Journal of Experimental & Clinical Cancer Research 2009, 28:135 http://www.jeccr.com/content/28/1/135 Introduction Prostate cancer (Pca) is the most frequently diagnosed malignancy and the second leading cause of cancer death among men in Western countries [1]. Notwithstanding the importance of this tumor, its causes remain largely unknown. Age, family history, race and country of resi- dence are the only established risk factors, but they explain only a small proportion of Pca incidence [2]. A considerable number of studies have addressed prostate sensitivity to androgens in relation to outcomes varying from normal prostate growth to benign and malignant diseases [3-5]. However, the role played by estrogens in the pathogenesis of a wide spectrum of prostate physio- logic and pathologic conditions is drawing increasing attention [6]. In regards to Pca, experimental data from studies conducted in Noble (NBL) rats strongly suggest a critical role for estrogens in prostate carcinogenesis. Indeed, in NBL rats chronically treated with testosterone, the addition of estrogens is associated with a 100% inci- dence of prostate adenocarcinomas, whereas the adminis- tration of testosterone as a single agent produces Pca in approximately 30 to 40% of treated animals [7,8]. The estradiol plus testosterone treatment also induces acinar lesions that are similar to human prostatic intraepithelial neoplasia, a well recognized pre-invasive stage of adeno- carcinoma [9]. Evidence is also mounting regarding the contribution of hydroxylated metabolites of estrone (E1) and estradiol (E2) to the overall estrogenic activity. The mutually exclu- sive hydroxylation of E1 and E2 at positions C-16α or C- 2 leads to the production of either biologically active estrogens (16α-hydroxyestrone/estradiol) or derivatives with virtually no estrogenic activity (2-hydroxyestrone/ estradiol), respectively [10-12]. The different profiles in terms of biological activity and genotoxic properties might have consequences in terms of Pca risk. However, the overall body of evidence remains particu- larly limited when considering estrogen metabolites in relation to Pca risk. Our prior case-control study, con- ducted in Buffalo, NY, suggested an increased risk of clin- ically evident Pca in men with a lower 2-OHE1/16α- OHE1 ratio [13]. Similar results from studies evaluating breast cancer, as another hormone-dependent tumor, support this observation [14-18]. In the current case-control study, we have further tested the hypothesis that the pathway favoring 2-hydroxylation over 16α-hydroxylation is associated with a reduction in Pca risk. We also conducted a systematic review of the lit- erature to evaluate the totality of the evidence of this research question. Material and methods From 1996 to 2001, 1961 men were enrolled in the West- ern New York Health Cohort Study (WNYHCS). A detailed description of the WNYHCS study design, meth- ods and participants' characteristics is available elsewhere [14]. In brief, all participants provided informed consent; the Human Subjects Review Board of the University at Buf- falo, School of Medicine and Biomedical Science approved procedures for protection of human subjects in the study. At the time of recruitment, trained interviewers collected extensive data on demographics and life style during in-person interviews. The use of a standardized protocol allowed for the collection of anthropometric data. The study participants donated morning spot urine which was kept at -80°C until biochemical determina- tions. From January 2003 through September 2004, we com- pleted the Western New York Health Cohort (WNYHC) re-call and follow-up. For the purposes of the present case- control study (PROMEN II study), the re-call process included male participants who met the following inclu- sion criteria: age at recruitment between 50 and 85, base- line history negative for malignancies, cardiovascular diseases and clinically defined type-2 diabetes. On this basis, the re-call and follow-up process involved 1092 cohort participants. Among them, 52 were not eligible for medical reasons other than Pca, 46 had died from causes other than Pca, 22 had moved out of Erie and Niagara Counties, and 117 were not able to be contacted by mail or phone. Among the remaining 855 study participants, 232 refused to join the study, 40 were scheduled but can- celled the appointment and 8 were still in-course of assessment at the end of the follow-up period. In this group of non-participating subjects, all of the cohort members referred to being free from Pca in their tele- phone interviews. Thus, 575 participants joined the study, accounting for an overall participation rate of 67% (575/ 855). Pca cases were men who had been diagnosed with inci- dent, histologically confirmed Pca within the time-frame between their recruitment in the WNYHC and the end of the follow-up period. Identifying Pca cases was based on the participants' reports at the re-call, which was subse- quently validated by clinical records provided by their urologists. We identified and validated a total number of 41 incident prostate cancer cases. The 534 control subjects were male members of the WNYHC who, based on their report, were free from clinically evident Pca at the time of diagnosis of the related case. The control status was vali- dated with a serum PSA assessment on a blood sample donated at the time of recall. We used a PSA cut-off value Page 2 of 8 (page number not for citation purposes)