Polymorphisms in Angiogenesis-Related Genes and Prostate Cancer

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Short Communication

Eric J. Jacobs,1 Ann W. Hsing,2 Elizabeth B. Bain,1 Victoria L. Stevens,1 Yiting Wang,1 Jinbo Chen,3 Stephen J. Chanock,2 S. Lilly Zheng,4 Jianfeng Xu,4 Michael J. Thun,1 Eugenia E. Calle,1 and Carmen Rodriguez1 1Department of Epidemiology and Surveillance Research, American Cancer Society, Atlanta, Georgia; 2Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Department of Health and Human Services, Bethesda, Maryland; 3Department of Epidemiology and Biostatistics, University of Pennsylvania, Philadelphia, Pennsylvania; and 4Center for Human Genomics, Wake Forest University School of Medicine, Winston-Salem, North Carolina

Abstract

Background: Angiogenesis is required for development EGF, LTA, HIF1AN, MMP9,orNOS2A. In the MMP2 and progression of prostate cancer. Potentially func- gene, three intronic SNPs, all in linkage disequilibri- tional single nucleotide polymorphisms (SNP) in genes um, were associated with overall and advanced prostate VEGF, HIF1A P important in prostate angiogenesis ( , and cancer (for overall prostate cancer, trend = 0.01 for NOS3 P P ) have previously been associated with risk or rs1477017, trend = 0.01 for rs17301608, trend = 0.02 for severity of prostate cancer. rs11639960). However, two of these SNPs (rs17301608 Methods: Prostate cancer cases (n = 1,425) and controls and rs11639960) were examined and were not associated (n = 1,453) were selected from the Cancer Prevention with prostate cancer in a recent genome-wide associa- Study II Nutrition Cohort. We examined associations tion study using prostate cancer cases and controls from between 58 SNPs in nine angiogenesis-related candi- the Prostate, Lung, Colorectal, and Ovary study cohort. date genes (EGF, LTA, HIF1A, HIF1AN, MMP2, MMP9, Furthermore, when we pooled our results for these two NOS2A, NOS3, VEGF) and risk of overall and advanced SNPs with those from the Prostate, Lung, Colorectal, prostate cancer. Unconditional logistic regression was and Ovary cohort; neither SNP was associated with used to estimate odds ratios, adjusted for matching prostate cancer. factors. Conclusion: None of the SNPs examined seem likely Results: Our results did not replicate previously to be importantly associated with risk of overall or observed associations with SNPs in VEGF, HIF1A,or advanced prostate cancer. (Cancer Epidemiol Bio- NOS3, nor did we observe associations with SNPs in markers Prev 2008;17(4):972–7)

Introduction

Angiogenesis, the growth of new blood vessels, is genes, described individually below, which are impor- required for the growth of microscopic cancers into tant in prostate angiogenesis. We then used cases and larger, clinically relevant tumors (1). The importance of controls from a large cohort of U.S. men to examine angiogenesis specifically in prostate carcinogenesis is associations between 58 polymorphisms in these genes supported by a large body of research, including studies and risk of advanced and overall prostate cancer. demonstrating altered expression of angiogenic factors Vascular endothelial growth factor (VEGF) plays a in prostate cancer, inhibition of tumor growth in animal central role in prostate angiogenesis (2). The G allele of a models after treatment with angiogenesis inhibitors, and VEGF promoter region single nucleotide polymorphism correlations between tumor blood vessel density and (SNP; rs1570360, also known as 1154 G/A), which both tumor characteristics and clinical outcome (2, 3). increases VEGF transcription (4), has been associated Proangiogenic factors important in prostate angiogenesis with significantly increased risk of prostate cancer in two have been reviewed (2, 3). We selected nine candidate small case control studies (5, 6). Hypoxia inducible factor 1 (HIF1A) is a transcription factor that is overexpressed even in early stage prostate cancer and increases transcription of VEGF (7). The Received 11/10/07; revised 1/15/08; accepted 1/21/08. minor allele of a SNP encoding an amino acid substitu- Grant support: Intramural Research Program of the NIH, National Cancer Institute. tion (rs11549465, also known as P582S) has been 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 associated with increased risk in a relatively small case with 18 U.S.C. Section 1734 solely to indicate this fact. control study of metastatic prostate cancer (8) and has Requests for reprints: Eric J. Jacobs, Epidemiology and Surveillance Research, also been detected as a somatic mutation in prostate American Cancer Society, National Home Office, 250 Williams Street, Atlanta, GA 30303-1002. Phone: 404-329-7916; Fax: 404-327-6450. E-mail: [email protected] cancers (9). However, no statistically significant associ- Copyright D 2008 American Association for Cancer Research. ation between this SNP and prostate cancer risk was doi:10.1158/1055-9965.EPI-07-2787 found in a recent larger study (10). Hypoxia inducible

Cancer Epidemiol Biomarkers Prev 2008;17(4). April 2008

factor l a subunit inhibitor (HIF1AN) is a potentially cluded because they were found not to have an adequate important inhibitor of HIF1A activity (11). sample available or because of handling or laboratory Epidermal growth factor (EGF) can increase VEGF errors. A total of 1,425 cases and 1,453 controls remained expression in prostate cancer cell lines (12). The minor for analysis. allele of a SNP in the 5¶ untranslated region (UTR) of the Of the cases included in this analysis, 62% were EGF gene (rs4444903, also known as 61 A/G) has been diagnosed before they provided a blood sample. How- associated with increased EGF expression as well as risk ever, important survival bias was considered unlikely and/or severity of melanoma and gastric cancer in some because only a small proportion (2.9%) of prostate cancer studies (reviewed in ref. 13). cases diagnosed during the time period included in Lymphotoxin a (LTA) increases synthesis of VEGF in this analysis died of prostate cancer before collection of prostate cancer cell lines (14). An LTA SNP (rs909253) blood samples from cohort members was completed. may increase transcription (15). We categorized 559 cases as advanced prostate cancer, Endothelial nitric oxide synthase (NOS3) and induc- defined as stage II cancers with a Gleason score of z7, ible nitric oxide synthase (NOS2A) both catalyze the syn- all stage III or stage IV cancers, and prostate cancers that thesis of nitric oxide, which can be proangiogenic (16), were listed as the underlying cause of death on a death and NOS2A is overexpressed in prostate cancer (17). A certificate. small case control study of a SNP in NOS3 (rs1799983, also known as D298E) found no overall association with SNP Selection and Genotyping. We selected SNPs prostate cancer risk (18) but reported that the minor allele based on results from previous epidemiologic studies was associated with reduced risk of advanced disease and potential functional importance. In addition, for se- EGF, HIF1A, HIF1AN, MMP2, MMP9 among prostate cancer cases (19). veral genes ( and ), Matrix metalloproteinase 2 (MMP2) and MMP9 we also selected all SNPs identified as haplotype-tagging degrade basement membranes and extracellular matrix, SNPs, using the method developed by Gabriel et al. (24), processes that are necessary for both angiogenesis and from HapMap data available in January 2006. tumor invasion (20). MMP2 is overexpressed in prostate Genotyping was done in two phases. The first phase cancer, with higher expression levels predicting poorer included genotyping of 10 SNPs at the National Cancer survival (21). The minor allele of a MMP2 promoter Institute’s Core Genotyping Facility, using a Taq Man LTA, VEGF region SNP (rs243865, also known as 1306 C/T) reduces assay. These included all SNPs for the , and transcription (22). NOS2A genes, and one of the EGF SNPs (rs4444903). Only cases diagnosed between enrollment in 1992 and June 2001 (n = 1,173) and matched controls (n = 1,187) Materials and Methods were included in the first phase, as follow-up through 2003 was not yet available at the time of genotyping. Study Population. Men in this analysis were selected The second phase included genotyping of an addi- from among the 86,404 male participants in the Cancer tional 48 SNPs at the Center for Human Genomics Prevention Study II Nutrition Cohort, a prospective (Wake Forest University), using the MassARRAY system study of cancer incidence among U.S. men and women (SEQUENOM). All prostate cancer cases (n = 1,425) established in 1992 (23). Approximately 99% of male diagnosed through August 2003 and matched controls participants were between the ages of 50 and 79 y at the (n = 1,453) were included in the second phase, including f time of enrollment, and 97% were White (23). Follow- all the cases and controls that were in the first phase. up questionnaires were sent to cohort members in 1997 All 48 SNPs were initially genotyped on a subset of and every 2 y thereafter to ascertain newly diagnosed 553 predominantly advanced prostate cancer cases cancers. Incident cancers reported on questionnaires (529 advanced cases that were verified at the time of were verified through medical records, linkage with genotyping as well as an additional 24 not advanced state cancer registries, or death certificates. From June cases) and matched controls (n = 553). To minimize 1998 through June 2001, participants in the Nutrition genotyping costs, 15 of these SNPs were then selected Cohort were invited to provide a blood sample. After for additional genotyping (based on previous literature obtaining informed consent, blood samples were collected and the initial genotyping results), using all remaining from 17,411 men. The recruitment, characteristics, and cases and matched controls. follow-up of the Nutrition Cohort are described in For quality control, 3.5% of the samples genotyped greater detail elsewhere (23). during both phases of the study were blinded replicates. From men who provided a blood sample, we Concordance for these replicates was 100% for all SNPs. identified 1,476 who had been diagnosed with prostate cancer between 1992 and 2003, and had not been Statistical Analysis. Odds ratios (OR) and 95% diagnosed with any other cancer (other than nonmela- confidence intervals (95% CI) for the association between noma skin cancer). For each case, we randomly selected each SNP and overall and advanced prostate cancer one control from among those who had provided a blood incidence were determined using unconditional logistic sample and had no history of cancer on the diagnosis regression. Our primary measure of association was date of the case. Each control was individually matched the per-allele OR, determined by entering a continuous to their case on birth date (F6 mo), date of blood variable for the number of minor alleles (0, 1, or 2), collection (F6 mo), and race/ethnicity (White, African- into the logistic regression model. All models were American, Hispanic, Asian, and other/unknown). adjusted for the study matching factors of birth year Twenty-nine cases were later excluded because their (single year categories), date of blood draw (single year initial self-report of prostate cancer could not be verified. categories), and race/ethnicity (White or other/unknown, An additional 22 cases and 23 controls were later ex- African-American, Hispanic, and Asian). In analyses

Cancer Epidemiol Biomarkers Prev 2008;17(4). April 2008

Table 1. ORs for prostate cancer associated with each additional minor allele, CPS-II Nutrition Cohort 1992 to 2003 b Gene/SNP* Location Major/ Controls Advanced c All prostate cancer minor prostate cancer allele x x k x k n MAF n MAF OR (95% CI), P n MAF OR (95% CI), P k k per minor allele per minor allele EGF { rs4444903 (61A/G) Exon 1 A/G 1,178 40.6 458 39.9 0.98 (0.84- 1.15) 0.82 1,163 39.8 0.98 (0.87-1.10) 0.68 rs881878 Intron 1 G/A 551 32.0 525 33.4 1.08 (0.90-1.29) 0.44 rs2250724** Intron 1 C/T 551 8.4 526 6.8 0.80 (0.58-1.10) 0.16 rs4698801 Intron 7 C/A 551 33.3 526 34.4 1.07 (0.89-1.29) 0.46 rs11568943 (R431K) Exon 8 G/A 1,441 7.6 557 6.3 0.82 (0.63-1.08) 0.16 1,419 7.5 0.99 (0.81-1.20) 0.90 rs2298991 Intron 11 T/G 552 42.7 526 41.2 0.95 (0.80-1.13) 0.58 rs11568993 Exon 13 C/T 553 8.5 526 7.4 0.87 (0.63-1.19) 0.38 rs11098057 Intron 13 G/C 550 39.7 526 38.3 0.96 (0.80-1.14) 0.61 rs2237051 (M708I)** Exon 14 G/A 551 39.9 526 38.4 0.95 (0.80-1.13) 0.59 rs11569017 Exon 15 A/T 551 7.2 526 5.3 0.73 (0.52-1.04) 0.081 rs2237052 Intron 18 A/G 551 39.5 525 38.2 0.96 (0.81-1.14) 0.65 rs2237054 Intron 18 T/A 550 8.6 526 6.5 0.75 (0.55-1.04) 0.084 rs2298999** Intron 18 T/C 551 42.7 523 41.7 0.97 (0.82-1.15) 0.71 rs7692976 Intron 18 A/G 546 42.1 521 41.8 1.00 (0.84-1.19) 0.99 rs4698803 (E920V) Exon 19 T/A 553 19.2 529 18.9 0.97 (0.78-1.20) 0.77 rs6533485 Intron 22 G/C 551 48.4 526 46.8 0.95 (0.80-1.12) 0.54 HIF1A rs7143164 Intron 1 G/C 551 10.3 525 9.7 0.94 (0.71-1.25) 0.68 rs12435848** Intron 1 G/A 553 20.2 528 19.4 0.94 (0.76-1.16) 0.56 rs4899056** Intron 4 C/T 553 10.9 527 11.2 1.04 (0.79-1.36) 0.80 rs1957757** Intron 6 C/T 551 9.3 527 9.1 0.99 (0.74-1.32) 0.93 rs11158358 Intron 6 C/G 553 16.8 527 15.8 0.91 (0.72-1.14) 0.40 rs2301111 Intron 7 C/G 553 22.1 528 21.0 0.93 (0.75-1.14) 0.47 rs2301113 Intron 10 A/C 549 23.0 527 22.1 0.93 (0.76-1.15) 0.52 rs11549465 (P582S)** Exon 12 C/T 1,450 11.7 556 9.6 0.80 (0.64-1.00) 0.047 1,420 9.7 0.80 (0.68-0.95) 0.010 HIF1AN rs1080698 5¶ UTR G/T 551 22.0 526 21.1 0.95 (0.78-1.17) 0.64 rs11190599 5¶ UTR C/T 551 22.1 526 21.1 0.95 (0.78-1.17) 0.64 rs2295778 (P41A) Exon 1 C/G 1,448 28.7 557 27.1 0.93 (0.79-1.08) 0.32 1,420 27.3 0.93 (0.83-1.04) 0.22 rs10883509 Intron 2 C/T 551 22.1 526 21.1 0.95 (0.78-1.17) 0.64 rs11190602 Intron 2 T/C 551 22.1 525 21.1 0.94 (0.77-1.16) 0.57 LTA { rs746868 Intron 1 G/C 1,176 40.4 457 41.4 1.04 (0.89-1.22) 0.61 1,159 39.7 0.97 (0.87-1.09) 0.64 { rs909253 (252A/G) Intron 1 A/G 1,184 32.6 460 32.5 1.00 (0.85-1.17) 0.96 1,166 33.8 1.06 (0.93-1.19) 0.39 { rs2239704 Exon 1 C/A 1,184 41.3 453 41.4 1.01 (0.86-1.18) 0.94 1,161 40.4 0.97 (0.86-1.09) 0.58 MMP2 cc (735C/T) 5¶ UTR C/T 551 10.4 525 10.4 1.00 (0.76-1.32) 0.98 rs243865 (1306C/T) 5¶ UTR C/T 1,449 24.5 557 27.5 1.15 (0.99-1.35) 0.074 1,418 25.2 1.03 (0.91-1.16) 0.66 rs865094 Intron 2 A/G 1,446 18.2 556 16.2 0.87 (0.72-1.05) 0.13 1,417 16.4 0.89 (0.77-1.02) 0.087 rs1477017 Intron 2 A/G 1,441 34.0 557 38.8 1.23 (1.07-1.42) 0.0047 1,417 37.3 1.15 (1.03-1.28) 0.011 rs11076101 Intron 3 C/T 1,453 8.7 556 7.2 0.81 (0.63-1.06) 0.12 1,419 7.3 0.83 (0.69-1.01) 0.064 rs17301608** Intron 3 C/T 1,432 35.4 552 40.6 1.25 (1.08-1.44) 0.0024 1,414 38.6 1.14 (1.03-1.27) 0.014 rs2192852 Intron 7 A/G 1,442 14.8 545 16.3 1.06 (0.93-1.22) 0.37 1,408 16.4 1.07 (0.96-1.18) 0.23 rs243840** Intron 9 A/G 550 18.4 527 17.0 0.92 (0.74-1.14) 0.44 rs9923304 Intron 9 C/T 551 43.1 527 43.2 1.01 (0.84-1.20) 0.96 rs243836 Intron 11 G/A 1,443 49.0 556 47.5 0.94 (0.81-1.08) 0.36 1,417 48.6 0.98 (0.88-1.09) 0.72 rs11639960** Intron 11 A/G 1,439 31.9 554 36.1 1.22 (1.05-1.41) 0.0095 1,410 34.8 1.14 (1.02-1.27) 0.020 rs11541998 Exon 12 C/G 1,440 10.8 555 11.4 1.07 (0.85-1.33) 0.57 1,411 10.6 0.99 (0.83-1.17) 0.86 rs7201 Exon 13 A/C 1,446 45.1 556 46.8 1.07 (0.93-1.24) 0.33 1,416 45.3 1.00 (0.90-1.12) 0.96 MMP9 rs17576 Exon 6 A/G 549 35.2 521 35.8 1.03 (0.86-1.22) 0.79 rs3918256 Intron 7 A/G 548 43.3 522 41.7 0.94 (0.79-1.12) 0.48 rs3787268 Intron 8 G/A 553 19.7 527 20.9 1.09 (0.88-1.34) 0.45 rs2250889 (R574P) Exon 10 C/G 1,441 5.5 555 4.4 0.81 (0.58-1.13) 0.21 1,415 5.1 0.95 (0.75-1.20) 0.65 rs2274756 (R668Q)** Exon 12 G/A 550 15.4 525 14.6 0.93 (0.73-1.18) 0.56 NOS2A { rs2297518 (S608L) ** Exon 16 C/T 1,178 18.7 459 19.2 1.03 (0.84-1.25) 0.80 1,166 20.5 1.11 (0.96-1.29) 0.16 NOS3 rs2070744 (786T/C) Intron 1 T/C 553 39.6 527 37.1 0.90 (0.76-1.07) 0.24 rs1799983 (D298E) Exon 7 G/T 1,446 32.1 556 30.9 0.95 (0.82-1.10) 0.51 1,420 31.3 0.96 (0.86-1.07) 0.48 VEGF { rs699947 (2578 C/A) 5¶ UTR C/A 1,177 48.8 460 49.9 1.05 (0.90-1.22) 0.57 1,165 47.9 0.96 (0.86-1.08) 0.53 { rs1570360 (1154G/A) 5¶ UTR G/A 1,172 31.6 458 33.0 1.06 (0.90-1.24) 0.50 1,158 32.2 1.02 (0.90-1.15) 0.73 { rs25648 Exon 1 C/T 1,161 16.8 450 17.2 1.04 (0.85-1.27) 0.70 1,142 16.2 0.96 (0.82-1.12) 0.57

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Table 1. ORs for prostate cancer associated with each additional minor allele, CPS-II Nutrition Cohort 1992 to 2003 (Cont’d) b Gene/SNP* Location Major/ Controls Advanced c All prostate cancer minor prostate cancer allele x x k x k n MAF n MAF OR (95% CI), P n MAF OR (95% CI), P k k per minor allele per minor allele { rs2010963 Exon 1 G/T 1,158 33.3 446 29.8 0.85 (0.71-1.00) 0.050 1,138 31.2 0.92 (0.81-1.04) 0.16 { rs3025039 (+936C/T) 3¶ UTR C/T 1,163 14.5 458 13.2 0.91 (0.73-1.14) 0.39 1,163 14.1 0.98 (0.83-1.15) 0.79

Abbreviation: CPS-II, Cancer Prevention Study II. *The name by which the SNP is commonly referred to in the literature, or the identity and location of the amino acid substitution, when one exists, is shown in parentheses. cAdvanced cases defined as having a Gleason score of z7, or stage III/IV, or fatal prostate cancer. bOnly selected subset of SNPs were genotyped for all cases and controls (see Materials and Methods). xMinor allele frequency. kORs and P values estimated from an unconditional logistic regression model using a continuous variable for the number of minor alleles (0, 1, or 2) and adjusted for matching factors (birth year, year of blood draw, and race/ethnicity). { Includes fewer cases and controls due to shorter study follow-up period at the time of genotyping. **SNP included in a recent genome-wide association study (25). ccNo rs number available; sequence information derived from reference (27). limited to matched pairs, conditional logistic regression with both advanced (P = 0.047) and overall prostate accounting for the original matched pair design yielded cancer (P = 0.010). Three SNPs in the MMP2 gene were similar results as unconditional logistic regression. also associated with both advanced and overall prostate P cancer [rs1477017 (located in intron 2), trend = 0.011 for P overall cancer; rs17301608 (intron 3), trend = 0.014 Results for overall prostate cancer; rs11639960 (intron 11), P trend = 0.020 for overall prostate cancer]. These three Table 1 shows per allele ORs for all 58 SNPs. One HIF1A MMP2 SNPs were in linkage disequilibrium and SNP, rs11549465, also known as P582S, was associated moderately highly correlated. Among controls, the

Table 2. ORs for prostate cancer by genotype for selected polymorphisms, CPS-II Nutrition Cohort 1992 to 2003

Gene/SNP Controls Advanced prostate cancer* All prostate cancer

n (%) n (%) OR (95% CI) n (%) OR (95% CI) HIF1A rs11549465 (P582S) CC 1,138 (78.5) 455 (81.8) 1.00 (Reference) 1,156 (81.4) 1.00 (Reference) CT 284 (19.6) 95 (17.1) 0.82 (0.64-1.07) 252 (17.7) 0.86 (0.71-1.04) TT 28 (1.9) 6 (1.1) 0.53 (0.22-1.29) 12 (0.9) 0.41 (0.21-0.82) P trend 0.047 0.010 MMP2 rs1477017 AA 639 (44.3) 201 (36.1) 1.00 (Reference) 566 (39.9) 1.00 (Reference) AG 624 (43.3) 280 (50.3) 1.43 (1.16-1.77) 645 (45.5) 1.17 (0.99-1.36) GG 178 (12.4) 76 (13.6) 1.36 (1.00-1.86) 206 (14.5) 1.30 (1.04-1.64) P trend 0.0047 0.011 MMP2 rs17301608 CC 600 (41.9) 188 (34.1) 1.00 (Reference) 541 (38.3) 1.00 (Reference) CT 650 (45.4) 280 (50.7) 1.38 (1.11-1.71) 655 (46.3) 1.12 (0.95-1.31) TT 182 (12.7) 84 (15.2) 1.47 (1.08-2.00) 218 (15.4) 1.33 (1.06-1.67) P trend 0.0024 0.014 MMP2 rs11639960 AA 675 (46.9) 212 (38.3) 1.00 (Reference) 597 (42.3) 1.00 (Reference) AG 610 (42.4) 284 (51.3) 1.49 (1.21-1.84) 645 (45.7) 1.20 (1.02-1.40) GG 154 (10.7) 58 (10.5) 1.21 (0.86-1.70) 168 (11.9) 1.24 (0.97-1.58) P trend 0.0095 0.020 NOS3 rs1799983 (D298E) GG 682 (47.2) 262 (47.1) 1.00 (Reference) 659 (46.4) 1.00 (Reference) GT 600 (41.5) 244 (43.9) 1.06 (0.86-1.31) 632 (44.5) 1.08 (0.93-1.27) TT 164 (11.3) 50 (9.0) 0.80 (0.56-1.13) 129 (9.1) 0.81 (0.62-1.04) P trend 0.51 0.48 VEGF rs1570360 (1154 G/A) GG 557 (47.5) 210 (45.9) 1.00 (Reference) 543 (46.9) 1.00 (Reference) GA 489 (41.7) 194 (42.4) 1.05 (0.83-1.32) 485 (41.9) 1.08 (0.85-1.20) AA 126 (10.8) 54 (11.8) 1.12 (0.78-1.61) 130 (11.2) 1.05 (0.80-1.38) P trend 0.50 0.73

NOTE: SNPs were selected based on previously reported associations and/or statistical significance. ORs were adjusted for matching factors (birth year, year of blood draw, and race/ethnicity). *Advanced cases defined as having a Gleason score of z7, or stage III/IV, or fatal prostate cancer.

Cancer Epidemiol Biomarkers Prev 2008;17(4). April 2008

squared correlation coefficient (r2) was 0.92 for the intron In addition to the MMP2 and HIF1A SNPs discussed 2 and intron 3 SNPs, 0.70 for the intron 2 and intron 11 above, nine additional SNPs included in this analysis SNPs, and 0.65 for the intron 3 and 11 SNPs. The linkage (footnoted in Table 1) were also examined in the PLCO disequilibrium coefficient (D’) was 0.99 for the intron 2 cohort (25). None of these SNPs were associated with and intron 3 SNPs, 0.88 for the intron 2 and intron 11 prostate cancer in either our analysis or in the PLCO SNPs, and 0.87 for the intron 3 and 11 SNPs. cohort. Analyses including only cases that were stage III, stage A limitation of this study is that we cannot rule out IV, or fatal (n = 148) yielded similar results. However, associations with more advanced prostate cancer (e.g., statistical precision was limited. stage III or above) because we had relatively few cases Table 2 shows more detailed genotype results for the this advanced. In addition, we could not examine four statistically significant SNPs described above, as associations separately by race or ethnicity because well as the VEGF SNP previously reported to be nearly all men in our study were White. Strengths of associated with prostate cancer (rs1570360) and the this study are its relatively large size and the inclusion of NOS3 SNP previously reported to be associated with a considerable number of SNPs for which an association severity among prostate cancer cases (rs1799983). with prostate cancer is biologically plausible and/or has Because the per allele analyses shown in Table 1 could been previously reported. miss associations limited to the homozygous variant In conclusion, our results do not support previously genotype, we also examined ORs for the homozygous reported associations between prostate cancer and SNPs variant genotype (compared with the homozygous in VEGF, HIF1A, and NOS3. Taking into account recent wild-type) for the 52 SNPs not shown in Table 2. results from other studies, none of the SNPs we The homozygous variant of one of these SNPs, examined seem likely to be strongly associated with risk HIF1AN rs2295778 (P41A), was associated with a of prostate cancer. statistically significant reduction in risk of advanced prostate cancer (OR for GG versus CC, 0.74; 95% CI, 0.56-0.99). Acknowledgments We thank Kimberly Walker-Thurmond and Cari Lichtman (American Cancer Society) for their contributions to making Discussion this study possible.

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Eric J. Jacobs, Ann W. Hsing, Elizabeth B. Bain, et al.

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