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Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. SERUM SEX STEROIDS DEPICT A NONLINEAR U-SHAPED ASSOCIATION WITH HIGH-RISK PROSTATE CANCER AT RADICAL PROSTATECTOMY Andrea Salonia, Firas Abdollah, Umberto Capitanio, Nazareno Suardi, Alberto Briganti, Andrea Gallina, Renzo Colombo, Matteo Ferrari, Giulia Castagna, Patrizio Rigatti, Francesco Montorsi Department of Urology, University Vita–Salute San Raffaele, Milan, Italy RUNNING TITLE: Sex steroids are nonlinearly associated with high-risk PCa KEY WORDS: prostate cancer; radical prostatectomy; testosterone; 17 estradiol; sex hormonebinding globulin SOURCE OF FUNDING: none CONFLICTS OF INTEREST: none TEXT WORD COUNT: 3386 NUMBER OF TABLES: 3 NUMBER OF FIGURES: 3 CORRESPONDING AUTHOR: Andrea Salonia, M.D. Department of Urology University Vita-Salute San Raffaele Via Olgettina 60 20132 Milan, Italy Tel. +39 02 2643 7286 Fax +39 02 2643 7298 Email: [email protected] Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. STATEMENT OF TRANSLATIONAL RELEVANCE Although a comprehensive serum hormonal milieu has scarcely been investigated in terms of prediction of pathologic outcomes in patients undergoing radical prostatectomy, virtually all previous reports assumed a linear relationship between the circulating hormonal milieu and the examined end points. Based on this concept of linearity, especially in the relationship between testosterone and prostate cancer, androgen deprivation therapy has always been confirmed as a standard treatment for metastatic prostate cancer, with the specific purpose of completely breaking down circulating testosterone levels and preventing any interaction with the various androgen receptors. The findings of this exploratory analysis demonstrate, for the first time, that preoperative serum total testosterone and estradiol levels and the total testosterone–estradiol ratio are independent predictors of high-risk prostate cancer, defined using the NCCN guidelines, but they depict a nonlinear U-shaped correlation (ie, both the lowest and the highest circulating levels of sex steroids are significantly associated with high-risk prostate cancer). This innovative finding could support the concept that the use of androgen deprivation therapy, as either a standard treatment for metastatic prostate cancer or as an adjuvant systemic therapy for high-risk men, could be even differently tailored according to the endocrine network at the root of the prostate cancer behavior, thus potentially supporting the newly-developed and evolving hormonal therapy which is aimed at reducing androgen signaling and controlling prostate cancer growth even in the presence of very low circulating androgen levels. Likewise, current findings may also contribute to support the use of androgen replacement therapy, which is undertaken commonly but cautiously in aging men with testosterone deficiency syndrome, since testosterone substitution might be titrated to a 'safe' level. 2 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT PURPOSE. Assess the association between preoperative serum total testosterone (tT), 17β estradiol (E2), sex hormone–binding globulin (SHBG), and tT–E2 ratio values with high-risk prostate cancer (PCa; as defined by the National Comprehensive Cancer Network practice guidelines) at radical prostatectomy (RP). METHODS. Serum E2, tT, and SHBG were dosed the day before surgery (7–11 AM) in a cohort of 724 candidates to RP. Restricted cubic spline functions tested the association between predictors (ie, model 1: age, body mass index [BMI], and serum tT, E2, and SHBG levels; Model 2: tT–E2 values instead of tT and E2 levels) and high-risk PCa. RESULTS. Low-, intermediate-, or high-risk PCa was found in 251 (34.7%), 318 (43.9%), and 155 (21.4%) patients, respectively. Patients in the high-risk class showed the lowest tT, E2, and tT–E2 ratio values (all p 0.02). At univariate analysis, only age, tT, E2, and tT–E2 ratio values were significantly associated with high-risk PCa (all p 0.006). At multivariate analyses considering model 1 variables, age (p = 0.03), serum tT (all p < 0.001), and E2 (all p 0.01) were associated with high-risk PCa; only tT–E2 ratios achieved independent predictor status for high-risk PCa (all p < 0.001) when considering model 2. Both the lowest and the highest tT, E2, and tT–E2 values depicted a nonlinear U-shaped significant association with high-risk PCa. CONCLUSIONS. These data showed that preoperative serum sex steroids are independent predictors of high-risk PCa, depicting a nonlinear U-shaped association. ABSTRACT WORD COUNT: 252 3 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. INTRODUCTION Data from several population-based studies failed to show a significant association between circulating levels of androgens (including total testosterone [tT]) and increased risk of prostate cancer (PCa) (1). In this context, despite long-held axiomatic convictions about T-centric and Tdependent prostate carcinogenesis (ie, the higher the level of circulating T, the greater the risk of developing PCa), low serum tT levels, rather than high levels, were associated with an increased risk of PCa in both animal and human studies (2-4). Similarly, data on radical prostatectomy (RP) populations have shown no unambiguous evidence reporting that preoperative circulating tT levels were associated with poor prognosis, including increased stage at presentation (5,6), advanced pathologic stages (5-9), higher rate of positive surgical margins (5,6), increased risk of biochemical failure (9.12), and worse survival (9,13). Likewise, controversies still exist regarding the relationship between serum tT levels and Gleason score; indeed, numerous studies (2,3,14-17) (but not all) (18) have reported low serum tT levels, rather than high levels, in association with advanced or high-grade PCa at surgery. A comprehensive serum hormonal milieu has scarcely been investigated in terms of prediction of pathologic outcomes in patients undergoing RP, with significant controversial findings (19-24). Virtually all previous reports assumed a linear relationship between the hormonal milieu and the examined end points (5-17). Based on this concept of linearity, especially in the relationship between T and PCa, androgen deprivation therapy (ADT) has always been confirmed as a standard treatment for metastatic PCa (25), with the specific purpose of completely breaking down circulating T levels and preventing any interaction with the various androgen receptors. However, previous reports suggested that high tT values, as well as low tT values, may be associated with adverse outcomes. This suggestion indicates a rather nonlinear relationship, with both extremes (high and low) predicting worse outcomes. 4 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. To test this hypothesis, we (1) analyzed the association between preoperative serum tT and highrisk PCa, as defined according to the National Comprehensive Cancer Network (NCCN) practice guidelines stratification (26), in a large cohort of nonscreened white European PCa patients undergoing RP at a single institution and (2) evaluated the association of 17β estradiol (E2), sex hormone–binding globulin (SHBG), and tT–E2 ratio values with high-risk PCa in the same cohort of men. 5 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. MATERIALS AND METHODS Patient population The analyses were based on a cohort of 838 nonscreened Caucasian-European PCa patients who underwent RP at a single academic referral center between June 2007 and May 2011. For the specific purpose of this analysis, none of the patients had uncontrolled diabetes, thyroid disease, hyperprolactinemia, hypoalbuminemia, or liver disease. Moreover, none of the patients had taken any hormonal neoadjuvant treatment or other hormonal preparations during the previous 12 mo. Symptoms of late-onset hypogonadism were not specifically collected for this cohort of men. All patients were comprehensively assessed with a detailed preoperative evaluation, including age; measured body mass index (BMI), defined as weight in kilograms by height in square meters; total serum prostate-specific antigen (PSA; Abbott Axym PSA assay; Abbott Laboratories, Abbott Park, IL, USA); biopsy Gleason sum; and clinical stage determined by a senior attending urologist, according to the 2002 American Joint Committee on Cancer staging system (27). Hypogonadsm was defined as tT <3 ng/ml (28). For the specific purpose of this analysis, candidates for RP were stratified for their risk of biochemical recurrence after definitive therapy according to the NCCN guidelines v.4.2011 (26) into low risk (tumors stage T1 or T2a, Gleason score 6, and serum PSA level <10 ng/ml), intermediate risk (tumors stage T2b or T2c, or Gleason score 7, or serum PSA level of 10–20 ng/ml), and high or very high risk (tumors stage T3a, or Gleason score 8–10, or serum PSA level >20 ng/ml) of recurrence. A total of 114 men were excluded because they lacked one or more of the entry criteria: preoperative PSA was missing (n = 12; 1.4%), clinical stage was missing or imprecise (n = 46; 5.5%), biopsy Gleason was imprecise (n = 22; 2.6%), or measured preoperative BMI was missing (n = 34; 4.1%). A sample of 724 patients (86.4%) was included in the analysis. 6 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. The study was approved by the local ethics committee; likewise, the assay of this protocol was approved by the local institutional review board. Informed consent was obtained from each patient before enrollment after full explanation of the purpose and nature of all procedures used. Hormone measurements To reflect the common practice of a clinical biochemistry laboratory, we elected to measure circulating hormones using commercially available analytic methods. In this context, a single preoperative venous blood sample was drawn from each participant at least 4 wk after transrectal ultrasound–guided prostate needle biopsy. Samples were drawn between 7 AM and 11 AM on the day before surgery (29) and were kept at 4°C until serum was separated by centrifugation at 4°C. Serum aliquots were then stored at 80°C until assay. In all cases, tT levels were measured via a direct chemiluminescence immunoassay (ADVIA Centaur; Siemens Medical Solutions Diagnostics, Deerfield, IL, USA); E2 was measured by a heterogeneous competitive magnetic separation assay (Bayer Immuno 1 System, Bayer Corp., Tarrytown, NY, USA); and SHBG levels were measured via a solid-phase, chemiluminescent immunometric assay on Immulite 2000 (Medical Systems SpA, Genoa, Italy). The same laboratory was used for all patients. The intra- and interassay coefficients of variation (CVs) were <6% and <8%, respectively, for both tT and E2. The intra- and interassay CVs for SHBG were <5% and <6%, respectively. Main Outcome Measures The primary end point of this analysis was to assess whether serum tT levels were differently distributed across NCCN guidelines risk classes in a nonscreened homogeneous cohort of Caucasian-European patients undergoing RP. The secondary end point was to assess the behavior of both circulating E2 and SHBG and tT–E2 ratio values in the same cohort of men according to NCCN guidelines stratification. Statistical analyses Data are presented as mean (median; range). The statistical significance of differences in means and proportions was tested with the one-way analysis of variance and the 2 trend test, respectively. 7 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Exploratory analyses were initially applied to all variables in a preliminary analysis, and variables were then kept where appropriate as significant to the results. Logistic regression models tested the association between predictors (eg, age; BMI; continuously coded tT, E2, SHBG levels; and tT–E2 values) and NCCN guidelines risk classes. Two models were developed: Model 1 included age, BMI, and continuously coded tT, E2, and SHBG levels; model 2 included age, BMI, and tT–E2 ratio values. Restricted cubic spline functions with three knots tested the potential nonlinear association between predictors and high-risk PCa and were used to display the data graphically (30,31). All statistical analyses were performed using the R statistical package (R Foundation for Statistical Computing, Vienna, Austria). All tests were two-sided, with a significance level set at 0.05. 8 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. RESULTS Table 1 lists the characteristics and descriptive statistics of the entire cohort of patients. Table 2 reports the characteristics and descriptive statistics examined according to NCCN guidelines riskclass segregation. Patients in the high-risk class showed the lowest serum tT and E2 levels as well as the lowest mean tT–E2 ratio (all p 0.02). In contrast, patients did not differ in terms of age, BMI, and serum SHBG values (all p 0.05) across the classes of risk. Hypogonadism was found in 177 (24.4%) of the whole cohort of patients. Hypogonadal patients were significantly more frequent across classes of increased risk (p 0.001) (Table 2). Figures 1a–1c depict the relationship between predictors (eg, serum tT, E2, and tT–E2 values) and high-risk PCa. In this context, high-risk PCa was significantly more frequent both for the lowest and the highest circulating levels of serum tT and E2, depicting a nonlinear U-shaped risk relationship (Fig. 1a and 1b). A similar finding was observed for the relationship between tT–E2 values and high-risk PCa (Fig. 1c). In contrast, the relationship of serum SHBG, age, and BMI with high-risk PCa did not demonstrate similar behavior (Fig. 1d, Fig. 2, and Fig. 3). According to univariate analysis, age at surgery, serum tT, E2, and tT–E2 ratio were significantly associated with high-risk PCa (all p 0.006), whereas BMI and circulating SHBG values were not (Table 3). At multivariate analyses, considering model 1 variables, patient age and circulating levels of both tT and E2 achieved independent predictor status for high-risk PCa (all p 0.03), whereas BMI and SHBG levels did not. Conversely, considering multivariate analyses for model 2 variables, only tT–E2 ratio values achieved independent predictor status for high-risk PCa (p < 0.001) (Table 3). Both univariate and multivariate analyses confirmed the nonlinear U-shaped relationship between the examined hormones and high-risk PCa, where the lowest (10th percentile) and highest (90th percentile) values predisposed to a higher risk of more aggressive PCa (Table 3). According to univariate analysis, age at surgery, serum tT, E2, and tT–E2 ratio were significantly associated with low-risk PCa (all p 0.02), whereas BMI and circulating SHBG values were not 9 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. (Table 3). At multivariate analyses, considering model 1 variables, circulating levels of tT achieved independent predictor status for low-risk PCa (all p 0.001), whereas all other variables did not. Conversely, considering multivariate analyses for model 2 variables, only tT–E2 ratio values achieved independent predictor status for low-risk PCa (all p 0.04) (Table 3). Both univariate and multivariate analyses confirmed the nonlinear inverse U-shaped relationship between the examined hormones and low-risk PCa, where the lowest (<10th percentile) and highest (>90th percentile) values predisposed to a lower probability of a low-risk PCa (Table 3). According to univariate analysis, serum E2, and tT–E2 ratio were significantly associated with intermediate-risk PCa (all p 0.02), whereas all the other variables were not (Table 3). At multivariate analyses, considering model 1 variables, only circulating levels of E2 showed a trend towards significance for intermediate-risk PCa (all p = 0.05), whereas all other variables did not. Conversely, considering multivariate analyses for model 2 variables, only tT–E2 ratio values achieved independent predictor status for low-risk PCa (all p 0.04) (Table 3) Both univariate and multivariate analyses confirmed the nonlinear inverse U-shaped relationship between the examined hormones and intermediate-risk PCa, where the lowest (<10th percentile) and highest (>90th percentile) values predisposed to a lower probability of intermediate-risk PCa (Table 3). 10 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. DISCUSSION We tested whether preoperative circulating tT, E2, and SHBG levels and tT–E2 ratio values were associated with high-risk PCa, defined according to the NCCN practice guidelines stratification, in a large, homogeneous cohort of nonscreened Caucasian-European men undergoing RP at a single academic institute. Our interest was fueled by the existing controversies regarding the role of circulating sex steroids as established predictors of pathologic outcomes at RP, usually stemming from the T-centric and T-dependent premise that there is a sort of linear relationship between the hormonal milieu and the specific PCa outcome. To the best of our knowledge, the findings of this exploratory analysis demonstrate, for the first time, that preoperative serum tT and E2 levels and the tT–E2 ratio are independent predictors of high-risk PCa, defined using the NCCN guidelines, but they depict a nonlinear U-shaped correlation (ie, both the lowest and the highest circulating levels of sex steroids are significantly associated with high-risk PCa). Conversely, these data showed that both BMI and SHBG levels are not multivariate predictors of high-risk PCa. One strength is that the current study was a single-institute survey with a large cohort of nonscreened, homogeneous, same-race patients for which all laboratory assessments, surgical procedures, and specimen evaluations were performed using a consistent method. A second strength is that the patients included in this study presented a wide variety of low- and high-risk tumors, possibly allowing for adequate variability in the baseline serum sex steroids to provide robust analysis. Third, all blood samples were correctly drawn after an overnight fast between 7 AM and 11 AM [level 2a, grade A (29)], thus avoiding a potential methodological flaw due to different collection times and diurnal variation of the steroid hormones. A further strength is that we rigorously excluded men with uncontrolled diabetes, thyroid disease, hyperprolactinemia, hypoalbuminemia, or liver diseases as well as those patients who had taken any form of hormonal preparation during the previous 12 mo (32). Similarly, the prevalence of hypogonadism was 24.4% 11 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. within our cohort of patients and is comparable with prevalence estimates from other studies (33). The latter observation certainly corroborates the validity of our cohort of men as a representative sample of PCa patients, although the results of our exploratory analysis may be optimal for this cohort of nonscreened patients. These results should be validated externally with men from different countries or ethnic backgrounds. Although contradictory findings have been reported, androgens are still believed to be critical determinants in normal and neoplastic growth and development of the prostate (16). Likewise, many data on RP populations have made it clear that there is a close correlation between circulating androgens—including the value of T as well as the condition of hypogonadism—and pathologic outcomes, although a unique direction that supports incontrovertible biology has not yet been identified (5-18). Moreover, although androgens and estrogens both play significant roles in the prostate, their specific balance seems to be even more critical in maintaining prostate health and tissue homeostasis in adulthood. Collectively, animal data have revealed that elevated T in the absence of estrogens may lead to the development of hypertrophy and hyperplasia of the prostate gland but not to malignancy (34). In contrast, high estrogen levels and low T have been shown to lead to the development of inflammation with aging and in premalignant lesions (34). Interestingly, T is the major precursor of E2 in men via a conversion mediated by the P450 aromatase enzyme (34,35). Aromatase is active in adipose tissue, adrenal glands, the testicles, and the prostate (22), meaning that it acts as a potential key regulator of the ratio of androgens to estrogens within the prostate (22,35). The local intraprostatic conversion of androgens to both reduced androgens and estrogens (35) is certainly of major importance according to the presence and function of local steroid metabolizing enzymes. Combined evidence supports the concept that aromatase expression and activity in the prostate may be upregulated at the tumor site, eventually resulting in an altered T-to-estrogen ratio. The almost complete lack of any observation of the potential significance of the T-to-estrogen ratio as a predictor of high-risk PCa could further stigmatize the value of our findings. 12 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Patient age emerged as an independent predictor of high-risk PCa at both univariate and multivariate analysis considering model 1 variables; restricted cubic spline functions depicted a trend toward a linear increase throughout years of age (Fig. 2). This result is coupled with the available controversial data regarding the impact of age on PCa; indeed, it is often suggested that older men are more likely to be diagnosed with high-risk PCa and subsequently have lower overall survival (36). Interestingly, Pierorazio et al (9) recently showed that higher levels of serum free T are associated with an increased risk of aggressive PCa among older men. In contrast, some data support even less aggressive PCa in older men (37). Our analysis found that preoperative BMI did not achieve independent predictor status for high-risk PCa, although restricted cubic spline functions depicted a sort of U-shaped correlation (Fig. 3). Our findings contrast with several previous studies showing that obesity is associated with an increased risk of aggressive disease (38-40). We are aware of the number of biases potentially contributing to these discrepancies, primarily including the multiple biological links that certainly exist between obesity, with its dramatic impact on the metabolism in general and on the metabolism of steroids specifically, and PCa. Our study is not devoid of limitations. The study reports the results of a sophisticated exploratory analysis that may be optimal for this cohort of nonscreened, same-race patients but that would deserve external validation with an independent sample and, possibly, with men from different countries or ethnic backgrounds. A second limitation is that our study did not use gas chromatography–mass spectrometry (41), which is considered the gold standard for measuring circulating tT levels; in contrast, to reflect common practice of a clinical biochemistry laboratory, we elected to measure circulating tT using commercially available analytic methods. A further major limitation comes from the idea that endocrine biology of prostate tissue is dependent on the exposure time at a given concentration of sex steroid, which, in turn, depends on serum fluctuations during the lifespan of the individual. In this context, one may speculate that a single serum assessment might not adequately represent the prostate’s hormonal environment throughout the 13 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. lifespan of each subject or at least for the duration of the malignant transformation and progression toward higher aggressiveness. However, we consider it almost clinically impossible to follow the circulating hormonal milieu of a sufficient number of men across their entire lives to assess the role of sex steroids as independent predictors of the eventual biology of PCa. Our analyses did not even consider the correlation between androgen concentrations in the circulation and actual concentrations in the prostate (42). Although the lack of that assessment might be considered a methodological flaw, it eventually exceeds the clinical applicability of biochemical parameters that may be of interest as routinely available predictors of pathologic outcomes at RP. As a final point, it is certainly of importance to emphasize that the study lacks a measurement of either circulating or intraprostatic dihydrotestosterone (DHT), the 5-reduced T product which binds androgen receptors (ARs) with high affinity both at the normal and the prostate tumor tissue level (43,44), eventually acting as a more active androgen than T (45). Overall, 5-reductase inhibitors (5ARIs) reduce serum DHT, and dutasteride demonstrated to cut circulating DHT values by at least 90% in men with localised PCa (46,47). Translationally, the two largest trials that investigated the use of 5ARIs showed an overall relative reduction of 23 to 25% in PCa diagnoses, with an absolute increase in the incidence of high-grade PCa in the chemoprevention group in both trials (48,49). More recently, dutasteride demonstrated to significantly delay PCa progression as compared with placebo (50), and these findings have supported the idea that using a 5ARI may become of benefit to reduce the need for aggressive treatment in men undergoing active surveillance for low-risk PCa (50). The absence of any DHT measurement makes it impossible to obtain a correct and refined assessment of what might objectively be the role of the U-shaped association described between circulating sex steroids and high-risk PCa in justifying how a variation of the serum levels of these extremely active androgens may affect the prostatic cell biology and the pathophysiological history of the prostate tissue itself. However, although further studies are certainly needed, we could speculate that for the lowest tT and tT–E2 ratio values, DHT hydrophobicity may strengthen human AR intermolecular interactions, slow the dissociation rate of 14 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. bound androgen, and stabilize the ligand-bound AR (45); moreover, at the lowest androgen levels DHT formation could be also secured and maintained by increased levels of 5a-reductase isoforms present at higher levels in PCa but less expressed in normal human physiology (45). Conversely, it emerges paradoxically more difficult to explain the correlation between the highest levels of sex steroids (mainly tT) and high risk PCa since debating such a relationship by stressing that for high values of those circulating hormones there is a greater substrate for 5-reductases, a greater possibility of activation of the intraprostatic ARs, and subsequently a greater likelihood of high risk Pca would certainly be too simplistic. 15 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. CONCLUSIONS Our data suggest that preoperative circulating tT and E2 levels and the tT–E2 ratio were associated with high-risk PCa, stratified according to the NCCN practice guidelines, in a large, homogeneous cohort of nonscreened, same-race men undergoing RP. The association between the serum sex steroids and PCa aggressiveness depicted a nonlinear U-shaped behavior (ie, both the lowest and the highest circulating levels of sex steroids resulted significantly associated with high-risk PCa). Patient age shows an overall trend toward significance. Conversely, these data showed that both BMI and SHBG levels are not multivariate predictors of high-risk PCa. Further studies are needed to more comprehensively understand the complex biology of the endocrine network in promoting different facets of PCa. 16 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ACKNOWLEDGMENTS The authors thank Dragonfly Editorial for reviewing the language in this manuscript. 17 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. REFERENCES 1. Endogenous Hormones and Prostate Cancer Collaborative Group, Roddam AW, Allen NE, Appleby P, Key TJ. Endogenous sex hormones and prostate cancer: a collaborative analysis of 18 prospective studies. J Natl Cancer Inst 2008;100:170-83. 2. Morgentaler A, Traish AM. 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Effect of dutasteride on the risk of prostate cancer. N Engl J Med 2010;362:1192-202. 50. Fleshner NE, Lucia MS, Egerdie B, Aaron L, Eure G, Nandy I, Black L, Rittmaster RS. Dutasteride in localised prostate cancer management: the REDEEM randomised, double-blind, placebo-controlled trial. Lancet 2012;379:1103-11 23 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. FIGURES LEGEND Fig. 1a-1c Figure 1a-1c depict the relationship between serum tT levels (ng/ml), E2 levels (pg/ml), and tT–E2 values and high-risk PCa at RP, respectively. [Y axis represents the risk (logarithmic scale) of high risk PCa at RP]. In this context, high-risk PCa was significantly more frequent both for the lowest and the highest circulating levels of serum tT and E2 (all p 0.03), depicting a nonlinear U-shaped risk behavior (Fig. 1a and 1b). Similar behavior was observed for the relationship between tT–E2 values and high-risk PCa (all p < 0.001) (Fig. 1c). Fig. 1d Figure 1d depicts the relationship between serum SHBG levels (nmol/l) and high-risk PCa at RP. [Y axis represents the risk (logarithmic scale) of high risk PCa at RP]. Fig. 2 Figure 2 depicts the relationship between patients age (years) and high-risk PCa at RP. [Y axis represents the risk (logarithmic scale) of high risk PCa at RP] Fig. 3 Figure 3 depicts the relationship between BMI values (kg/m2) and high-risk PCa at RP. [Y axis represents the risk (logarithmic scale) of high risk PCa at RP] Keys: tT = total testosterone; E2 = 17β estradiol; tT–E2 = total testosterone17β–estradiol ratio; BMI = body mass index; SHBG = sex hormone binding globulin 24 Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Table 1 – Patients’ characteristics and descriptive statistics No. of patients 724 Age (years) Mean (median) 64.4 (64.8) Range 41.4 – 82.3 10th; 90th percentile values 54.7; 73.8 BMI (kg/m2) Mean (median) 26.2 (26.0) Range 17.3 – 41.2 10th; 90th percentile values 22.6; 29.9 PSA (ng/mL) Mean (median) 11.7 (6.8) Range 0.4 – 150.0 Clinical stage [No. (%)] T1c 432 (59.7) T2 212 (29.3) T3 80 (11.0) Biopsy Gleason sum [No. (%)] 6 442 (61.0) 7 211 (29.1) 8 71 (9.8) Pathological stage [No. (%)] pT2 523 (72.2) pT3 195 (26.9) pT4 6 (0.8) Pathological Gleason sum [No. (%)] 6 280 (38.7) 7 343 (47.4) 8 101 (14.0) tT (ng/mL) Mean (median) 4.3 (4.3) Range 0.02 – 13.6 10th; 90th percentile values 1.4; 6.9 tT 3 ng/mL [No. (%)] 177 (24.4) E2 (pg/mL) Mean (median) 33.1 (32.0) Range 6.0 – 89.1 10th; 90th percentile values 18.5; 49.2 tT/E2 ratio Mean (median) 0.1 (0.1) Range 0.0 – 0.6 0.06; 0.10 10th; 90th percentile values SHBG (nmol/L) Mean (median) 36.7 (34.0) Range 6.0 – 132.0 10th; 90th percentile values 21.0; 56.0 Keys: BMI = body mass index; PSA = prostate specific antigen; tT = total testosterone; E2 = 17β estradiol; tT/E2 = total testosterone/17β estradiol ratio; SHBG = sex hormone binding globulin Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. 63.5 (63.5) 50.6 – 80.0 26.2 (26.1) 20.0 – 40.1 4.4 (4.2) 0.0 – 10.6 46 (18.3) 33.0 (32.0) 1.0 – 71.0 0.14 (0.14) 0.01 – 0.5 35.0 (34.0) 6.0 – 106.0 Age [years; mean (median)] Range BMI [kg/m2; mean (median)] Range tT [ng/mL; mean (median)] Range tT <3ng/mL [No. (%)] E2 [pg/mL; mean (median)] Range tT/E2 ratio [mean (median)] Range SHBG [nmol/L; mean (median)] Range 64.4 (64.8) 41.4 – 82.3 26.3 (26.0) 19.5 – 39.4 4.5 (4.5) 0.0 – 13.0 70 (22.0) 34.3 (33.2) 8.0 – 89.1 0.13 (0.13) 0.0 – 0.6 37.0 (34.0) 7.0 – 115.0 318 (43.9) Intermediate 68.5 (66.4) 44.8 – 82.0 26.0 (25.6) 17.3 – 41.2 3.9 (3.9) 0.0 – 13.6 61 (39.4) 30.5 (28.0) 3.0 – 77.0 0.12 (0.12) 0.0 – 0.3 39.0 (36.0) 11.0 – 132.0 155 (21.4) High 0.09 0.69 0.023 <0.001 0.008 0.005 0.69 0.37 3.78 (2, 24.76) 4.85 5.35 2.22 p – value 4.7 F Keys: BMI = body mass index; tT = total testosterone; E2 = 17β estradiol; tT/E2 = total testosterone/17β estradiol ratio; SHBG = sex hormone binding globulin p value according to ANOVA or 2 test, as indicated. 251 (34.7) Patients [No. (%)] * Risk classes Low Variable Table 2 – Patients’ characteristics and descriptive statistics according to NCCN guidelines™ risk classes after definitive therapy Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. [0.96 - 1.00] 1.01; 0.84 [0.95 - 1.06] --; <0.001 [0.96 - 1.00] 1.00; 1.00 [0.95 - 1.05] --; <0.001 --; 0.05 --; 0.69 --; 0.002 --; 0.04 [1.29 – 1.98] [1.62 – 2.30] [0.80 - 1.19] [0.78 - 1.07] --; 0.005 [0.47 - 0.77] [0.45 - 0.69] --; 0.006 1.60; <0.001 vs. [0.98 - 1.05] [0.95 - 1.01] [0.90 - 1.06] 1.03; 0.15 1.00; 0.66 [0.99 – 1.02] [1.00 – 1.02] 1.01; 0.15 Restricted cubic spline functions with three knots tested the potential nonlinear association between predictors and NCCN guidelines PCa risk classes. Two models were developed: Model 1 included age, BMI, and continuously coded tT, E2, and SHBG levels; Model 2 included age, BMI, and tT–E2 ratio values. [1.00 – 1.02] 1.01; 0.07 [0.94 - 1.02] [0.92 - 0.99] 1.03; 0.13 0.97; 0.12 0.98; 0.41 0.96; 0.05 1.02; 0.29 [1.10 – 1.24] [0.95 - 1.01] 1.17; <0.001 [1.10 – 1.24] [0.47 - 1.85] [0.49 -3.94] [0.52 - 1.29] [1.26 -2.41] percentile [0.80 – 0.90] 0.85; <0.001 1.17; <0.001 [0.80 – 0.90] 0.85; <0.001 0.81; 0.01 [0.68 - 1.15] 1.55; 0.02 0.91; 0.24 10th [1.13 - 3.69] 1.15; 0.07 0.85; 0.04 vs. [1.10 - 1.33] 1.27; 0.003 1.30; 0.02 1.00; 0.001 --; <0.001 [0.91 – 1.02] 90th vs. --; <0.001 [1.02 – 1.12] --; 0.12 [1.10 - 1.17] [0.92 - 1.00] [0.93 - 0.99] --; 0.003 1.07; 0.005 1.13; <0.001 0.96; 0.05 0.96; 0.02 [0.94 - 1.03] [0.90 – 0.98] 0.94; 0.002 --; 0.008 0.98; 0.43 [0.87 – 0.92] 0.90; <0.001 [0.90 - 0.97] [1.01 - 1.08] 1.05; 0.02 0.93; 0.001 [1.02 - 1.08] 1.05; 0.002 [0.99 - 1.05] 0.96; 0.23 [0.88 - 4.37] th 10 [0.97 - 1.05] 1.01; 0.59 --; <0.001 Model 2 1.03; 0.06 10th percentile >10 -<90 th percentile 90 th [1.02 - 1.09] 10th percentile th 1.05; 0.002 >10th-<90th vs. percentile [0.52 – 0.80] 1.93; <0.001 0.98; 0.81 0.91; 0.24 [0.44 – 0.62] 0.60; <0.001 10th [0.85 - 1.27] 0.56; <0.001 vs. [0.99 - 1.34] 90th [1.25 - 2.01] 0.64; <0.001 --; <0.001 [0.91 – 1.03] 0.97; 0.29 [1.00 – 1.06] 1.03; 0.03 Model 1 MVA models [1.37 - 2.04] 0.52; <0.001 --; <0.001 [0.92 – 1.03] 0.98; 0.43 [1.01 – 1.06] 1.04; 0.006 UVA model 10th percentile 1.04; 0.68 --; 0.89 1.15; 0.07 --; 0.12 [0.43 - 1.07] 1.02; 0.47 [0.79 - 1.02] 1.00; 0.99 Model 2 1.67; <0.001 [0.97 - 1.07] 1.01; 0.54 [0.98 - 1.02] 1.00; 0.96 Model 1 MVA models [0.97 - 1.06] 1.02; 0.51 [0.98 - 1.02] 0.99; 0.92 UVA model High risk >10th-<90th vs. 1.59; <0.001 [0.57 1.64] 1.01; 0.69 [0.96 - 1.00] 0.98; 0.11 Model 2 risk Intermediate Keys: BMI = body mass index; tT = total testosterone; E2 = 17β estradiol; tT/E2 = total testosterone/17β estradiol ratio; SHBG = sex hormone binding globulin SHBG ratio tT/E2 E2 tT BMI 0.98; 0.08 0.98; 0.02 Model 1 Age MVA models UVA model Predictor Low risk Table 3 – Univariate (UVA) and multivariate (MVA) logistic regression analyses with restricted cubic spline functions with three knots predicting low, intermediate and high risk class PCa (OR; p – value [95%CI]) among the whole cohort of patients. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 15, 2012; DOI: 10.1158/1078-0432.CCR-11-2799 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. SERUM SEX STEROIDS DEPICT A NONLINEAR U-SHAPED ASSOCIATION WITH HIGH-RISK PROSTATE CANCER AT RADICAL PROSTATECTOMY Andrea Salonia, Firas Abdollah, Umberto Capitanio, et al. Clin Cancer Res Published OnlineFirst May 15, 2012. 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