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Interleukin-6 Sequence Variants Are not Associated with Prostate Cancer Risk

¹ Center for Human Genomics, Wake Forest University School of Medicine, Winston-Salem, North Carolina; ² Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden; ³ Department of Radiation Sciences, Oncology, University of Umeå, Umeå, Sweden; ⁴ Regional Oncologic Center Stockholm-Gotland, Karolinska Sjukhuset, Stockholm, Sweden; and ⁵ Department of Urology, Johns Hopkins Medical Institutions, Baltimore, Maryland

Requests for reprints: Jianfeng Xu, Center for Human Genomics, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. Phone: 336-713-7500; Fax: 336-713-7566. E-mail: jxu{at}wfubmc.edu

	Introduction

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Prostate cancer is the most common cancer in the United States and in western Europe. Although genetic susceptibility seems to play a stronger role in prostate cancer risk than in any other common cancer, including breast and colon cancer, the largely unknown genetic mechanism of prostate cancer is likely to be complex (1). Recently, the role of chronic inflammation in the initiation and progression of various cancers, including prostate cancer, has gained increasing attention (2).

Many genes, including interleukin-6 (IL-6), are involved in the regulation of inflammation. IL-6, a pleiotropic cytokine with critical roles in inflammation and immune responses, also acts as a growth and differentiation factor for prostate cancer cells (3). Increased levels of IL-6 have been observed in patients with hormone-refractory prostate cancer (3). Moreover, IL-6 may contribute to the initiation, or facilitate the transition, of prostate cancer cells from a hormone-dependent to a hormone-independent state (4). Although multiple pieces of evidence support a potential role of IL-6 in prostate cancer initiation and progression, the overall effect of IL-6 may be complicated due to its dual properties, both as a suppressor of inflammation and as a growth factor. Although the complexity of IL-6 presents challenges, one effective approach to assess the overall role of this gene is a genetic association study. If IL-6 plays a role in prostate cancer susceptibility and progression, sequence variants of the gene should have different frequencies between prostate cancer cases and controls and between patients with localized and advanced disease.

To test for genetic association, we systematically genotyped multiple IL-6 sequence variants in a large population-based prostate cancer case-control study in Sweden. Several strengths of this study population make it ideal to assess the genetic association, including a large number of participants, a low degree of genetic heterogeneity in Sweden, and a significant portion of cases having advanced disease due to the lack of widespread prostate-specific antigen screening.

	Subjects and Methods

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Participants
We used a large-scale population-based case-control population in Sweden, named Cancer of the Prostate in Sweden. The case participants were recruited from four of six regional cancer registries that cover the entire population of Sweden. Reporting of newly diagnosed cancer cases to the registries is required by law for both the attending physician and the pathologist.

The source–person–time in the Cancer of the Prostate in Sweden study is composed of men living in the area of rebro and the northern part of Sweden (Västernorrland, Jämtland, Västerbotten, and Norrbotten) from January 1 as well as men living in the area of Västmanland, Södermanland, Gävleborg, Dalarna, Värmland, and Uppland from July 1, 2001 to September 2002 (except for Jämtland and the county of Lycksele in Västerbotten where the source person time ended March 1, 2002). The source–person–time was divided into two age-specific study bases. The first study base included men ages 35 to 65 years living in all of the regions mentioned above. The second study base included men ages 66 to 79 years living in the area of Örebro, Västmanland, Södermanland, and the northern part of Sweden.

The inclusion criterion for cases in Cancer Prostate in Sweden study was pathologic or cytologic verified adenocarcinoma of the prostate (International Classification of Diseases for Oncology C61). In total, 1,961 prostate cancer cases were invited to participate in the Cancer Prostate in Sweden study, and of those, 1,444 (73.6%) agreed to participate by donating a blood sample and completing the questionnaire. DNA samples are available for 1,383 cases. Because clinical data are not included in the Cancer Registry, we obtained this information from the National Prostate Cancer Registry (http://www.roc.se). For 95.3% of the cases, we were able to obtain and link their clinical information, including tumor-node-metastasis stage, Gleason grade, prostate-specific antigen level at the time of diagnosis, means of diagnosis, and primary treatment. The cases were thereafter classified as either localized (T1-T2N0M0 or grade I-II or Gleason sum 2-7) or advanced (T3/T4 or N+ or M+ or grade III or Gleason sum 8-10).

Control subjects were randomly selected from the updated Swedish Population Registry and frequency matched according to the age (within 5 years) and geographic origin of the cases. Of the 1,697 randomly selected controls that were invited for participation in the study, 866 (52.0%) agreed to participate and to complete a blood draw and questionnaire. DNA samples are available for 780 control subjects. The cancer status of the controls was verified by linking them to the National Prostate Cancer Registry; eight controls that turned out to have a prostate cancer diagnosis prior to inclusion were thereby excluded from analysis. The same questionnaire and blood samples were obtained from cases and controls. Written informed consent was obtained from each subject and the study was approved by the Ethical Committee both at Karolinska Institutet and at Umeå University.

Genotyping
The IL-6 gene is 5 kb at 7p21 and is composed of five exons. Our aim was to choose single nucleotide polymorphisms (SNP) with a frequency of at least 5% at a resolution of 1 SNP/kb across the target genomic region of IL-6, including 2 kb of the promoter, all exons, introns, and the 3' untranslated region. We also included additional functional and coding SNPs. These SNPs were identified through the public database (IIPGA and National Center for Biotechnology Information dbSNP). Initially, 14 SNPs were selected, including 2 reported nonsynonymous changes (P32S and D162V). After genotyping these 14 SNPs among 96 randomly selected samples from control men, we then excluded 3 SNPs that were monomorphic (including P32S). An additional SNP (1826G/T) was added to maintain a density of 1 SNP/kb. Haplotypes of these SNPs were inferred using the PHASE computer program (5). Haplotype tagging SNPs that capture at least 95% of inferred haplotypes were selected using the htSNP2 computer program (http://www-gene.cimr.cam.ac.uk/clayton/software/stata). Six haplotype tagging SNPs were identified and then genotyped in all 1,383 cases and 780 controls.

Primers for the six SNPs were designed using Spectra Design computer software. The primer information and PCR conditions are available at the author's Web site (http://www.wfubmc.edu/genomics). Genotyping was done using the MassARRAY system (Sequenom, San Diego, CA).

Statistical Analysis
Tests of Hardy-Weinberg equilibrium and pair-wise linkage disequilibrium for each of the six SNPs were carried out using an exact test as implemented in the GDA computer software (6). Allele frequency differences between the two groups were tested for each SNP using the ² test (1 df). Genotype frequency differences were also tested using the ² test (2 df). Both tests were done using the SAS/Genetics computer program. Odds ratios of prostate cancer for the variant allele carriers (homozygous or heterozygous) versus homozygous common allele carriers were estimated for each SNP using unconditional logistic regression and adjusted for age and geographic regions. Haplotype frequency differences were tested using the Haplo.score computer program (7).

	Results

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All six SNPs were in Hardy-Weinberg equilibrium in the case and control groups (all P > 0.27). These SNPs were in strong linkage disequilibrium, as most of the pair-wise D' estimates were 1.0, with the lowest one at 0.96.

The distributions of all six SNPs among prostate cancer cases and controls are shown in Table 1. We observed no significant differences in the allele and genotype frequencies between cases and controls (data not shown). No significant increased or decreased risk for prostate cancer was detected among men carrying variant genotypes (heterozygous or homozygous) for any of the SNPs (Table 1). Haplotype analysis of the six SNPs revealed four major haplotypes, including AGCCGA, GGGGAA, GCGGAA, and GGGGGA, of SNPs –661G/A, –636G/C, –237G/C, 1027G/C, 3268G/A, and 4157A/T (D162V). However, we observed no significant difference in the frequency of these four haplotypes between cases and controls.

	Statistical Power, Study Limitations, and Conclusions

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Because of the large number of subjects in this study, we have excellent power to detect sequence variants even if they have relatively low prevalence in the population and confer only a moderate excess risk to prostate cancer susceptibility or to prostate cancer progression. For example, we have >80% power to detect risk genotypes present in 10% of the population that confer an odds ratio of 1.47 to prostate cancer susceptibility (at a significance level of 5%, two-sided test). We also have >80% power to detect risk genotypes present in 10% of the population that confer an odds ratio of 1.58 to the progression of prostate cancer (at a significance level of 5%, two-sided test). In addition, because we have genotyped six haplotype tagging SNPs across the gene and did haplotype analysis, it is unlikely that we missed any common risk variant, even if the potential risk SNP was not directly genotyped. Therefore, we conclude that sequence variants in the IL-6 gene are unlikely to play a major role in prostate cancer susceptibility and prostate cancer progression.

	Acknowledgments

 
We thank all the study participants in the Cancer Prostate in Sweden study; Ulrika Lund for coordinating the study at Karolinska Institute; all urologists who recruited their patients to this study and provided clinical data to the National Prostate Cancer Registry; Karin Andersson, Susan Okhravi-Lindh, Gabriella Thorén-Berglund, and Margareta åswärd at the regional cancer registries in Umeå, Uppsala, Stockholm-Gotland, and Lindköping; and Sören Holmgren and the personnel at the Medical Biobank in Umeå for skillfully handling the blood samples.

	Footnotes

 
Grant support: Swedish Cancer Foundation and Spear grant from the Umeå University Hospital, Umeå, Sweden. This study was also partially funded by Center for Human Genomics at Wake Forest University School of Medicine.

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10/20/03; accepted 12/ 3/03.

	References

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1. Nelson WG, De Marzo AM, Isaacs WB. Prostate cancer. N Engl J Med 2003;349:366–81.[Free Full Text]
2. Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow? Lancet 2001;357:539–45.[CrossRef][Medline]
3. Smith PC, Hobisch A, Lin DL, Culig Z, Keller ET. Interleukin-6 and prostate cancer progression. Cytokine Growth Factor Rev 2001;12:33–40.[CrossRef][Medline]
4. Lee SO, Lou W, Hou M, de Miguel F, Gerber L, Gao AC. Interleukin-6 promotes androgen-independent growth in LNCaP human prostate cancer cells. Clin Cancer Res 2003;9:370–6.[Abstract/Free Full Text]
5. Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 2001;68:978–89.[CrossRef][Medline]
6. Weir BS. Genetic data analysis II: methods for discrete population genetic data. Sunderland (MA): Sinauer Association; 1996.
7. Schaid DJ, Rowland CM, Tines DE, Jacobson RM, Poland GA. Score tests for association between traits and haplotypes when linkage phase is ambiguous. Am J Hum Genet 2002;70:425–34.[CrossRef][Medline]

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