Replication Study for the Association of Seven Genome- Gwas-Identified Loci With Susceptibility to Ovarian Cancer in the Polish Population

We investigated the previously-demonstrated association of seven genome-wide association studies (GWAS) single nucleotide polymorphisms (SNPs), including rs2072590 (HOXD-AS1), rs2665390 (TIPARP), rs10088218 and rs10098821 (8q24), rs3814113 (9p22), rs9303542 (SKAP1) and rs2363956 (ANKLE1), as risk factors of epithelial ovarian tumors (EOTs). These SNPs were genotyped in two hundred seventy three patients with EOTs and four hundred sixty four unrelated healthy females from the Polish population. We observed the lowest p values of the trend test for the 9p22 rs3814113 and 8q24 rs10098821 SNPs in patients with all subtypes of ovarian cancer (ptrend = 0.010 and ptrend = 0.014, respectively). There were also significant p values for the trend of the 9p22 rs3814113 and the 8q24 rs10098821 SNPs for serous histological subtypes of ovarian cancer (ptrend = 0.006, ptrend = 0.033, respectively). Moreover, stratification of the patients based on their histological type of cancer demonstrated, in the dominant hereditary model, a significant association of the 9p22 rs3814113 SNP with serous ovarian carcinoma OR = 0.532 (95 % CI = 0.342 - 0.827, p = 0.005, pcorr = 0.035). Despite the relatively small sample size of cases and controls, our studies confirmed some of the previously-demonstrated GWAS SNPs as genetic risk factors for EOTs. Electronic supplementary material The online version of this article (doi:10.1007/s12253-014-9822-6) contains supplementary material, which is available to authorized users.


Patients and Controls
The patient group consisted of 273 women with histologically diagnosed ovarian carcinoma according to the International Federation of Gynecology and Obstetrics (FIGO). They were enrolled into the study from the University Hospital, Clinic of Gynecological Surgery and Chair of Gynecologic Oncology at Poznan University of Medical Sciences. Histopathological classification, describing the stage, grade and tumor type, was carried out by an experienced pathologist ( Table 1). The controls included 464 unrelated healthy female volunteers who were matched by age to the cancer patients ( Table 1). The patients and healthy female volunteers were Caucasian from the Wielkopolska area of Poland. Written informed consent was obtained from all participating individuals. The study design was accepted by the Local Ethical Committee of Poznań University of Medical Sciences.

Statistical Analysis
Hardy-Weinberg equilibrium (HWE) was evaluated by Pearson's goodness-of-fit Chi-squared (χ 2 ) statistic. The data were tested for association with ovarian cancer using the Cochran-Armitage trend test. The distinction in the allele and genotype frequencies between cancer patients and healthy female volunteers were determined using standard χ 2 or Fisher tests. The odds ratio (OR) and associated 95 % confidence intervals (95%CI) were also calculated. SNPs were assessed under recessive and dominant inheritance models. To adjust for the multiple testing, we used a Bonferroni correction. High order gene-gene interactions among all tested polymorphic loci were evaluated by the multifactor dimensionality reduction (MDR) approach (MDR version 2.0 beta 5) [25]. Based on the obtained testing balanced accuracy and cross-validation consistency values, the best statistical gene-gene interaction

Results
Contribution of rs2072590 (HOXD-AS1), rs2665390 (TIPARP), rs10088218 and rs10098821 (8q24), rs3814113 (9p22), rs9303542 (SKAP1) and rs2363956 (ANKLE1) SNPs to Ovarian Cancer Development The prevalence of HOXD-AS1, TIPARP, 8q24, 9p22, SKAP1 and ANKLE1 genotypes did not display deviation from HWE between the patient and control groups (p>0.05). The number of genotypes, OR, and 95 % CI values for the seven HOXD-AS1, TIPARP, 8q24, 9p22, SKAP1 and ANKLE1 polymorphisms are presented in Table 2. The lowest p values of the trend test in patients with all histological EOT subtypes were found for the 9p22 rs3814113 and 8q24 rs10098821 SNPs (p trend =0.010 and p trend =0.014, respectively) ( Table 2). Moreover, we observed significant p values of the trend for the 9p22 rs3814113 and 8q24 rs10098821SNPs for serous histological subtypes of ovarian cancer (p trend =0.006 and p trend = 0.033, respectively) ( Table 2). The statistical significance for multiple testing determined by correction of gene number was p=0.007. Therefore, none of the seven HOXD-AS1, TIPARP ,8q24, 9p22, SKAP1,and ANKLE1 polymorphisms displayed a significant association with all subtypes of ovarian cancer either in dominant or recessive inheritance models ( Table 2). Stratification of the patients based on histological type of cancer revealed, in the dominant hereditary model, a significant association of the 9p22 rs3814113 SNP with serous ovarian carcinoma, OR= 0.532 (95 % CI=0.342 -0.827, p=0.005). However, the 9p22 rs3814113 polymorphism did not display significant association with other histological types and any histological grade and clinical stage. Furthermore, there was no significant association between the HOXD-AS, TIPARP, 8q24, SKAP1 and ANKLE1 polymorphisms with clinical stage, histological grade and subtype.

Discussion
Family and twin investigations have provided us with concrete evidence indicating that there are inherited genetic factors involved in susceptibility to the development of EOTs [17,18]. GWAS have been performed in order to identify common low-penetrance ovarian cancer susceptibility genes [22][23][24]. The GWAS conducted by Song et al. (2009) demonstrated the 9p22 rs3814113 SNP to be a significant genetic risk factor contributing to all histological subtypes of EOTs [22]. In addition to this finding, GWAS analysis performed by Goode et al. (2010) found genome-wide significant association for the 3q25 rs2665390, 17q21 rs9303542, 8q24 rs10088218 and 2q31 rs2072590 SNPs with all EOTs subtypes [23]. The GWAS by Bolton et al. (2010) demonstrated that SNPs rs8170 and rs2363956 on 19p13 displayed genomewide significance for susceptibility of serous ovarian cancer but not all histological subtypes of EOTs [24].
Our follow-up studies, conducted in Caucasian women with ovarian cancer enrolled in the Wielkopolska area of Poland, identify a significant p trend of rs3814113 on 9p22 with all sybtypes of EOTs. In addition to this finding, we observed that rs3814113 on 9p22 may play a protective role from the development of serous histological subtypes of ovarian carcinoma. The stratification of the GWAS by Song et al. (2009) that was based on histological subtypes also indicated that rs3814113 exhibited the greatest association with serous  [22]. Moreover, the 9p22 rs3814113 SNP has been demonstrated to be a protective genetic factor of ovarian cancer for carriers of BRCA1 or BRCA2 mutations [26]. There has also been a recent evaluation of the functional role of seven ovarian cancer susceptibility GWAS polymorphisms in association with microRNAs (miRNAs) presence [27]. This study demonstrated the highest numbers of miRNAs, 68 significantly linked to the rs3814113 SNP [27]. Moreover, the rs3814113 polymorphism was significantly associated with miR-17-92 cluster, which is considered the most remarkable cluster involved in tumorigenesis [27]. Additionally, cell carriers of the rs3814113 SNP displayed prominence of several elementary biological pathways such as cellular response to stress, adenyl nucleotide binding, intracellular organelle lumen, and others [27]. Other functional studies assessed the relationship between changeability of gene expression and the presence of seven ovarian cancer susceptibility GWAS SNPs [28]. These studies demonstrated significant association between the 9p22 rs3814113 SNP and changes in the levels of 274 mRNAs [28]. However, the strongest association of the rs3814113 SNP was observed for increased levels of MT1G and ATL2 mRNAs, which respectively encode metallothionein 1G (OMIM *156353) and atlastin GTPase (OMIM *609368) [28]. Our studies also found significant p trend values for the 8q24 rs10098821 SNP for all patients with ovarian cancer, and also specifically for serous histological subtype. The Goode et al. (2010) GWAS analysis also demonstrated a generally greater association of the 8q24 rs10098821 SNPs with serous as compared to other ovarian EOTs subtypes [23]. Moreover, the 8q24 locus was found to be a risk for several malignancies encompassing breast, prostate, and colorectal cancer [29,30]. A functional association study between GWAS SNPs and whole genome mRNA expression profiles revealed that the 8q24 rs10098821 SNP had the largest number of significant associations, specifically 38 [28]. The study also indicated possible cis-associations between rs10098821 and MYC expression [28]. The 8q24 polymorphisms linked to EOTs and other carcinomas are situated approximately 700 kb 3′ of the MYC protooncogene, and these SNPs probably control the expression of this oncogene distally [28,31].
Presently, genetic risk evaluation for ovarian cancer can be conducted for subjects with a family history of some cancer and/or BRCA1/2 mutations identified within families. However, the usage of low-penetrance SNPs in screening for the risk of ovarian cancer in various ethnicities has not yet been employed. This is in contrast to colorectal and breast cancers, where combinations of low penetrance risk genetic variants are already employed for susceptibility screening in some populations [32,33].
It was demonstrated that the 9p22 rs3814113 and 8q24 rs10098821 variants were associated with the risk of EOTs in subjects of European ancestry [22,23]. In the subjects of non-European ancestry (African or Asian ethnic group), these SNPs did not show statistically significant correlations with the risk of EOTs; however, these results could be due to small sample size [22]. Our study found a significant association of the 9p22 rs3814113 SNP with serous subtypes, and significant trend pvalues for the 9p22 rs3814113 and 8q24 rs10098821 SNPs with all EOTs and serous subtypes in Caucasian patients from the Wielkopolska region of Poland. However, our replication studies have been conducted in relatively small patient and control groups, resulting in a possible missed significant association for the other studied SNPs in ovarian cancer. Therefore, this study should be replicated in other independent cohorts to validate the role of low penetrance SNPs in EOTs development and also in their use as screening tools in the evaluation of ovarian cancer susceptibility.