 936CC and the variant 634CC genotypes constitute inherited determinants of SBC and SBC aggressiveness in Brazil, but are not significant predictors of circulating VEGF levels.

CYP1A1, GSTM1 and GSTT1 polymorphisms, tobacco and alcohol status and risk of head and neck squamous cell carcinoma

We examined the influence of the CYP1A1 A4889G and T6235C, GSTM1 and GSTT1 polymorphisms, involved in carcinogen metabolism, on the head and neck (HN) squamous cell carcinoma (SCC) risk. DNA from 142 HNSCC patients and 142 controls was analysed by polymerase chain reaction (PCR)–restriction fragment length polymorphism or multiplex-PCR for the polymorphisms analyses. Excesses of the CYP1A1 4889AG+GG and 4889AG+GG plus GSTT1 null genotype were seen in patients with heavy tobacco habit compared with controls (41.9% versus 26.8%, P = 0.03; 26.2% versus 10.3%, P = 0.04, respectively). Carriers of the referred genotypes and heavy tobacco consumption were under a 2.0-fold and 2.8-fold increased risks for HNSCC than others, respectively. The CYP1A1 6235TC+CC plus GSTM1 and GSTT1 null genotypes were more common in pharyngeal SCC patients than in controls (5.3% versus 0.7%, P = 0.04). Carriers of the combined genotype had 16.0-fold increased risk for the disease than others. The frequency of one null genotype of the GSTM1 or GSTT1 gene was higher in patients with pharyngeal SCC and heavy smoking status than in controls (76.3% versus 57.7%, P = 0.04). Carriers of the referred genotype and heavy tobacco status had a 2.4-fold increased risk for pharyngeal SCC than others. In contrast, the CYP1A1 6235TC+CC genotype was more common in controls than in laryngeal SCC patients (35.9% versus 21.6%, P = 0.01). Carriers of the genotype had a 0.2-fold decreased risk for the disease than others. Our data present preliminary evidence that inherited combined CYP1A1 A4889G and T6235C abnormalities and GSTM1 and GSTT1 pathways are important determinants of HNSCC, particularly pharyngeal SCC in heavy smoking individuals from south-eastern Brazil.

Glutathione S-transferase mu 1 (GSTM1) and theta 1 (GSTT1) genetic polymorphisms and atopic asthma in children from Southeastern Brazil

Xenobiotics can trigger degranulation of eosinophils and mast cells. In this process, the cells release several substances leading to bronchial hyperactivity, the main feature of atopic asthma (AA). GSTM1 and GSTT1 genes encode enzymes involved in the inactivation of these compounds. Both genes are polymorphic in humans and have a null variant genotype in which both the gene and corresponding enzyme are absent. An increased risk for disease in individuals with the null GST genotypes is therefore, but this issue is controversial. The aim of this study was to investigate the influence of the GSTM1 and GSTT1 genotypes on the occurrence of AA, as well as on its clinical manifestations. Genomic DNA from 86 patients and 258 controls was analyzed by polymerase chain reaction. The frequency of the GSTM1 null genotype in patients was higher than that found in controls (60.5% versus 40.3%, p = 0.002). In individuals with the GSTM1 null genotype the risk of manifested AA was 2.3-fold higher (95%CI: 1.4-3.7) than for others. In contrast, similar frequencies of GSTT1 null and combined GSTM1 plus GSTT1 null genotypes were seen in both groups. No differences in genotype frequencies were perceived in patients stratified by age, gender, ethnic origin, and severity of the disease. These results suggest that the inherited absence of the GSTM1 metabolic pathway may alter the risk of AA in southeastern Brazilian children, although this must be confirmed by further studies with a larger cohort of patients and age-matched controls from the distinct regions of the country.

Polymorphisms of glutathione S‐transferase mu1 (GSTM1) and theta 1 (GSTT1) genes in chronic myeloid leukaemia

To the Editor: The environmental exposure to cytotoxic and genotoxic agents, particularly those derived from benzene, may be associated with an increased risk of chronic myeloid leukaemia (CML) (1). The enzymes of the glutathione S-transferase (GST) system reduce molecules of chemical agents to less toxic levels. Gene codings for the GST mu1 (GSTM1) and theta 1 (GSTT1) proteins are polymorphic in humans and are homozygous null in approximately 50% and 20% of normal individuals, respectively, which results in a lack of the active proteins (2). An increase in homozygous GSTT1 null genotype was observed in CML patients from India (3). As GSTT1 is involved in the metabolism of ethylene oxide (EO), a genotoxic agent capable of producing heritable translocations and the frequency of spontaneous chromosome abnormalities, presumably because of EO, is higher in GSTT1 null individuals (4), it was hypothesised that the loss of the GSTT1 genes might influence the production of Philadelphia (Ph) chromosome, leading to CML. Cancer is the second most common cause of death in south-eastern Brazil and chemical agentsrelated diseases are a serious health problem in this area of the country (5, 6). For this reason, the identification of the GSTM1 and GSTT1 gene polymorphisms in CML patients, from an area in which there is a potential exposure to cytotoxic and genotoxic agents, was considered necessary in order to test whether homozygous null genotypes influenced the risk for disease. Genomic DNA obtained from bone marrow samples of 125 CML patients [73 male, 52 female; age (mean ± SD) 39.0 ± 16.4 yr; 108 White people, 17 African–Americans] and peripheral blood of 341 racially matched controls [198 male, 143 female; age (mean ± SD) 53.0 ± 4.3 yr; 296 White people, 45 African–Americans] was analysed using the multiplex-polymerase chain reaction (7, 8). The diagnosis of CML was based on haematological, cytogenetic and molecular analyses. The statistical significance of the differences between groups was calculated by chi-square or Fischer’s exact test. Crude odds ratio (OR) was given within 95% confidence intervals (CI) and was ageand gender-adjusted. We observed similar frequencies of GSTM1, GSTT1 and combined null genotypes in patients and controls (43.2% vs. 43.7%, P 1⁄4 1.00; 18.4% vs. 17.6%, P 1⁄4 0.95; and 7.2% vs. 7.9%, P 1⁄4 0.75 respectively). Individuals with GSTM1 (OR 1⁄4 0.98; 95% CI: 0.65–1.50), GSTT1 (OR 1⁄4 1.06; 95% CI: 0.62–1.80) and combined null genotypes (OR 1⁄4 0.84; 95% CI: 0.36–1.96) were at similar risk of CML compared with those without the null genotypes. We also examined the frequency of the polymorphisms in patients in relation to clinical variables (Table 1). Similar frequencies of the GSTM1, GSTT1 and combined null genotypes in patients under 50 yr or older, gender and ethnic origin were seen. The frequency of the GSTM1 null genotype was lower in patients in the accelerated phase or with blast crisis than those seen in patients with chronic phase (20.0% vs. 49.0%, P 1⁄4 0.01). Similar frequency of the GSTT1 and combined null genotypes was found in patients with distinct phases of the disease. In conclusion, our results indicate that the inherited absence of this carcinogen detoxification pathway could be unimportant for the Ph chromosome production and aetiology of CML. However, the role of the GSTM1 null genotype in different phases of this disease requires confirmation by large-scale molecular epidemiological studies.

Polymorphisms of glutathione S-transferase mu 1, theta 1, and pi 1 genes and prognosis in Hodgkin lymphoma

We examined the influence of the glutathione S-transferase mu 1 (GSTM1), theta 1 (GSTT1), and pi 1 (GSTP1) polymorphisms, which are involved in the metabolism of alkylating agents and anthracyclines, on the outcome of patients with Hodgkin lymphoma (HL) treated with conventional chemotherapy. Genomic DNA from 125 consecutive cases was analyzed by polymerase chain reaction and enzymatic digestion for polymorphism determination. The GSTM1 undeleted genotype was associated with more advanced tumor stage and worse disease-free survival. The GSTT1 undeleted genotype was associated with higher recurrence rate. In contrast, higher toxicity of chemotherapy was attributed to absence of the GSTT1 gene. Concerning overall survival, lower tumor stage (p = 0.006) and International Prognostic Score (p = 0.02), lower peripheral leukocyte count (p = 0.0003), higher serum albumin level (p = 0.08), and GSTT1 undeleted genotype (p = 0.04) were predictive of a better outcome of patients. In multivariate analysis comparing staging and GST polymorphism, only tumor stage and GSTT1 genotype remained in the model. Our results suggest that the GSTT1 polymorphism influences the outcome of Brazilian patients with HL. However, studies of toxicity, pharmacokinetics, and protein function may clarify whether carriers of the distinct genotypes should receive different doses of chemotherapeutic agents.

Polymorphisms of glutathione S-transferase Mu 1 (GSTM1), Theta 1 (GSTT1), and Pi 1 (GSTP1) genes and epithelial ovarian cancer risk

Background: Exposure of ovarian cells to estrogen, which is detoxified by glutathione S-transferases (GSTs), has been associated with epithelial ovarian cancer (EOC) development. Objectives: We tested in this study whether the GSTM1, GSTT1 and GSTP1 Ile105Val polymorphisms alter the risk of EOC. Materials and methods: Genomic DNA from 132 EOC patients and 132 controls was analyzed by polymerase chain reaction and restriction fragment length polymorphism methods. The differences between groups were analyzed by χ2 or Fisher’s exact test. Results: The frequencies of GSTP1 Ile/Ile (57.6% versus 45.5%, P = 0.03), GSTM1 null plus GSTP1 Ile/Ile (43.5% versus 25.8%; P = 0.03) and GSTM1 null plus GSTT1 null plus GSTP1 Ile/Ile (30.3% versus 7.7%; P = 0.007) genotypes were higher in patients than in controls. Individuals with the respective genotypes had a 1.80 (95% CI: 1.06–3.06), 2.38 (95% CI: 1.08–5.24) and 11.28 (95%CI: 1.95–65.30)-fold increased risks of EOC than those with the remaining genotypes. Conclusions: Our data present preliminary evidence that GSTM1, GSTT1 and GSTP1 polymorphisms, particularly in combination, constitute important inherited EOC determinants in individuals from Southeastern Brazil.

High risk of ‘de novo’ acute myeloid leukaemia in individuals with cytochrome P450 A1 (CYP1A1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) gene defects

To the Editor, The environmental exposure to benzene and tobacco’s polycyclic aromatic hydrocarbons (PAH) has been associated with an increased risk for acute myeloid leukaemia (AML). The ability to metabolise carcinogens varies among human beings and, therefore, healthy individuals may be at distinct risks of the disease. The cytochrome P450 (CYP) proteins are the first line of defence against toxic chemicals since participating in the activation of the agents in order to make their inactivation easier. PAH are activated by the CYP1A1 enzymes, coded by the CYP1A1 polymorphic gene, resulting in the formation of substances, which can act as carcinogens. The variant alleles of the CYP1A1 T6235C and A4889G polymorphism give rise to enzymes with increased activities in PAH activation (1). The variant CC and GG genotypes were found in 1–20% and 4–40% of healthy individuals of distinct ethnic populations, respectively, but their roles for the risk of AML are controversial (2–5). The NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme of the second line of defence against toxic chemicals, which detoxifies benzene-derived quinones. A polymorphism with a C fi T substitution at nucleotide 609 of the NQO1 gene was related to decreased activity of the coded enzyme (6). The variant TT genotype was found in 5–25% of different ethnic groups and seems to increase the AML risk (4, 7–11). The environmental diseases resulting from exposure to benzene and tobacco-related diseases were described as serious health problems in south-eastern Brazil. Thus, the identification of the genotypes of the CYP1A1 and the NQO1 polymorphisms in healthy and AML individuals from this region was considered necessary to test their influences, if any, on the risk for disease or on its clinical features in our population. Genomic DNA from peripheral blood of 133 consecutive de novo AML patients (70 males, 63 females; median age: 47 years, range: 11–89; 112 Caucasians, 17 Blacks, 4 no classified) and 133 controls (70 males, 63 females; median age: 53 years, range: 25–60; 116 Caucasians, 17 Blacks) was analysed by the polymerase chain reaction and the restriction fragment length polymorphism assays for identification of the CYP1A1 T6235C and A4889G (12), and the NQO1 C609T (9) genotypes. Both the patients and controls samples confirmed the Hardy-Weinberg expectations at the CYP1A1 T6235C (X = 2.20, P = 0.14; X = 0.82, P = 0.36) and A4889G (X = 2.54, P = 0.12; X = 0.89, P = 0.34) loci. Controls’ (X = 3.69, P = 0.08) but not patients’ samples (X = 6.52, P < 0.02) were in Hardy-Weinberg equilibrium at the NQO1 C609T locus. The frequency of the CYP1A1 6235TC+CC and NQO1 609CT+TT genotypes was higher in patients than in the controls. Carriers of the variant allele C of the CYP1A1 gene and the allele T of the NQO1 gene were under a 2.75-fold and a 1.92-fold increased risk of

Association between genetic polymorphisms in DNA mismatch repair-related genes with risk and prognosis of head and neck squamous cell carcinoma

We examined the influence of MLH1 c.−93G>A, MSH2 c.211 + 9C>G, MSH3 c.3133G>A and EXO1 c.1765G>A polymorphisms, involved in DNA mismatch repair (MMR), on head and neck (HN) squamous cell carcinoma (SCC) risk and prognosis. Aiming to identify genotypes, DNA from 450 HNSCC patients and 450 controls was analyzed by PCR‐RFLP or real time PCR. MSH2 GG plus MSH3 GG (31.7% vs. 18.7%, p = 0.003) genotypes were higher in laryngeal SCC (LSCC) patients than in controls. Carriers of the respective combined genotype were under a 3.69 (95% CI: 1.54–8.81)‐fold increased risk of LSCC. Interactions of tobacco and tobacco plus all the above‐mentioned polymorphisms on HNSCC and LSCC risk were also evident in study (p = 0.001). At 60 months of follow‐up, relapse‐free survival (RFS) was shorter in patients with EXO1 GG genotype (54.8% vs. 61.1%, p = 0.03) and overall survival (OS) was shorter in patients with MSH3 GG genotype (42.8% vs. 52.5%, p = 0.02) compared to those with other genotypes, respectively. After multivariate Cox analysis, patients with EXO1 GG and MSH3 GG genotypes had worst RFS (HR: 1.50, 95% CI: 1.03–2.20, p = 0.03) and OS (HR: 1.59, 95% CI: 1.19–2.13, P = 0.002) than those with the remaining genotypes, respectively. Our data present, for the first time, evidence that inherited MLH1 c.‐93G>A, MSH2 c.211 + 9C>G, MSH3 c.3133G>A, and EXO1 c.1765G>A abnormalities of DNA MMR pathway are important determinants of HNSCC, particularly among smokers, and predictors of patient outcomes.

Características mamográficas do câncer de mama associadas aos polimorfismos GSTM1 e GSTT1

INTRODUCTION: Enzymes of the Glutathione S-transferase system (GST) modulate the effects of exposure to several cytotoxic and genotoxic agents. The GSTM1 and GSTT1 genes are polymorphic in humans and their deletions have been associated to increased risk of many cancers, including breast cancer. OBJECTIVE: To evaluate the occurrence of homozygous deletions of the GSTM1 and GSTT1 genes in women with sporadic breast cancer and in women without cancer and to compare breast cancer mammographic features between patients with and without these deletions. METHODS: The study evaluated 100 patients with sporadic breast cancer treated from September 2004 to June 2005 and 169 women without cancer, determining the frequency of the above-mentioned deletions by PCR and calculating the odds ratios and their 95% confidence intervals. Medical files and mammograms of 100 patients with breast cancer were evaluated and correlated with mammographic features such as density, mammographic findings and the BI-RADS classification. These findings were correlated with the genetic deletions by the PR (Prevalence-Ratio) with their respective 95% confidence intervals. RESULTS: The GSTM1 gene was deleted in 40% of the cancers and in 44.4% of controls (OR = 1.20; CI 95% 0.70 - 2.04; p=0.5659) while the GSTT1 gene was deleted in 20% and 19.5%, respectively (OR = 0.73; CI 95% 0.37-1.44; p=0.4124). High mammographic density had been associated with GSTM1 deletion (PR 2.43; CI 1.11 to 4.08). GST deletions were not associated with predominant mammographic findings and the BI-RADS classification. CONCLUSION: GSTM1 homozygous deletion was associated with high mammographic density.

Polymorphisms of glutathione S-transferase Mu 1, glutathione S-transferase theta 1 and glutathione S-transferase Pi 1 genes in Hodgkin's lymphoma susceptibility and progression.

We tested in this study whether the polymorphisms of the glutathione S-transferase Mu1 (GSTM1), glutathione S-transferase Theta 1 (GSTT1) and glutathione S-transferase Pi 1 (GSTP1), involved in metabolism of chemical agents, cell proliferation and cell survival, alter the risk for Hodgkin lymphoma (HL). Genomic DNA from 110 consecutive patients with HL and 226 controls was analysed by polymerase chain reaction and restriction digestion for the polymorphism analyses. Similar frequencies of the GSTM1 and GSTT1 genotypes were seen in patients and controls. In contrast, the frequency of the GSTP1 wild genotype (59.1%versus 36.3%, P = 0.004) was higher in patients than in controls. Individuals with the wild genotype had a 2.68 (95%CI: 1.38–5.21)-fold increased risk for the disease than others. An excess of the GSTP1 wild genotype was also observed in patients with tumors of stages III + IV when compared with those with tumors of stages I + II (39.1%versus 20.0%, P = 0.03). These results suggest that the wild allele of the GSTP1 gene is linked to an increased risk and high aggressiveness of the HL in our cases but they should be confirmed by further studies with larger cohorts of patients and controls.