ANTICANCER RESEARCH 28 : 3079-3082 (2008)

Association Between Genetic Polymorphisms and Ovarian Cancer Risk LAETITIA DELORT 1,2,3 , NASSÉRA CHALABI 1,2,3 , SAMIR SATIH 1,2,3 , NADÈGE RABIAU 1,2,3 , FABRICE KWIATKOWSKI 1, YVES-JEAN BIGNON 1,2,3,4 and DOMINIQUE J BERNARD-GALLON 1,2,3

1Département d’Oncogénétique du Centre Jean Perrin, CBRV, 3EA 2416, Université d’Auvergne-CJP and 4Univ-Clermont 1, 63001 Clermont-Ferrand; 2CRNH, 63009 Clermont-Ferrand; France

Abstract. Background: The etiology of ovarian cancer is and estrone at ovulation. These compounds could in turn not fully understood. Polymorphisms in low penetrance genes stimulate the proliferation of ovarian cells (3). Indeed, estrogen involved in carcinogen and estrogen metabolism are metabolism produces reactive estrogen metabolites hypothesized to play a role in the initiation of (catecholestrogens, CE) by phase I enzymes which are then carcinogenesis. Patients and Methods: A case –control study metabolized to semi-quinones and quinones. Two phase I was conducted to investigate the role of these polymorphisms enzymes are involved in this pathway: cytochrome P450 1A1 in ovarian cancer risk. The participants were genotyped for (CYP1A1) produces 2-OH-E2, a CE which can form stable eleven polymorphisms in seven genes involved in estrogen adducts; cytochrome P450 1B1 (CYP1B1) produces 4-OH-E2, and xenobiotic metabolism (CYP1A1, CYP1B1, COMT, which forms depurinating agents which may lead to the GSTP1, NAT2, estrogen receptor ESR, and progesterone initiation of a carcinogenic process. Phase II enzymes such as receptor PGR). Results and Conclusion: The odds ratios for catechol- O-methyl transferase (COMT) and glutathione-S- ovarian cancer risk were 2.02 (95% confidence interval [CI] transferase (GSTP1) intervene by inactivating CE and quinones = 1.14-3.56) in the NAT2 intermediate acetylators and 4.07 respectively. These carcinogens can also be detoxified by (95% CI = 1.30-12.70) in the slow acetylators. At least three another phase II enzyme, N-acetyltransferase 2 (NAT2). The cumulative high-risk genotypes increased ovarian cancer presence of single nucleotide polymorphisms (SNPs) in this risk, but not significantly. More studies are needed in order enzyme’s gene defines different acetylation phenotypes to define genetic ovarian risk factors. (fast/intermediate/slow) (4, 5). The presence of polymorphisms in the cited genes modifies the enzymatic activities, leading to Ovarian cancer is the fifth cause of cancer among French a less efficient detoxication of reactive compounds and thus women, with 4,488 new cases and 3,508 deaths reported in could be involved in ovarian carcinogenesis. 2000 (1). The etiology of this pathology is poorly understood The association between polymorphisms in the genes and most risk factors are related to hormonal exposure and involved in estrogen and xenobiotic metabolism and ovarian reproduction. Two major hypotheses have been formulated in cancer risk was investigated. order to explain the etiology of ovarian cancer. The first is that of “incessant ovulation” in which cell proliferation is Patients and Methods stimulated and malignant transformation of the ovarian A case –control study consisting of 51 ovarian cancer cases with no epithelium occurs (2). The second is the “gonadotropin BRCA mutation and no familial history of ovarian cancer and 1,000 hypothesis” which implicates the role of hormonal stimulation healthy women was conducted. All the participants came from the on ovarian epithelial cells. The ovaries secrete estradiol (E2) Auvergne region in France. Written informed consent was obtained from all the participants . A blood sample was collected and a questionnaire concerning reproductive, anthropometric and lifestyle characteristics was completed. The 5’ nuclease assay (Taqman) Correspondence to: Pr ofessor Yves-Jean Bignon, Département (Applied Biosystems, Foster City, CA, USA) was used to genotype d’Oncogénétique, Centre Jean Perrin, 58 rue Montalembert, B.P. eleven SNP s in seven low penetrance genes: CYP1A1 Ile 462 Val 392, 63011 Clermont-Ferrand, France. Tel: +33 0473278201, Fax: [dbSNP: rs1048943]; CYP1B1 Val 432 Leu [dbSNP: rs1056836] and +33 0473278042, e-mail: [email protected] CYP1B1 Asn 453 Ser [dbSNP: rs1800440]; NAT2-341 Ile 114 Thr [dbSNP: rs1801280]; NAT2-481 Leu 161 Leu [dbSNP: rs1799929]; Key Words: Ovarian cancer, polymorphisms, estrogen and xenobiotic NAT2-803 Arg 268 Lys [dbSNP: rs1208]; NAT2-590 Arg 197 Gln metabolism. [dbSNP: rs1799930]; COMT Val 158 Met [dbSNP: rs4680] and

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Table I. Distribution of the genotype of each polymorphism among Table II. Evaluation of ovarian cancer risk according to the number of ovarian cancer cases and controls. high-risk genotypes in the CYP1A1 Ile 462 Val, CYP1B1 Val 432 Leu, COMT Val 158 Met and NAT2 acetylator phenotype. Genotype No. of No of OR a 95% CI b cases (%) controls (%) No. high-risk genotype a No. of No. of OR 95% CI cases controls CYP1A1 Ile 462 Val Ile-Ile 48 (94.1) 932 (93.6) 1.00 0122 Ile-Val 3 (5.9) 59 (5.9) 0.85 0.28-2.60 15166 0.66 0.08-5.85 Val-Val 0 (0) 5 (0.5) 0.72 0.08-6.75 2 17 434 0.86 0.11-6.76 CYP1B1 Val 432 Leu 3 26 344 1.66 0.22-12.56 Val-Val 6 (11.8) 203 (20.3) 1.00 4215 2.93 0.27-32.20 Val-Leu 27 (52.9) 475 (47.5) 1.26 0.84-1.88 Leu-Leu 18 (35.3) 322 (32.2) 1.58 0.70-3.55 aLow-risk genotype is defined as the wild-type homozygous for CYP1B1 Asn 453 Ser CYP1A1, CYP1B1, COMT and the (fast-intermediate) NAT2 acetylator Asn-Asn 32 (62.7) 660 (15.5) 1.00 phenotype. Asn-Ser 18 (35.2) 301 (47.0) 1.26 0.63-1.70 Ser-Ser 1 (2) 39 (37.5) 1.58 0.40-2.90 Acetylation Fast 1 (1.9) 52 (5.2) 1.00 acetylator phenotype (Table I). Unfortunately, the results Intermediate 13 (25.5) 395 (39.5) 2.02 1.14-3.56 Slow 37 (72.6) 547 (54.7) 4.07 1.30-12.70 concerning the other studied SNP, were not significant, COMT Val 158 Met certainly due to the small number of cases. However, some Val-Val 11 (21.6) 237 (23.7) 1.00 trends could be observed and, in particular, an increased risk Val-Met 22 (43.1) 480 (48.0) 1.19 0.80-1.77 associated with CYP1B1 Leu 432 and COMT Met 158 carriers, Met-Met 18 (35.3) 283 (28.3) 1.42 0.65-3.13 whereas a decreased risk could be attributed to the PGR and ESR Thr 594 Thr ACA/ACA 0 (0) 40 (4.0) 1.00 GSTP1 variants. ACA/ACG 15 (29.4) 275 (27.7) 1.25 0.72-2.17 The impact of the SNP was assessed according to the ACG/ACG 36 (70.6) 679 (68.3) 1.57 0.52-4.72 NAT2 acetylation phenotype, since a significant increased PGR +331 G>A risk had previously been shown, and to the COMT GG 48 (94.1) 916 (91.6) 1.00 genotype in order to compare with other studies (7, 8). As GA 3 (5.9) 80 (8.0) 0.67 0.21-2.12 AA 0 (0) 4 (0.4) 0.45 0.05-4.50 only one ovarian cancer case presented a fast acetylation GSTP Ile 105 Val phenotype, the fast and intermediate acetylators were Ile/Ile 26 (46.2) 459 (45.9) 1.00 grouped together for further analysis. An increased risk Ile/Val 20 (45.6) 434 (43.4) 0.88 0.57-1.35 seemed to be associated with the combination CYP1A1 Ile- Val/Val 5 (8.2) 107 (10.7) 0.77 0.32-1.83 Val/ COMT Met-Met (OR=3.53, 95% CI=1.00-12.55) and aOR, odds ratios adjusted for age; b95% CI, 95% confidence interval. with the combination NAT2 slow acetylator/ COMT Val- Met (OR=5.09, 95% CI=1.67-15.51) compared to the basic OR. When the ovarian cancer risk was explored according to the number of high-risk genotypes, the 462 432 158 GSTP1 Ile 105 Val [dbSNP: rs1695]. Polymorphisms in the estrogen CYP1A1 Ile Val, CYP1B1 Val Leu, COMT Val Met and progesterone receptors ( ESR Thr 594 Thr [dbSNP: rs2228480]; and NAT2 acetylation phenotype, a non-significant increase PGR +331G/A [dbSNP: rs10895068]) were also investigated in risk was observed with at least three high-risk genotypes because of their major role in hormone metabolism. The software (Table II). SEM (Centre Jean-Perrin, Clermont Ferrand, France) was used for data analyses (6). Odds ratios (ORs) adjusted for age and 95% These results suggested a possible effect of polymorphisms confidence interval (95% CI) were calculated by unconditional in COMT and NAT2 in ovarian cancer risk. These two genes logistic regression for the analysis of allelic frequencies and encode phase II enzymes and regulate the level of produced distribution of genotypes between the two populations. carcinogens. The studied polymorphisms lead to reduced enzymatic activity, reduced elimination of carcinogens and Results and Discussion catecholestrogens, and thus could affect the initiation of a carcinogenic process. No difference in allelic frequency was observed between the Few previous studies have determined the association ovarian cancer cases and the controls. The analysis of the between polymorphisms in the cited genes and ovarian genotype distribution showed a significant increased risk cancer risk. Holt et a l.’ s (8) was the only study to show an with the NAT2 intermediate and slow acetylator phenotypes, increased risk for CYP1B1 Leu 432 carriers, with an OR of with ORs equal to 2.02 (95% CI=1.14-3.56) and 4.07 (95% 1.5 (95% CI=1.1-2.3) compared to Val 432 homozygotes. CI=1.30-12.70) respectively in comparison with the fast When the results were stratified according to the COMT

3080 Delort et al : Ovarian Cancer and Genetic Polymorphisms genotype, a 2.6-fold increase in risk for the homozygous References CYP1B1 Leu 432 and COMT Met 158 individuals was found (8). In contrast, Goodman et al . (7), found an increased 1 Tretarre B, Remontet L, Menegoz F et al : Ovarian cancer: risk for CYP1B1 Val 432 carriers, which was more in incidence and mortality in France. J Gynecol Obstet Biol Reprod accordance with results obtained for breast cancer for (Paris) 34 : 154-161, 2005. 2 Fathalla MF: Incessant ovulation-a factor in ovarian neoplasia? example, and suggested a joint effect between CYP1B1 432 158 Lancet 2: 163, 1971. Val and COMT Met carriers. Gene –environment and 3 Lukanova A and Kaaks R: Endogenous hormones and ovarian gene –gene interactions were investigated, but no significant cancer: epidemiology and current hypotheses. Cancer Epidemiol results were obtained (7). A large case –control study in the Biomarkers Prev 14 : 98-107, 2005. United States failed to show an association between 4 Cheng TC, Chen ST, Huang CS et al : Breast cancer risk polymorphisms in CYP1A1, CYP1B1, COMT and SULT1A1 associated with genotype polymorphism of the catechol (sulfotransferase 1A1), and ovarian cancer risk. A estrogen-metabolizing genes: a multigenic study on cancer susceptibility. Int J Cancer 113 : 345-353, 2005. multigenic model was constructed which demonstrated 5 Mitrunen K and Hirvonen A: Molecular epidemiology of sporadic some evidence that SNP s in these four genes breast cancer. The role of polymorphic genes involved in oestrogen simultaneously were associated with ovarian cancer (9). biosynthesis and metabolism. Mutat Res 544 : 9-41, 2003. More studies are needed in order to identify ovarian 6 Kwiatkowski F, Girard M, Hacène K and Berlie J: Sem: a cancer genetic susceptibility. The strengths of the present suitable statistical software adaptated for research in oncology. study were the population genetic homogeneity and the Bull Cancer 87 : 715-721, 2000. number of studied polymorphisms. Indeed, the studies 7 Goodman MT, McDuffie K, Kolonel LN et al : Case –control study of ovarian cancer and polymorphisms in genes involved in previously cited were composed of a mix between Asian, catecholestrogen formation and metabolism. Cancer Epidemiol Caucasian or other populations, which could explain the Biomarkers Prev 10 : 209-216, 2001. differences in the results. The present analysis was limited 8 Holt SK, Rossing MA, Malone KE, Schwartz SM, Weiss NS and by the small number of ovarian cancer cases, revealing Chen C: Ovarian cancer risk and polymorphisms involved in only trends in ovarian cancer risk and limited gene –gene estrogen catabolism. Cancer Epidemiol Biomarkers Prev 16 : interactions. 481-489, 2007. 9 Sellers TA, Schildkraut JM, Pankratz VS et al : Estrogen bioactivation, genetic polymorphisms, and ovarian cancer. Acknowledgements Cancer Epidemiol Biomarkers Prev 14 : 2536-2543, 2005.

This study was funded by La Ligue Nationale Française de Lutte Contre le Cancer (Puy-de-Dôme, Allier, Cantal). Laetitia Delort is a recipient of L’Association pour la Recherche sur le Cancer. The Received April 18, 2008 authors thank Centre République for the enrollment of the control Revised June 10, 2008 population. Accepted June 16, 2008

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