response of PCa to testosterone deprivation in the early 1940s, testosterone has been considered as fuel to the fire of PCa.

BJUI BJU INTERNATIONAL Low testosterone levels are related to poor prognosis factors in men with prostate cancer prior to treatment Eduardo Garc í a-cruz, Marta Piqueras, Jorge Huguet, Lluis Peri, Laura Izquierdo, Mireia Musquera, Agustin Franco, Ricardo Alvarez-Vijande, Maria Jose Ribal and Antonio Alcaraz Urology Department, Hospital Clinic de Barcelona, Barcelona, Spain Accepted for publication 29 February 2012 Study Type Prognosis (case series) Level of Evidence 4 OBJECTIVES Low testosterone levels have been related to a higher diagnosis of prostate cancer (PCa). Hormonal levels have been related to poor prognosis factors in men with PCa, mainly after radical prostatectomy. Our aim was to determine the relationship between hormonal levels and PCa prognosis factors in men with PCa prior to the onset of treatment. PATIENTS AND METHODS We prospectively analysed 137 males diagnosed in our centre with PCa with 5 +5 core prostate biopsies from February 2007 to December 2009. As part of our clinical protocol, we performed hormonal determination (testosterone and sex hormone binding globulin) following International Society of Andrology, International Society for the Study of the Aging Male and European Association of Urology recommendations. Free testosterone and bioavailable testosterone were calculated using Vermeulen s formula. What s known on the subject? and What does the study add? Prostate growth is ruled by testosterone. Nevertheless, the paradigm that high testosterone levels induce prostate cancer development or lead to a poor prognosis in prostate cancer is not supported by evidence. A growing number of studies suggest that, on the contrary, low testosterone levels are related to poor prognosis features in prostate cancer such as higher prostate-specific antigen or higher Gleason score. Our experience shows that testosterone levels are related to risk of progression of prostate cancer those men with lower testosterone levels are at higher risk of progression of their prostate cancer after treatment delivery. Age, prostate-specific antigen (PSA), free to total PSA, PSA density, number of previous biopsies, digital rectal examination staging, Gleason score, percentage of tumour in the biopsy sample, bilaterality of the tumour and risk of progression group were prospectively recorded. RESULTS Higher testosterone levels were related to lower digital rectal examination staging ( P = 0.02) and lower PSA level ( P = 0.05). Higher testosterone was not related to lower Gleason score ( P = 0.08). Testosterone was inversely related to PCa bilaterality ( P < 0.01) and percentage of tumour in the biopsy ( P < 0.01). High testosterone levels were found in patients allocated to the low risk of progression group and inversely ( P = 0.03). In multivariate analysis, higher age and lower testosterone were related to higher D Amico risk of progression. CONCLUSION Patients with PCa and lower testosterone levels have poor prognosis factors and higher tumour burden before treatment onset. These findings reinforce the idea that low testosterone levels pretreatment are related to a poor prognosis in PCa. KEYWORDS testosterone levels, prognosis factors, prostate cancer INTRODUCTION The relationship between testosterone and prostate cancer (PCa) is controversial and the evidence is conflicting. Since Huggins et al. [1,2 ] published their studies on the response of PCa to testosterone deprivation in the early 1940s, testosterone has been considered as fuel to the fire of PCa. However, this relationship is challenged by the fact that testosterone serum levels decrease with age as prostate cancer detection increases [3 ]. Up to now, no study has related high testosterone with increased risk of PCa development. On the contrary, some data show that PCa might be found at lower bioavailable testosterone levels [4 6 ]. 2012 BJU INTERNATIONAL 110, E541 E546 doi:10.1111/j.1464-410x.2012.11232.x E541

GARCÍA-CRUZ ET AL. Moreover, there is evidence that low serum testosterone levels may be related to worse prognosis and aggressiveness factors in PCa patients after radical prostatectomy (RP) [7 14 ]. Most of the available data about PCa and testosterone arise from studies of patients who have undergone RP. There are few data about the hormonal pattern and factors affecting PCa diagnosis and prognosis at prostate needle biopsies [15 18 ] before treatment was applied. After the diagnosis of PCa has been obtained, D Amico risk of progression classification and others are of help in predicting PCa prognosis using clinical data and information from the TRUS prostatic biopsy, before treatment is delivered and final pathological data are available. Our aim was to analyse the hormonal pattern and its correlation with broadly established PCa prognosis factors pretreatment (such as PSA, DRE and Gleason score, and specifically D Amico risk of progression) in order to determine the value of the hormonal pattern as an indicator of subsequent PCa outcome. Age (years) (mean ± SD ) 68.8 ± 9 PSA (ng/ml) (mean ± SD ) 65 ± 497 PSA < 10 87/137 (65.9%) PSA 10 20 30/137 (22.7%) PSA > 20 15/137 (11.4%) DRE T1 87/137 (63.5%) T2 45/137 (32.8%) T3 5/137 (3.6%) Number of biopsy First biopsy 95/122 (77.9%) Second biopsy 13/122 (10.7%) Third or further biopsy 14/122 (11.4%) Gleason score Gleason 2 6 80/137 (58.4%) Gleason 7 40/137 (29.2%) Gleason 8 10 17/137 (12.4%) D Amico risk of progression Low 46/137 (33.6%) Median 67/137 (48.9%) High 24/137 (17.5%) Percentage of tumour in the sample (mean ± SD ) 38 ± 44 Bilaterality 47/137 (34.3%) Testosterone (ng/dl) (mean ± SD ) 456 ± 152 SHBG (nmol/l) (mean ± SD ) 52 ± 27 Free testosterone (ng/dl) 7.2 ± 2.8 Bioavailable testosterone (ng/dl) 168 ± 66 TABLE 1 Main clinical, biochemical, pathological and hormonal data of the 137 men included in the study D Amico risk of progression groups: low risk, PSA < 10, DRE T2a and Gleason score 6; intermediate risk, PSA 10 20, DRE = T2b and Gleason score 7; high risk, PSA > 20 or DRE T2c or Gleason score 8. PATIENTS AND METHODS We prospectively analysed 137 males with PCa diagnosed in our centre after TRUS prostate biopsy from February 2007 to December 2009. The indication for prostate biopsy was suspicious DRE or PSA elevation. Diagnosis was carried out with 5 +5 core TRUS-guided prostate biopsy. As part of our clinical protocol, we determined the hormonal pattern (testosterone and sex hormone binding globulin [SHBG ] ) in these patients. Serum samples were obtained by venipuncture of fasting patients between 7:00 and 11:00 am. Testosterone and SHBG (automated immunometric chemoluminescent method, Cobas Roche) were determined. Free testosterone and bioavailable testosterone were calculated using Vermeulen s formula. Men with testosterone < 346 ng/dl were considered to have low testosterone levels. We prospectively recorded age, number of previous biopsies, PSA, free-to-total PSA (fpsa), PSA density (dpsa), DRE, prostate volume and pathological data (Gleason score, percentage of biopsy affected by tumour, bilaterality of the tumour). To describe the tumour burden, in our centre the amount of tumour in the biopsy is given as a percentage ranging from > 0% to 100% in each prostatic lobe (percentage of tissue from the total amount of tissue of the biopsy; i.e. men with 5% tumour in the left prostatic lobe and 40% tumour in the right prostatic lobe would have a total of 45% tumour in the biopsy sample). Age and prostate volume were analysed as continuous variables. PSA and its surrogates (dpsa, fpsa) were analysed as continuous variables and the PSA as a categorical variable (PSA < 10, PSA 10 20 and PSA > 20). DRE was analysed as a categorical variable (T1, normal DRE; T2, suspicious of organ confined prostate cancer; T3, suspicious of non-organ confined prostate cancer). Specifically we analysed the D Amico risk of progression classification as a reliable and widely spread pretreatment classification of risk of PCa progression. Tumours were subclassified depending on their risk of progression: low risk, PSA < 10, DRE T2a and Gleason score 6; intermediate risk, PSA 10 20, DRE = T2b and Gleason score 7; high risk, PSA > 20 or DRE T2c or Gleason score 8 [19 ]. A multivariate analysis between hormonal pattern (specifically testosterone and SHBG) and age was performed to define which variables were related to D Amico risk of progression. SPSS software (SPSS Inc., Chicago, IL, USA) was used to analyse the study data. P < 0.5 was considered statistically significant. RESULTS Clinical, pathological, biochemical and hormonal data of the 137 patients enrolled in the study are shown in Table 1. The results of the analysis between independent variables and the hormonal pattern are displayed in Table 2. The relationship between hormonal pattern and tumour burden (percentage of tumour E542 2012 BJU INTERNATIONAL

LOW T IS RELATED TO HIGHER PROSTATE CANCER RISK TABLE 2 Result of the univariate analysis between clinical, pathological and biochemical variables and hormonal variables and their P values Free testosterone (ng/dl) P Bioavailable testosterone (ng/dl) P Testosterone (ng/dl) P SHBG (nmol/l) P PSA 0.05 0.108 0.01 0.01 <10 464 ± 162 48 ± 18 7.9 ± 3.1 187 ± 72 10 20 418 ± 159 61 ± 45 6.1 ± 2.1 144 ± 50 >20 350 ± 174 53 ± 13 5.3 ± 2.6 124 ± 63 DRE 0.02 0.622 0.013 0.012 <T2b 451 ± 164 52 ± 27 7.4 ± 2.7 175 ± 64 T2b 408 ± 194 57 ± 82 6.1 ± 3 144 ± 71 >T2b 305 ± 152 46 ± 19 5 ± 2.2 117 ± 52 Gleason 0.08 0.436 0.154 0.149 2 6 452 ± 180 54 ± 30 7.2 ± 2.5 169 ± 59 7 441 ± 159 49 ± 27 7.4 ± 3.4 175 ± 80 8 10 347 ± 115 46 ± 10 5.8 ± 2.4 137 ± 57 D Amico risk of progression group 0.03 0.441 0.022 0.022 Low risk 470 ± 171 51 ± 19 7.8 ± 2.8 182 ± 66 Intermediate risk 445 ± 151 55 ± 33 7.1 ± 2.8 168 ± 66 High risk 365 ± 162 46 ± 16 5.6 ± 2.1 132 ± 48 Bilaterality 0.000 0.076 0.029 0.029 Yes 359 ± 167 45 ± 23 6.3 ± 2.9 149 ± 69 No 474 ± 160 54 ± 29 7.4 ± 2.7 175 ± 63 0.126 Percentage of positive biopsy B 0.067, β = 0.258 0.006 B 0.180, β = 0.115 0.180 B 1.956, β = 0.129 0.128 B 0.084, β = 0.129 D Amico risk of progression groups: low risk, PSA < 10, DRE T2a and Gleason score 6; intermediate risk, PSA 10 20, DRE = T2b and Gleason score 7; high risk, PSA > 20 or DRE T2c or Gleason score 8. Bilaterality: presence of tumour in both prostatic lobes. Percentage of positive biopsy: percentage of tissue affected by tumour from the total tissue sample. FIG. 1. Testosterone levels and percentage of biopsy affected by tumour. Linear regression between the variables testosterone and percentage of tumour representation in the biopsy sample is displayed ( P < 0.01). In our centre, the tumour burden in the biopsy is expressed as the percentage of tumour in each prostatic lobe, ranging from > 0% to 100%. Thus, the percentage of tumour represented in the biopsy adding the two prostatic lobes ranges from >0% to 200%. Tumour burden, percentage of tumour in the biopsy sample 200 150 100 50 0 0 200 400 600 800 Testosterone, ng/dl Sqr lineal = 0.046 1000 FIG. 2. Testosterone levels and progression risk. Tumours were subclassified depending on their D Amico risk of progression: low risk, PSA < 10, DRE T2a and Gleason score 6; intermediate risk, PSA 10 20, DRE = T2b and Gleason score 7; high risk, PSA > 20 or DRE T2c or Gleason score 8. Low risk, testosterone 470 ± 171 ng/dl; intermediate risk, testosterone 445 ± 151 ng/dl; high risk, testosterone 365 ± 162 ng/dl; P = 0.03. Testosterone, ng/dl 1000 800 600 400 200 0 Low Intermediate High D Amico risk of progression in the biopsy) is displayed in Fig. 1. Finally, a visual representation of the analysis between testosterone levels and D Amico risk of progression is shown in Fig. 2. Lower testosterone levels were related to higher PSA ( P = 0.05), higher clinical staging ( P = 0.022), poorer D Amico risk of progression ( P = 0.03), higher rate of bilaterality ( P = 0.000) and higher tumour burden ( P = 0.006). No relationship was found between hormonal pattern and Gleason score when analysed as a threecategories variable, although a certain trend was appreciated ( P = 0.08). We analysed hormonal pattern in relationship to Gleason score analysed as a two-categories variable: Gleason 7 or Gleason > 7. Testosterone was found to be related to Gleason score (testosterone Gleason 7, 448 ± 173 ng/dl, vs testosterone Gleason > 7, 347 ± 115 ng/ dl; P = 0.026). Low testosterone levels (testosterone < 346 ng/dl) were found to be related to 2012 BJU INTERNATIONAL E543

GARCÍA-CRUZ ET AL. TABLE 3 Summary of the studies analysing the relationship between PCa and hormonal pattern Author Patients Findings Testosterone cut-point Hoffman et al. [11] 117 PCa (57 RRP), 7/64 patients had higher Gleason score in low testosterone patients vs 0/48 in 300 ng/dl retrospective normal testosterone patients Low free testosterone patients had higher stage Schatzl et al. [ 12 ] 156 PCa, prospective Gleason score 7.2 in low testosterone patients vs 6.4 normal testosterone patients 300 ng/dl Yamamoto et al. [ 14 ] 272 RPP, retrospective Low testosterone patients had higher 5-year PSA recurrence rate (OR 2.7) 300 ng/dl Teloken et al. [ 13 ] 64 RRP, retrospective Low testosterone patients had higher rate of positive margins (39% vs 14.6%) 270 ng/dl Lane et al. [ 10 ] 455 RRP, prospective Low testosterone patients had higher risk of patterns 4 5 (OR 2.4) 220 ng/dl Isom-Batz et al. [7] 326 RRP, retrospective Low testosterone patients had higher stage and had higher rate of PSA > 10 300 ng/dl Non-statistical trend towards higher PSA failure in low testosterone patients (34% vs 16%) Testosterone cut-point used to define hypogonadism. RRP, radical retropubic prostatectomy; OR, odds ratio. D Amico risk of progression (testosterone < 346 ng/dl, low risk 38%, intermediate risk 51%, high risk 11%, vs testosterone > 346 ng/dl, low risk 29%, intermediate risk 41%, high risk 30%; P = 0.043). Moreover, low testosterone was related to tumour bilaterality (25.5% of bilateral tumours in > 346 ng/dl vs 50% of bilateral tumours in >346 ng/dl; P = 0.009) and tumour burden (32% of tumour in men with testosterone > 346 ng/dl vs 53% in <346 ng/dl; P = 0.001). When comparing the statistical analysis between testosterone and free and bioavailable testosterone with clinical end-points, testosterone and bioavailable and free testosterone were found to be significantly related to the same clinical variables (PSA classification, DRE, D Amico risk of progression and bilaterality) except for percentage of positive biopsy, where testosterone was statistically related ( P = 0.006) to the burden of tumour but free and bioavailable testosterone were not ( P = 0.128 and P = 0.126). A multivariate analysis between testosterone and age in relationship to D Amico risk of progression was carried out. Testosterone ( P = 0.038) and age ( P = 0.000) but not SHBG ( P = 0.816) were related to D Amico risk of progression. We found no statistical significance between age groups and Gleason score: Gleason < 7, mean age 67 ± 8.7, vs Gleason 7, mean age 69 ± 9.7; P = 0.324. DISCUSSION Although prostate growth is regulated by androgens and androgenic deprivation causes PCa to regress [1,20 ], there is a shift in the paradigm of considering testosterone as a risk factor for prostate cancer [3,21 23 ]. In this sense the literature shows that high testosterone levels are not related to a higher risk of PCa and, on the contrary, low testosterone levels might be related to more prevalence of PCa [5 ] and poor PCa prognosis [7 14 ]. We analysed a cohort of men diagnosed in our centre with PCa after prostate biopsy due to PSA elevation or abnormal DRE, before a decision on treatment was discussed. In this subset of patients, our study shows that low testosterone levels are related to higher PSA, higher DRE staging and higher tumour burden (represented by tumour bilaterality and higher percentage of tumour representation in the biopsy). When we analysed hormonal pattern in relation to D Amico risk of progression, lower testosterone levels were related to higher D Amico stages. Thus, lower testosterone men harbour larger tumours (higher percentage of biopsy affected by tumour, higher rate of bilateral tumours and higher DRE staging), have higher PSA levels and show a certain trend towards higher Gleason scores (specifically Gleason score > 7). Thus, men with low testosterone are more likely to have a higher risk of progression after treatment is delivered. In previous literature, in men submitted to RP, preoperative testosterone levels have been related to higher Gleason score [10 12 ], higher pathological stage [7 9 ], higher rate of PSA > 10 [7 ], higher rate of positive margins [13 ] and higher risk of biochemical failure [14 ] (Table 3 ). Nevertheless, there are few data about pretreatment testosterone levels and their relationship with clinical pretreatment prognostic factors [16 ] (Table 3 ). In our opinion, analysing only RP patients might induce a selection bias, taking into consideration that RP patients are younger and fit compared with those submitted for external beam radiotherapy, cryotherapy or androgen suppression. Thus, our patients include not only young patients in good health with a low tumour stage but also older, unfit and higher stage patients who would probably have lower testosterone levels. In our opinion our population is more representative of patients with PCa than if we had only considered patients undergoing RP. Our data are in agreement with previous literature, as they support the notion that low testosterone levels are related to higher PSA and higher staging, and subsequently higher risk of treatment failure and PSA relapse. A certain trend was observed when analysing low testosterone in relation to higher Gleason scores, a relationship that has already been published. To our knowledge, the link between risks of progression groups, tumour burden in the E544 2012 BJU INTERNATIONAL

LOW T IS RELATED TO HIGHER PROSTATE CANCER RISK biopsy sample and hormone levels has not been published to date. We hypothesize that testosterone level is useful as a pretreatment tool to help us define prognosis and risk of progression, and thus help us in better counselling patients. Low testosterone levels seem to be related to PCa and with worse prognosis factors. On the other hand, castration induces metastatic prostate cancer to regress. The proposed hypothesis for this apparent contradiction [3,21 23 ] suggests that cancer cells are stimulated to dedifferentiate in a testosterone-deficient environment, leading to more aggressive tumours and a higher Gleason stage. From our point of view and according to our data, our opinion is that both age and hormones play a role in the development of high risk PCa. As hormones are deeply dependent on age, it could be said that aging is relevant and hormones are only incidentally related to PCa. However, we found that the difference in testosterone values for high and low grade tumours was not likely to be due to age, as these do not vary between groups ( P non-significant). Some limitations of our study must be taken into consideration. First, ours is a singlecentre, rather short series. Thus, selection bias might be present. Although the sample is relatively small, we were able to reach statistical significance in showing the link between PCa prognosis factors and hormonal pattern. Second, our study was cross-sectional and this fact should be noted: PCa and testosterone are evolving entities through years and decades. Thus, our static analysis may be incomplete as no follow-up was considered. In conclusion, in our study patients with adenocarcinoma of the prostate and lower testosterone levels are affected by more extensive disease (higher clinical staging), higher tumour burden (higher rate of bilateral tumours and higher biopsy affected by tumour) and poorer prognosis (higher PSA, higher D Amico stages). Pretreatment testosterone determination could contribute to determination of the optimum treatment in men with adenocarcinoma of the prostate. ACKNOWLEDGEMENTS The authors would like to acknowledge Nizam Mamode, from Guy s and St Thomas, and Andrea Sallent for reviewing the manuscript. CONFLICT OF INTEREST None declared. REFERENCES 1 Huggins C, Stevens RE, Hodges CV. Studies on prostatic cancer. II. The effects of castration on advanced carcinoma of the prostate gland. Arch Surg 1941 ; 43 : 209 23 2 Huggins C. Effect of orchiectomy and irradiation on cancer of the prostate. Ann Surg 1942 ; 115 : 1192 200 3 Isbarn H, Pinthus JH, Marks LS et al. 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The clinical potential of pretreatment serum testosterone level to improve the efficiency of prostate cancer screening. Eur Urol 2007 ; 51 : 375 80S 19 D Amico AV, Whittington R, Malkowicz SB et al. Biochemical outcome after radical prostatectomy, external beam radiation therapy, or interstitial radiation therapy for clinically localized prostate cancer. JAMA 1998 ; 280 : 969 74 20 Huggins C, Hodges CV. Studies on prostatic cancer. I. The effect of castration, of estrogen and of androgen 2012 BJU INTERNATIONAL E545

GARCÍA-CRUZ ET AL. injection on serum phosphatases in metastatic carcinoma of the prostate. Cancer Res 1941 ; 1 : 293 7 21 Morgentaler A. Testosterone deficiency and prostate cancer: emerging recognition of an important and troubling relationship. Eur Urol 2007 ; 52 : 623 5 22 Morgentaler A. Guilt by association: a historical perspective on Huggins, testosterone therapy, and prostate cancer. J Sex Med 2008 ; 5 : 1834 40 23 Morgentaler A. Testosterone and prostate cancer: an historical perspective on a modern myth. Eur Urol 2006 ; 50 : 935 9 Correspondence: Eduard Garc í a-cruz, Urology Department, Hospital Clinic de Barcelona, 170 Villarroel st., 08036 Barcelona, Spain. e-mail: edu_garcia_cruz@yahoo.com Abbreviations : PCa, prostate cancer ; RP, radical prostatectomy. E546 2012 BJU INTERNATIONAL