A Comprehensive Analysis of Steroid Hormones and Progression of Localized High-Risk Prostate Cancer

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Published OnlineFirst February 7, 2019; DOI: 10.1158/1055-9965.EPI-18-1002

Research Article Cancer Epidemiology, Biomarkers A Comprehensive Analysis of Steroid Hormones & Prevention and Progression of Localized High-Risk Prostate Cancer Eric Levesque 1, Patrick Caron2, Louis Lacombe1,Veronique Turcotte2, David Simonyan3, Yves Fradet1, Armen Aprikian4, Fred Saad5, Michel Carmel6, Simone Chevalier4, and Chantal Guillemette2

Abstract

Background: In men with localized prostate cancer who are terone levels were higher in low-risk disease. Associations were undergoing radical prostatectomy (RP), it is uncertain whether observed between adrenal precursors and risk of cancer pro- their systemic hormonal environment is associated with out- gression. In high-risk patients, a one-unit increment in log- comes. The objective of the study was to examine the associ- transformed androstenediol (A5diol) and dehydroepiandros- ation between the circulating steroid metabolome with prog- terone-sulfate (DHEA-S) levels were linked to DFS with HR of nostic factors and progression. 1.47 (P ¼ 0.0017; q ¼ 0.026) and 1.24 (P ¼ 0.043; q ¼ 0.323), Methods: The prospective PROCURE cohort was recruited respectively. Although the number of metastatic events was from 2007 to 2012, and comprises 1,766 patients with local- limited, trends with metastasis-free survival were observed for ized prostate cancer who provided blood samples prior to RP. A5diol (HR ¼ 1.51; P ¼ 0.057) and DHEA-S levels (HR ¼ 1.43; The levels of 15 steroids were measured in plasma using mass P ¼ 0.054). spectrometry, and their association with prognostic factors and Conclusions: In men with localized prostate cancer, our disease-free survival (DFS) was established with logistic regres- data suggest that the preoperative steroid metabolome is sion and multivariable Cox proportional hazard models. associated with the risk of recurrence of high-risk disease. Results: The median follow-up time after surgery was 73.2 Impact: The associations of adrenal androgens with pro- months. Overall, 524 patients experienced biochemical failure gression of localized high-risk disease could help reﬁne hor- and 75 developed metastatic disease. Testosterone and andros- monal strategies for these patients.

Introduction (DHT), dehydroepiandrosterone-sulfate (DHEA-S), androstenedi- one (4-Dione or AD), androstanediol-glucuronide (3a-diol-G) and Prostate cancer is the most commonly diagnosed cancer and estradiol (E )—and the risk of developing prostate cancer (2). Also, the second most common cause of cancer-related deaths among 2 another large study did not find any association between prediag- North American men (1). Currently, approximately 85% of nostic circulating hormone levels (testosterone, DHT, E and 3a- newly diagnosed cases of prostate cancer are localized within 2, diol-G) and lethal prostate cancer (3). In the latter study, the the prostate. Despite the fact that sex steroid hormones play a authors did, however, find an association between an increased central role in prostate biology, a collaborative analysis of risk of lethal prostate cancer with higher prediagnostic levels of 18 prospective studies failed to demonstrate an association DHT in men with aggressive Gleason score (GS) 8 and stage T3 between six circulating steroids—testosterone, dihydrotestosterone disease (3). Results from phase III chemoprevention trials further support 1Centre Hospitalier Universitaire (CHU) de Quebec Research Centre and Faculty the role of sex steroid hormones in prostate cancer carcinogen- fi a of Medicine, Laval University, Quebec, Canada. 2CHU de Quebec Research esis (4, 5). Indeed, nasteride (a 5 -reductase type I inhibitor) Centre and Faculty of Pharmacy, Laval University, Quebec, Canada. 3Statistical and dutasteride (a dual 5-AR inhibitor targeting 5a-reductase type and Clinical Research Platform, CHU de Quebec Research Centre, Quebec, I and type II), both of which block the conversion of testosterone Canada. 4McGill University Health Centre, McGill University, Faculty of Medicine, into DHT in target cells, reduced the occurrence of prostate cancer 5 Quebec, Canada. Centre Hospitalier de l'Universite de Montreal, Faculty of by approximately 25% (4, 5). However, this reduction in prostate 6 Medicine, Universite de Montreal, Quebec, Canada. Universite de Sherbrooke, cancer risk comes at the expense of a controversial but potentially Faculty of Medicine, Quebec, Canada. increased risk of developing higher-grade prostate cancer (4, 5). Note: Supplementary data for this article are available at Cancer Epidemiology, In agreement with the finasteride and dutasteride findings, in Biomarkers & Prevention Online (http://cebp.aacrjournals.org/). men with a confirmed diagnosis of prostate cancer, previous Corresponding Authors: Eric Levesque, CHU de Quebec Research Centre and studies have suggested that low pretreatment levels of serum Laval University, 2705 Blvd. Laurier, Quebec G1V 4G2, Canada. Phone: 418-525- testosterone are associated with disease aggressiveness and clin- 4444; Fax: 418-654-2761; E-mail: [email protected]; and ical outcomes (6, 7). Chantal Guillemette, [email protected] However, other circulating hormones may contribute to clin- doi: 10.1158/1055-9965.EPI-18-1002 ical outcomes in men with localized disease. This is reinforced by 2019 American Association for Cancer Research. the fact that cancer cells possess the intracrine biosynthesis

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surgeons. Serial prostate-specific antigen (PSA) measurements and clinical data were gathered during follow-up. A total of 1,908 single preoperative blood samples were available for steroid analysis. One hundred and forty-two patients were documented to have a prior exposure to a 5a-reductase inhibitor and, therefore, were excluded from the analyses leaving a total of 1,766 patients for this study. No other forms of hormonal manipulation were administered before blood sampling. The objective of the study was the assessment of associations of steroid hormone levels with prostate cancer prognostic factors and progression. After prostatectomy, patients were seen regularly and PSA was measured every 3 months for 2 years, every 4–6 months for 2 years, and then every 6–12 months or at the discretion of the physicians. Disease-free survival (DFS) was defined as the occurrence of biochemical recurrence (BCR), metastasis, and/or death (all-causes). BCR was defined as (i) the occurrence of a first PSA >0.2 ng/mL any time after surgery or (ii) a detectable PSA of <0.2 ng/mL that triggered initiation of salvage radiation or androgen abla- tion therapy (10–13). Before surgery, each patient provided written informed consent for research and the protocol was evaluated and approved by the Centre Hospitalier Universitaire (CHU) de Quebec (Quebec, Canada) and local Ethical Research Committees. Figure 1. Simplified schematic representation of the 15 steroid hormones measured in the PROCURE cohort. Steroids were profiled by multiplex MS assays in Plasma steroid measurements by MS preoperative plasma samples except for androstanedione (A-dione) and Blood samples were collected and processed at a preoperative androstane-3a-17b-diol (3a-diol). Abbreviations: testo, testosterone; 3b-diol, visit and banked at 80 C until analyzed. LC/MS-MS and GC-MS androstane-3b,17b-diol; 3a-diol-17G, androstane-3a,17b-diol-17-glucuronide. were used to measure plasma steroid levels as described previously (Fig. 1; ref. 14). Ten unconjugated steroids were measured in a single assay using 250 mL of plasma, whereas sulfates and machinery to locally synthesize active androgens from precursors glucuronides were measured in two independent assays using such as DHEA-S, dehydroepiandrosterone (DHEA), 4-Dione or 20 mL and 100 mL of plasma, respectively. Analyses were per- AD, and androstenediol (A5diol) originating from extragonadal formed in a blinded fashion. Reference steroids were purchased peripheral sources, such as the adrenal glands (Fig. 1; refs. 8, 9). To from Steraloids. Internal standards (deuterated steroids) were our knowledge, a comprehensive assessment of the circulating added to samples and quality controls were included in each hormonal environment including precursors associated with run. The measured steroids and their limits of quantification were disease aggressiveness and clinical outcomes in men with newly as follows: DHEA, 100 pg/mL; progesterone (Prog), 50 pg/mL; diagnosed localized prostate cancer has not been assessed. This A5diol, 50 pg/mL; testosterone, 30 pg/mL; DHT, 10 pg/mL; lack of information is mostly due to the fact that the majorities of androsterone (AST), 50 pg/mL; androstane-3b,17b-diol (3b- previous studies conducted in localized disease were mostly Diol), 10 pg/mL; estrone (E1), 5 pg/mL; estradiol (E2), 1 pg/mL; focused on testosterone and relied on low specificity and accuracy 4-Dione or AD, 50 pg/mL; AST-glucuronide (AST-G), 1 ng/mL; radioimmunoassays measuring a single hormone at a time. The androstane-3a,17b-diol-3-glucuronide (3a-diol-3G), 0.25 ng/ use of gold standard mass spectrometry (MS) methods for accu- mL; androstane-3a,17b-diol-17-glucuronide (3a-diol-17-G), rate and specific assessment of various steroids is thus critical. 0.25 ng/mL; DHEA-S, 0.075 mg/mL; estrone-sulfate (E1-S), In this study, we analyzed the preoperative plasma levels of 15 0.075 ng/mL. Three low and three high hormone concentration steroids using sensitive and specific MS assays in a prospective quality control replicates were included in each run and all cohort of 1,766 men undergoing radical prostatectomy (RP) for metabolite coefficients of variation were <10%. newly diagnosed prostate cancer. We then evaluated the effect of the association between these steroids and clinical and pathologic Statistical analysis prognostic factors and also the independent prognostic value of Quantitative variables are described as mean, SD, 95% confi- individual circulating hormones. dence interval (CI), median, and range, and categorical variables as frequencies and percentages. Parametric or nonparametric (F test or Wilcoxon–Mann–Whitney test) tests were used to compare Materials and Methods continuous data by groups after normality verification; x2 or exact The PROCURE prostate cancer cohort tests were used for categorical data comparisons. Log-transformed The study included 1,908 patients with localized prostate data were used to account for the abnormal hormonal distribu- cancer who were recruited between 2007 and 2012 at four tion and were used in subsequent analyses as performed in studies university hospital centers in the Province of Quebec in Canada similar in scope (13, 15–18). Data generated with untransformed (Montreal, McGill, Quebec, and Sherbrooke). All patients had hormone concentration are also provided for completeness in the localized prostate cancer at the time of diagnosis and underwent Supplementary section. Spearman correlations were estimated RP. Lymph-node dissection was performed at the discretion of the between serum concentrations to evaluate correlations between

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steroids. Cox proportional hazard models (HR) were used to characteristics of the studied cohort. Clinicopathologic features estimate the association between hormone levels and DFS. In incorporated in multivariable models and the associated risk of multivariable analyses, the statistical model included age, PSA recurrence after radical prostatectomy are showed in Supplemen- level (continuous), the pathologic GS (GS6, GS3þ4, GS4þ3, tary Fig. S1. GS 8), pathologic T staging [extracapsular extension (pT3a) and seminal vesicle invasion (pT3b)], margin, and nodal sta- Relationship between circulating steroids and established tus (19, 20). On the basis of the high recurrence rate in high-risk prognostic factors (n ¼ 1,766) patients, the clinical impact of the steroid metabolome on Mean hormone values of the PROCURE cohort are depicted progression was also speciﬁcally evaluated in this subgroup. in Supplementary Table S1. All steroids and their relationships Interactions between high-risk disease and each hormone were with PSA and GS are depicted in Supplementary Tables S2 and tested in separate Cox proportional hazard models, which S3. Higher GS was associated with a stepwise increase (>30%) included risk group, hormones, and interaction term as inde- in AST-G levels (P ¼ 0.036; q ¼ 0.180), 3a-diol-3G (P ¼ 0.036; pendent factors. Because we studied 15 hormones, FDRs q ¼ 0.180) and DHEA-S (P ¼ 0.023; q ¼ 0.180; Supplementary (q-values) were calculated to determine the degree to which Table S3). None of the steroid hormones examined were the results were prone to false positives with the use of the associated with pathologic T staging (Supplementary Table RQVALUE package (http://genomics.princeton.edu/storeylab/ S4). The 323 patients in the lower risk category (GS 6/PSA qvalue/; ref. 21). The q value represents the rate at which < 10/ pT2c) displayed higher mean preoperative testosterone hormones data are null. All analyses were performed done levels (4.10 ng/mL, 95% CI, 3.74–4.45) compared to the using SAS 9.4 by the biostatistician involved in the study (DS). 312 patients in the high-risk category (GS 8and/orPSA> 20 and/or > pT3a;3.76ng/mL;95%CI,3.59–3.93; P ¼ 0.049; Results q ¼ 0.367). A similar observation was made for AST (P ¼ 0.040; q ¼ 0.367; Supplementary Table S5). Clinical and pathologic characteristics of the PROCURE prostate cancer cohort We studied 1,766 men with localized prostate cancer who were Associations between circulating steroids and DFS (n ¼ 1,766) undergoing RP as part of the multi-institutional PROCURE Pros- In multivariable Cox analyses adjusted for established prog- tate Cancer Biobank project (22). The median follow-up time was nostic factors shown in Supplementary Fig. S1, patients with 73.2 months after surgery. Five hundred twenty-four patients increasing levels of the adrenal precursor A5diol had an increased experienced BCR (29.6%), occurring at a median time of 19.0 risk of progression (Fig. 2; Supplementary Table S6). Results for months after surgery. Seventy-ﬁve patients developed metastasis regression analyses with A5diol modeled as quartiles (Q) also and 111 deaths (all-cause) were documented. Table 1 depicts exposed an increased risk of recurrence (Supplementary Table S7). Compared with men in the lowest quartile (Q1), those in highest Table 1. Clinical and pathologic characteristics of the PROCURE cohort quartiles (Q4) had increased risk of progression (Q4/Q1: HR ¼ Characteristics n ¼ 1,766 (%) 1.34; 95% CI, 1.06–1.70; P ¼ 0.015; q ¼ 0.120). Restricting the Mean age at diagnosis (year) 62.7 SD 6.4 Range 34–81 Median follow-up time (mo) 73.2 Biochemical recurrence (BCR) 524 (29.6) Median time to BCR (mo) 19.0 PSA at diagnosis, ng/mL 10 1,469 (83.3) >10 to 20 222 (12.7) >20 63 (3.7) Unknown 12 (0.1) Pathologic GS 6 408 (23.1) 7 3þ4 781 (44.2) 4þ3 407 (23.0) 8 170 (9.6) Pathologic T stage pT2c 1,118 (63.3) pT3a 466 (26.4) pT3b 181 (10.3) Unknown 1 (0.1) Nodal invasion

pN0/pNx 1,694 (95.9) pN1 72 (4.1) Margin status Positive 626 (35.4) Figure 2. Negative 1,054 (59.7) Sex steroid hormone levels and DFS after prostatectomy (n ¼ 1,766). Boxes Unknown 86 (4.9) represent HRs and their 95% CIs in multivariable analyses. Each unit Metastasis 75 (4.2) increment in log-transformed hormone levels is associated with the indicated Abbreviations: mo, months; pNx, nodal status unknown. changes in HR values.

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analyses of quartiles to BCR/metastasis generate similar data (not shown). Similarly, A5diol levels (as a continuous variable) were associated with DFS. Indeed, a one-unit increment in log-transformed A5diol levels was associated with a HR of 1.19 (95% CI, 1.03–1.37; P ¼ 0.017; q ¼ 0.255; Supplementary Table S6). Results for regression analyses with DHEA-S modeled as quartile also suggested an increased risk of recurrence (Q4/Q1: HR ¼ 1.34; 95% CI, 1.06–1.69; P ¼ 0.016; q ¼ 0.120; Supplementary Table S7). Trends for a shorter DFS were observed with higher levels (continuous) of the adrenal precursor DHEA-S (HR ¼ 1.12; 95% CI, 0.99–1.27; P ¼ 0.071; q ¼ 0.410) as well as of the DHT metabolite 3a-diol-3G (HR ¼ 1.13; 95% CI, 0.99–1.29; P ¼ 0.082; q ¼ 0.410; Fig. 2; Supplementary Table S6). Restrict- ing the analyses to BCR/metastasis generate similar data (Sup- plementary Table S8). Performing the analysis with untransformed hormone values identify similar associations. Indeed, the associated change in HR per SD variation in untransformed hormone concentration is depicted in Supplementary Table S9. Correlation between all measured hormones is shown in Sup- plementary Fig. S2.

Analyses of the prognostic significance of circulating hormones in high-risk disease (n ¼ 312) Analyses were then conducted in 312 patients with high-risk disease comprising 1 high-risk features: GS 8, PSA > 20, or > pT3a. A positive interaction between A5diol and disease risk was observed (P ¼ 0.022). In these patients, a one-unit increment in log-transformed A5diol levels was linked to DFS with a HR of 1.47 (95% CI, 1.15–1.87; P ¼ 0.0017; q ¼ 0.026; Fig. 3A). When analyzed as quartiles, A5diol levels were also associated with DFS (log-rank P ¼ 0.036; Fig. 3B). Results obtained for DHEA-S displayed a HR of 1.24 (95% CI, 1.01–1.52; P ¼ 0.043; q ¼ 0.323; log-rank P ¼ 0.099; Fig. 3C; Supplementary Table S10). Restricting the analyses to BCR/metastasis generate similar data (Supplementary Table S11). Performing the analysis with Figure 3. untransformed hormone values in this subgroup identify addi- Sex steroid hormone levels and DFS after prostatectomy in patients with tional associations with DHEA and 3a-diol-17G (Supplementary high-risk disease (n ¼ 312). A, Boxes represent HRs and their 95% CIs in Table S12). Finally, trends were observed for associations between multivariable analyses. Each unit increment in log-transformed hormone levels is associated with the indicated changes in HR values. Kaplan–Meier A5diol, DHEA-S levels, and metastasis-free survival. Increment in curves of quartiles (Q1–Q4) of A5diol (B) and DHEA-S (C). log-transformed A5diol levels was associated with a HR of 1.51 (95% CI, 0.98–2.30; P ¼ 0.057; q ¼ 0.335) and DHEA-S with a HR of 1.43 (95% CI, 0.99–2.05; P ¼ 0.054; q ¼ 0.335; Supplementary prostate cancer. Indeed, low pretreatment testosterone levels Table S13). The association with testosterone did not reach were previously associated with adverse pathologic factors significance with a HR of 1.41 (95% CI, 0.84–2.38; P ¼ 0.196; defining an aggressive disease phenotype (6, 7, 23–26). In q ¼ 0.490). The association between metastasis-free survival and agreement, in this analysis of the PROCURE cohort of patients untransformed hormone values is depicted in Supplementary with prostate cancer with localized disease, we also observed an Table S14. association between lower levels of testosterone and AST and tumor aggressiveness based on the combination of PSA values at diagnosis, GS, and pT staging. In addition, we showed that Discussion DHEA-S and inactive androgen glucuronide end products The evaluation of circulating steroids in relation to prostate progressively increased with higher GS. For instance, high cancer progression has been largely focused on the potent andro- circulating levels of inactive AST-G, which is the major DHT gens testosterone and DHT. To our knowledge, a comprehensive metabolite found in circulation, is observed in patients with a assessment of preoperative circulating steroids from gonadal, GS 8. This suggests that proficient intracellular formation of adrenal, and peripheral sources in relation to prognostic factors 5a-reduced androgens and their glucuronides occurs in these and DFS in men with localized disease in a sizeable prospective patients,thelatterbeingproposedtobetterreflect androgen cohort like PROCURE has never been evaluated, and especially exposure (27). Intraprostatic androgen measurements of tumor not with an accurate and specific MS method. cells and their microenvironment will be required to establish Several studies have demonstrated a correlation between whether circulating glucuronide derivatives reflect increased testosterone levels and prognostic factors in men with localized local androgen exposure (27, 28) and/or the increased

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androgen inactivation capacity of cancer cells previously help further stratify this population of patients. Further research is reported in aggressive disease (9, 29, 30). necessary to confirm the associations observed between A5diol, Beyond few associations observed with prognostic factors, our DHEA-S, and clinical outcomes. Indeed, large studies are required data expose an added prognostic value particularly for the adrenal in men with localized prostate cancer, especially in those at high precursors A5diol and DHEA-S on progression in this clinical risk of recurrence, where circulating levels of these hormones context. For instance, increment in A5diol levels was associated might (i) improve prognostication/stratification, (ii) help refine with an independent additional risk of progression. This associ- the circulating hormonal milieu associated with adverse clinical ation is clinically apparent in high-risk disease (Pinteraction ¼ outcomes, and (iii) advance hormonal strategies of patients with 0.022; Fig. 3). This observation is of biologic significance, as progressive disease. On the basis of the natural history of prostate A5diol, a major metabolite of DHEA in prostate homoge- cancer progression following biochemical recurrence (41), and nates (31, 32) has been shown to have dual actions in cancer knowing that intracellular levels of A5diol and DHEA-S remain cells binding both the androgen and estrogen receptors (33, 34). at significant levels in patients on androgen-deprivation ther- We also observed a trend for an association between A5diol levels apy (32, 35, 36), the ongoing follow-up of the PROCURE and the development of metastatic disease. Besides, the com- cohort will ultimately allow, in an approximately 5–10-year monly used drug, bicalutamide, fails to block A5diol androgen timeframe horizon, to assess the impact of these hormones on receptor transactivation in prostate cancer cells in vitro (33), and the development of metastasis, castration-resistant disease, and A5diol levels in the prostate are unaffected (35, 36) or only prostate cancer mortality. partially reduced (32) by androgen-deprivation therapy. There- fore, it is plausible that this adrenal precursor contributes to progression after prostatectomy, supports development of metas- Disclosure of Potential Conflicts of Interest tasis, and thus progression to the lethal castration-resistant E. Levesque reports receiving a commercial research grant from Janssen Inc. and Astellas Inc., and has received speakers bureau honoraria from and state (37). The ongoing follow-up of the PROCURE cohort will hasprovidedexperttestimonyforPfizerInc.M.Carmelreportsreceivinga help clarify this possibility. Results obtained with DHEA-S suggest commercial research grant from Abbvie and Janssen and is consultant/ a tendency toward an increased risk of progression with higher advisory board member for Bayer. No potential conflicts of interest were preoperative levels (Fig. 3A and C). DHEA-S, present in the disclosed by the other authors. micromolar range, is the most abundant circulating steroid in men and acts as a main steroid reservoir for cancer cells (38, 39). Indeed, tumor cells are capable of converting DHEA-S to active Authors' Contributions steroids following the successive action of multiple enzymes Conception and design: E. Levesque, C. Guillemette Development of methodology: C. Guillemette including steroid sulfatase, 3b-hydroxysteroid dehydrogenase, b a Acquisition of data (provided animals, acquired and managed patients, 17 -hydroxysteroid dehydrogenase, and 5 -reductase enzymes. provided facilities, etc.): P. Caron, L. Lacombe, V. Turcotte, Y. Fradet, The associations of DHEA-S with GS, and the trend observed with A. Aprikian, F. Saad, M. Carmel, S. Chevalier, C. Guillemette progression of high-risk disease in multivariable models, suggest Analysis and interpretation of data (e.g., statistical analysis, biostatistics, that peripheral sources of steroids originating from the adrenals computational analysis): E. Levesque, L. Lacombe, D. Simonyan, F. Saad, may contribute to progression in those men (39). Because A5diol C. Guillemette Writing, review, and/or revision of the manuscript: E. Levesque, L. Lacombe, and DHEA-S originate essentially from the adrenals, it is unlikely D. Simonyan, Y. Fradet, A. Aprikian, F. Saad, S. Chevalier, C. Guillemette that the variations observed are the consequence of altered steroid Administrative, technical, or material support (i.e., reporting or organizing metabolism associated with disease aggressiveness, perhaps data, constructing databases): E. Levesque, L. Lacombe, V. Turcotte, Y. Fradet, except for testosterone, as previously suggested (3), and the A. Aprikian, C. Guillemette inactive androgen glucuronides known to be formed in peripheral Study supervision: E. Levesque, C. Guillemette tissues including the prostate (40). The strengths of this study include a large and multi-institu- Acknowledgments The authors are thankful to all participating patients and staff at each site tional prospective cohort of patients, with pathologically confi fi associated with the PROCURE Biobank who have made this scienti c contri- rmed localized prostate cancer, all of whom were treated by bution possible. The personnel involved in the Biobank at each site are the radical prostatectomy and the ongoing clinical follow-up. In employees of their Centre or Research Institute and are not PROCURE employ- addition, the measurement of steroid hormones was performed ees. This work was supported by research grants from the Fonds de Recherche du using sensitive, specific, and validated MS assays. To our knowl- Quebec-Sante (FRQ-S) Innovation fund to the CHU Research Centre (grant no. edge, this study is one of the largest studies to assess a compre- 26678, to C. Guillemette, E. Levesque, L. Lacombe, Y. Fradet) and Prostate hensive relationship between circulating steroid hormones and Cancer Canada (grant no. DS2013-55, to E. Levesque), the Cancer Research Society (grant no. 16181, to C. Guillemette), and the Canada Research Chair progression in men with localized prostate cancer. Limitations Program (grant no. 950-203962, to C. Guillemette). E. Levesque holds a CIHR include the exploratory nature of the study and the limited Clinician-Scientist Award. C. Guillemette holds the Canada Research Chair in number of metastatic events. Longer follow-up periods will be Pharmacogenomics (Tier I). Results are based on samples and patient data required to more adequately address the associations with met- obtained from the PROCURE Biobank, supported by donations in a partnership astatic disease. with the Cancer Research Society of Canada.

Conclusions The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked In this large prospective cohort of men with localized prostate advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate cancer, we showed that the steroid metabolome is associated with this fact. prognostic factors. The importance of the steroid metabolome is clinically apparent in high-risk disease where the circulating pool Received September 8, 2018; revised November 1, 2018; accepted February 2, of adrenal precursors may support disease progression, and may 2019; published ﬁrst February 7, 2019.

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References 1. DeSantis CE, Lin CC, Mariotto AB, Siegel RL, Stein KD, Kramer JL, et al. 22. Brimo F, Aprikian A, Latour M, Tetu B, Doueik A, Scarlata E, et al. Strategies Cancer treatment and survivorship statistics, 2014. CA Cancer J Clin 2014; for biochemical and pathologic quality assurance in a large multi-institu- 64:252–71. tional biorepository; the experience of the PROCURE Quebec Prostate 2. Endogenous H, Prostate Cancer Collaborative Group, Roddam AW, Allen Cancer Biobank. Biopreserv Biobanking 2013;11:285–90. NE, Appleby P, Key TJ. Endogenous sex hormones and prostate cancer: a 23. Salonia A, Abdollah F, Capitanio U, Suardi N, Briganti A, Gallina A, et al. collaborative analysis of 18 prospective studies. J Natl Cancer Inst 2008; Serum sex steroids depict a nonlinear u-shaped association with high-risk 100:170–83. prostate cancer at radical prostatectomy. Clin Cancer Res 2012;18: 3. Gershman B, Shui IM, Stampfer M, Platz EA, Gann PH, Sesso HL, et al. 3648–57. Prediagnostic circulating sex hormones are not associated with mortality 24. Tu H, Gu J, Meng QH, Kim J, Strom S, Davis JW, et al. Low serum for men with prostate cancer. Eur Urol 2014;65:683–9. testosterone is associated with tumor aggressiveness and poor prognosis 4. Thompson IM, Goodman PJ, Tangen CM, Lucia MS, Miller GJ, Ford LG, in prostate cancer. Oncol Lett 2017;13:1949–57. et al. The influence of finasteride on the development of prostate cancer. 25. Garcia-Cruz E, Piqueras M, Huguet J, Peri L, Izquierdo L, Musquera M, et al. N Engl J Med 2003;349:215–24. Low testosterone levels are related to poor prognosis factors in men with 5. Andriole GL, Bostwick DG, Brawley OW, Gomella LG, Marberger M, prostate cancer prior to treatment. BJU Int 2012;110:E541–6. Montorsi F, et al. Effect of dutasteride on the risk of prostate cancer. 26. Schatzl G, Madersbacher S, Thurridl T, Waldmuller J, Kramer G, Haitel A, N Engl J Med 2010;362:1192–202. et al. High-grade prostate cancer is associated with low serum testosterone 6. Salonia A, Abdollah F, Capitanio U, Gallina A, Suardi N, Briganti A, et al. levels. Prostate 2001;47:52–8. Preoperative sex steroids are significant predictors of early biochemical 27. Labrie F, Belanger A, Belanger P, Berube R, Martel C, Cusan L, et al. recurrence after radical prostatectomy. World J Urol 2013;31:275–80. Androgen glucuronides, instead of testosterone, as the new markers of 7. Leon P, Seisen T, Cussenot O, Drouin SJ, Cattarino S, Comperat E, et al. Low androgenic activity in women. J Steroid Biochem Mol Biol 2006;99:182–8. circulating free and bioavailable testosterone levels as predictors of high- 28. Belanger A, Brochu M, Cliche J. Levels of plasma steroid glucuronides in grade tumors in patients undergoing radical prostatectomy for localized intact and castrated men with prostatic cancer. J Clin Endocrinol Metab prostate cancer. Urol Oncol 2015;33:384. 1986;62:812–5. 8. Labrie F. Adrenal androgens and intracrinology. Semin Reprod Med 2004; 29. Li H, Xie N, Chen R, Verreault M, Fazli L, Gleave ME, et al. UGT2B17 22:299–309. expedites progression of castration-resistant prostate cancers by promoting 9. Montgomery RB, Mostaghel EA, Vessella R, Hess DL, Kalhorn TF, Higano ligand-independent AR signaling. Cancer Res 2016;76:6701–11. CS, et al. Maintenance of intratumoral androgens in metastatic prostate 30. Belledant A, Hovington H, Garcia L, Caron P, Brisson H, Villeneuve L, et al. cancer: a mechanism for castration-resistant tumor growth. Cancer Res The UGT2B28 sex-steroid inactivation pathway is a regulator of steroido- 2008;68:4447–54. genesis and modifies risk of prostate cancer progression. Eur Urol 2016;69: 10. Lalonde E, Ishkanian AS, Sykes J, Fraser M, Ross-Adams H, Erho N, et al. 601–9. Tumour genomic and microenvironmental heterogeneity for integrated 31. Mitamura K, Nagaoka Y, Shimada K, Honma S, Namiki M, Koh E, et al. prediction of 5-year biochemical recurrence of prostate cancer: a retro- Simultaneous determination of androstenediol 3-sulfate and dehydroepi- spective cohort study. Lancet Oncol 2014;15:1521–32. androsterone sulfate in human serum using isotope diluted liquid chro- 11. Lalonde E, Alkallas R, Chua MLK, Fraser M, Haider S, Meng A, et al. matography-electrospray ionization-mass spectrometry. J Chromatogr B Translating a prognostic DNA genomic classifier into the clinic: retrospec- Analyt Technol Biomed Life Sci 2003;796:121–30. tive validation in 563 localized prostate tumors. Eur Urol 2017;72:22–31. 32. Zang T, Taplin ME, Tamae D, Xie W, Mesaros C, Zhang Z, et al. Testicular vs 12. Briollais L, Ozcelik H, Xu J, Kwiatkowski M, Lalonde E, Sendorek DH, et al. adrenal sources of hydroxy-androgens in prostate cancer. Endocr Relat Germline mutations in the Kallikrein 6 region and predisposition for Cancer 2017;24:393–404. aggressive prostate cancer. J Natl Cancer Inst 2017;109:djw258. 33. Miyamoto H, Yeh S, Lardy H, Messing E, Chang C. Delta5-androstenediol is 13. Levesque E, Laverdiere I, Audet-Walsh E, Caron P, Rouleau M, Fradet Y, a natural hormone with androgenic activity in human prostate cancer cells. et al. Steroidogenic germline polymorphism predictors of prostate cancer Proc Natl Acad Sci U S A 1998;95:11083–8. progression in the estradiol pathway. Clin Cancer Res 2014;20:2971–83. 34. Hackenberg R, Turgetto I, Filmer A, Schulz KD. Estrogen and androgen 14. Caron P, Turcotte V, Guillemette C. A chromatography/tandem mass receptor mediated stimulation and inhibition of proliferation by androst- spectrometry method for the simultaneous profiling of ten endogenous 5-ene-3 beta,17 beta-diol in human mammary cancer cells. J Steroid steroids, including progesterone, adrenal precursors, androgens and estro- Biochem Mol Biol 1993;46:597–603. gens, using low serum volume. Steroids 2015;104:16–24. 35. Higashi T, Takayama N, Kyutoku M, Shimada K, Koh E, Namiki M. Liquid 15. Levesque E, Huang SP, Audet-Walsh E, Lacombe L, Bao BY, Fradet Y, et al. chromatography-mass spectrometric assay of androstenediol in prostatic Molecular markers in key steroidogenic pathways, circulating steroid levels, tissue: influence of androgen deprivation therapy on its level. Steroids and prostate cancer progression. Clin Cancer Res 2013;19:699–709. 2006;71:1007–13. 16. Petrick JL, Hyland PL, Caron P, Falk RT, Pfeiffer RM, Dawsey SM, et al. 36. Arai S, Miyashiro Y, Shibata Y, Tomaru Y, Kobayashi M, Honma S, et al. Associations between prediagnostic concentrations of circulating sex ste- Effect of castration monotherapy on the levels of adrenal androgens in roid hormones and esophageal/gastric cardia adenocarcinoma among cancerous prostatic tissues. Steroids 2011;76:301–8. men. J Natl Cancer Inst 2019;111:34–41. 37. Mizokami A, Koh E, Fujita H, Maeda Y, Egawa M, Koshida K, et al. The 17. Eliassen AH, Missmer SA, Tworoger SS, Spiegelman D, Barbieri RL, Dowsett adrenal androgen androstenediol is present in prostate cancer tissue after M, et al. Endogenous steroid hormone concentrations and risk of breast androgen deprivation therapy and activates mutated androgen receptor. cancer among premenopausal women. J Natl Cancer Inst 2006;98:1406–15. Cancer Res 2004;64:765–71. 18. Eliassen AH, Missmer SA, Tworoger SS, Hankinson SE. Circulating 38. Labrie F. Intracrinology. Mol Cell Endocrinol 1991;78:C113–8. 2-hydroxy- and 16alpha-hydroxy estrone levels and risk of breast cancer 39. Taplin ME, Montgomery B, Logothetis CJ, Bubley GJ, Richie JP, Dalkin BL, among postmenopausal women. Cancer Epidemiol Biomarkers Prev 2008; et al. Intense androgen-deprivation therapy with abiraterone acetate plus 17:2029–35. leuprolide acetate in patients with localized high-risk prostate cancer: 19. Brajtbord JS, Leapman MS, Cooperberg MR. The CAPRA score at 10 years: results of a randomized phase II neoadjuvant study. J Clin Oncol 2014; contemporary perspectives and analysis of supporting studies. Eur Urol 32:3705–15. 2017;71:705–9. 40. Belanger A, Hum DW, Beaulieu M, Levesque E, Guillemette C, Tchernof A, 20. Audet-Walsh E, Bellemare J, Nadeau G, Lacombe L, Fradet Y, Fradet V, et al. et al. Characterization and regulation of UDP-glucuronosyltransferases in SRD5A polymorphisms and biochemical failure after radical prostatecto- steroid target tissues. J Steroid Biochem Mol Biol 1998;65:301–10. my. Eur Urol 2011;60:1226–34. 41. Pound CR, Partin AW, Eisenberger MA, Chan DW, Pearson JD, Walsh PC. 21. Storey JD, Tibshirani R. Statistical significance for genomewide studies. Natural history of progression after PSA elevation following radical pros- Proc Natl Acad Sci U S A 2003;100:9440–5. tatectomy. JAMA 1999;281:1591–7.

706 Cancer Epidemiol Biomarkers Prev; 28(4) April 2019 Cancer Epidemiology, Biomarkers & Prevention

A Comprehensive Analysis of Steroid Hormones and Progression of Localized High-Risk Prostate Cancer

Eric Lévesque, Patrick Caron, Louis Lacombe, et al.

Cancer Epidemiol Biomarkers Prev 2019;28:701-706. Published OnlineFirst February 7, 2019.

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