Serum levels of Sex hormone Binding globulin ( ShBg ) are not predictive of prostate cancer diagnosis and aggressiveness : results from an italian biopsy cohort

ARtIclE INFO _________________________________________________________ ___________________

of PCa (3,(6)(7)(8)(9).The role of these hormones in the pathogenesis of PCa is complex and no real consensus has been reached regarding their possible clinical implications.
The use and identification of new PCa biomarkers would help urologists in decision making and developing normograms with the aim of better identifying patients at risk of PCa and particularly high grade disease (3).The aim of our study was to evaluate the relationship between levels of SHBG and PCa in a group of patients undergoing transrectal prostate biopsies in order to identify a possible relationship between SHBG serum levels and PCa diagnosis and aggressiveness.

MATERIALS AND METHODS
After an Internal Review Board approval, between 2006 and 2012, a consecutive series of patients with no known history of PCa were referred to our department to undergo initial prostate biopsy because of an abnormal finding on digital rectal examination (DRE) and/or an elevation of serum levels of PSA (> 4ng/mL).Patients with uncontrolled diabetes, thyroid disease, hyperprolactinemia, hypoalbuminemia or liver disease were excluded from the cohort.DRE was performed by a senior staff urologist and judged positive if suggestive of cancer.Body mass index (BMI) was calculated as kg/m 2 .Every patient underwent 12core TRUS (Transrectal Ultrasound) biopsy with the same scheme, following our department's protocol (10).All biopsies were performed and pathologically reviewed in the same institute.Low grade disease was defined as Gleason ≤ 6 and high grade disease as Gleason ≥ 7.
On the day of biopsy, after obtaining a dedicated informed consent, blood specimens were drawn before the procedure at 8:00 AM (all men were fasting from the night before).PSA, SHBG and testosterone were measured.Blood samples were kept at 4° until serum was separated by centrifugation.Serum and plasma aliquots were then stored at -80° until assay.The IMMULITE 2000 (Siemens, Germany) chemoluminescent enzyme immunoassay method which is a solid phase, sequential chemiluminescent immunometric assay was used to determine PSA (ng/mL), SHBG (pg/ mL) and testosterone (ng/mL) concentrations.However, testosterone serum levels were not evaluated in this study as they have been previously investigated by our group (9).

Statistical analysis
Statistical analysis was performed using SPSS software.Evaluation of data distribution confirmed a not normal distribution of the study dataset.Differences between groups of patients in medians for quantitative variables and differences in distributions for categorical variables were tested with the Kruskal Wallis one way analysis of variance and Chi-square test, respectively.Using multiple logistic regression with the enter method, the statistical significant variables as assessed in univariate analysis were entered and investigated as predictors of prostate cancer presence vs. absence and in a separate model comparing high grade vs. low-grade among men with cancer at biopsy.The logistic regression analysis was carried out using date from patients whose complete data were available.The variables considered for entry into the model included Age, PSA, BMI, DRE and SHBG.Receiver operator characteristic curves (ROC) were produced to evaluate the area under the curve (AUC) and the diagnostic performance of PSA, SHBG and BMI for the detection of PCa and high grade PCa.An alpha value of 5% was considered as the threshold for significance.Data are presented as median with Inter Quartile Range (IQR) and mean ± standard deviation (SD).Odds ratios and 95% Confidence Interval (CI) were calculated for the parameters in each group using PCa negative and Gleason 6 as reference group.Patients with PCa were significantly older (p = 0.000), presented a higher PSA (p = 0.000) when compared to patients with a negative biopsy.No significant differences were observed in terms of BMI and SHBG levels (Table -2).On the multivariate analysis Age, PSA and DRE were found to be independent predictors of prostate cancer risk (Table -3).Risk of having prostate cancer is increased by 2.7% per unit of age, by 239% if DRE was positive and by 7.8% per unit of PSA.

Overall
Patients with high grade disease were significantly older (p = 0.000), presented a high-er PSA (p = 0.035) and higher BMI (p = 0.050) while no difference was found in terms of SHBG serum levels (Table -2).On multivariate analysis Age, DRE, BMI and PSA were found to be independent predictors of high grade disease (Table-3).Risk of high grade disease is increased 5.1% per unit of age, by 152% if DRE, by 9.8% per unit of BMI and by 7.8% per unit of PSA.In addition ROC analyses were applied to evaluate SHBG, BMI and the presence of cancer (Fig- ure-1).SHBG produced an area under the curve (AUC = 0.517; CI: 0.474-0.561;p = 0.428) that did not seem to qualify to predict PCa.BMI produced an area under the curve that did not seem to qualify to predict PCa (AUC = 0.503 ;CI: 0.459-0.547;p = 0.882).PSA served as a control (AUC = 0.595; CI: 0.553-0.638;p = 0.000).Furthermore, ROC analyses were applied to evaluate 17BE levels, BMI and the presence of high grade PCa (Figure -1).SHBG produced an area under the curve (AUC = 0.524; CI: 0.453-0.596;p = 0.596) that did not seem to qualify to predict high grade PCa.BMI produced an area under the curve (AUC = 0.569; CI: 0.500-0.639;p = 0.05) that qualified to predict high grade disease.PSA served as a control (AUC = 0.575; CI: 0.505-0.645;p = 0.035).

DISCUSSION
Our experience confirmed how older patients with higher PSA are at increased risk of PCa diagnosis.Moreover higher PSA and higher BMI in patients scheduled for prostate biopsy have an increased risk of developing high grade disease as showed by our previous studies (11).Clearly the strong association between DRE and prostate cancer risk at biopsy in our series confirms the internal validity of our results and it is in line with the peer review literature (3,12).
In our series obesity is a prevalent condition and it is associated with an increased risk of high grade disease in patients with PCa.This data confirmed our previous experiences where we observed how obesity is a significant independent risk factor for high grade Gleason score when PCa is diagnosed by prostate biopsy (3,9,13).Furthermore, the same observations were reported in other studies evaluating the possible relationship between obesity and PCa development and progression (14,15).Although further studies are needed to confirm these findings and to evaluate which  a population of patients at high risk of prostate cancer while most of the available evidence considered patients scheduled for RRP.
Case-control studies have also evaluated the relationship between hormonal status and PCa diagnosis.Sawada et al. analysed plasma testosterone and SHBG and the risk of PCa among Japanese men and found that neither testosterone neither SHBG levels were associated with an increased risk for total PCa (respectively: p = 0.49; p = 0.89) (18).In the same way Heikkila et al. in their case control study found no correlation between SHBG levels and PCa risk (23).On the other hand, Gann et al. made a prospective study of sex hormone levels and risk of PCa and found an inverse trend of risk for PCa with increasing levels of SHBG (p = 0.07) (24).Finally, a metanalysis from Roddam et al., considering 18 prospective studies on sex hormones and PCa found no correlation between serum concentrations of sex hormones and PCa risk and a modest inverse association between SHBG levels and PCa risk with a relative risk reduction of 14% (25).It is important to point out that one of the possible limitations in comparing these studies is the time of venous sampling.SHBG levels are quite stable during the day although variations in SHBG levels in a longer period of time hasn't been evaluated yet (26).Moreover serum levels of SHBG are influentiated by IGF-1 levels and BMI (26).Data analysed by the available studies differ in these terms and so far are difficult to compare.From all these studies it is evident that the exact role of SHBG in PCa genesis and progression is still an open debate and more standardised and multicentric studies are needed to clarify these preliminary observations.
It is correct to point out the limitation of our study.Cancer aggressiveness was classified according to biopsy specimens and TRUS-guided prostate biopsy is associated with a certain degree of under and overestimating of PCa, however with a single centre study and a single uropathologist reviewing all biopsies this possible bias was reduced.Another limitation of our study is that a single assessment of circulating SHBG concentrations may not be reliable clinically; however, as reported by Salonia et al., a single fasting morning venous blood sample determines the assessment of the hormone status more user friendly in every day clinical practice biological factors are responsible for the observed results, our experience add further evidence on the relationship between obesity and PCa.
SHBG is a serum globulin that binds testosterone in plasma regulating though its free component.SHBG has been associated with PCa genesis (16)(17)(18).Berndt et al. (16) found a strong relationship between SHBG gene polymorphism and risk of developing PCa.Moreover SHBG receptors have been found on prostate cells and their activation leads to an increase in cAMP possibly activating oncological pathways (17,18).
However, in our experience, no significant difference in SHBG serum levels was found between patients with or without PCa.Specifically SHBG serum levels were not associated with an increased risk PCa diagnosis on biopsy or with an increased risk of high grade disease in patients with PCa.Moreover the ROC analysis showed how SHBG predicts PCa or high PCa no more accurately than a coin toss.Although our experience is preliminar to obtain a definitive conclusion, it is the largest cohort study available regarding the association between SHBG and PCa diagnosis in patients with elevated PSA and/or abnormal DRE scheduled for prostate biopsy.Most of the available studies have evaluated the role of SHBG exclusively in patients scheduled for radical retropubic prostatectomy (RRP).Lee et al. found that SHBG preoperative levels are predictive of extraprostatic extension of tumour, SHBG serum levels achieved the role of independent predictor of extraprostatic extension on multivariate analysis (OR = 1.342, p = 0.039) (19).Salonia et al. in the same settings found that SHBG levels were independent predictors of extracapsular extension in men undergoing radical prostatectomy (OR = 1.01, p < 0.001) (20).Moreover high SHBG levels increase the risk of nodal metastases by 11% per unit (OR = 1.11, p < 0.001) and the addition of SHBG increases the predictive accuracy of the base model using clinically established predictors from 72.7% to 82.8% (21).However as observed in our experience they found no correlation between SHBG levels and high grade disease (p = 0.61) (22).Although all these studies differ from our series, a complete comparison is not possible as we evaluated the serum levels of SHBG in (20).However, at present our study is the largest prospective series evaluating the association between SHBG levels and PCa risk among men with elevated PSA or/and abnormal digital rectal examination scheduled for prostate biopsy and using the same biopsy template for the entire cohort.
We have also considered the possibility of running a separate analysis for patients with Gleason 8-10 (excluding patients with Gleason 7) but for the sake of homogeneity with papers published in the peer review literature (7,9,10,11,13,21), we decided to run a dichotomous analysis grouping in the high risk population also men with a Gleason score 7. Furthermore, our approach to define high grade prostate cancer by Gleason score ≥ 7 is also in line with a number of published papers (27,28).However, we acknowledge that further research and large number of patients are needed to explore potential differences in the different Gleason score subgroups.

CONCLUSIONS
In our cohort of patients at risk of PCa, the serum level of SHBG is not a significant predictor of prostate cancer diagnosis and aggressiveness on initial prostate biopsy.According to our experience, SHBG, should not be considered a reliable marker to predict poorly differentiated cancer in the setting of initial prostate biopsy.Further studies are needed to confirm our findings in other populations and to evaluate which biological factors related to serum SHBG levels are involved to prostate cancer development and progression.

Figure 1 -
Figure 1 -Receiver operating characteristic analysis curve comparing SHBG serum levels and BMI in patients with and without prostate cancer (ROC Curve 1) and in patients with low vs high grade disease (ROC Curve 2).

PCa =
Prostate Cancer SHBG = Sex Hormone Binding Globulin DRE = Digital Rectal Examination BMI = Body Mass index ROC = Receiver Operator Characteristics AUC = Area Under the Curve IQR = Inter Quartile Range CI = Confidence interval RRP = Radical retropubic prostatectomy TRUS = Transrectal Ultrasound