Abstract
Mounting evidence supports the notion that inflammatory processes play a role in carcinogenesis, and interleukin-10 (IL10) is an important inflammatory cytokine. This study aimed to evaluate the contribution of IL10 A-1082G (rs1800896), T-819C (rs3021097) and A-592C (rs1800872) genotypes to the risk of childhood acute lymphoblastic leukemia (ALL) in Taiwan. Associations of these IL10 polymorphic genotypes with ALL risk were analyzed in 266 patients with childhood ALL patients and 266 non-cancer healthy controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. The results showed that CC genotype carriers at IL10 T-819C were at lower risk for childhood ALL (odds ratio=0.33, 95% confidence interval=0.16-0.68). On the contrary, AC and CC genotype carriers at IL10 A-592C were at higher risk for childhood ALL (odds ratio=1.73 and 6.34, 95% confidence interval=1.19-2.51 and 3.16-12.72, respectively). There was no difference in the distribution of A-1082G genotypes between childhood ALL and control groups. The genotypes at IL10 T-819C and A-592C may serve as predictive biomarkers for childhood ALL in Taiwan.
Acute lymphoblastic leukemia (ALL), a type of pediatric leukemia, accounts for 25-30% of all childhood malignancies (1). Worldwide, the annual incidence of childhood ALL is approximately 10 cases per 100,000, with a peak incidence occurring at approximately 2 to 5 years of age (2). Although the pathological, immunophenotypic and diagnostic features of ALL are well documented, the genetic contribution to ALL has not been fully clarified (1). Accumulating evidence suggests that genetic factors may play a significant role in the development of childhood ALL. For instance, inherited genetic human diseases, such as Down syndrome and Fanconi anemia, have been associated with enhanced ALL risk (3, 4). Additionally, genetic mutations in several oncogenes and tumor-suppressor genes, such as p53, neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS), and PHD finger protein 6 (PHF6), have frequently been identified in patients with ALL (5); and finally, only a small fraction of children who are exposed to ionic radiation developed ALL, indicating individual susceptibility.
Interleukin-10 (IL10), also known as human cytokine synthesis inhibitory factor, is produced mainly by monocytes and lymphocytes. The genetic polymorphisms found in the regulatory sites, especially the promoter region of IL10 gene, are believed to affect the expression of IL10 protein and possibly be associated with leukemia susceptibility and prognosis. In literature, Yao et al. found the T allele of IL10 T-819C was not only associated with higher risk of AML, but also synergistically acts with the A allele of IL10 A-592C, contributing to enhanced expression of IL10 at mRNA level among the patients (6). In 2015, Fei and colleagues confirmed that the genotypes of IL10 T-819C and A-592C have synergistic effects on determining personal AML susceptibility (7). However, both reports investigated the contribution of IL10 genotypes to adult AML, not ALL, and the investigated patients were all adults. In addition, the sample size of their cases were rather small (less than 200) and their findings need to be validated in other populations as well.
To validate whether IL10 genotype is a risk factor for childhood leukemia, we sought to investigate the association of polymorphisms in the promoter region of IL10 A-1082G, T-819C and A-592C with childhood leukemia in a Taiwanese population.
Materials and Methods
Study population and sample collection. Two hundred and sixty-six patients diagnosed with childhood ALL (all under 18 years of age) were recruited during 2005-2010 from the General Surgery Outpatient Clinics within the Pediatric Departments at China Medical University Hospital and the National Taiwan University Hospital, Taiwan, Republic of China. All clinical characteristics of these individuals, including their histological details, were identified and recorded by expert surgeons. All participants completed a questionnaire with the assistance and approval of their parents and provided peripheral blood samples. An equal number of age-matched individuals without cancer were selected for use as a control group following initial random sampling from the Health Examination Cohort. Our study was approved by The Institutional Review Board of China Medical University Hospital, and all participants provided written informed consent. Selected characteristics of all the participants are provided in Table I.
Genotyping assays. Genomic DNA was prepared from peripheral blood leucocytes using a QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan) and further processed according to previous methods (8-10). Briefly, the polymerase chain reaction (PCR) cycling conditions for IL10 genotyping included the following: one cycle at 94°C for 5 min, 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, along with a final extension at 72°C for 10 min as described in our previous publications (11-14). The success rate of PCR-restriction fragment length polymorphisms (RFLP) is 100%, and the genotypes of 5% of the participants in both the control and patient groups were analyzed by direct sequencing PCR (Genomics BioSci & Tech Co., Taipei, Taiwan) and the consistency was 100% between PCR-RFLP and direct sequencing. Pairs of PCR primer sequences and the restriction enzyme for each DNA product are all listed in Table II.
Statistical analyses. To ensure that the controls used were representative of the general population and to exclude the possibility of genotyping error, the deviation of the genotypic frequencies of IL10 SNPs in the controls from those expected under the Hardy–Weinberg equilibrium was assessed using the goodness-of-fit test. All of them fit Hardy–Weinberg equilibrium (p>0.05). Pearson's Chi-square test or Fisher's exact test (when the expected number in any cell was less than five) was used to compare the distribution of the IL10 genotypes between cases and controls. Leukemia risk associated with the genotypes was estimated as odds ratio (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression. Data were recognized as significant when the statistical p-value was less than 0.05.
Results
The distributions of age and gender among the patients with ALL and the controls are shown in Table I. The age and gender of the patients and controls were all well-matched and none of the differences between the groups was statistically significant (p>0.05) (Table I).
The genotypic frequencies of IL10 promoter A-1082G (rs1800896), T-819C (rs3021097) and A-592C (rs1800872) polymorphisms among the patients and the controls are shown in Table III. Using wild-type -819T as the reference group, there was an obvious association between homozygosity for - 819C of IL10 and childhood ALL risk (OR=0.33, p=0.0034). Combination of the homozygotes and heterozygotes for the C allele (TC + CC) showed that the C allele at IL10 T-819C conferred a 0.65-fold risk for childhood ALL (p=0.0175) (Table III). Using wild-type -592A as the reference group, heterozygosity and homozygosity of -592C for IL10 conferred an increased risk of childhood ALL (OR=1.73 and 6.34, p=0.0053 and 0.0001, respectively). Combination of the homozygotes and heterozygotes for the C allele (AC + CC) showed that the C allele at IL10 T-592C conferred an increased risk for childhood ALL of 1.49-fold (p=0.0001) (Table III). On the contrary, neither hetero- nor homozygosity for -1082G of IL10 seemed to be risky for childhood ALL (Table III). Overall, among the three IL10 polymorphisms investigated, -819C of IL10 seems to be protective, while - 592C seems to be a genotypic marker indicating increased risk for childhood ALL.
The distributions of allelic frequencies for IL10 promoter A-1082G (rs1800896), T-819C (rs3021097) and A-592C (rs1800872) polymorphisms among the patients and the controls are shown in Table IV. Consistent with the findings in Table III, C alleles at IL10 T-819C and A-592C were significantly associated with childhood ALL risk (p=0.0013 and 1.01×10−9, respectively). In contrast, allele A or G at IL10 A-1082G were not differently distributed between the childhood ALL and control groups (p>0.05).
Discussion
IL10, which is produced by activated T-cells, monocytes, B-cells and thymocytes, was found to be an important immunoregulatory cytokine playing a modulating role in activating and suppressing immunoresponses (15). IL10 not only regulates the differentiation and proliferation of several types of immune cells, but has both tumor-promoting and tumor-inhibiting properties, and thus may play a critical role in tumor development and metastasis (16). Polymorphisms in IL10, especially those in the promoter region, can alter the function of this cytokine, altering the downstream signaling and cellular behaviors, and greatly regulate the development of human disorders. In the literature, the genotype at IL10 A-1082G was associated with osteosarcoma (17), lymphoma (18), gastric cancer (13, 19, 20), oral cancer (14), nasopharyngeal carcinoma (21) and papillary thyroid cancer (22). In addition, the genotype at IL10 A-1082G has been implicated in development of chronic lymphoid leukemia among Russians, especially in the late stages (23). For IL10 T-819C, genotype was associated with lung cancer (12, 24), gastric cancer (25), and adult leukemia (6, 7), while genotype for IL10 A-592C was associated with lung (24), gastric (26, 27) and esophageal (28) cancer, and adult leukemia (6). Little is known about the contribution of IL10 genotypes to childhood leukemia.
The present study investigated the role of genotypes of IL10 gene, a gene which has not been reported to be associated with childhood leukemia risk. Among the three IL10 promoter polymorphisms examined in this study, T-819C (rs3021097) and A-592C (rs1800872), but not A-1082G (rs1800896), were found to be associated with childhood ALL (Tables III and IV). This is the first study to examine the contribution of IL10 genotypes to childhood cancer. The sample size of the patient group is relatively large among childhood cancer studies, and concise data were analyzed without any modification, strengthening the accuracy and reliability of the current study.
Similarly among adults in a Han population, IL10 T-819C and A-592C genotypes were found to be associated with adult AML (6, 7). However, there are many differences between ALL and AML, and more differences in the development for childhood and adult leukemia. From the molecular perspective, polymorphisms at IL10 T-819C and A-592C may determine subtle differences in the capacity of IL10 to alter the functions of IL10 itself and its downstream signaling and cellular behaviors, contributing to individual susceptibilities to ALL and AML among children and adults. It was reported that the T allele of IL10 T-819C was not only associated with higher risk of AML, but also synergistically acts with A allele of IL10 A-592C, contributing to enhanced expression of IL10 at the mRNA level among patients (7). In the future, the joint effects of IL10 genotypes with other genes need to be further investigated in other populations.
In conclusion, to our knowledge, this is the first report to investigate the association between IL10 gene polymorphisms and childhood leukemia. Our findings suggest that genotypes at IL10 promoter T-819C (rs3021097) and A-592C (rs1800872) are potentially predictive of risk for childhood leukemia.
Acknowledgements
The Authors are grateful to Shin-Ting Lee and Shiou-Ting Yen for their technical assistance. All the participants in this study are appreciated. This study was partially supported by research grant from Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW105-TDU-B-212-133019).
Footnotes
↵* These Authors contributed equally to this study.
Conflict of Interest
The Authors declare no conflicts of interest.
- Received December 31, 2015.
- Revision received February 22, 2016.
- Accepted February 23, 2016.
- Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved