Genetic variants in fibroblast growth factor receptor 2 (FGFR2) contribute to susceptibility of breast cancer in Chinese women

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<ul><li><p>Carcinogenesis vol.29 no.12 pp.23412346, 2008doi:10.1093/carcin/bgn235Advance Access publication October 8, 2008</p><p>Genetic variants in fibroblast growth factor receptor 2 (FGFR2) contribute tosusceptibility of breast cancer in Chinese women</p><p>Jie Liangy, Peizhan Chen1,y, Zhibin Hu, Xiaoyi Zhou, LuChen1, Mian Li1, Yan Wang, Jinhai Tang2, Hui Wang1 andHongbing Shen</p><p>Laboratory of Reproductive Medicine, Cancer Center, Nanjing MedicalUniversity, 140 Hanzhong Road, Nanjing 210029, China, 1Institute forNutritional Sciences, Shanghai Institutes for Biological Sciences, ChineseAcademy of Sciences, Shanghai 200031, Peoples Republic of China and2Department of General Surgery, Jiangsu Cancer Hospital, Nanjing 210009,China</p><p>To whom correspondence should be addressed. Tel/Fax: 86 25 868 62756;Email: hbshen@njmu.edu.cnCorrespondence may also be addressed to Hui Wang.Tel/Fax: 86 21 5492 0941;Email: huiwang@sibs.ac.cn</p><p>Fibroblast growth factor receptor 2 (FGFR2) belongs to theFGFR family, which plays an important role in cell growth, in-vasiveness, motility and angiogenesis. In human breast cancer,expression of FGFR2 is estrogen receptor (ER)-dependent andcorrelates with a lower rate of apoptosis. Recently, whole-genomeassociation studies have identified several single-nucleotide poly-morphisms (SNPs) of FGFR2 as novel breast cancer susceptibilityloci. In the present study of 1049 breast cancer patients and 1073cancer-free controls, we assessed whether polymorphisms ofFGFR2 are associated with breast cancer risk in Chinese womenand whether these associations are stronger in women with areproductive history suggestive of greater exposure to endogenousestrogens. We genotyped three FGFR2 polymorphisms(rs2981582C/T, rs1219648A/G and rs2420946C/T) using theSNPstream 12-plex platform. Each of the three SNPs was signif-icantly associated with increased breast cancer risk in a dose-dependent manner. Compared with women with 02 risk loci,those with 3 risk loci had a 1.36-fold increased odds of breastcancer (95% confidence interval 5 1.131.62, P 5 0.001). Instratified analyses, associations between the presence of 3 risk lociand breast cancer were stronger among women with ER- and/orprogesterone receptor-positive cancers, premenopausal womenand women with an older age at first live birth. Furthermore,there was a significant additive interaction between risk genotypesand menopausal status (P for multiplication interaction/additiveinteraction: 0.083/0.037). These findings indicate that genetic var-iants in FGFR2 may contribute to breast cancer occurrence inChinese women, possibly through pathways related to estrogenand/or progesterone.</p><p>Introduction</p><p>Breast cancer is the most commonly occurring cancer and the leadingcause of cancer death among women worldwide, accounting for 23% ofall cancers and .400 000 deaths annually (1). In China, the incidencerate of breast cancer has increased dramatically in recent years, witha sharp rise of 38.5% between 2000 and 2005 (2). The etiology of breastcancer appears to entail a complex combination of genetic, environ-mental and lifestyle factors; of these, menstrual and reproductive fac-tors, such as earlier menarche, nulliparity, later age at first birth andlater menopause, have a dominant relation to risk (35). In developingcountries, breast cancer tends to be diagnosed at an earlier age than in</p><p>European and American population, and the rapid rate of increase inincidence before menopause is not maintained afterwards, possiblyreflecting diminished levels of circulating estrogens (5).</p><p>Twin- and family-based studies indicate that a substantial propor-tion of breast cancer is due to inherited susceptibility, but only a smallfraction of cases are associated with inherited mutations of knownhigh-penetrance genes (e.g. BRCA1, BRCA2, ATM, etc.). Commonvariants in other lower penetrance genes may be more importantand may account for higher attributable risks (6). Recently, a large-scale genome-wide association study (7) identified five new indepen-dent low-penetrance susceptibility loci that were strongly associatedwith breast cancer in populations of diverse ethnicity. Four of these[fibroblast growth factor receptor 2 (FGFR2), TNRC9, MAP3K1 andLSP1] contain plausible causative genes, among which FGFR2 hasa prior relevance to breast cancer.</p><p>FGFR2 belongs to the FGFR family, which contributes to cellgrowth, invasiveness, motility and angiogenesis (8). Overexpressionof FGFR2 is observed in breast cancer cell lines (9), as well as inbreast tumor tissues (10). The FGFR2 gene, located at chromosome10q26, contains at least 22 exons (11). Recently, Easton et al. (7)found that rs2981582 in intron 2 of FGFR2 was associated with anincreased risk of breast cancer in a two-stage genome-wide associa-tion study among 4398 cases and 4316 controls, followed by a thirdstage of 21 860 cases and 22 578 controls from 22 studies. In addi-tion, in a genome-wide association study of 1145 post-menopausalwomen and 1142 controls of European ancestry, Hunter et al. (12)subsequently reported that four single-nucleotide polymorphisms(SNPs) (rs2420946, rs1219648, rs2981579 and rs11200014) in intron2 of FGFR2 were associated with breast cancer risk. Interestingly, thefive SNPs were in high linkage disequilibrium (LD) in people withEuropean ancestry (all pairwise r2 . 0.96).</p><p>Prior studies have not clearly documented possible relationshipsbetween FGFR2 polymorphisms and hormone receptor-defined sub-sets of breast cancer nor have possible interactions between FGFR2polymorphisms and breast cancer risk factors been reported. Weposited that FGFR2 polymorphisms may contribute to breast cancersusceptibility by acting through hormonal pathways, and we hypoth-esized that the risk associated with these polymorphisms would varydepending on the hormone receptor status of the breast tumor and onthe presence of known reproductive risk factors. To test this hypoth-esis, we performed genotyping analyses of rs2981582C/T (fromEaston et al.), rs1219648A/G and rs2420946C/T (the two mostsignificant SNPs from Hunter et al.) in FGFR2 in a casecontrolstudy of 1049 breast cancer cases and 1073 cancer-free controls ina Chinese population.</p><p>Materials and methods</p><p>Study subjects</p><p>This study was approved by the institutional review board of Nanjing MedicalUniversity. The hospital-based casecontrol study included 1058 breast cancercases and 1078 cancer-free controls, and the process of participant recruitmenthas been described previously (13). In brief, incident breast cancer patientswere consecutively recruited from the First Affiliated Hospital of NanjingMedical University, the Cancer Hospital of Jiangsu Province and the GulouHospital, Nanjing, China, between January 2004 and May 2007. Exclusioncriteria included reported previous cancer history, metastasized cancer fromother organs and previous radiotherapy or chemotherapy. All breast cancercases were newly diagnosed and histopathologically confirmed without restric-tions of age or histological type. Cancer-free control women, frequency-matched to the cases on age (5 years) and residential area (urban or rural),were randomly selected from a cohort of .30 000 participants in a commu-nity-based screening program for non-infectious diseases conducted from 2004to 2006 in the same Jiangsu Province, China. We determined the participants inthe cohort as cancer free if they did not report having cancer history at the time</p><p>Abbreviations: CI, confidence interval; ER, estrogen receptor; FGFR2, fibro-blast growth factor receptor 2; LD, linkage disequilibrium; OR, odds ratio; PR,progesterone receptor; SNP, single-nucleotide polymorphism.</p><p>yThese authors contributed equally to this work.</p><p> The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org 2341</p><p> at J.N. D</p><p>esmarais L</p><p>ibrary, Laurentian U</p><p>niversity on October 6, 2014</p><p>http://carcin.oxfordjournals.org/D</p><p>ownloaded from</p><p>http://carcin.oxfordjournals.org/</p></li><li><p>of interview and blood donation. All participants were genetically unrelated,ethnic Han Chinese women.</p><p>After providing informed consent, each woman was personally inter-viewed face-to-face by trained interviewers using a pretested standard ques-tionnaire to obtain information on demographic data, menstrual andreproductive history, environmental exposure history and family history ofany cancer in first-degree relatives (parents, siblings and children). The datacollection methods were similar for the cases and controls in terms of theinvestigation of reproductive history including number of births, numberof pregnancies, age at menopause and type of menopause. Natural post-menopausal was determined in both cases and controls for women withoutmenstrual flow for .12 months and also no history of sex hormone-relatedtreatment (e.g. surgery, radiation and drug treatment). After the interview,each subject provided 5 ml of venous blood. The estrogen receptor (ER) andprogesterone receptor (PR) status of breast cancers was determined from theresults of immunohistochemistry examinations contained in the medicalrecords of the hospitals. ER/PR statuses were carried out for the breast cancercases by established immunohistochemical method of avidinbiotinperoxidase complex assay. Immunoreactivity scoring was performed by eval-uating the percentage of cancer cell nuclear staining, and the percentage ofstaining cells 10% was considered positive.</p><p>Genotype analyses</p><p>Genomic DNA was isolated from leukocyte pellets from venous blood byproteinase K digestion and followed by phenolchloroform extraction. Thethree SNPs (rs2981582C/T, rs1219648A/G and rs2420946C/T) were geno-</p><p>typed on the GenomeLab SNPstream high-throughput 12-plex genotypingplatform (Beckman Coulter, Fullerton, CA) following the manufacturers in-structions. This platform uses a single-base pair extension reaction to incorpo-rate two-color fluorescence terminal nucleotides that are detected bya specialized imager (14).</p><p>Ge3notyping was performed without the knowledge of case or control status.Approximately equal numbers of case and control specimens were assayed ineach 384-well polymerase chain reaction plate with four blanks and 24 randomlyselected duplicates, and the coincidence rate was .99%. Genotyping failed innine cases (0.85%) and five controls (0.46%) due to DNA quality or quantity; alldata from these 14 women were excluded from analyses. Therefore, 1049 breastcancer cases and 1073 controls were included in the final analyses.</p><p>Statistical analyses</p><p>Differences between cases and controls in demographic characteristics, risk fac-tors and frequencies of FGFR2 variants were evaluated by v2 tests (for categor-ical variables) or Students t-test (for continuous variables). Associationsbetween FGFR2 genotypes and risk of breast cancer were estimated by com-puting odds ratios (ORs) and their 95% confidence intervals (CIs) from logisticregression analyses with adjustment for age, age at menarche, menopausal statusand family history of cancer. HardyWeinberg equilibrium was evaluated bya goodness-of-fit v2 test to compare the observed genotype frequencies with theexpected ones among the controls. We used the PHASE 2.0 program to inferhaplotype frequencies based on the observed FGFR2 genotypes. To performtests of possible generisk factor interactions for additive models, we imple-mented bootstrapping using Stata software (version 8.2; StataCorp LP, College</p><p>Table I. Distributions of select variables in breast cancer cases and cancer-free controls</p><p>Variables Cases, n 5 1049 Controls, n 5 1073 P OR (95% CI)</p><p>Age, year (mean SD) 51.59 11.18 51.77 11.10 0.712Age at menarche, year (mean SD) 15.29 1.94 16.03 1.92 ,0.0001Age at menopause, year (mean SD)a 48.99 4.42 49.26 4.23 0.318Age at first live birth, year (mean SD)b 25.58 3.42 24.94 3.20 ,0.0001Menopausal status 0.330</p><p>Premenopausal 509 (48.52%) 498 (46.41%)Post-menopausal 540 (51.48%) 575 (53.59%)</p><p>Family history of cancer ,0.0001Positive 305 (28.88%) 231 (21.53%)Negative 744 (70.92%) 842 (78.47%)</p><p>Parity 0.0041 589 (58.14) 528 (52.54) 1.002 236 (23.30) 259 (25.77) 0.82 (0.661.01)3 114 (11.25) 104 (10.35) 0.98 (0.741.31)4 74 (7.31) 114 (11.34) 0.58 (0.430.80)</p><p>Pregnancy 0.0011 167 (17.04) 221 (22.37) 1.002 284 (28.98) 309 (31.28) 1.22 (0.941.57)3 246 (25.10) 186 (18.83) 1.75 (1.332.31)4 283 (28.88) 272 (27.53) 1.38 (1.061.79)</p><p>Type of menopause ,0.0001Natural 458 (84.81) 571 (99.30) 1.00Unusualc 82 (15.19) 4 (0.70) 25.56 (9.3070.25)</p><p>ERd</p><p>Positive 439 (54.00)Negative 374 (46.00)</p><p>PRd</p><p>Positive 446 (54.86)Negative 367 (45.14)</p><p>Menopausal status Premenopausal Post-menopausal ,0.0001ER/PR 189 (48.46) 175 (41.37)ER/PR 49 (12.56) 33 (7.80)ER/PR 20 (5.13) 55 (13.00)ER/PR 132 (33.85) 160 (37.83)</p><p>Age ,50 50 0.0001ER/PR 182 (47.52) 182 (42.33)ER/PR 53 (13.84) 29 (6.74)ER/PR 25 (6.53) 50 (11.63)ER/PR 123 (32.11) 169 (39.30)</p><p>aAge at menopause information was available in 522 breast cancer cases (96.7%) and 518 controls (90.1%) with post-menopausal status.bAge at first live birth information was available in 1006 breast cancer cases (95.9%) and 1043 controls (97.2%).cUnusual menopause include operation, radiotherapy or other status.dInformation of ER and PR status was available in 813 breast cancer cases (77.5%).</p><p>J.Liang et al.</p><p>2342</p><p> at J.N. D</p><p>esmarais L</p><p>ibrary, Laurentian U</p><p>niversity on October 6, 2014</p><p>http://carcin.oxfordjournals.org/D</p><p>ownloaded from</p><p>http://carcin.oxfordjournals.org/</p></li><li><p>Station, TX). All the statistical analyses were performed with Statistical AnalysisSystem software (9.1.3; SAS Institute, Cary, NC), unless indicated otherwise.</p><p>Results</p><p>The 1049 breast cancer cases and 1073 cancer-free controls weresimilar with regard to age at interview, menopausal status and age atmenopause (Table I). However, cases differed from controls in havingan earlier age at menarche, a later age of first live birth, a greaterlikelihood of a positive family history of any cancer, less parities andmore pregnancies. Unusual menopause was also a major risk factorfor breast cancer. Among 813 breast cancer cases with known ER andPR status, 364 (44.8%) were positive with both ER and PR and 292(33.9%) were negative for both. When we stratified ER/PR status bymenopause status and age, post-menopausal and aged women weremore likely to be ER/PR breast cancer patients (Table I).</p><p>The genotype distributions of FGFR2 rs2981582C/T, rs1219648A/Gand rs2420946C/T polymorphisms in the breast cancer cases andcontrols are shown in Table II. The call rates of the three SNPs wereall .97%. The observed genotype frequencies for these three SNPswere all in agreement with HardyWeinberg equilibrium in the con-trols (P 5 0.974, 0.270 and 0.413 for rs2981582, rs1219648 andrs2420946, respectively).</p><p>In the multivariate logistic regression models (Table II), each of thevariant genotypes of FGFR2 rs2981582 was associated with a signif-icantly increased breast cancer risk compared with the wild-type CCgenotype (adjusted OR 5 1.31, 95% C...</p></li></ul>