Interactions of IL-12 A and IL-12 B Polymorphisms on the Risk of Cervical Cancer in ChineseWomen

Purpose: Accumulative evidence suggests that interleukin-12 (IL-12) plays a central role in the Th1 responses and thus participates in the carcinogenesis of human papillomavirus ^ related cervical cancer.We hypothesized that potentially functional polymorphisms in IL12A and IL12B may individually and jointly contribute to cervical cancer risk. Experimental Design:We genotyped IL12A rs568408 [3¶ untranslated region (UTR) G>A] and rs2243115 (5¶UTR T>G) and IL12B rs3212227 (3¶UTR A>C) in a hospital-based study of 404 cervical cancer cases and 404 cancer-free controls. Results:The IL12A rs568408 GA/AA and IL12B rs3212227 AC/CC variant genotypes were associatedwith a significantly increased riskof cervical cancer [adjusted odds ratio,1.43; 95%confidence interval (CI), 1.06-1.93; and adjusted odds ratio, 1.30; 95% CI, 0.97-1.75, respectively], compared with their corresponding wild-type homozygotes. Moreover, a significant gene-gene interaction of these 2 loci were evident in the risk of cervical cancer, and subjects carrying variant genotypes of both loci hada1.82-fold (95%CI,1.28-2.57) increased riskof cervical cancer. In the stratified analyses, the combined genetic effect was more pronounced in patients whohad earlystage tumors or more parities. Subjects carrying rs568408 AG/AA and rs3212227 AC/CC genotypes andhaving >2 parities showed a 6.00-fold (95%CI, 2.86-12.56) elevated cervical cancer risk (P for multiplicative interaction = 0.046). Conclusion:These findings suggest that IL12A rs568408 and IL12B rs3212227may individually and jointly contribute to the riskof cervical cancer andmaymodify cervical cancer risk associated with parity, but these data need further validation. Cervical cancer is the second most common cancer among women worldwide, with an estimated 493,000 new cases and 274,000 deaths in the year 2002, and 83% of the cases had occurred in developing countries (1). Although there have been substantial declines in both incidence and mortality rates in the past 30 years, >100,000 cervical cancer cases are diagnosed in China every year (2). Epidemiologic and laboratory-based studies have identified the infection with one of 15 high-risk, or oncogenic, HPV types as a necessary but not sufficient cause of cervical cancer (3–5). The prevalence of genital HPV infection in developing countries is very high in young women, but most of the infected regress without intervention (6). Given the mounting evidence that long-term HPV infection is a prerequisite for cervical carcinogenesis (7, 8), genetic variations that influence the host primary immune response may determine the outcome of high-risk HPV infection. Recently, cell-mediated immunity has been thought as one of the important control mechanisms in the HPV-associated carcinogenesis. There is evidence that the cell-mediated immunity response of the host, both systemically and locally, are important determinants for the course of the infection (9). The cell-mediated immunity response to the NH2-terminal E6 and E7 peptides has been shown to be associated with regression of cervical intraepithelial neoplasia and loss of associated HPV infection (10). Persistently infected individuals, usually with focally high levels of HPV DNA replication, have an increased probability of progressing to a high-grade cervical intraepithelial neoplasia and an invasive carcinoma (6, 7, 11). Th type 1 (Th1) cells, which promote cell-mediated immunity responses to intracellular pathogens by cytokine production, are necessary for the clearance of HPV (12). Interleukin-12 (IL-12) is a heterodimeric proinflammatory cytokine formed by a 35,000 dalton light chain (known as p35 encoded by IL12A) and a 40,000 dalton heavy chain (known as Cancer Prevention and Susceptibility Authors’Affiliations: Laboratory of Reproductive Medicine, Nanjing Medical University, Department of Epidemiology and Biostatistics, Cancer Center of Nanjing Medical University, Department of Gynecology, The First Affiliated Hospital of Nanjing Medical University, and Nanjing Maternity and Child Health Hospital of Nanjing Medical University, Nanjing, China; Tumor Hospital of Nantong City, Nantong, China; and Department of Epidemiology,The University of TexasM. D. Anderson Cancer Center, Houston,Texas Received 7/16/08; revised 9/8/08; accepted 9/9/08. Grant support: Innovative Key Grant of Ministry of Education of China (#705023); National Key Basic Research Program Grants (2002CB512908), Key Development Program of Nanjing Medical University (07NMUZ011), and Natural Science Basic Research Program of Jiangsu Colleges (08KJB330002). Program for Changjiang Scholars and Innovative ResearchTeam in University (IRT0631); Key Development Program of Nanjing Medical University (07NMUZ011); and Natural Science Basic Research Program ofJiangsu Colleges (08KJB330002). The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints:ZhibinHuorHongbing Shen, Department of Epidemiology and Biostatistics, Cancer Center of Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China. Phone: 86-25-868-62756; Fax: 86-25-86527613; E-mail: zhibin_____hu@njmu.edu.cn or hbshen@njmu.edu.cn. F2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-1829 www.aacrjournals.org Clin Cancer Res 2009;15(1) January1, 2009 400 Research. on August 29, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from p40 encoded by IL12B), which induces the production of IFN-g, favors the differentiation of Th1 cells and forms a link between innate resistance and adaptive immunity (13). Studies showed that IL-12 had a central role in Th1 responses (14–16), and it is required for optimal Th1 cell development during the immune response to pathogens (17, 18). Besides the activity of antivirus, IL-12 is also important for the host resistance to tumors. The antitumor activity of IL-12 has been extensively reported in mouse models of cancer, where it has been shown to inhibit tumorigenesis and induce regression of established tumors (19–21). The major antitumor activities of IL12 rely on its ability to promote Th1 adaptive immunity and CTL responses (13). The two subunits of IL12, p35 and p40 encoded by IL12A and IL12B , respectively, are located on separate chromosomes (3p12-q13.2 and 5q31-33). Because of the functional relevance of IL12, several molecular epidemiologic studies have been done to investigate the association between the IL12A and IL12B polymorphisms and risk of cancers, including non– Hodgkin lymphoma (22–24), hepatocellular carcinoma (25), lung cancer (26), gastric cancer (27, 28), and others (29–32). In the present study, we hypothesized that IL12A and IL12B polymorphisms were associated with cervical cancer risk. To test this hypothesis, we did a genotyping analysis for rs2243115 (5¶UTR T>G) and rs568408 (3¶UTR G>A) in IL12A and rs3212227 (3¶UTR A>C) in IL12B in a hospital-based study of 404 cervical cancer cases and 404 age frequency–matched controls in Chinese women. Materials and Methods Study population. This hospital-based case-control study was approved by the institutional review board of Nanjing Medical University. Four hundred and four newly diagnosed, histologically confirmed cervical cancer patients were consecutively recruited between March 2006 and April 2007 from the First Affiliated Hospital of Nanjing Medical University and the Nantong Tumor Hospital, Jiangsu, China with a response rate of 93.1% (404 of 434). A total of 404 controls were randomly selected from a pool of >30,000 individuals (overall response rate, 86.3%) who participated in a community-based screening program for noninfectious diseases conducted in Jiangsu Province during the same time period as the cases were recruited. These control subjects had no self-reported cancer history and were frequencymatched to the cases on age (F5 y) and residential areas (urban and rural). The cases and control subjects were all genetically unrelated Han Chinese women. After informed consent was obtained, each subject was personally interviewed to provide information on demographic data, tobacco smoking, menstrual and reproductive history, and family history of cancer (any reported cancer in first-degree relatives). After interview, a 5-mL venous blood sample was collected from each subject and used for genotyping assays. Single nucleotide polymorphism selection and genotyping. Because no common (minor allele frequency, >0.05) nonsynonymous single nucleotide polymorphism (SNP) was found in the dbSNP database (build 129) for both IL12A and IL12B , we searched all the common potentially functional SNPs reported for Asians, which were located at 5¶-flaking regions, 5¶-, and 3¶-untranslated regions (UTR). As a result, we selected three common SNPs: rs2243115 (5¶UTR T>G) and rs568408 (3¶UTR G>A) in IL12A and rs3212227 (3¶UTR A>C) in IL12B . Genomic DNA was extracted from a leukocyte pellet by traditional proteinase K digestion and was followed by phenol-chloroform extraction and ethanol precipitation. The PCR-RFLP assay was used to detect all the three SNPs. The primers of rs3212227 were 5¶-GATATCTTTGCTGTATTTGTATAGTT-3¶ (forward) and 5¶-AATATTTAAATAGCATGAAGGC-3¶ (reverse), which generated a 118-bp fragment. The fragment was then digested by TaqI (NewEngland BioLabs) and separated on a 3% agarose gel. The variant allele rs3212227C produced 2 fragments of 92 and 26 bp, and the wild-type allele rs3212227A resulted in a single 118bp fragment. For rs2243115 and rs568408, the forward primers were introduced a mismatched A to replace C and a mismatched T to replace C, respectively, at -3 bp from the polymorphic sites to create BsenI and NdeI (New England BioLabs) restriction sites. The primers were 5¶-AGAAAAGACCTGTGAACAAAACGACT-3¶ (forward) and 5¶-AGATGGCTC