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. Author manuscript; available in PMC: 2014 Jul 1.
Published in final edited form as: Int J Cancer. 2013 Feb 12;133(1):10.1002/ijc.28000. doi: 10.1002/ijc.28000

Nucleotide variation in IL10 and IL12 and their receptors and cervical and vulvar cancer risk: a hybrid case-parent triad and case-control study

Shehnaz K Hussain 1,2, Margaret M Madeleine 3,4, Lisa G Johnson 3, Qin Du 5, Denise A Galloway 6, Janet R Daling 3,4, Mari Malkki 5, Effie W Petersdorf 5, Stephen M Schwartz 3,4
PMCID: PMC3833442  NIHMSID: NIHMS435314  PMID: 23280621

Abstract

Given the important role of cell mediated immunity in viral clearance and control of premalignant lesions, we hypothesize that variation in the IL12/IL10 cytokine and cytokine receptor genes may influence cervical and vulvar cancer risk. We evaluated 76 tagSNPs from 7 candidate genes (IL10, IL12A, IL12B, IL10RA, IL10RB, IL12RB1, and IL12RB2) in case-parent sets (n=43 cervical squamous cell carcinoma (SCC), n=96 cervical adenocarcinoma, n=53 vulvar SCC), additional cases (n=356 cervical SCC, n=406 cervical adenocarcinoma, and n=473 vulvar SCC) and population based controls (1,111). We calculated log-additive odds ratios (ORs) and 95% confidence intervals (CIs) for the association between tagSNP and cancer risk using a pseudo-likelihood based method which combined genotype information on cases, parents, and population controls. After correction for multiple comparisons, we identified several statistically significant SNP associations. Cervical SCC risk was associated with the minor alleles of the IL10RA rs9610 3′ UTR SNP (OR=1.76, 95% CI= 1.15–2.68) and two synonymous IL12RB2 SNPs (rs4297265, OR=0.46, 95% CI=0.26–0.82; rs2229546, OR=0.43, 95% CI=0.21–0.87). Cervical adenocarcinoma risk was associated with the minor alleles of the IL10RA rs4252314 intronic SNP (OR=2.23, 95% CI = 1.26–3.96) and IL12RB1 rs11575934 non-synonymous SNP (OR=1.51, 95% CI=1.12–2.05). Finally, the minor allele of the IL12B rs3181224 3′ UTR SNP was associated with a reduced risk of vulvar SCC (OR=0.30, 95% CI=0.12–0.74). These results raise the possibility that a shift in the balance of the immune response due to genetic variants in key cytokine genes could influence the development of cervical and vulvar cancer.

Keywords: Cervical cancer, vulvar cancer, case-parent, interleukin 12, interleukin 10

Introduction

Over the last decade, there has been substantial progress in our understanding of the etiology and prevention of anogenital cancers. Despite this fact, these cancers are still responsible for considerable mortality and morbidity worldwide.1 Furthermore, since the 1970’s, the incidence of invasive cervical adenocarcinoma and vulvar squamous cell carcinoma has been on the rise in the United States.2, 3 Persistent oncogenic human papillomavirus (HPV) infection is a necessary cause of all cervical cancers and a large subset of vulvar cancers.4 Those vulvar cancers that are etiologically linked to HPV primarily affect younger women and are associated with nonkeratinizing basaloid or warty vulvar intraepithelialneoplasia. These neoplasias are very similar to cervical squamous intraepithelialneoplasia, and are associated with similar HPV types and co-factors.5, 6

The immune response is important in cervical and vulvar cancer pathogenesis. Although neutralizing antibodies have a pivotal role in the prevention of initial HPV infection, cell-mediated immune response is important for viral clearance and control of premalignant lesions. The homeostatic balancing of key T helper (Th) cytokines controls lymphocyte development and differentiation, influencing the type of cell mediated immune response. Th1 cytokines, such as IL12, enhance T-cell mediated immunity, while IL10 (a Th2 cytokine) down-regulates the expression of IL12 and other Th1cytokines. There is a large body of literature showing a shift in circulating and cervical cytokine profiles from Th1 to Th2, specifically decreased IL12 and increased IL10, with HPV infection/persistence and increasing grade of cervical disease.7–11 A growing body of literature also highlights the important role of immunogenetics in HPV-related carcinogenesis.12–15

Studies show that IL10 and IL12 cytokine expression is partly genetically determined.16–19 However, whether IL10 and IL12 SNPs are associated with the risk of HPV associated anogenital cancers has not been adequately addressed. Thus, we investigated the association of common variation in these genes and their receptors with cervical and vulvar cancer risk in a population-based, hybrid case-control and case-parent study.

Materials and Methods

Study design

We designed a candidate gene association study combining case-parent triad and traditional case-control approaches. Given the genotypes of biological parents, the distribution of genotypes for a polymorphism among probands should conform to Mendelian expectation if there is no association between the polymorphisms and disease risk. Evidence for a genetic association is inferred from a non-Mendelian distortion in the probands’ genotype frequencies. The precision of this association is enhanced by including cases without parents (non-proband cases) and unrelated controls.20 Homogeneity of the ORs in the case-parent controls and case-unrelated controls is an assumption of this method.

Study population

This study was conducted within a large population-based case-control study of host and environmental factors related to anogenital cancer risk.5, 21 Briefly, the case-control study included 18 to 74 year-old Seattle metropolitan residents, with incident invasive squamous cell carcinoma (SCC) of the cervix (ICD-O 8010–8081), invasive or in situ adenocarcinoma (AC) of the cervix (ICD-O 8140–8480) and invasive or in situ SCC of the vulva (ICD-O 8010, 8070–8077, 8081) diagnosed between January 1986 and June 1998, or between January 2000 and December 2004. Cases were ascertained through the Cancer Surveillance System, a population-based registry that is a part of the National Cancer Institute’s Surveillance, Epidemiology, and End Results program.22 Controls were identified and selected using a one-step modification of the Waksberg-Mitofsky method of random-digit telephone dialing and frequency matched to cases on age and county of residence.23

Probands are a subset of the cases described in the previous paragraph. Eligible probands were cases diagnosed at ages 18 to 49 between January 2001 and December 2004 for cervical cancer, and January 2001 and December 2003 for vulvar cancer. Ages and diagnosis dates were restricted in an attempt to improve the likelihood that parents were alive and thus available for participation when parent recruitment began in May, 2003. A biological parent of a proband was eligible for this study if his or her daughter provided consent to contact him/her and parental contact information. Parents were not eligible if they resided outside of the United States or did not speak English.

Data and specimen collection

Standardized interviews were administered to collect information on demographic and other characteristics with a known or suspected relationship to anogenital cancer. Venous blood samples were collected into EDTA-containing tubes to provide buffy coats from which DNA could be extracted for genetic polymorphism assays, and stored at −80°C. A small proportion of study participants (3%) preferred to donate a buccal rinse sample in place of blood.

Response proportions

Among the 1,189 eligible cervical SCC patients identified for the case-control study, 744 (62.6%) were interviewed and among those interviewed 674 (90.6%) provided a specimen from which DNA could be obtained. For cervical adenocarcinoma (in situ and invasive), among the 805 eligible patients, 553 (68.7%) were interviewed and 520 (94.0%) provided a specimen. A similar proportion, 807 of the 1194 eligible vulvar SCC patients (67.6%) was interviewed, and DNA was collected from 73.4% of participating patients. Among the eligible population-based control women who were approached, 67 % agreed to participate in the interview, and 83.9% (N=1,372) of those interviewed donated a blood sample from which DNA could be obtained. Sixty-five % of eligible probands who provided a specimen provided consent to contact at least one biological parent. Among the 337 parents for whom we had complete contact information and who resided within the U.S., 1 (0.3%) died prior to contact, 31 (9.2%) refused to participate, 6 (1.8%) consented to participate but did not submit a specimen, and 299 (88.7%) consented to participate and provided a blood or buccal specimen. DNA was successfully isolated from samples belonging to 295 of these 299 parents.

Women whose self-reported race was anything other than White were excluded from the current study because they comprised less than 10% of the study population, precluding meaningful sub-group analyses stratified by race while increasing the possibility of bias due to population stratification if combined in an analysis without genomic adjustment. All tables present data and results from analysis of the White study population only.

Candidate gene tagSNP selection & genotyping

TagSNPs were selected within each candidate gene including all exons, introns, and approximately 5 kb of 3′ and 5′ flanking sequence with an attempt to cover all known common nucleotide variation within those regions. We included genes coding for all subunits of IL10 (IL10), IL12 (IL12A, IL12B), the IL10 receptor (IL10RA, IL10RB), and the IL12 receptor (IL12RB1, IL12RB2). Information on nucleotide variation for these genes was obtained from the European descent population in the SeattleSNPs Variation Discovery Resource and the International HapMap Project.24, 25 To select tagSNPs, a pairwise r2 ≥ 0.80 was used to delineate groups of highly correlated SNPs with a minor allele frequency of 5% or greater, primarily using Tagger and Snagger.26, 27 Generally one SNP per group of correlated SNPs was selected for genotyping, however, at times redundant SNPs were selected to ensure adequate gene coverage in case of SNP genotyping failure. Some additional SNPS were also selected with MAF < 5% if they were located in an exon or of interest based on previous literature.

Genomic DNA was extracted from buffy coat aliquots from blood samples, or cell pellets from buccal samples, using a modified phenol-chloroform method. SNP genotyping was performed primarily using the Illumina GoldenGate® custom assay. A smaller proportion of SNPs were genotyped using TaqMan® Assays (Applied Biosystems Incorporated) or KASPar SNP Genotyping Assays (an allele-specific PCR system, Kbiosciences). Several SNPs were genotyped using more than one of these assays for quality control purposes. Call rates for passing SNPs were very high for all three assays: GoldenGate assay (mean=0.998, range=0.977 to 1.00), TaqMan (mean=0.976, range=0.958 to 0.987), and KASPar (mean=0.989, range=0.984 to 0.994), and the concordance between assays was very high (0.987–0.999). The final set of tagSNPs included in this study was selected based on successful genotyping on ≥90% of the samples and genotype concordance within pairs of duplicate samples ≥90%. After excluding SNPs failing our quality control requirements and monomorphic SNPs, genotype data were available on 100 tagSNPs in our candidate genes: 73 genotyped using the GoldenGate assay, 15 genotyped using the TaqMan assays, and 12 genotyped using the KASPar assay. We further screened our genotyped SNPs to identify pairs or groups of SNPs that were in high linkage disequilibrium (LD, r2 ≥0.80) in our study population. We selected only one SNP per group of SNPs in high LD to present in our tables, for a total of 76 tagSNPs.

Specimens from study participants, including 7% replicate DNA aliquots, were distributed randomly throughout the reaction plates. Laboratory technicians were blinded to all research information about the samples, including the identities of the quality control replicate aliquots. Analysis of our replicate samples revealed a low discordance proportion ranging from 0 to 0.4%. A total of 8 cervical cancer cases, 1 vulvar cancer case, and 8 population controls were dropped due to genotyping failure for the majority of SNP.

HPV typing

Tissue blocks from 265 out of 399 (66.4%) cervical SCC cases, 346 out of 502 (68.9%) cervical adenocarcinoma cases, and 400 out of 526 (76.0%) vulvar SCC cases were obtained for HPV typing. PCR-RFLP methods were used for typing, with MY09/MY11 L1 consensus and HPV16 and HPV18 E6 type-specific primers, and coamplification of 236 or 536 bp fragments of β-globin to test for sample integrity.28

Data analysis

Case-parent sets found to violate Mendelian transmission for more than two SNPs were excluded from all analyses. TagSNP genotypes were tested for consistency with Hardy-Weinberg equilibrium (HWE) within the non-parental control sample using a Pearson’s χ2 p value.

To obtain odds ratio (ORs) and 95% confidence intervals (CIs) for the association of each tagSNP with cervical and vulvar cancer risk on a log-additive scale, we used a pseudo-likelihood based method that utilized genotype information on probands, parents, non-proband cases, and population controls implemented in the software package Hybrid.29 The cervical cancer control group was restricted to women without prior hysterectomy, thus reflecting the population from which the cases arose. In families where only one parent was available (dyads), missing parental genotypes were imputed by Hybrid through a combination of the EM algorithm and multiple imputation methods. To prioritize our results for interpretation and future studies, we chose to implement the Holm procedure, a sequentially rejective adaptation to the Bonferroni method, to correct for multiple testing.30 Holm-adjusted p-values are included in Table 3 corresponding to associations where uncorrected p-values are less than or equal to 0.05. Associations with uncorrected p-values less than or equal to 0.05 were considered nominally significant, while associations with Holm-adjusted p-values less than 0.05 were considered statistically significant. ORs for tagSNPs with nominally or statistically significant associations utilizing all of the genotype information described in the previous paragraph were also calculated separately for case-parent and case-unrelated controls in Hybrid (Supplementary Table).

Table 3.

IL10, IL12A, IL12B, IL10RA, IL10RB, IL12RB1, and IL12RB2 tagSNP associations with cervical and vulvar cancer, Seattle-Puget Sound region, 1986–2004

Gene SNP Cervical squamous cell carcinoma
Cervical adenocarcinoma
Vulvar squamous cell carcinoma
MAF in population controls
16 triads, 27 dyads, 357 unrelated cases, and 861 population controls
55 triads, 41 dyads, 406 unrelated cases, and 861 population controls
17 triads, 36 dyads, 474 unrelated cases, and 1,111 population controls
MAF1 (cases) Odds ratio 95% CI Holm-adjusted p-value MAF (cases) Odds ratio 95% CI Holm-adjusted p-value MAF (cases) Odds ratio 95% CI Holm-adjusted p-value
IL10 rs3024505 0.14 0.70 (0.37,1.36) 0.14 1.21 (0.8,1.82) 0.16 0.57 (0.27,1.17) 0.15
rs3024498 0.26 1.31 (0.82,2.08) 0.26 0.90 (0.63,1.27) 0.25 0.89 (0.54,1.45) 0.27
rs3024509 0.07 1.09 (0.48,2.48) 0.07 0.78 (0.4,1.52) 0.06 1.64 (0.84,3.19) 0.07
rs3021094 0.07 0.45 (0.15,1.35) 0.08 1.11 (0.64,1.9) 0.08 1.28 (0.66,2.48) 0.08
rs1800871 0.23 1.11 (0.65,1.89) 0.24 1.10 (0.79,1.53) 0.25 1.62 (1.04,2.54) 0.060 0.23
rs1800894 0.04 0.86 (0.27,2.79) 0.03 0.51 (0.17,1.58) 0.03 1.16 (0.42,3.25) 0.04
rs1800896 0.46 1.22 (0.79,1.89) 0.48 1.00 (0.75,1.33) 0.46 0.91 (0.58,1.43) 0.50
rs1800890 0.38 1.26 (0.81,1.96) 0.36 1.10 (0.82,1.47) 0.37 0.78 (0.53,1.15) 0.39
IL10RA rs4252301 0.09 0.57 (0.03,9.55) 0.11 1.05 (0.25,4.34) 0.11 NE 0.11
rs4252253 0.33 0.38 (0.16,0.91) 0.100 0.31 1.13 (0.74,1.71) 0.38 1.02 (0.58,1.79) 0.33
rs2256111 0.16 0.67 (0.4,1.12) 0.15 1.21 (0.88,1.68) 0.19 0.95 (0.61,1.48) 0.17
rs3135932 0.10 1.11 (0.56,2.17) 0.11 1.21 (0.8,1.83) 0.11 0.79 (0.41,1.51) 0.11
rs4252279 0.04 0.64 (0.25,1.61) 0.04 1.02 (0.61,1.7) 0.04 0.95 (0.45,1.99) 0.03
rs4252314 0.02 1.57 (0.63,3.93) 0.03 2.23 (1.26,3.96) 0.002 0.02 1.11 (0.33,3.72) 0.03
rs4252286 0.31 0.52 (0.1,2.74) 0.31 0.71 (0.24,2.08) 0.36 0.28 (0.03,2.59) 0.32
rs2229113 0.05 1.01 (0.62,1.67) 0.05 1.16 (0.84,1.6) 0.05 0.95 (0.58,1.56) 0.05
rs2229114 0.46 0.48 (0.11,2.07) 0.46 1.03 (0.48,2.22) 0.41 1.92 (0.88,4.15) 0.45
rs9610 0.33 1.76 (1.15,2.68) 0.001 0.31 0.75 (0.53,1.06) 0.37 1.16 (0.72,1.87) 0.32
IL10RB rs999788 0.30 0.90 (0.5,1.61) 0.29 0.94 (0.63,1.41) 0.28 0.72 (0.39,1.31) 0.28
rs2239573 0.25 1.12 (0.68,1.83) 0.26 1.07 (0.78,1.47) 0.25 1.13 (0.71,1.81) 0.26
rs2834167 0.20 0.97 (0.54,1.75) 0.22 0.97 (0.68,1.39) 0.21 0.70 (0.42,1.18) 0.22
rs2284552 0.44 0.85 (0.45,1.61) 0.43 0.96 (0.66,1.41) 0.46 0.75 (0.42,1.34) 0.45
rs2849997 0.11 1.02 (0.61,1.72) 0.12 0.98 (0.7,1.37) 0.12 0.94 (0.64,1.39) 0.12
rs8178474 0.30 0.73 (0.34,1.6) 0.30 0.98 (0.61,1.58) 0.30 0.64 (0.32,1.3) 0.32
rs2834168 0.47 0.70 (0.43,1.15) 0.42 0.98 (0.71,1.34) 0.45 1.05 (0.7,1.58) 0.45
rs2247526 0.47 1.24 (0.76,2.01) 0.43 0.87 (0.63,1.2) 0.46 1.27 (0.85,1.89) 0.45
rs2248118 0.30 1.09 (0.69,1.71) 0.33 0.93 (0.66,1.31) 0.31 1.16 (0.8,1.67) 0.33
rs2843944 0.50 0.50 (0.28,0.91) 0.120 0.48 0.97 (0.7,1.34) 0.46 0.97 (0.62,1.52) 0.47
rs999259 0.05 1.32 (0.91,1.92) 0.05 0.86 (0.64,1.15) 0.06 1.23 (0.82,1.83) 0.06
rs8178556 0.38 0.83 (0.28,2.42) 0.38 1.23 (0.69,2.19) 0.39 0.85 (0.28,2.59) 0.39
rs1058867 0.19 0.89 (0.53,1.47) 0.19 1.06 (0.77,1.46) 0.19 1.15 (0.74,1.77) 0.20
rs7281762 0.20 0.73 (0.38,1.41) 0.19 1.11 (0.76,1.61) 0.19 0.74 (0.44,1.25) 0.19
IL12A rs582537 0.42 0.64 (0.38,1.08) 0.44 0.97 (0.72,1.31) 0.46 0.98 (0.65,1.47) 0.45
rs2243135 0.40 1.17 (0.72,1.91) 0.38 0.94 (0.69,1.29) 0.39 0.86 (0.55,1.35) 0.40
rs2243154 0.09 1.03 (0.41,2.61) 0.10 0.70 (0.39,1.26) 0.09 1.19 (0.67,2.1) 0.09
IL12B rs6870828 0.49 1.53 (0.86,2.73) 0.49 1.05 (0.75,1.49) 0.48 0.91 (0.6,1.4) 0.48
rs3181225 0.20 1.85 (1.07,3.21) 0.080 0.20 1.14 (0.76,1.71) 0.21 1.16 (0.69,1.96) 0.17
rs3181224 0.11 1.16 (0.58,2.32) 0.11 1.08 (0.63,1.85) 0.09 0.30 (0.12,0.74) 0.001 0.11
rs1368439 0.17 0.84 (0.41,1.68) 0.19 0.96 (0.64,1.43) 0.18 0.92 (0.55,1.54) 0.19
rs3212227 0.21 0.50 (0.23,1.09) 0.18 0.87 (0.59,1.29) 0.20 0.98 (0.61,1.58) 0.20
rs3213119 0.03 0.52 (0.11,2.48) 0.03 0.66 (0.2,2.13) 0.02 0.50 (0.14,1.84) 0.03
rs2853694 0.49 0.80 (0.49,1.33) 0.49 1.03 (0.75,1.42) 0.49 1.23 (0.81,1.85) 0.48
rs2569253 0.46 0.64 (0.39,1.04) 0.46 0.93 (0.68,1.28) 0.44 0.69 (0.45,1.07) 0.47
rs3181216 0.31 0.90 (0.52,1.53) 0.31 1.00 (0.72,1.4) 0.33 1.22 (0.8,1.86) 0.33
rs1433048 0.17 0.88 (0.45,1.73) 0.22 0.85 (0.58,1.26) 0.18 1.12 (0.67,1.88) 0.18
rs2546893 0.50 1.23 (0.78,1.93) 0.49 1.12 (0.82,1.52) 0.48 1.09 (0.73,1.63) 0.50
rs730691 0.36 0.57 (0.32,1.01) 0.450 0.36 0.86 (0.62,1.2) 0.36 0.98 (0.66,1.44) 0.37
rs2546890 0.48 1.16 (0.75,1.79) 0.47 0.99 (0.74,1.32) 0.47 0.89 (0.58,1.36) 0.48
rs10052709 0.14 1.11 (0.61,2.03) 0.14 0.96 (0.59,1.57) 0.10 0.67 (0.34,1.32) 0.13
rs10045431 0.28 0.90 (0.53,1.51) 0.30 1.02 (0.73,1.43) 0.26 0.65 (0.41,1.03) 0.29
IL12RB1 rs445521 0.40 1.08 (0.65,1.79) 0.40 1.00 (0.74,1.34) 0.39 0.98 (0.65,1.49) 0.42
rs17885963 0.06 2.00 (1.03,3.91) 0.320 0.05 0.70 (0.34,1.48) 0.05 1.07 (0.45,2.53) 0.05
rs3746190 0.40 1.65 (0.97,2.8) 0.37 1.04 (0.77,1.43) 0.37 0.70 (0.49,1.01) 0.150 0.39
rs383483 0.50 0.78 (0.47,1.3) 0.50 0.75 (0.55,1.01) 0.150 0.49 1.31 (0.88,1.94) 0.48
rs438421 0.30 0.77 (0.48,1.24) 0.28 0.82 (0.57,1.17) 0.28 1.03 (0.63,1.68) 0.28
rs2045386 0.40 1.13 (0.73,1.74) 0.42 0.79 (0.58,1.09) 0.40 0.84 (0.57,1.25) 0.42
rs11575934 0.32 0.94 (0.56,1.6) 0.32 1.51 (1.12,2.05) 0.001 0.33 1.16 (0.79,1.71) 0.32
rs11575926 0.18 0.97 (0.51,1.85) 0.16 1.39 (0.97,2.00) 0.18 0.79 (0.48,1.32) 0.17
rs11673460 0.07 1.59 (0.7,3.62) 0.05 0.72 (0.36,1.42) 0.06 0.64 (0.28,1.46) 0.06
rs11086087 0.09 0.54 (0.2,1.45) 0.13 1.49 (0.96,2.3) 0.13 1.44 (0.83,2.52) 0.12
IL12RB2 rs3762315 0.18 0.70 (0.38,1.27) 0.22 0.70 (0.46,1.07) 0.18 1.18 (0.75,1.85) 0.20
rs3762317 0.13 0.71 (0.33,1.52) 0.17 0.70 (0.42,1.15) 0.13 1.18 (0.67,2.09) 0.15
rs12142823 0.14 0.61 (0.27,1.34) 0.18 0.76 (0.46,1.27) 0.14 1.19 (0.67,2.13) 0.16
rs7544381 0.33 0.70 (0.41,1.18) 0.36 0.92 (0.66,1.28) 0.34 1.08 (0.7,1.67) 0.36
rs17129741 0.04 0.71 (0.20,2.59) 0.03 0.66 (0.26,1.63) 0.04 1.16 (0.45,3) 0.04
rs12138219 0.18 0.64 (0.33,1.25) 0.21 0.70 (0.45,1.1) 0.19 1.31 (0.77,2.22) 0.20
rs17129794 0.16 0.59 (0.29,1.2) 0.18 1.03 (0.7,1.52) 0.19 0.88 (0.5,1.55) 0.17
rs1495963 0.02 0.73 (0.13,4.14) 0.02 1.42 (0.51,3.93) 0.02 1.56 (0.48,5.04) 0.01
rs12145984 0.14 0.47 (0.16,1.39) 0.17 1.02 (0.66,1.58) 0.15 0.70 (0.39,1.24) 0.17
rs3790565 0.18 0.49 (0.25,0.95) 0.210 0.18 0.89 (0.6,1.31) 0.18 0.86 (0.48,1.55) 0.18
rs10749775 0.12 0.53 (0.22,1.27) 0.13 1.00 (0.64,1.55) 0.13 0.90 (0.44,1.85) 0.12
rs3790567 0.23 0.58 (0.32,1.03) 0.600 0.23 0.86 (0.6,1.22) 0.24 0.91 (0.56,1.48) 0.24
rs2307153 0.02 0.86 (0.17,4.44) 0.02 1.18 (0.48,2.89) 0.03 1.60 (0.44,5.77) 0.02
rs17129908 0.12 0.66 (0.32,1.38) 0.10 0.79 (0.48,1.33) 0.12 0.86 (0.47,1.59) 0.12
rs4297265 0.40 0.46 (0.26,0.82) 0.002 0.42 1.01 (0.74,1.37) 0.42 0.84 (0.57,1.23) 0.43
rs2229546 0.33 0.43 (0.21,0.87) 0.030 0.35 1.12 (0.8,1.57) 0.33 0.86 (0.57,1.31) 0.34
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1

MAF: Minor allele frequency

TagSNPs with nominally or statistically significant associations in either the cervical SCC or cervical adenocarcinoma models were also included in a post hoc analysis with HPV16 and HPV18 positive cervical cancer as an outcome. Cases positive for both HPV16 and HPV18 were included in both analyses. Similarly, tagSNPs with nominally or statistically significant associations in the vulvar SCC models were included in HPV positive only vulvar SCC analysis. Holm’s correction was not applied to these post hoc analyses.

Results

Two case-parent sets were dropped due to non-Mendelian transmission. Cervical SCC analyses included 43 invasive cervical SCC proband cases with 59 parents (16 triads/27 dyads), 356 non-proband cases, and 861 population-based controls. Cervical AC analyses included 79 in situ proband cases and 17 invasive proband cases with 151 parents (55 triads/41 dyads), 241 in situ non-proband cases and 165 invasive non-proband cases, and 861 population-based controls. Vulvar SCC analyses included 49 in situ proband cases and 4 invasive proband cases with 70 parents (17 triads/36 dyads), 402 in situ non-proband cases and 71 invasive non-proband cases, and 1,111 population-based controls. There were higher proportions of in situ cervical AC and vulvar SCC proband cases compared with non-proband cases because probands were intentionally selected to be young, and in situ carcinomas tend to be diagnosed at a younger age (Table 1).

Table 1.

Selected characteristics of the proband cervical and vulvar cancer cases, non-proband cervical and vulvar cancer cases, and population controls, Seattle-Puget Sound Region, 1986–2004

Cervical squamous cell carcinoma
Cervical adenocarcinoma
Vulvar squamous cell carcinoma
Population controls
Proband cases (n=43) Non-proband cases (n=356) Proband cases (n=96) Non-proband cases (n=406) Proband cases (n=53) Non-proband cases (n=473) Cervical cancer1 (n=861) Vulvar cancer (n=1,111)
Characteristic
Tumor behavior, N (%)
 In situ - - 79 (82.3) 241 (59.4) 49 (92.5) 402 (85.0) - -
 Invasive 43 (100) 356 (100) 17 (17.7) 165 (40.6) 4 (7.5) 71 (15.0) - -
HPV DNA, N (%)
 Not tested 29 (67.4) 105 (29.5) 54 (56.2) 102 (25.1) 20 (37.7) 106 (22.4) - -
 Negative 3 (7.0) 19 (5.3) 3 (3.1) 42 (10.3) 3 (5.7) 39 (8.3) - -
 HPV 16+/18− 11 (25.6) 164 (46.1) 24 (25.0) 118 (29.0) 23 (43.4) 271 (57.3) - -
 HPV 16−/18+ 0 21 (5.9) 10 (10.4) 101 (24.9) 2 (3.8) 20 (4.2)
 HPV 16+/18+ 0 21 (5.9) 5 (5.2) 28 (6.9) 1 (1.9) 10 (2.1)
 Other type (s) 0 26 (7.3) 0.0 15(3.7) 4 (7.6) 27 (5.7) - -
Mean age (years) 38.0 43.1 32.8 39.7 39.3 48.3 42.6 45.8
Cigarette smoking, N (%)
 Never 24 (55.8) 126 (35.4) 56 (58.3) 210 (51.7) 15 (28.3) 92 (19.5) 468 (54.4) 577 (51.9)
 Former 9 (20.9) 100 (28.1) 21 (21.9) 109 (26.9) 6 (11.32) 111 (23.4) 223 (25.9) (303) 27.3
 Current 10 (23.3) 130 (36.5) 19 (19.8) 87 (21.4) 32 (60.38) 270 (57.1) 170 (19.7) 231 (20.8)
Number of lifetime sexual partners, N (%)
 1 3 (7.0) 34 (9.6) 6 (6.3) 52 (12.8) 1 (1.9) 42 (8.9) 204 (23.7) 295 (26.5)
 2 to 4 13 (30.2) 109 (30.6) 18 (18.8) 116 (28.6) 6 (11.3) 105 (22.2) 258 (30.0) 344 (30.9)
 5 to 14 22 (51.2) 150 (42.1) 46 (47.9) 171 (42.1) 29 (54.7) 204 (43.1) 291 (33.8) 344 (30.9)
 ≥ 15 5 (11.6) 63 (17.7) 26 (27.1) 67 (16.5) 17 (32.1) 121 (25.6) 108 (12.5) 126 (11.3)
Number of births, N (%)
 0 11 (25.6) 66 (18.5) 43 (44.8) 126 (31.3) 19 (35.9) 132 (27.9) 224 (26.0) 248 (22.3)
 1 8 (18.6) 58 (16.25) 14 (14.6) 72 (17.9) 11 (20.8) 90 (19.0) 135 (15.7) 154 (13.9)
 2 10 (23.3) 108 (30.25) 30 (31.3) 121 (30.0) 18 (34.0) 117 (24.7) 255 (29.6) 338 (30.4)
 ≥ 3 14 (32.5) 124 (34.8) 9 (9.4) 84 (20.8) 5 (9.4) 134 (28.3) 247 (28.7) 371 (33.4)
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Excluding women with prior hysterectomy

Vulvar SCC cases were more than twice as likely as controls to be current cigarette smokers, while cervical SCC cases were slightly more likely, and cervical AC cases were slightly less likely, to be current smokers compared to controls (Table 1). All cases tended to have more lifetime sexual partners and a history of oral contraceptive use compared to controls. Among the 76 SNPs included in this study, 21(27.6%) were located in UTRs or exons, 18 (23.7%) were in flanking regions, and 37 (48.7%) were intronic (Table 2). The MAF in controls ranged from 0.01 to 0.50 (median=0.24). One SNP (IL12RB2 rs3762315) had a HWE p value ≤0.001.

Table 2.

Characteristics of tagSNPs in IL10, IL12A, IL12B, IL10RA, IL10RB, IL12RB1, and IL12RB2

HUGO gene name (region) dbSNP reference sequence number SNP context (amino acid change)1 Alleles (major/minor) Minor allele frequency in controls HWE p-value2 Genotyping method3 SNPs in high LD4
IL10 (1q31) rs3024505 flanking 3′UTR G/A 0.15 0.636 OPA rs3024493
rs3024498 3′UTR A/G 0.27 0.037 OPA
rs3024509 intron A/G 0.07 0.880 OPA
rs3021094 intron A/C 0.08 1.000 OPA
rs1800871 flanking 5′UTR G/A 0.23 1.000 OPA rs1800872
rs1800894 flanking 5′UTR G/A 0.04 1.000 KBIO
rs1800896 flanking 5′UTR A/G 0.50 0.039 OPA rs3024496
rs1800890 flanking 5′UTR T/A 0.39 0.009 OPA
IL10RA (11q23) rs4252301 SYNON G/C 0.11 1.000 OPA
rs4252253 intron C/A 0.33 0.942 OPA
rs2256111 SYNON G/A 0.17 0.655 KBIO
rs3135932 NONSYN (Ser/Gly) A/G 0.11 1.000 KBIO
rs4252279 intron G/A 0.03 0.718 OPA rs4252246
rs4252314 intron A/G 0.03 0.627 OPA
rs4252286 intron G/A 0.32 0.935 TAQMAN
rs2229113 NONSYN (Gly/Arg) G/A 0.05 0.253 TAQMAN rs2512143, rs2512148
rs2229114 NONSYN (Gly/Arg) G/A 0.45 1.000 OPA
rs9610 3′UTR G/A 0.32 0.397 KBIO
IL10RB (21q22) rs999788 flanking 5′UTR G/A 0.28 0.970 OPA
rs2239573 intron A/G 0.26 0.537 KBIO
rs2834167 NONSYN (Lys/Glu) A/G 0.22 0.107 OPA
rs2284552 intron C/A 0.45 0.376 OPA
rs2849997 intron C/T 0.12 0.696 KBIO
rs8178474 intron A/G 0.32 0.679 OPA
rs2834168 intron A/G 0.45 0.733 OPA
rs2247526 intron A/G 0.45 0.952 OPA rs2843701, rs765429
rs2248118 intron G/A 0.33 0.951 OPA
rs2843944 intron G/A 0.47 0.966 OPA
rs999259 intron G/A 0.06 0.339 OPA
rs8178556 intron A/C 0.39 0.329 OPA
rs1058867 3′UTR A/G 0.20 0.103 OPA rs3171425
rs7281762 flanking 3′UTR G/A 0.19 0.127 OPA rs81878562
IL12A (3q25) rs582537 intron C/A 0.45 0.652 OPA rs2227314
rs2243135 intron C/G 0.40 0.431 OPA
rs2243154 flanking 3′UTR G/A 0.09 0.972 OPA
IL12B (5q31) rs6870828 flanking 3′UTR T/C 0.48 0.600 KBIO
rs3181225 flanking 3′UTR G/A 0.17 0.995 OPA rs2569254
rs3181224 flanking 3′UTR A/G 0.11 1.000 OPA
rs1368439 3′UTR A/C 0.19 0.624 OPA
rs3212227 3′UTR A/C 0.20 1.000 OPA rs3212220
rs3213119 NONSYN (Val/Phe) C/A 0.03 1.000 OPA
rs2853694 intron A/C 0.48 0.037 KBIO
rs2569253 intron C/T 0.47 0.001 TAQMAN
rs3181216 intron T/A 0.33 0.266 OPA
rs1433048 intron A/G 0.18 0.855 OPA
rs2546893 intron G/A 0.50 0.636 OPA
rs730691 intron G/A 0.37 0.220 OPA
rs2546890 flanking 5′UTR A/G 0.48 0.079 OPA
rs10052709 flanking 5′UTR G/C 0.13 0.745 OPA
rs10045431 flanking 5′UTR C/A 0.29 0.481 OPA
IL12RB1 (19p13) rs445521 flanking 3′UTR A/C 0.42 0.740 OPA
rs17885963 flanking 3′UTR GC 0.05 1.000 OPA
rs3746190 3′UTR G/A 0.39 0.600 OPA
rs383483 intron A/G 0.48 0.195 OPA
rs438421 intron G/A 0.28 0.339 OPA
rs2045386 intron G/A 0.42 0.872 OPA
rs11575934 NONSYN (Gln/Arg) A/G 0.32 0.293 TAQMAN rs375947, rs391410, rs17852635
rs11575926 NONSYN (Arg/His) G/A 0.17 0.638 KBIO
rs11673460 intron G/T 0.06 1.000 KBIO
rs11086087 SYNON G/C 0.12 0.194 OPA rs17879591, rs3761041
IL12RB2 (1p31) rs3762315 flanking 5′UTR A/G 0.20 0.000 OPA
rs3762317 flanking 5′UTR A/G 0.15 0.409 OPA
rs12142823 5′UTR C/T 0.16 1.000 TAQMAN
rs7544381 intron G/A 0.36 0.914 OPA
rs17129741 intron TA 0.04 0.506 OPA rs2030071
rs12138219 intron A/G 0.20 0.846 OPA
rs17129794 intron A/C 0.17 0.400 OPA
rs1495963 SYNON G/A 0.01 0.328 OPA
rs12145984 intron G/C 0.17 0.766 OPA
rs3790565 intron A/G 0.18 0.322 OPA rs6660836
rs10749775 intron A/C 0.12 0.064 OPA
rs3790567 intron G/A 0.24 0.776 OPA rs3977726
rs2307153 NONSYN (Gly/Asp) G/A 0.02 1.000 OPA
rs17129908 intron A/T 0.12 0.092 OPA
rs4297265 SYNON A/G 0.43 0.862 OPA rs881086
rs2229546 SYNON A/C 0.34 0.215 TAQMAN rs6699474, rs7555183
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Location of polymorphism within gene: flanking 5′UTR is upstream of the first exon of the gene, 5′ UTR is in the upstream untranslated region, 3′ flanking is downstream of the last exon of the gene, 3′ UTR is in the downstream untranslated region, exon is in coding region of the gene, intron is between coding regions of the gene. All SNPs are listed in the 5′ to 3′ direction on the forward strand

2

Pearson’s chi-square p-value for compliance with Hardy-Weinberg equilibrium

3

OPA = Illumina GoldenGate Custom Assay, KBIO = KASPar SNP Genotyping Assays, TAQMAN = TaqMan Probe Applied or Custom SNP Genotyping Assays

4

SNPs listed in this column represent SNPs that were genotyped in cases, parents, and population controls in this study and found to be in high linkage disequilibrium (LD), defined as r2≥0.80, and were therfore excluded from further analyses

None of the IL10 SNPs were significantly associated with cervical SCC or AC, but one flanking 5′ UTR SNP was associated with vulvar SCC (rs1800871, OR=1.62, 95% CI=1.04–2.54, Table 3). In IL10RA, one intronic SNP was associated with cervical SCC (rs4252253, OR=0.38, 95% CI=0.16–0.91), a second intronic SNP was statistically significantly associated with cervical AC (rs4252314, OR=2.23, 95% CI = 1.26–3.96), and a 3′ UTR SNP was statistically significantly associated with increased risk of cervical SCC (rs9610, OR=1.76, 95% CI= 1.15–2.68). None of the IL10RB SNPs were significantly associated with cervical or vulvar cancer, with the exception of one intronic SNP whose minor allele was associated with a 50% reduced risk of cervical SCC (rs2843944, OR=0.50, 95% CI= 0.28–0.91).

Several SNPs were associated with cervical and vulvar cancer in the genes coding for IL12 and IL12 receptor. Among the most notable associations were a non-synonymous SNP in IL12B (rs3213119) whose minor allele was associated with a non-significant 44–55% reduced risk of cervical SCC, cervical AC, and vulvar SCC. Another IL12B intronic SNP (rs2569253, minor allele) was associated with a non-significant reduced risk of both cervical SCC (OR=0.64, 95% CI=0.39–1.04) and vulvar SCC (OR=0.69. 95% CI=0.45–1.07). The minor allele of a flanking 3′UTR SNP was associated with an increased risk of cervical SCC (rs3181225, OR=1.85, 95% CI=1.07–3.21), while the minor allele of another flanking 3′UTR SNP was associated with a statistically significant reduced risk of vulvar SCC (rs3181224, OR=0.30, 95% CI=0.12–0.74). Cervical SCC was also inversely associated with the minor allele of another intronic SNP (rs730691, OR=0.57, 95% CI=0.32–1.01).

In IL12RB1, the minor allele of a flanking 3′UTR SNP was associated with an increased risk of cervical SCC (rs17885963, OR=2.0, 95% CI=1.03–3.91), while the minor allele of another 3′UTR SNP was associated with a reduced risk of vulvar SCC (rs3746190, OR=0.70, 95% CI=0.49–1.01). Additionally, cervical AC risk was inversely associated with the minor allele of an intronic SNP (rs383483, OR=0.75, 95% CI=0.55–1.01) and positively statistically significantly associated with the minor allele of a non-synonymous SNP (rs11575934, OR=1.51, 95% CI=1.12–2.05). Cervical SCC risk was inversely statistically significantly associated with the minor allele of two synonymous SNPs (rs4297265, OR=0.46, 95% CI=0.26–0.82 and rs2229546, OR=0.43, 95% CI=0.21–0.87).

In general, the HPV16 positive stratified cervical cancer SNP associations were similar to results from the cervical SCC models given the high proportion of HPV16 positive versus HPV18 positive cervical SCC (49% versus 10%, Table 4). An exception was a strong inverse association seen between the minor alleles of each of the two synonymous SNPs in IL12RB2 (rs4297265 and rs2229546) and cervical SCC in contrast to HPV18 positive cervical cancers being positively associated with the minor alleles of these SNPs. Also, the strong positive association that was observed between IL10RA SNP rs4252314 minor allele and cervical AC, was seen in both HPV16 positive and HPV18 positive cervical cancers. The HPV positive vulvar SCC SNP associations for rs1800871, rs3181224, and rs3746190 were in the same direction and of similar magnitude to results for all vulvar SCC (data not shown).

Table 4.

Selected tagSNP associations with cervical cancer, by HPV DNA type detected in tissue

Gene SNP HPV 161
HPV 181
19 triads, 21 dyads, 331 unrelated cases, and 861 population controls
9 triads, 6 dyads, 171 unrelated cases, and 861 population controls
Odds ratio 95% CI Odds ratio 95% CI
IL10RA rs4252253 0.84 (0.41, 1.71) 1.98 (0.75, 5.19)
IL10RA rs4252314 2.73 (1.15, 6.47) 3.92 (1.38, 11.13)
IL10RA rs9610 1.16 (0.74, 1.82) 0.51 (0.26, 0.99)
IL10RB rs2843944 0.98 (0.57, 1.68) 0.84 (0.34, 2.03)
IL12B rs3181225 1.66 (0.95, 2.91) 0.63 (0.20, 1.99)
IL12B rs730691 0.65 (0.38, 1.12) 1.01 (0.43, 2.38)
IL12RB1 rs17885963 1.26 (0.59, 2.73) 0.92 (0.17, 4.99)
IL12RB1 rs383483 0.69 (0.40, 1.18) 0.88 (0.42, 1.83)
IL12RB1 rs11575934 1.38 (0.85, 2.22) 1.10 (0.56, 2.18)
IL12RB2 rs3790565 0.60 (0.30, 1.20) 1.05 (0.51, 2.18)
IL12RB2 rs3790567 0.62 (0.34, 1.13) 0.72 (0.33, 1.56)
IL12RB2 rs4297265 0.59 (0.36, 0.95) 1.85 (0.90, 3.81)
IL12RB2 rs2229546 0.59 (0.33, 1.06) 2.73 (1.21, 6.13)
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Includes cases that were both HPV16 and HPV18 positive

Discussion

We report the first comprehensive study of cervical and vulvar cancer risk in relation to variation in genes related to IL12 and IL10, two immunoregulatory cytokines with opposite effects on the Th1/Th2 balance. The underlying hypothesis of this study is that a cell mediated immune response associated with increased expression of Th1 cytokines, such as IL12, combats pathological events in the cervix and vulva that may be associated with HPV persistence and pathogenesis of cancer; IL10 inhibits this response, and the ability to mount a Th1 response is partly genetically determined.

IL12 is an important modulator of the immune response and provides the stimulus for CD4+ T cells and NK cells to differentiate toward a cell mediated immunity Th1 phenotype. IL12 is produced primarily by antigen-presenting cells and its functions are mediated through high-affinity binding to the IL12 receptor expressed on activated T and NK cells. The biologically active form of IL12 is a heterodimer (p70) composed of two unrelated disulfide-linked subunits, p35 and p40, which are encoded by IL12A and IL12B, respectively.31

Among the most notable findings from this study was the observation that IL12B SNPs, particularly SNPs in the 3′ region (rs3181225, rs3181224, rs3212227) and a non-synonymous SNP (rs3213119) were associated with risk of cervical and vulvar cancers. These results are particularly interesting in the context of literature describing functional and disease associations of IL12B SNPs. The rs3212227 minor allele is associated with increased secretion of IL12 p70 by stimulated human monocytes.17 Thus, the 50% reduced risk of cervical SCC is consistent with our hypothesis that variation in this pro-Th1 cytokine gene bolsters the functional immune response against HPV infection and related carcinogenesis. The rs3212227 minor allele has also been associated with a decreased risk of a number of different infectious and Th1/Th17-mediated autoimmune diseases.32–34

A few studies to date have examined the association IL12B SNPs and cervical cancer. Women who were heterozygous for the rs3212227 minor allele were at increased cervical cancer risk in one study,35 but not in other studies.36, 37 Carriers of the rs2569254 minor allele (not genotyped in our study) were at significantly increased risk for cervical cancer.37 Rs2569254 is an intronic IL12B SNP which is in strong LD with rs3181225, a 3′flanking SNP whose minor allele was significantly increased risk of cervical SCC in our study. The minor allele of another 3′flanking SNP, rs3181224, was associated with reduced risk of vulvar SCC in our study. Also, the minor allele of intronic SNP rs730691, which is in LD with 3′flanking SNP rs2116821in HapMap (not genotyped in our study), was associated with reduced risk of cervical SCC in our study. Taken together, these findings suggest that IL12B SNPs, particularly those in the 3′ region, are important in cervical and vulvar carcinogenesis.

Although there are some differences in the implicated SNP, it is notable that several studies, including ours, have found an association between 3′ region SNPs in IL12B and diseases related to infections or autoimmunity, including cervical and vulvar cancer, given the importance of this region in post-transcriptional regulation. Furthermore, chromosome 5q31, where IL12B is located, contains numerous immunoregulatory genes, and the true disease associated variant could be in one of those genes and in LD with SNPs in the 3′ region of IL12B. For example, EBF1, a principal regulator of B cell development, is about 200kb downstream of IL12B and according to HapMap CEU data there is LD between several pairs of SNPs in IL12B and EBF1 (r2 ranging from 0.52–0.73). Importantly, genetic variation in EBF1 has been associated with several infections and immune related disorders.38

The minor allele of IL12B rs3213119 was associated with a 44–50% reduced risk of cervical and vulvar cancers in our study, although these associations did not reach statistical significance. This SNP results in valine to phenylalanine substitution, which is predicted to affect protein function and designated “possibly damaging” by Polyphen and “damaging” by SIFT.39, 40 Further studies are needed to elucidate the functional consequences of this SNP, including effects on protein folding, formation of IL12 homodimers/heterodimers, and binding to the IL12 receptor.

We found several SNPs in the IL12 receptor genes to be significantly associated with cervical cancer risk. One of the most noteworthy findings was that the minor allele of a non-synonymous SNP in IL12RB1 (rs11575934, glutamine to arginine) was associated with a significant increased cervical AC risk. In previous studies, the minor allele of rs11575934 was associated with a more than 2-fold increased risk of tuberculosis and functional data show that IFNγ (another pro-Th1 immunity cytokine) production, T lymphocyte proliferation, and NK lytic activity in response to IL12 were significantly lower in minor allele homozygotes compared to major allele homozygotes.41 Importantly, this SNP is in near complete LD (r2=0.97–0.99) with several other interesting SNPs in IL12RB1 including a non-synonymous SNP (rs375947) and synonymous SNP (rs17852635). However, neither non-synonymous SNPs (rs11575934 and rs375947) are predicted to have functional effects.39, 40 In IL12RB2, the minor alleles of several SNPs were associated with reduced risk of cervical SCC, including two synonymous SNPs, rs4297265 and rs2229546. Interestingly, the minor allele of rs4297265 was associated with a similar reduced risk of HPV16 positive cervical cancer, while rs2229546 was associated with an increased risk of HPV18 positive cervical cancer. Further studies are needed to confirm these associations and elucidate the potential role of these SNPs on IL12 receptor function.

There were also a few notable associations between cervical and vulvar cancer risk and SNPs in IL10 and its receptor in our study. IL10 is a Th2 cytokine with suppressive effects on cell-mediated immunity, including the inhibition of Th1 cytokines such as IL12. In addition to its immunsuppressive effects, IL10 can induce transcription of the HPV16 E7 oncoprotein.42 Fifty percent of the variability of IL10 expression is thought to be due to genetic variance, and functional studies indicate that certain haplotypes made up of several IL10 5′ region SNPs are associated with IL10 levels in circulation.18, 19 SNPs in IL10, IL10RA, or IL10RB may also influence IL10 levels in the cervix.43

Rs1800871, the minor allele of which was associated with an increased risk of vulvar SCC in our study, is a 5′ region SNP. Rs1800871, and other SNPs that are in high LD with this SNP (including rs1800872 and rs100893) have been associated with HPV clearance/persistence and risk of cervical cancer and precursor lesions in some studies,44–46 but not others.47, 48 To our knowledge, no previous studies have examined the association between genetic variation in IL10RA and IL10RB and cervical or vulvar cancer risk. Interestingly, one study found that several polymorphisms in IL10RA were associated with cervical IL10 levels.43 These SNPs did not include, nor were they in high LD with, the intronic (rs4252314) and 3′ UTR (rs9610) SNPs that we found to be positively associated with cervical cancer.

In our study, several SNPs were associated with cervical SCC, cervical AC, or vulvar SCC, but rarely were similar associations observed across all three sites. Although these three cancers have a common HPV etiology, they exhibit differences in strength, and in some cases direction, of association with major risk factors including specific HPV types and cigarette smoking.21, 49–51 The vast majority of papers examining the immune response to HPV have focused on cervical cancer, and there is some indication that the immune environment in the squamous epithelium (where cervical SCCs develop) differs from that in the glandular cells (where cervical ACs develop).52 Additionally, HPV16 positive high grade vulvar lesions and cervical cancer appear to have unique associations certain class I and II human leukocyte antigen alleles,15, 53 loci that play an important role in regulating T lymphocyte responses to viral proteins. Thus, there may be immunological differences that explain the associations observed in our study across tumor types.

A major assumption of the hybrid design is homogeneity of the ORs in case-parent and case-unrelated controls. For all associations found to be nominally or statistically significant, this assumption appears to be valid (Supplementary Table). Strengths of this study include the population-based recruitment of cases and controls and attempted coverage of all common genetic variation in our candidate genes. Additionally, the use of a hybrid case-parent triad/case-control study design represents a relatively innovative feature of this study, making use of all available samples thus maximizing statistical power. It is possible that one or more of our SNP associations is modified by risk factors, such as cigarette smoking. However, due to the small number of study subjects and large number of additional hypothesis tests, we opted not to explore effect modification by risk factors to limit the possibility of reporting false positive associations.

In summary, our results raise the possibility that a shift in the balance of the immune response due to genetic variants in key cytokine genes could have consequences for the pathogenesis of cervical and vulvar cancer. As uptake of the prophylactic HPV vaccines continues to rise, the burden of HPV infection and associated neoplasms will be reduced. However, there is a large number of women who will not benefit from the vaccine as they have already acquired HPV infection, are beyond the target age of vaccination, or live in low-resource countries that are challenged by the high cost and distribution of the vaccine. Identification of immunogenetic risk factors that contribute to cervical and vulvar cancer risk may help shed light on the biological mechanisms leading to, and potentially identify women who are at increased risk of developing, these malignancies.

Supplementary Material

Supp Table S1

Acknowledgments

Financial Support: This research was supported by grants from the U.S. National Institutes of Health (R01CA112512; P01CA042792, R03CA108363, R25CA094880, P30ES07033, R25CA087949, K07CA140360) as well as institutional funds from the Fred Hutchinson Cancer Research Center.

Abbreviations Used

HPV

human papillomavirus

Th

T helper

SCC

squamous cell carcinoma

AC

adenocarcinoma

OR

odds ratio

CI

confidence intervals

LD

linkage disequilibrium

Footnotes

Novelty Statement: This is the first manuscript reporting on cervical and vulvar cancer risk in relation to genetic variation in IL12 and IL10 and their receptors; two immunoregulatory cytokines with opposite effects on the Th1/Th2 cytokine balance. We employed a non-traditional study design which utilizes genetic data from cases, their biological parents, and population controls.

Conflicts of Interest: None of the authors have a conflict of interest to report.

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