Mol Biol Rep (2014) 41:3091–3097 DOI 10.1007/s11033-014-3169-7
Interleukin-10-1082 gene polymorphism is associated with papillary thyroid cancer ¨ zer Esra C ¸ il • Alkın Kumral • Mu¨ge Kanmaz-O Pervin Vural • Semra Dog˘ru-Abbasog˘lu • Yu¨ksel Altuntas¸ • Mu¨jdat Uysal
•
Received: 22 January 2013 / Accepted: 16 January 2014 / Published online: 28 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The etiopathogenesis of thyroid cancer has not been clearly elucidated although the role of chronical inflammation and the imbalance between pro- and antiinflammatory cytokines may play a role in the etiology. The aim of the present study was to investigate whether cytokine gene polymorphisms are associated with papillary thyroid cancer (PTC), and to evaluate the relationship between genotypes and clinical/laboratory manifestation of PTC. Tumor necrosis factora (TNFa) G-308A (rs 1800629), interleukin-6 (IL-6) G-174C (rs 1800795) and IL-10 A-1082G (rs 1800896) single nucleotide polymorphisms in DNA from peripheral blood leukocytes of 190 patients with thyroid cancer and 216 healthy controls were investigated by real-time PCR combined with melting curve analysis. There was no notable risk for PTC afflicted by TNFa-308 and IL-6-174 alone. However, IL-10-1082 G allele frequency were higher among PTC patients than healthy controls (p = 0.009). The patients with IL-10-1082 GG geotype have twofold increased risk of developing thyroid cancer according to AA genotype (OR 2.07, 95 % CI 1.21–3.55). In addition, the concomitant presence of IL-10-1082 G allele (GG ? AG genotypes) together with IL-6 -174 GG genotype has a nearly twofold increased risk for thyroid cancer (OR 1.75 with 95 % CI 1.00–3.05, p = 0.049). We suggest that IL-10-1082 G allele is associated with an increased risk
E. C¸il Y. Altuntas¸ Division of Endocrinology, Department of Internal Medicine, S¸ is¸ li Etfal Research and Training Hospital, S¸ is¸ li, Istanbul, Turkey ¨ zer P. Vural (&) A. Kumral M. Kanmaz-O S. Dog˘ru-Abbasog˘lu M. Uysal Department of Biochemistry, Istanbul Faculty of Medicine, Istanbul University, C¸apa, Istanbul 34093, Turkey e-mail: [email protected]
of PTC. The polymorphism of IL-10 gene can improve our knowledge about the pathogenesis of PTC, and could provide to estimate people at the increased risk for PTC. Keywords Papilary thyroid cancer TNFa IL-6 IL10 Polymorphism
Introduction Papillary thyroid carcinoma (PTC) is the most common type of endocrine malignancy. It accounts for more than 70 % of all thyroid cancers [1]. Although the exact pathophysiologic mechanisms of PTC remain elusive, there is growing evidence that the disease is a consequence of interaction between genetic and environmental factors [2]. Cytokines are molecules that influence activation, growth, and differentiation of several target cells [3, 4]. They are pro-inflammatory and anti-inflammatory mediators that participate in the induction and effector phases of the inflammatory and immune responses, and modulate development and growth of both normal and neoplastic thyroid cells. A variety of cytokines including tumor necrosis factor a (TNFa), interleukin (IL)-1a, IL-6, IL-8, IL-10, IL-12 and interferon-c (IFNc) have been shown to be produced in thyroid follicular cells and intrathyroid inflammatory cells [5]. TNFa and IL-6 are main cytokines that play a central role in initiation and regulation of the cytokine cascade during an inflammatory response. With respect to tumorigenesis, TNFa and IL-6 are secreted by several cancer cells and are necessary to sustain cancer cell growth and recruitment of leukocytes to tumor sites [2]. On the other hand, interleukin-10 is a major immunomodulatory cytokine suppressing synthesis of pro-inflammatory cytokines as TNFa, IL-1, IL-8, and IFNc [6], and
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potentiating the up-regulation of B cell production and differentiation [7, 8]. IL-10 is an immunosuppressive cytokine which may facilitate development of cancer by supporting tumor escape from the immune response [9]. Several single nucleotide polymorphisms (SNPs) in the regulatory regions of TNFa, IL-6 and IL-10 have been implicated to modulate the risk of various malignancies, possibly by influencing the expression of the protein [10– 16]. Therefore, in the present study, we aimed to investigate whether TNFa G-308A, IL-6 G-174C and IL-10 G-1082A polymorphisms could predispose to PTC, and to evaluate the possible relationship between genotypes and clinical/biochemical characteristics of PTC.
Materials and methods One hundred and ninety patients with the diagnosis of PTC were included in this study. Informed consent was obtained from each subject. The study was approved by the Institutional Review Board at S¸ is¸ li Etfal Research and Training Hospital. PTC had been either diagnosed or suspected in these patients for clinical or epidemiological reasons, including the results of fine-needle aspiration cytology and/ or histological analysis. None of the patients had received chemotherapy, radiotherapy, or surgery before admission to our department. All of the patients underwent total or near-total thyroidectomy. The control group consisted of 216 individuals matched for age and sex. None of the controls had personal or family history of thyroid disease and goiter on examination; they had normal thyroid functions and were negative for thyroid autoantibodies. Exclusion criteria (for both study group and controls) were the existence of any comorbid cardiac, infectious, musculoskeletal or malignant disease and a recent history of operation or trauma. None of the patients and controls are consuming alcohol. Height (m) and weigh (kg) were measured after fasting, without shoes and wearing light clothes. All measurements were conducted with the patient in a standing position. Body mass index (BMI) was calculated by dividing the weight by the height squared. Blood samples were taken in the morning subsequent to an overnight (12 h) fast. Peripheral venous blood samples were collected in plain tubes for routine biochemical analysis, and in EDTA-K3 for genotype analysis. Serum triglyceride, cholesterol, HDL-, LDL- and VLDL-cholesterol measurements were performed on 1800 DPP Roche autoanalyzer (Roche Diagnostics, Mannheim, Germany). Serum TSH, free T3, free T4, anti-Tg (anti-thyroglobulin antibody) and anti-TPO (anti-thyroid peroxidase antibody) were measured on Modular EEE Electrod Elecsys Roche autoanalyzer (Roche Diagnostics, Mannheim, Germany).
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Genomic DNA was isolated from peripheral blood leukocytes by using high pure PCR template preparation kit (Roche Diagnostics GmbH, Mannheim, Germany). For detection of polymorphisms light SNIP assays were used. Light SNIP assays are based on simple probe melting curve analysis. They consist of pre-mixed primers and probes. They were developed and optimized according to NCBI ‘‘rs’’ numbers of studied SNP’s by Tib MolBiol (Berlin, Germany). The detection of polymorphisms was performed in a LightCycler (Roche Diagnostics, Mannheim, Germany). Melting curves were evaluated by two independent observers who were blinded to the analysis of the clinical data. In addition, 10 % of randomly selected samples were repeated independently to verify genotyping results and 100 % concordance was found. Differences in genotype distributions and allele frequencies in the cases and the controls were compared for statistical significance using the Chi square (v2) test. The statistical significance for deviations from Hardy–Weinberg Equilibrium (HWE) was determined using the Pearson v2-test. Odds ratios (ORs) were calculated and given with 95 % confidence intervals (CIs). Multiple logistic regression analysis was applied to evaluate the effects of genotypes of studied polymorphisms on PTC susceptibility after adjustment for gender, age, smoking status. In addition, multiple logistic regression analysis including TNFa, IL-6 and IL-10 gene polymorphisms as the exposure variable and the presence or absence of concurrent chronic lymphocytic thyroiditis as the dependent variable was performed. We examined also the association between combined polymorphisms and PTC in carriers and noncarriers of mutant allele of above mentioned genes, and computed interaction terms between TNFa and IL-10; TNFa and IL-6; IL-6 and IL-10 by using v2-test. Mann– Whitney U, Kruskal–Wallis and Spearman correlation tests were used for the evaluation of clinical and biochemical parameters. All statistical analyses were performed with SPSS 15.0 for Windows (Chicago, IL, USA). In addition the NCSS 2000 statistical package (Kaysville, Utah, USA) was used to evaluate the power analysis. We had a 96 % power to detect an effect size (W) of 0.20 using a 2 degrees of freedom (a = 0.05).
Results TNFa-308, IL-6-174 and IL-10-1082 promoter polymorphisms were analyzed in 190 patients with PTC (146 women and 44 men) and 216 unrelated healthy controls (160 women and 56 men). The clinical characteristics of controls and patients with PTC were given in Table 1. There were no significant differences among study and control groups in terms of mean age and sex distribution.
Mol Biol Rep (2014) 41:3091–3097 Table 1 Characteristics of controls and patients with papillary thyroid cancer (PTC)
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Control (n = 216)
PTC (n = 190)
Mean ± SD
46.0 ± 9.9
47.2 ± 12.02
Range
28–79
28–79
56 (25.9)
44 (23.2)
Age (years)
Gender Male, n (%) Female, n (%) Family history, n (%)
160 (74.1)
146 (76.8)
–
76 (40.0)
Smoking, n (%)
70 (32.4)
64 (33.7)
Exposure to radiation history
None
None
Ethnic differences
None
None
I
–
133 (70.0)
II
–
21 (11.1)
III
–
29 (15.2)
–
7 (3.7)
TNM stage
IV Tumor size C1 cm
–
146 (76.8)
\1 cm
–
44 (23.2)
–
26 (13.7)
Man–Whitney U test
BMI (kg/m2) (mean ± SD)
CLT
25.70 ± 4.69
25.55 ± 4.69
BMI body mass index, anti-Tg anti-thyroglobulin antibody, anti-TPO anti-thyroid peroxidase antibody, CLT concurrent chronic lymphocytic thyroiditis, TSH thyroidstimulating hormone, HDL-C high density lipoproteinecholesterol, LDL-C low density lipoproteine-cholesterol, VLDL-C very low density lipoproteine-cholesterol
Anti-TPO (IU/mL) (mean ± SD)
–
614.5 ± 398.9
* p \ 0.05
Anti-Tg (IU/mL) (mean ± SD)
–
575.2 ± 390.5
TSH (mIU/L) (mean ± SD)
1.78 ± 0.9
6.32 ± 7.65*
FreeT3 (pmol/L) (mean ± SD)
3.7 ± 0.32
2.92 ± 0.86
FreeT4 (pmol/L) (mean ± SD)
14.6 ± 2.2
13.2 ± 3.6
Cholesterol (mg/dL) (mean ± SD)
172.87 ± 37.50
201.65 ± 41.80
Triglyceride (mg/dL) (mean ± SD)
110.73 ± 53.20
123.23 ± 70.79
HDL-C (mg/dL) (mean ± SD)
60.25 ± 13.16
55.14 ± 12.29
LDL-C (mg/dL) (mean ± SD)
105.83 ± 35.41
121.35 ± 34.68
VLDL-C (mg/dL) (mean ± SD)
20.16 ± 11.98
25.75 ± 15.49
No significant differences were observed between female and male patients with respect to clinical and hormonal parameters. The genotypic and allelic distributions of TNFa-308, IL6-174 and IL-10-1082 polymorphisms for cases and controls are shown in Table 2. All genotype distributions were in accordance with the HWE among the controls. No notable differences were observed in allele or genotype frequencies for TNFa-308 and IL-6-174 genes alone (Table 2). With regard to IL-10-1082 polymorphism, the GG genotype versus AA in the PTC group differed significantly from that in the control group (p = 0.008) with an OR of 2.07 (95 % CI 1.21–3.55). We found also that in PTC patients there was a significant increase of IL-10-1082 G allele frequency (p = 0.009). To check wether the factors such as age, gender and smoking status influence the association between studied polymorphisms and PTC, we performed a multiple logistic regression analysis. The
results are shown in Table 3. Accordingly, IL-10-1082 was still significantly associated with PTC after adjustement age, gender and smoking status (p = 0.007, Table 3). The concurrent chronic lymphocytic thyroiditis was present in 26 (13.7) of PTC patients. None of the polymorphisms were found to be related to concurrent chronic lymphocytic thyroiditis (Table 4). We also investigated whether any combinations of TNFa-308, IL-6-174 and IL-10-1082 variant alleles affected the risk for PTC. The concomitant presence of IL10-1082 GG and AG genotypes (GG ? AG) together with IL-6-174 GG had a nearly twofold increased risk for thyroid canser (OR 1.75 with 95 % CI 1.00–3.05). However, the difference was at borderline significance (p = 0.049) (Table 5). In addition, the possible relation between studied polymorphisms and certain clinical phenotypes, including tumor size, stage, TSH, free T3, free T4, antiTPO, anti-Tg and lipid profile parameters were evaluated in
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Table 2 Distribution of genotypes and allele frequencies in patients with papillary thyroid cancer (PTC) and control group
Controls n (%)
PTC n (%)
OR (95 % CI)
p
TNFa-308 rs 1800629 GG
174 (80.6)
153 (80.5)
Reference
–
AG
37 (17.1)
36 (19.0)
1.11 (0.67–1.84)
0.70
AA
5 (2.3)
1 (0.5)
0.23 (0.03–1.97)
0.14
AG ? AA
42
37
1.00 (0.61–1.64)
0.99
G allele frequency
0.89
0.90
Reference
–
A allele frequency
0.11
0.10
0.91 (0.58–1.43)
0.62
IL-6 -174 rs 1800795 GG
113 (52.3)
110 (57.9)
Reference
–
CG
85 (39.4)
63 (33.2)
0.76 (0.50–1.16)
0.20
CC
18 (8.3)
17 (8.9)
0.97 (0.48–1.98)
0.93
GG ? CC
103
80
0.80 (0.54–1.18)
0.25
G allele frequency C allele frequency
0.72 0.28
0.74 0.26
Reference 0.88 (0.65–1.20)
– 0.43
AA
80 (37.0)
58 (30.5)
Reference
–
AG
100 (46.3)
78 (41.1)
1.08 (0.69–1.69)
0.75
GG
36 (16.7)
54 (28.4)
2.07 (1.21–3.55)
0.008 0.17
IL-10 -1082 rs 1800896
Each p value was based on Chi square (v2) analysis CI confidence interval, IL-6 interleukin-6, IL-10 interleukin10, OR Odds ratio, TNFa tumor necrosis factor a
AG ? GG
136
132
1.34 (0.89–2.03)
A allele frequency
0.60
0.51
Reference
–
G allele frequency
0.40
0.49
1.45 (1.10–1.92)
0.009
Table 3 Association between PTC and studied cytokine polymorphisms after adjustment for age, gender and smoking status Adjusted OR
95 % CI
Table 4 Association between concurrent chronic lymphocytic thyroiditis and studied cytokine polymorphisms in PTC patients
p value
Adjusted OR
95 % CI
p value
TNFa G -308A
1.09
0.56–2.61
0.80
TNFa G -308A
1.95
0.50–7.62
0.33
IL-6 G-174C
0.89
0.60–1.32
0.56
IL-6 G-174C
1.41
0.56–3.58
0.47
IL-10 A-1082G
1.66
1.15–2.4
0.007
IL-10 A-1082G
0.72
0.26–1.99
0.53
patients with PTC and we could not find any difference (data not shown).
Discussion We have carried out a genetic association study of TNFa G-308A, IL-6 G-174C and IL-10 G-1082A polymorphisms in PTC—the most common malignancy of thyroid gland. The frequencies of alleles and genotypes of selected polymorphisms were similar to those in previous studies in Caucasian populations as well as in our previous studies [17–20]. There is increasing evidence that chronical inflammation and the imbalance between pro- and anti-inflammatory
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cytokines are implicated in the etiopathogenesis of thyroid cancer [2]. TNFa is a pro-inflammatory cytokine produced by macrophages, monocytes, epithelial cells and lymphocytes, and induces the production of IFNc and IL-6 [21]. TNFa exhibits cytotoxic as well as cytostatic effects on thyrocytes, and further, TNFa up-regulates HLA class I expression, with subsequent damage of thyrocytes [21]. IL-6 is another pro-inflammatory cytokine mainly produced by mononuclear phagocytes under the stimulation of IL-1 and TNFa. IL-6 is also an important growth and differentiation factor for T, B lymphocytes and thyroid cells, and is able to modulate both cellular and humoral immunity [22]. Thyroid cancer cells secrete several cytokines including TNFa and IL-6 which are necessary to sustain cancer cell growth and recruit leukocytes to tumorous area [2, 4]. Moreover, TNFa
Mol Biol Rep (2014) 41:3091–3097 Table 5 Combined effect of TNFa-308, IL-6 -174 and IL-10 -1082 polymorphisms on the risk for papillary thyroid cancer (PTC)
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Controls n (%)
PTC n (%)
OR (95 % CI)
p
TNFa-308 GG combined with IL-10 -1082 AA IL-10 -1082 AG ? GG
67 (31.0)
44 (23.2)
–
108 (50.0)
106 (55.8)
1.50 (0.94–2.38)
0.09
TNFa-308 GG combined with IL-6 -174 GG
93 (43.1)
91 (47.9)
–
IL-6 -174 CG ? CC
80 (37.0)
62 (36.2)
0.80 (0.51–1.23)
0.29
TNFa-308 AG ? AA combined with IL-10 -1082 AA
14 (6.5)
14 (7.4)
–
IL-10 -1082 AG ? GG
27 (12.5)
26 (13.7)
0.96 (0.38–2.41)
0.93
21 (11.1) 16 (8.4)
– 0.66 (0.27–1.60)
0.36
TNFa-308 AG ? AA combined with IL-6 -174 GG IL-6 -174 CG ? CC
20 (9.3) 23 (10.6)
IL-6 -174 GG combined with ORs and 95 % CIs were based on Chi square (v2) analysis CI confidence interval, IL-6 interleukin-6, IL-10 interleukin10, OR Odds ratio, TNFa tumor necrosis factor a
IL-10 -1082 AA
45 (20.8)
32 (16.8)
–
IL-10 -1082 AG ? GG
66 (30.6)
82 (43.2)
1.75 (1.00–3.05)
0.049
IL-6 -174 CG ? CC combined with IL-10 -1082 AA
38 (17.6)
26 (13.7)
–
IL-10 -1082 AG ? GG
67 (31.0)
50 (26.3)
1.09 (0.59–2.03)
and IL-6 and tumor-associated leukocytes have been shown to contribute to invasive phenotype [4]. IL-10 is a pleiotropic cytokine with both anti-inflammatory and antiangiogenic functions, and may have both pro-tumoral and ati-tumoral properties. It is produced by activated T cells, B cells, monocytes, thymocytes, acting as growth factor, and stimulates humoral immune response [6–8]. Thyroid cancer cells produce high amounts of IL-10, which play an important role in many pathological features of thyroid cancer [23]. Moreover, it was shown that the presence of IL10 in the thyroid cancer environment contributes to thyroid cancer cell survival and proliferation [23]. The gene encoding TNFa is located in the short arm of chromosome 6 (6p21.3) [24], which also contains genes encoding HLA molecules. Owing to the biological effects of TNFa upon the thyroid gland and also its gene location, TNFa should be able to influence an individual’s susceptibility to thyroid cancer. Several studies have suggested a role for the importance of TNFa promoter region polymorphism in the pathogenesis of colon [25] and cervical cancers [26], and hepatocellular [27], head and neck squamous cell [28], nasopharyngeal carcinomas [29]. The gene encoding IL-6 is located in the short arm of chromosome 7 (7p21) [24]. The polymorphisms of the promoter region -174 correlate well with the development of prostate [30], colorectal [16] cancers and PTC [31], although there were some studies with conflicting results [32, 33]. With regard to IL-10 gene, it comprises 5 exons, and is located on chromosome 1 at 1q31–1q32 [34]. IL-101082 polymorphism is reported to be a genetic risk factor
0.78
for many cancer types, including PTC [35–39]. However, these results were not replicated in other studies [40, 41]. The pathogenic impact of TNFa, IL-6 and IL-10 in thyroid cancer is underscored by the effect of the functional polymorphisms in the promoter regions of TNFa, IL-6 and IL-10 genes associated with different transcriptional and expressional rates [10–16]. In our study, we hypothesize that TNFa-308, IL-6-174 and IL-10-1082 polymorphisms could influence the risk for PTC development through altered production of TNFa, IL-6 and IL-10, resulting in imbalance between pro- and anti-inflammatory cytokines with subsequent tumorigenesis. To our knowledge, the present study is the first one examining the possible relationship between TNF a G-308A and PTC. We found that polymorphisms in the promoter regions of TNFa and IL-6 genes are not significant risk factors for development of PTC alone. On the other hand, we found that IL-10-1082 G allele and GG genotype frequencies (associated with high production of IL-10) were higher in PTC patients in comparison with healthy controls. The individuals with IL10-1082 GG genotype have twofold increased risk of developing thyroid cancer according to AA genotype even after adjustment for age, gender and smoking status. Our results are in line with other studies reporting that IL-101082 polymorphism may be a genetic risk factor for PTC [35, 36]. The one of the most interesting finding of our study is the fact that concomitant presence of IL-10-1082 G allele (GG ? AG) together with IL-6 -174 GG genotype has a nearly twofold increased risk for thyroid cancer. It was
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shown that reporter gene constructs containing the G allele of the -1082 of IL-10 gene appears to have higher transcriptional activity than with the -1082 A allele [11, 12]. There are conflicting results about the impact of IL-6-174 on plasma IL-6 levels. According to some authors, C allele is related with higher plasma IL-6 levels [10, 12], others postulate that G allele is associated with increased production [13–16]. We suspect that IL-10 G allele combined with IL-6 GG genotype results in high production of IL-10 and IL-6 which may lead to accentuation of inflammatory response with immunosuppression, and propagation of tumorigenesis. However, the exact mechanisms for transcripted and expressed products of these polymorphisms interacting with other genetic and environmental factors remain to be elucidated. As a conclusion, we suggest that IL-10-1082 G allele could predispose to PTC. The polymorphism of IL-10 gene may have important implications for pathogenesis of PTC. IL-10-1082 polymorphism can be used as a marker and could provide to estimate people at the increased risk for PTC. In addition, this polymorphism may lead to development of alternative therapeutic approach. When it is identified, high-risk subjects could undergo more detailed examinations to detect obscure PTC. Moreover, individualized treatment and prevention programs can be developed to a high-risk population for PTC. Acknowledgments This work was supported by the Research Fund of Istanbul University Project No: 8763.
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Interleukin-10-1082 gene polymorphism is associated with papillary thyroid cancer ¨ zer Esra C ¸ il • Alkın Kumral • Mu¨ge Kanmaz-O Pervin Vural • Semra Dog˘ru-Abbasog˘lu • Yu¨ksel Altuntas¸ • Mu¨jdat Uysal
•
Received: 22 January 2013 / Accepted: 16 January 2014 / Published online: 28 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The etiopathogenesis of thyroid cancer has not been clearly elucidated although the role of chronical inflammation and the imbalance between pro- and antiinflammatory cytokines may play a role in the etiology. The aim of the present study was to investigate whether cytokine gene polymorphisms are associated with papillary thyroid cancer (PTC), and to evaluate the relationship between genotypes and clinical/laboratory manifestation of PTC. Tumor necrosis factora (TNFa) G-308A (rs 1800629), interleukin-6 (IL-6) G-174C (rs 1800795) and IL-10 A-1082G (rs 1800896) single nucleotide polymorphisms in DNA from peripheral blood leukocytes of 190 patients with thyroid cancer and 216 healthy controls were investigated by real-time PCR combined with melting curve analysis. There was no notable risk for PTC afflicted by TNFa-308 and IL-6-174 alone. However, IL-10-1082 G allele frequency were higher among PTC patients than healthy controls (p = 0.009). The patients with IL-10-1082 GG geotype have twofold increased risk of developing thyroid cancer according to AA genotype (OR 2.07, 95 % CI 1.21–3.55). In addition, the concomitant presence of IL-10-1082 G allele (GG ? AG genotypes) together with IL-6 -174 GG genotype has a nearly twofold increased risk for thyroid cancer (OR 1.75 with 95 % CI 1.00–3.05, p = 0.049). We suggest that IL-10-1082 G allele is associated with an increased risk
E. C¸il Y. Altuntas¸ Division of Endocrinology, Department of Internal Medicine, S¸ is¸ li Etfal Research and Training Hospital, S¸ is¸ li, Istanbul, Turkey ¨ zer P. Vural (&) A. Kumral M. Kanmaz-O S. Dog˘ru-Abbasog˘lu M. Uysal Department of Biochemistry, Istanbul Faculty of Medicine, Istanbul University, C¸apa, Istanbul 34093, Turkey e-mail: [email protected]
of PTC. The polymorphism of IL-10 gene can improve our knowledge about the pathogenesis of PTC, and could provide to estimate people at the increased risk for PTC. Keywords Papilary thyroid cancer TNFa IL-6 IL10 Polymorphism
Introduction Papillary thyroid carcinoma (PTC) is the most common type of endocrine malignancy. It accounts for more than 70 % of all thyroid cancers [1]. Although the exact pathophysiologic mechanisms of PTC remain elusive, there is growing evidence that the disease is a consequence of interaction between genetic and environmental factors [2]. Cytokines are molecules that influence activation, growth, and differentiation of several target cells [3, 4]. They are pro-inflammatory and anti-inflammatory mediators that participate in the induction and effector phases of the inflammatory and immune responses, and modulate development and growth of both normal and neoplastic thyroid cells. A variety of cytokines including tumor necrosis factor a (TNFa), interleukin (IL)-1a, IL-6, IL-8, IL-10, IL-12 and interferon-c (IFNc) have been shown to be produced in thyroid follicular cells and intrathyroid inflammatory cells [5]. TNFa and IL-6 are main cytokines that play a central role in initiation and regulation of the cytokine cascade during an inflammatory response. With respect to tumorigenesis, TNFa and IL-6 are secreted by several cancer cells and are necessary to sustain cancer cell growth and recruitment of leukocytes to tumor sites [2]. On the other hand, interleukin-10 is a major immunomodulatory cytokine suppressing synthesis of pro-inflammatory cytokines as TNFa, IL-1, IL-8, and IFNc [6], and
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potentiating the up-regulation of B cell production and differentiation [7, 8]. IL-10 is an immunosuppressive cytokine which may facilitate development of cancer by supporting tumor escape from the immune response [9]. Several single nucleotide polymorphisms (SNPs) in the regulatory regions of TNFa, IL-6 and IL-10 have been implicated to modulate the risk of various malignancies, possibly by influencing the expression of the protein [10– 16]. Therefore, in the present study, we aimed to investigate whether TNFa G-308A, IL-6 G-174C and IL-10 G-1082A polymorphisms could predispose to PTC, and to evaluate the possible relationship between genotypes and clinical/biochemical characteristics of PTC.
Materials and methods One hundred and ninety patients with the diagnosis of PTC were included in this study. Informed consent was obtained from each subject. The study was approved by the Institutional Review Board at S¸ is¸ li Etfal Research and Training Hospital. PTC had been either diagnosed or suspected in these patients for clinical or epidemiological reasons, including the results of fine-needle aspiration cytology and/ or histological analysis. None of the patients had received chemotherapy, radiotherapy, or surgery before admission to our department. All of the patients underwent total or near-total thyroidectomy. The control group consisted of 216 individuals matched for age and sex. None of the controls had personal or family history of thyroid disease and goiter on examination; they had normal thyroid functions and were negative for thyroid autoantibodies. Exclusion criteria (for both study group and controls) were the existence of any comorbid cardiac, infectious, musculoskeletal or malignant disease and a recent history of operation or trauma. None of the patients and controls are consuming alcohol. Height (m) and weigh (kg) were measured after fasting, without shoes and wearing light clothes. All measurements were conducted with the patient in a standing position. Body mass index (BMI) was calculated by dividing the weight by the height squared. Blood samples were taken in the morning subsequent to an overnight (12 h) fast. Peripheral venous blood samples were collected in plain tubes for routine biochemical analysis, and in EDTA-K3 for genotype analysis. Serum triglyceride, cholesterol, HDL-, LDL- and VLDL-cholesterol measurements were performed on 1800 DPP Roche autoanalyzer (Roche Diagnostics, Mannheim, Germany). Serum TSH, free T3, free T4, anti-Tg (anti-thyroglobulin antibody) and anti-TPO (anti-thyroid peroxidase antibody) were measured on Modular EEE Electrod Elecsys Roche autoanalyzer (Roche Diagnostics, Mannheim, Germany).
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Genomic DNA was isolated from peripheral blood leukocytes by using high pure PCR template preparation kit (Roche Diagnostics GmbH, Mannheim, Germany). For detection of polymorphisms light SNIP assays were used. Light SNIP assays are based on simple probe melting curve analysis. They consist of pre-mixed primers and probes. They were developed and optimized according to NCBI ‘‘rs’’ numbers of studied SNP’s by Tib MolBiol (Berlin, Germany). The detection of polymorphisms was performed in a LightCycler (Roche Diagnostics, Mannheim, Germany). Melting curves were evaluated by two independent observers who were blinded to the analysis of the clinical data. In addition, 10 % of randomly selected samples were repeated independently to verify genotyping results and 100 % concordance was found. Differences in genotype distributions and allele frequencies in the cases and the controls were compared for statistical significance using the Chi square (v2) test. The statistical significance for deviations from Hardy–Weinberg Equilibrium (HWE) was determined using the Pearson v2-test. Odds ratios (ORs) were calculated and given with 95 % confidence intervals (CIs). Multiple logistic regression analysis was applied to evaluate the effects of genotypes of studied polymorphisms on PTC susceptibility after adjustment for gender, age, smoking status. In addition, multiple logistic regression analysis including TNFa, IL-6 and IL-10 gene polymorphisms as the exposure variable and the presence or absence of concurrent chronic lymphocytic thyroiditis as the dependent variable was performed. We examined also the association between combined polymorphisms and PTC in carriers and noncarriers of mutant allele of above mentioned genes, and computed interaction terms between TNFa and IL-10; TNFa and IL-6; IL-6 and IL-10 by using v2-test. Mann– Whitney U, Kruskal–Wallis and Spearman correlation tests were used for the evaluation of clinical and biochemical parameters. All statistical analyses were performed with SPSS 15.0 for Windows (Chicago, IL, USA). In addition the NCSS 2000 statistical package (Kaysville, Utah, USA) was used to evaluate the power analysis. We had a 96 % power to detect an effect size (W) of 0.20 using a 2 degrees of freedom (a = 0.05).
Results TNFa-308, IL-6-174 and IL-10-1082 promoter polymorphisms were analyzed in 190 patients with PTC (146 women and 44 men) and 216 unrelated healthy controls (160 women and 56 men). The clinical characteristics of controls and patients with PTC were given in Table 1. There were no significant differences among study and control groups in terms of mean age and sex distribution.
Mol Biol Rep (2014) 41:3091–3097 Table 1 Characteristics of controls and patients with papillary thyroid cancer (PTC)
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Control (n = 216)
PTC (n = 190)
Mean ± SD
46.0 ± 9.9
47.2 ± 12.02
Range
28–79
28–79
56 (25.9)
44 (23.2)
Age (years)
Gender Male, n (%) Female, n (%) Family history, n (%)
160 (74.1)
146 (76.8)
–
76 (40.0)
Smoking, n (%)
70 (32.4)
64 (33.7)
Exposure to radiation history
None
None
Ethnic differences
None
None
I
–
133 (70.0)
II
–
21 (11.1)
III
–
29 (15.2)
–
7 (3.7)
TNM stage
IV Tumor size C1 cm
–
146 (76.8)
\1 cm
–
44 (23.2)
–
26 (13.7)
Man–Whitney U test
BMI (kg/m2) (mean ± SD)
CLT
25.70 ± 4.69
25.55 ± 4.69
BMI body mass index, anti-Tg anti-thyroglobulin antibody, anti-TPO anti-thyroid peroxidase antibody, CLT concurrent chronic lymphocytic thyroiditis, TSH thyroidstimulating hormone, HDL-C high density lipoproteinecholesterol, LDL-C low density lipoproteine-cholesterol, VLDL-C very low density lipoproteine-cholesterol
Anti-TPO (IU/mL) (mean ± SD)
–
614.5 ± 398.9
* p \ 0.05
Anti-Tg (IU/mL) (mean ± SD)
–
575.2 ± 390.5
TSH (mIU/L) (mean ± SD)
1.78 ± 0.9
6.32 ± 7.65*
FreeT3 (pmol/L) (mean ± SD)
3.7 ± 0.32
2.92 ± 0.86
FreeT4 (pmol/L) (mean ± SD)
14.6 ± 2.2
13.2 ± 3.6
Cholesterol (mg/dL) (mean ± SD)
172.87 ± 37.50
201.65 ± 41.80
Triglyceride (mg/dL) (mean ± SD)
110.73 ± 53.20
123.23 ± 70.79
HDL-C (mg/dL) (mean ± SD)
60.25 ± 13.16
55.14 ± 12.29
LDL-C (mg/dL) (mean ± SD)
105.83 ± 35.41
121.35 ± 34.68
VLDL-C (mg/dL) (mean ± SD)
20.16 ± 11.98
25.75 ± 15.49
No significant differences were observed between female and male patients with respect to clinical and hormonal parameters. The genotypic and allelic distributions of TNFa-308, IL6-174 and IL-10-1082 polymorphisms for cases and controls are shown in Table 2. All genotype distributions were in accordance with the HWE among the controls. No notable differences were observed in allele or genotype frequencies for TNFa-308 and IL-6-174 genes alone (Table 2). With regard to IL-10-1082 polymorphism, the GG genotype versus AA in the PTC group differed significantly from that in the control group (p = 0.008) with an OR of 2.07 (95 % CI 1.21–3.55). We found also that in PTC patients there was a significant increase of IL-10-1082 G allele frequency (p = 0.009). To check wether the factors such as age, gender and smoking status influence the association between studied polymorphisms and PTC, we performed a multiple logistic regression analysis. The
results are shown in Table 3. Accordingly, IL-10-1082 was still significantly associated with PTC after adjustement age, gender and smoking status (p = 0.007, Table 3). The concurrent chronic lymphocytic thyroiditis was present in 26 (13.7) of PTC patients. None of the polymorphisms were found to be related to concurrent chronic lymphocytic thyroiditis (Table 4). We also investigated whether any combinations of TNFa-308, IL-6-174 and IL-10-1082 variant alleles affected the risk for PTC. The concomitant presence of IL10-1082 GG and AG genotypes (GG ? AG) together with IL-6-174 GG had a nearly twofold increased risk for thyroid canser (OR 1.75 with 95 % CI 1.00–3.05). However, the difference was at borderline significance (p = 0.049) (Table 5). In addition, the possible relation between studied polymorphisms and certain clinical phenotypes, including tumor size, stage, TSH, free T3, free T4, antiTPO, anti-Tg and lipid profile parameters were evaluated in
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Table 2 Distribution of genotypes and allele frequencies in patients with papillary thyroid cancer (PTC) and control group
Controls n (%)
PTC n (%)
OR (95 % CI)
p
TNFa-308 rs 1800629 GG
174 (80.6)
153 (80.5)
Reference
–
AG
37 (17.1)
36 (19.0)
1.11 (0.67–1.84)
0.70
AA
5 (2.3)
1 (0.5)
0.23 (0.03–1.97)
0.14
AG ? AA
42
37
1.00 (0.61–1.64)
0.99
G allele frequency
0.89
0.90
Reference
–
A allele frequency
0.11
0.10
0.91 (0.58–1.43)
0.62
IL-6 -174 rs 1800795 GG
113 (52.3)
110 (57.9)
Reference
–
CG
85 (39.4)
63 (33.2)
0.76 (0.50–1.16)
0.20
CC
18 (8.3)
17 (8.9)
0.97 (0.48–1.98)
0.93
GG ? CC
103
80
0.80 (0.54–1.18)
0.25
G allele frequency C allele frequency
0.72 0.28
0.74 0.26
Reference 0.88 (0.65–1.20)
– 0.43
AA
80 (37.0)
58 (30.5)
Reference
–
AG
100 (46.3)
78 (41.1)
1.08 (0.69–1.69)
0.75
GG
36 (16.7)
54 (28.4)
2.07 (1.21–3.55)
0.008 0.17
IL-10 -1082 rs 1800896
Each p value was based on Chi square (v2) analysis CI confidence interval, IL-6 interleukin-6, IL-10 interleukin10, OR Odds ratio, TNFa tumor necrosis factor a
AG ? GG
136
132
1.34 (0.89–2.03)
A allele frequency
0.60
0.51
Reference
–
G allele frequency
0.40
0.49
1.45 (1.10–1.92)
0.009
Table 3 Association between PTC and studied cytokine polymorphisms after adjustment for age, gender and smoking status Adjusted OR
95 % CI
Table 4 Association between concurrent chronic lymphocytic thyroiditis and studied cytokine polymorphisms in PTC patients
p value
Adjusted OR
95 % CI
p value
TNFa G -308A
1.09
0.56–2.61
0.80
TNFa G -308A
1.95
0.50–7.62
0.33
IL-6 G-174C
0.89
0.60–1.32
0.56
IL-6 G-174C
1.41
0.56–3.58
0.47
IL-10 A-1082G
1.66
1.15–2.4
0.007
IL-10 A-1082G
0.72
0.26–1.99
0.53
patients with PTC and we could not find any difference (data not shown).
Discussion We have carried out a genetic association study of TNFa G-308A, IL-6 G-174C and IL-10 G-1082A polymorphisms in PTC—the most common malignancy of thyroid gland. The frequencies of alleles and genotypes of selected polymorphisms were similar to those in previous studies in Caucasian populations as well as in our previous studies [17–20]. There is increasing evidence that chronical inflammation and the imbalance between pro- and anti-inflammatory
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cytokines are implicated in the etiopathogenesis of thyroid cancer [2]. TNFa is a pro-inflammatory cytokine produced by macrophages, monocytes, epithelial cells and lymphocytes, and induces the production of IFNc and IL-6 [21]. TNFa exhibits cytotoxic as well as cytostatic effects on thyrocytes, and further, TNFa up-regulates HLA class I expression, with subsequent damage of thyrocytes [21]. IL-6 is another pro-inflammatory cytokine mainly produced by mononuclear phagocytes under the stimulation of IL-1 and TNFa. IL-6 is also an important growth and differentiation factor for T, B lymphocytes and thyroid cells, and is able to modulate both cellular and humoral immunity [22]. Thyroid cancer cells secrete several cytokines including TNFa and IL-6 which are necessary to sustain cancer cell growth and recruit leukocytes to tumorous area [2, 4]. Moreover, TNFa
Mol Biol Rep (2014) 41:3091–3097 Table 5 Combined effect of TNFa-308, IL-6 -174 and IL-10 -1082 polymorphisms on the risk for papillary thyroid cancer (PTC)
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Controls n (%)
PTC n (%)
OR (95 % CI)
p
TNFa-308 GG combined with IL-10 -1082 AA IL-10 -1082 AG ? GG
67 (31.0)
44 (23.2)
–
108 (50.0)
106 (55.8)
1.50 (0.94–2.38)
0.09
TNFa-308 GG combined with IL-6 -174 GG
93 (43.1)
91 (47.9)
–
IL-6 -174 CG ? CC
80 (37.0)
62 (36.2)
0.80 (0.51–1.23)
0.29
TNFa-308 AG ? AA combined with IL-10 -1082 AA
14 (6.5)
14 (7.4)
–
IL-10 -1082 AG ? GG
27 (12.5)
26 (13.7)
0.96 (0.38–2.41)
0.93
21 (11.1) 16 (8.4)
– 0.66 (0.27–1.60)
0.36
TNFa-308 AG ? AA combined with IL-6 -174 GG IL-6 -174 CG ? CC
20 (9.3) 23 (10.6)
IL-6 -174 GG combined with ORs and 95 % CIs were based on Chi square (v2) analysis CI confidence interval, IL-6 interleukin-6, IL-10 interleukin10, OR Odds ratio, TNFa tumor necrosis factor a
IL-10 -1082 AA
45 (20.8)
32 (16.8)
–
IL-10 -1082 AG ? GG
66 (30.6)
82 (43.2)
1.75 (1.00–3.05)
0.049
IL-6 -174 CG ? CC combined with IL-10 -1082 AA
38 (17.6)
26 (13.7)
–
IL-10 -1082 AG ? GG
67 (31.0)
50 (26.3)
1.09 (0.59–2.03)
and IL-6 and tumor-associated leukocytes have been shown to contribute to invasive phenotype [4]. IL-10 is a pleiotropic cytokine with both anti-inflammatory and antiangiogenic functions, and may have both pro-tumoral and ati-tumoral properties. It is produced by activated T cells, B cells, monocytes, thymocytes, acting as growth factor, and stimulates humoral immune response [6–8]. Thyroid cancer cells produce high amounts of IL-10, which play an important role in many pathological features of thyroid cancer [23]. Moreover, it was shown that the presence of IL10 in the thyroid cancer environment contributes to thyroid cancer cell survival and proliferation [23]. The gene encoding TNFa is located in the short arm of chromosome 6 (6p21.3) [24], which also contains genes encoding HLA molecules. Owing to the biological effects of TNFa upon the thyroid gland and also its gene location, TNFa should be able to influence an individual’s susceptibility to thyroid cancer. Several studies have suggested a role for the importance of TNFa promoter region polymorphism in the pathogenesis of colon [25] and cervical cancers [26], and hepatocellular [27], head and neck squamous cell [28], nasopharyngeal carcinomas [29]. The gene encoding IL-6 is located in the short arm of chromosome 7 (7p21) [24]. The polymorphisms of the promoter region -174 correlate well with the development of prostate [30], colorectal [16] cancers and PTC [31], although there were some studies with conflicting results [32, 33]. With regard to IL-10 gene, it comprises 5 exons, and is located on chromosome 1 at 1q31–1q32 [34]. IL-101082 polymorphism is reported to be a genetic risk factor
0.78
for many cancer types, including PTC [35–39]. However, these results were not replicated in other studies [40, 41]. The pathogenic impact of TNFa, IL-6 and IL-10 in thyroid cancer is underscored by the effect of the functional polymorphisms in the promoter regions of TNFa, IL-6 and IL-10 genes associated with different transcriptional and expressional rates [10–16]. In our study, we hypothesize that TNFa-308, IL-6-174 and IL-10-1082 polymorphisms could influence the risk for PTC development through altered production of TNFa, IL-6 and IL-10, resulting in imbalance between pro- and anti-inflammatory cytokines with subsequent tumorigenesis. To our knowledge, the present study is the first one examining the possible relationship between TNF a G-308A and PTC. We found that polymorphisms in the promoter regions of TNFa and IL-6 genes are not significant risk factors for development of PTC alone. On the other hand, we found that IL-10-1082 G allele and GG genotype frequencies (associated with high production of IL-10) were higher in PTC patients in comparison with healthy controls. The individuals with IL10-1082 GG genotype have twofold increased risk of developing thyroid cancer according to AA genotype even after adjustment for age, gender and smoking status. Our results are in line with other studies reporting that IL-101082 polymorphism may be a genetic risk factor for PTC [35, 36]. The one of the most interesting finding of our study is the fact that concomitant presence of IL-10-1082 G allele (GG ? AG) together with IL-6 -174 GG genotype has a nearly twofold increased risk for thyroid cancer. It was
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shown that reporter gene constructs containing the G allele of the -1082 of IL-10 gene appears to have higher transcriptional activity than with the -1082 A allele [11, 12]. There are conflicting results about the impact of IL-6-174 on plasma IL-6 levels. According to some authors, C allele is related with higher plasma IL-6 levels [10, 12], others postulate that G allele is associated with increased production [13–16]. We suspect that IL-10 G allele combined with IL-6 GG genotype results in high production of IL-10 and IL-6 which may lead to accentuation of inflammatory response with immunosuppression, and propagation of tumorigenesis. However, the exact mechanisms for transcripted and expressed products of these polymorphisms interacting with other genetic and environmental factors remain to be elucidated. As a conclusion, we suggest that IL-10-1082 G allele could predispose to PTC. The polymorphism of IL-10 gene may have important implications for pathogenesis of PTC. IL-10-1082 polymorphism can be used as a marker and could provide to estimate people at the increased risk for PTC. In addition, this polymorphism may lead to development of alternative therapeutic approach. When it is identified, high-risk subjects could undergo more detailed examinations to detect obscure PTC. Moreover, individualized treatment and prevention programs can be developed to a high-risk population for PTC. Acknowledgments This work was supported by the Research Fund of Istanbul University Project No: 8763.
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