Elevated periprostatic venous testosterone correlates with prostate cancer progression after radical prostatectomy

BACKGROUND Generally, clinical assessment of gonadal testosterone (T) in human physiology is determined using concentrations measured in peripheral blood. Prostatic T exposure is similarly thought to be determined from peripheral T exposure. Despite the fact that androgens drive prostate cancer, peripheral T has had no role in the clinical evaluation or treatment of men with localized prostate cancer. METHODS To assess the role of local androgen delivery in prostate cancer, we obtained blood from the (periprostatic) prostatic dorsal venous complex in 266 men undergoing radical prostatectomy from July 2014 to August 2021 and compared dorsal T (DT) levels with those in circulating peripheral blood (PT) and prostatic tissue. Comprehensive targeted steroid analysis and unbiased metabolomics analyses were performed. The association between the DT/PT ratio and progression-free survival after prostatectomy was assessed. RESULTS Surprisingly, in some men, DT levels were enriched several-fold compared with PT levels. For example, 20% of men had local T concentrations that were at least 2-fold higher than peripheral T concentrations. Isocaproic acid, a byproduct of androgen biosynthesis, and 17-OH-progesterone, a marker of intratesticular T, were also enriched in the dorsal vein of these men, consistent with testicular shunting. Men with enriched DT had higher rates of prostate cancer recurrence. DT/PT concentration ratios predicted worse outcomes even when accounting for known clinical predictors. CONCLUSIONS These data suggest that a large proportion of men have a previously unappreciated exposure to an undiluted and highly concentrated T supply. Elevated periprostatic T exposure was associated with worse clinical outcomes after radical prostatectomy. FUNDING National Cancer Institute (NCI), NIH grants R01CA172382, R01CA236780, R01CA261995, R01CA249279, and R50CA251961; US Army Medical Research and Development Command grants W81XWH2010137 and W81XWH-22-1-0082.

To extract T, DHT, and AST from the prostate tissue, 5 to 25 mg tissue was homogenized with 500 µL methanol (Fisher) containing the internal standard (5 ng/ml of T-d3, DHT-d3, and AST-d2).The androgens were extracted from the homogenate as follows: after derivatization with 0.75 µM hydroxylamine, the samples were loaded onto solid-phase cartridges that were conditioned with 1 mL methanol and 1 mL water; then, after washing with water, the analytes were eluted with 1 mL methanol.The samples were evaporated and reconstituted with methanol:water, 50:50.
The LC-MS/MS system was an ultra-pressure liquid chromatography system (Shimadzu Corporation, Japan) consisting of two LC-30AD pumps, a DGU-20A5R degasser, a CTO-30A column oven, SIL-30AC autosampler, and a system controller CBM-20A coupled with a Qtrap 5500 mass spectrometer (AB Sciex).Data acquisition and processing were performed using Analyst software (version 1.6.2) from ABSciex.
Steroids were ionized using electrospray ionization in positive mode.Analytes were quantified using multiple reaction monitoring.The steroids and their internal standards were separated using a Zorbax Eclipse plus C18 column (Agilent) with a mobile phase consisting of (A) 0.1% formic acid in water:methanol, 70:30 and (B) 0.1% formic acid in acetonitrile:methanol:water, 50:30:20, with a gradient program at a flow rate of 0.2 mL/min.Sample injection volume was 10 µL.

Steroid Analysis
The multicollinearity assessment App within the suite of MILO's (Machine Intelligence Learning Optimizer) automated machine learning and statistical software tools, was used to generate and assess the initial correlations between the steroid concentrations in peripheral blood, dorsal blood, as well as the ratio of peripheral to dorsal blood samples for each steroid profiled.
The correlation heat map shown is a graphical representation of the correlations between the different variables in this dataset.Each square on the grid represents the relationship between two variables.The color of each square indicates the strength of the relationship, with darker colors indicating a stronger relationship.The r value is the Pearson correlation coefficient.

Untargeted metabolomics analysis
Peripheral and dorsal samples from 40 patients (20 with DT/PT ratio < 0.87 and 20 with DT/PT ratio > 4.50) were subject to untargeted metabolomics.The analysis was carried out by Metabolon, Inc., (https://www.metabolon.com).In brief, samples were prepared using the automated MicroLab STAR system (Hamilton Company).Proteins were precipitated with methanol followed by centrifugation.The resulting extract was divided into five fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup.Samples were placed briefly on a TurboVap (Zymark) to remove the organic solvent.The sample extracts were stored overnight under nitrogen before preparation for analysis.Data analysis was performed using MetaboAnalyst (V5.0).Any feature with more than 50% missing values was excluded from the analysis, and missing values were replaced by 1/5 of the minimum value.Data were normalized to the median, log transformed, and auto-scaled.Data were assumed to be nonparametric, and significance of the difference between the groups was tested using the Mann Whitney U test.

Clinical Analysis
For the clinical outcomes analysis, we performed a chart review of the 266 patients with both peripheral and dorsal vein blood samples.Of these patients, 57 were excluded because they enrolled after October 2019, which would not have allowed for adequate follow up time to establish clinical outcomes, lack of post-operative follow-up, or enrollment in a separate trial of adjuvant enzalutamide.The final cohort of 209 patients was used for analysis of clinical outcomes.
The clinical endpoint for this study was biochemical recurrence as defined by the National Comprehensive Cancer Network (NCCN) criteria: an undetectable PSA level after prostatectomy with a subsequent detectable PSA level that increases on 2 or more determinations, or progression to adjuvant radiation therapy (XRT) due to a high-risk Decipher score.Our patients were recommended for adjuvant XRT due to an intermediate-or high-risk Decipher score based on evaluation of the prostatectomy specimen.The Genomic Decipher Score is a prostate cancer geneexpression classifier that predicts an individual patient's risk of metastasis and prostate cancer-specific mortality.We decided to include progression to adjuvant XRT due to the Decipher score value as a part of the clinical endpoint because radiation therapy limits the evaluation of those patients for biochemical recurrence.

Statistical Analysis
We used the Kolmogorov-Smirnov test for common distribution to test the assumption that peripheral testosterone and dorsal testosterone are the same concentration.We divided our cohort into two groups based on the dorsal testosterone / peripheral testosterone ratio: DT/PT >1 (n= 122) and DT/PT ≤1 (n=144).Tissue androgens were compared between the two groups using the Mann-Whitney U test.Given that the PT and DT distributions differed the most above their medians, comparisons were also made between the top two quartiles of the data using the Mann-Whitney U test.Where necessary, data were transformed only for clear visualization.All data analyses were done using the statistical software R (www.r-project.org), and results were considered significant at the =0.05 level.Unlike the comparison between the dorsal vein blood in the same men, isocaproic acid is not among the metabolites that differ significantly between the groups.

Fig. S1 .
Fig. S1.Peripheral blood metabolomics comparison between men who have high vs. low DT/PT.

Fig. S3 .
Fig. S3.A DT/PT ratio > 2 is associated with adverse clinical outcomes in men after radical

Table S1 :
Patient characteristics and tumor pathology

Table S2 :
Testosterone concentration in peripheral and dorsal venous blood at various DT/PT cut points

Table S3 :
Comparison of tissue androgens in the 3 rd and 4 th quartiles in low versus high groups across a range of DT/PT cut points.