Tumor Biol. (2014) 35:12075–12082 DOI 10.1007/s13277-014-2508-6
RESEARCH ARTICLE
Correlation of CLPTM1L polymorphisms with lung cancer susceptibility and response to cisplatin-based chemotherapy in a Chinese Han population Yiqian Liang & Asmitananda Thakur & Lei Gao & Ting Wang & Shuo Zhang & Hui Ren & Junhui Meng & Tingting Geng & Tianbo Jin & Mingwei Chen
Received: 21 July 2014 / Accepted: 14 August 2014 / Published online: 26 August 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract The implication of genetic factors in predisposition to cancer is a recognized fact. The Cleft lip and palate transmembrane 1-like (CLPTM1L) gene resides in a locus in the chromosome 5p15.33 region that is associated with lung cancer susceptibility and has a role in carcinogenesis. We conducted a case-control study in a Chinese population of 309 pathologically confirmed lung cancer patients and 310 controls to investigate the effect of variant genotypes within the CLPTM1L locus on susceptibility to lung cancer and sensitivity to cisplatin-based chemotherapy. We genotyped nine single nucleotide polymorphisms (SNPs) within the CLPTM1L locus and examined their correlation with lung cancer risk and treatment response using χ2 and unconditional logistic regression analysis. We identified rs451360 as a novel SNP associated with lung cancer risk in the Chinese Han population. The “T” allele of rs451360 was associated with decreased risk of lung cancer (p=0.007, odd ratio (OR)=0.59, 95 % confidence interval (CI): 0.40–0.87). Significant multiplicative interactions were observed between gender and Electronic supplementary material The online version of this article (doi:10.1007/s13277-014-2508-6) contains supplementary material, which is available to authorized users. Y. Liang : A. Thakur : L. Gao : T. Wang : S. Zhang : H. Ren : J. Meng : M. Chen (*) Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of School of Medicine of Xi’an Jiaotong University, 277 Yanta West Street, Xi’an 710061, People’s Republic of China e-mail: [email protected] M. Chen e-mail: [email protected] T. Geng : T. Jin National Engineering Research Center for Miniaturized Detection Systems, College of Life Sciences, Northwest University, Xi’an 710069, Shaanxi Province, People’s Republic of China
polymorphisms of rs402710, the “T/T” genotype of which was associated with decreased lung cancer risk in male patients (p=0.016, OR=0.35, 95 % CI: 0.17–0.73). CLPTM1L polymorphisms did not affect the tumor sensitivity to cisplatin combination chemotherapy in our study patients. The results of the present study suggest a potential association between CLPTM1L variants and lung cancer risk in the Chinese Han populations. Keywords Lung cancer . Single nucleotide polymorphism . CLPTM1L . Susceptibility . Chemotherapy
Introduction Lung cancer is a major health issue across the globe [1, 2]. In China, the incidence of lung cancer is 540,000 cases annually, and it has been identified as the primary cause of cancerrelated deaths in both genders [3, 4]. Epidemiological studies have demonstrated that tobacco smoking is a primary etiologic factor for lung cancer, although only 10–15 % of lifetime smokers develop lung cancer [5]. Heritable factors contribute 26 % to the risk of lung carcinogenesis [6] and may have a detrimental role in susceptibility to lung cancer. Despite considerable advances in the field of tumor biology, the majority of patients with lung cancer are diagnosed at an already advanced stage, and thus surgical resection is not a feasible treatment option. Platinum-based doublet chemotherapy is the current standard of therapy in this situation, and cisplatin is the most commonly used agent [7]. However, the response to cisplatin in lung cancer patients varies significantly owing to drug resistance [8]. Single nucleotide polymorphisms (SNPs) are the most common genetic variants in human genomes and have been proved to be useful genetic markers for the detection of cancer variants via linkage
12076
disequilibrium [9]. Recent genome-wide association studies (GWAS) have shown that polymorphic variation at chromosome 5p15.33 influences the risk of developing various types of cancer [10–14]. This region contains two candidate susceptibility genes, telomerase reverse transcriptase (TERT) and Cleft lip and palate transmembrane 1-like (CLPTM1L). CLPTM1L encodes a protein that has been found to be upregulated in cisplatin-resistant ovarian tumor cell lines and may be associated with apoptosis [14]. Furthermore, CLPTM1L has been observed to be overexpressed in human lung tumors and lung tumor cell lines [15, 16]. SNPs in the gene may modulate antiapoptotic capacity and contribute to individual variations in chemotherapy response. McKay et al. [10] identified rs402710 as a potential susceptibility locus for lung cancer (p=2×10−7) by conducting a GWAS in a total of 3,259 cases and 4,159 controls. The rs31484, rs421629, rs467095, rs380286, rs4975616, rs401681, rs10073340, and rs451360 within the CLPTM1L locus were also found to be significantly associated with lung cancer risk. Pande et al. [17] confirmed the association between the “A” allele of rs451360 and decreased risk of lung cancer in another study involving a European population (p= 4.9×10−4). Wang et al. [18] carried out a GWA study of lung cancer in European populations and identified rs4975616 and rs401681 showing an association with lung cancer risk at p18 years, adequate bone marrow reserve, and satisfactory liver and renal function. These patients were in clinical stage III or IV and had a measurable lesion on computed tomography scan at the beginning of treatment. The patients received cisplatinbased chemotherapy every 3 weeks, for a maximum of 6 cycles or until disease progression or unacceptable toxicity occurred. Responses to treatment were determined according to the Response Evaluation Criteria in Solid Tumor Group (RECIST) guidelines after every 2 cycles of chemotherapy [19]. For data analysis, patients achieving complete response (CR) or partial response (PR) were considered “responders,” and patients with stable disease (SD) or progressive disease (PD) were defined as “non-responders” [20]. SNP selection and genotyping Candidate tagging SNPs (tSNPs) in CLPTM1L gene were selected from previously established polymorphisms associated with lung cancer or other cancers [10, 13, 18, 21–23]. Validated SNPs were selected with a minor allele frequency
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(MAF) >5 % in the HapMap Chinese Han Beijing (CHB) population. Finally, a total of nine tSNPs (i.e., rs4975616, rs451360, rs421629, rs380286, rs402710, rs10073340, rs401681, rs467095, and rs31484) were selected for further genotyping. Genomic DNA was prepared from peripheral blood samples using the GoldMag whole blood genomic DNA purification kit (GoldMag Co. Ltd., Xi’an, China), following the manufacturer’s instructions. DNA concentrations were measured by absorbance at 260 nm with the NanoDrop 2000 (Thermo Scientific, Waltham, MA) whereas detection for contamination by proteins was done by measuring absorbance at 280 nm and calculating the 260/280 ratio. Primers for amplification process and single base extension reactions were designed with Sequenom MassARRAY Assay Design 3.0 software [24]. The PCR primers for the nine selected tSNPs are showed in Supplement Table S1. SNP genotyping were performed in 384-well plate by Sequenom MassARRAY RS1000 (Sequenom, San Diego, CA) according to the standard protocol recommended by the manufacturer [25]. Sequenom Typer 4.0 software (Sequenom) was used to perform data management and analyses [24–26]. Statistical analysis Allele frequency of each SNP in the control subjects was analyzed using χ2 test to evaluate departure from HardyWeinberg equilibrium (HWE). The genotype and allele distributions for tSNPs in lung cancer patients and control subjects were compared by χ2 test [27]. In multivariate analyses, we performed unconditional logistic regression analysis to assess the association between each tSNP and the risk of lung cancer while adjusting for age, gender, and smoking status. The odds ratios (ORs) and 95 % confidence intervals (CIs) were calculated using the logistic regression model [28]. The association between tSNP genotype and lung cancer risk was assessed in gender-specific populations by stratification and interaction analysis. The χ2 test or Fisher’s exact test were used to Table 1 tSNPs in CLPTM1L gene
SNP single nucleotide polymorphism, HWE Hardy-Weinberg equilibrium
compare cisplatin combination chemotherapy response rates according to genotype and allele, and ORs and 95 % CIs were tested by unconditional logistic regression analysis with adjustment for gender, age, smoking status, histology, and TNM stage. In the regression analysis, the outcome variable was patient response to treatment, and patients who were nonresponders to treatment (SD + PD) were compared to patients who responded to treatment (CR + PR). A twosided p value
RESEARCH ARTICLE
Correlation of CLPTM1L polymorphisms with lung cancer susceptibility and response to cisplatin-based chemotherapy in a Chinese Han population Yiqian Liang & Asmitananda Thakur & Lei Gao & Ting Wang & Shuo Zhang & Hui Ren & Junhui Meng & Tingting Geng & Tianbo Jin & Mingwei Chen
Received: 21 July 2014 / Accepted: 14 August 2014 / Published online: 26 August 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract The implication of genetic factors in predisposition to cancer is a recognized fact. The Cleft lip and palate transmembrane 1-like (CLPTM1L) gene resides in a locus in the chromosome 5p15.33 region that is associated with lung cancer susceptibility and has a role in carcinogenesis. We conducted a case-control study in a Chinese population of 309 pathologically confirmed lung cancer patients and 310 controls to investigate the effect of variant genotypes within the CLPTM1L locus on susceptibility to lung cancer and sensitivity to cisplatin-based chemotherapy. We genotyped nine single nucleotide polymorphisms (SNPs) within the CLPTM1L locus and examined their correlation with lung cancer risk and treatment response using χ2 and unconditional logistic regression analysis. We identified rs451360 as a novel SNP associated with lung cancer risk in the Chinese Han population. The “T” allele of rs451360 was associated with decreased risk of lung cancer (p=0.007, odd ratio (OR)=0.59, 95 % confidence interval (CI): 0.40–0.87). Significant multiplicative interactions were observed between gender and Electronic supplementary material The online version of this article (doi:10.1007/s13277-014-2508-6) contains supplementary material, which is available to authorized users. Y. Liang : A. Thakur : L. Gao : T. Wang : S. Zhang : H. Ren : J. Meng : M. Chen (*) Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of School of Medicine of Xi’an Jiaotong University, 277 Yanta West Street, Xi’an 710061, People’s Republic of China e-mail: [email protected] M. Chen e-mail: [email protected] T. Geng : T. Jin National Engineering Research Center for Miniaturized Detection Systems, College of Life Sciences, Northwest University, Xi’an 710069, Shaanxi Province, People’s Republic of China
polymorphisms of rs402710, the “T/T” genotype of which was associated with decreased lung cancer risk in male patients (p=0.016, OR=0.35, 95 % CI: 0.17–0.73). CLPTM1L polymorphisms did not affect the tumor sensitivity to cisplatin combination chemotherapy in our study patients. The results of the present study suggest a potential association between CLPTM1L variants and lung cancer risk in the Chinese Han populations. Keywords Lung cancer . Single nucleotide polymorphism . CLPTM1L . Susceptibility . Chemotherapy
Introduction Lung cancer is a major health issue across the globe [1, 2]. In China, the incidence of lung cancer is 540,000 cases annually, and it has been identified as the primary cause of cancerrelated deaths in both genders [3, 4]. Epidemiological studies have demonstrated that tobacco smoking is a primary etiologic factor for lung cancer, although only 10–15 % of lifetime smokers develop lung cancer [5]. Heritable factors contribute 26 % to the risk of lung carcinogenesis [6] and may have a detrimental role in susceptibility to lung cancer. Despite considerable advances in the field of tumor biology, the majority of patients with lung cancer are diagnosed at an already advanced stage, and thus surgical resection is not a feasible treatment option. Platinum-based doublet chemotherapy is the current standard of therapy in this situation, and cisplatin is the most commonly used agent [7]. However, the response to cisplatin in lung cancer patients varies significantly owing to drug resistance [8]. Single nucleotide polymorphisms (SNPs) are the most common genetic variants in human genomes and have been proved to be useful genetic markers for the detection of cancer variants via linkage
12076
disequilibrium [9]. Recent genome-wide association studies (GWAS) have shown that polymorphic variation at chromosome 5p15.33 influences the risk of developing various types of cancer [10–14]. This region contains two candidate susceptibility genes, telomerase reverse transcriptase (TERT) and Cleft lip and palate transmembrane 1-like (CLPTM1L). CLPTM1L encodes a protein that has been found to be upregulated in cisplatin-resistant ovarian tumor cell lines and may be associated with apoptosis [14]. Furthermore, CLPTM1L has been observed to be overexpressed in human lung tumors and lung tumor cell lines [15, 16]. SNPs in the gene may modulate antiapoptotic capacity and contribute to individual variations in chemotherapy response. McKay et al. [10] identified rs402710 as a potential susceptibility locus for lung cancer (p=2×10−7) by conducting a GWAS in a total of 3,259 cases and 4,159 controls. The rs31484, rs421629, rs467095, rs380286, rs4975616, rs401681, rs10073340, and rs451360 within the CLPTM1L locus were also found to be significantly associated with lung cancer risk. Pande et al. [17] confirmed the association between the “A” allele of rs451360 and decreased risk of lung cancer in another study involving a European population (p= 4.9×10−4). Wang et al. [18] carried out a GWA study of lung cancer in European populations and identified rs4975616 and rs401681 showing an association with lung cancer risk at p18 years, adequate bone marrow reserve, and satisfactory liver and renal function. These patients were in clinical stage III or IV and had a measurable lesion on computed tomography scan at the beginning of treatment. The patients received cisplatinbased chemotherapy every 3 weeks, for a maximum of 6 cycles or until disease progression or unacceptable toxicity occurred. Responses to treatment were determined according to the Response Evaluation Criteria in Solid Tumor Group (RECIST) guidelines after every 2 cycles of chemotherapy [19]. For data analysis, patients achieving complete response (CR) or partial response (PR) were considered “responders,” and patients with stable disease (SD) or progressive disease (PD) were defined as “non-responders” [20]. SNP selection and genotyping Candidate tagging SNPs (tSNPs) in CLPTM1L gene were selected from previously established polymorphisms associated with lung cancer or other cancers [10, 13, 18, 21–23]. Validated SNPs were selected with a minor allele frequency
Tumor Biol. (2014) 35:12075–12082
12077
(MAF) >5 % in the HapMap Chinese Han Beijing (CHB) population. Finally, a total of nine tSNPs (i.e., rs4975616, rs451360, rs421629, rs380286, rs402710, rs10073340, rs401681, rs467095, and rs31484) were selected for further genotyping. Genomic DNA was prepared from peripheral blood samples using the GoldMag whole blood genomic DNA purification kit (GoldMag Co. Ltd., Xi’an, China), following the manufacturer’s instructions. DNA concentrations were measured by absorbance at 260 nm with the NanoDrop 2000 (Thermo Scientific, Waltham, MA) whereas detection for contamination by proteins was done by measuring absorbance at 280 nm and calculating the 260/280 ratio. Primers for amplification process and single base extension reactions were designed with Sequenom MassARRAY Assay Design 3.0 software [24]. The PCR primers for the nine selected tSNPs are showed in Supplement Table S1. SNP genotyping were performed in 384-well plate by Sequenom MassARRAY RS1000 (Sequenom, San Diego, CA) according to the standard protocol recommended by the manufacturer [25]. Sequenom Typer 4.0 software (Sequenom) was used to perform data management and analyses [24–26]. Statistical analysis Allele frequency of each SNP in the control subjects was analyzed using χ2 test to evaluate departure from HardyWeinberg equilibrium (HWE). The genotype and allele distributions for tSNPs in lung cancer patients and control subjects were compared by χ2 test [27]. In multivariate analyses, we performed unconditional logistic regression analysis to assess the association between each tSNP and the risk of lung cancer while adjusting for age, gender, and smoking status. The odds ratios (ORs) and 95 % confidence intervals (CIs) were calculated using the logistic regression model [28]. The association between tSNP genotype and lung cancer risk was assessed in gender-specific populations by stratification and interaction analysis. The χ2 test or Fisher’s exact test were used to Table 1 tSNPs in CLPTM1L gene
SNP single nucleotide polymorphism, HWE Hardy-Weinberg equilibrium
compare cisplatin combination chemotherapy response rates according to genotype and allele, and ORs and 95 % CIs were tested by unconditional logistic regression analysis with adjustment for gender, age, smoking status, histology, and TNM stage. In the regression analysis, the outcome variable was patient response to treatment, and patients who were nonresponders to treatment (SD + PD) were compared to patients who responded to treatment (CR + PR). A twosided p value
