Polymorphisms of GSTs in Lung Adenocarcinoma Patients Followed in the Context of a Biobank ()
1. Introduction
Lung cancer was the first cause of cancer-related deaths in 2012 in the European Union with 353,000 deaths [1]. Tobacco smoking is the major attributable risk factor for the increasing prevalence of lung cancer across the world [2]. However, not all smokers are equally susceptible to tobacco-related carcinogens [3]. Thus, the identification of genes responsible for lung carcinogenesis susceptibility may allow researchers to perform screening programs and chemoprevention trials in subgroups of chronic smokers.
Lung Adenocarcinoma (ADC) has replaced squamous cell carcinoma as the most frequent histological subtype of lung cancer [4-7]. The Nancy’s Centre of Biological Resources (“Centre des Ressources Biologiques”, CRB) is an ISO 9001-2000 certified biobank with biological material and follow-up data from lung cancer patients, which collected during the last 20 years. Nancy’s CRB has been developed in the Nancy Central Hospital, aiming to recruit lung cancer patients willing to donate their samples for cancer research [8].
The French National Institute of Medical Research (INSERM) carried out this study aiming at the identification of biological tests associated with lung cancer risk. The tests included single nucleotide polymorphisms (SNP) of a family of detoxifying enzymes called Glutathione-S Transferases (GSTs). The activity of GSTs is dependent upon a steady supply of glutathione (GSH).
The primary role of GSTs is to detoxify xenobiotics by catalyzing the nucleophilic attack by GSH on xenobiotic substrates, thereby preventing their interaction with crucial cellular proteins and nucleic acids [9,10].
The aim of this study was to estimate and compare the frequency of GSTs polymorphisms in a French population of ADC patients. Therefore, we analysed genomic DNA from peripheral blood samples of lung adenocarcinoma patients and healthy controls.
2. Materials and Methods
2.1. Patients
A retrospective study was conducted by the CRB between 1988 and 2007: 743 subjects were evaluated (296 consecutive patients operated upon for ADC and 447 healthy subjects who were used as the control group).
The diagnosis of ADC was established by bronchial biopsies, cytological examination of sputum, bronchial washings and brushings and/or transthoracic fine needle aspiration of the lung lesion. All histological diagnoses had been confirmed by the same pathologists’ panel. The histological classification was based upon the 2004 World Health Organization guidelines [11]. The sixth edition of the TNM (tumor, node, and metastasis) classification was used [12].
The CRB collection was initiated in 1988 to store tissue samples of patients operated for lung tumors as a resource for research purposes. From 2002, all survivors—new patients and control subjects—were invited to give their written informed consent, offer access to their medical records, and to donate biological material for research. Standard operating procedures were followed for all steps of sample collection and storage and for the retrieval of clinical annotations culminating in ISO 9001 certification of the biobank.
All the procedures were carried out according to the principles of the institutional guidelines and the study was approved by the local Ethics Committee.
2.2. Methods
Genomic DNA was obtained from peripheral blood samples (12 ml) collected in EDTA tubes. The DNA was extracted using proteinase K digestion and phenol: chloroform purification. The Mu class GST (GSTM) and the Theta class GST (GSTT) SNPs were studied with duplex SYBR Green q PCR using the primers described in Table 1 and results being read on melt curves.
2.3. Statistical Analysis
The Hardy-Weinberg equilibrium was tested with the χ2 statistic for the goodness-to-fit (one degree of freedom). Statistical differences between groups were calculated by the χ2 or Fischer’s exact test. Conditional analysis was used to obtain race, age and gender-adjusted crude odds ratios (OR). A p-value of <0.05 was considered significant. All analyses were performed using the statistical package SPSS version 17.0.
3. Results
Table 2 summarizes the main epidemiological and clinical characteristics of the study population. ADC patients were found to present a higher incidence of GSTM (p < 0.0001, 95%CI 1.63 - 3.24) and a lower incidence of GSTT (p < 0.0001, 95%CI 0.31 - 0.66) genotypes comparing to control. The null and GSTMT genotype had no significant statistical differences between the two groups (Table 3).