BJUI KEYWORDS. prostate cancer, benign prostatic hyperplasia, androgens, testosterone, dihydrotestosterone, measurement

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1 BJUI Intraprostatic testosterone and dihydrotestosterone. Part I: concentrations and methods of determination in men with benign prostatic hyperplasia and prostate cancer Tim M. van der Sluis, Andr é N. Vis, R. Jeroen A. van Moorselaar, Hong N. Bui *, Marinus A. Blankenstein *, Eric J.H. Meuleman and Annemieke C. Heijboer * Departments of Urology and *Clinical Chemistry, VU University Medical Centre, Amsterdam, The Netherlands Accepted for publication 26 July 2011 Owing to inconsistencies and methodological differences, the present peer-reviewed literature lacks conclusive data on the intraprostatic levels of androgens, in particular dihydrotestosterone (DHT), in untreated benign prostatic hyperplasia (BPH) and prostate cancer. To date, no difference has been shown between DHT concentrations in normal prostatic tissue and BPH, and nor has a difference been shown in DHT concentrations between the histologically distinct regions of the prostate. Recent literature has also failed to show a consistent difference in androgen level between BPH and prostate cancer. The role of intraprostatic DHT in the pathogenesis of BPH and in the initiation and progression of prostate cancer thus remains to be established. Increased What s known on the subject? and What does the study add? The male steroid hormone metabolism is an important target in the treatment of prostatic diseases. However, present literature is inconsistent about intraprostatic concentrations of steroid hormones and the role of intraprostatic steroid hormones in the arise and the course of prostatic disease is yet to be determined. Part I of this two-piece reviews the different methods of steroid hormone measurement and gives an overview of steroid hormone concentrations in normal and diseased prostatic tissue. More accurate methods of measurement and increased knowledge of androgenic steroid hormonal pathways of the prostate could lead to better treatment of prostatic diseases. knowledge of the mechanisms of the androgenic steroid pathways in prostatic diseases, with a special focus on intraprostatic androgen levels may lead to more optimized and more personalized forms of treatment, and probably new therapeutic targets as well. KEYWORDS prostate cancer, benign prostatic hyperplasia, androgens, testosterone, dihydrotestosterone, measurement INTRODUCTION During a man s lifetime, the prostate is continuously affected by steroid androgens, derived from both the testes and adrenal cortex. While dihydrotestosterone (DHT) is the principal androgen in the prostate gland, testosterone is the abundant androgen in the blood, when adrenal androgens are not taken into account. Both androgens competitively bind the androgen receptor (AR). The androgen-ar complex, in turn, stimulates androgenregulated processes, e.g. cell proliferation, cell differentiation and transcription of androgen-regulated genes [1 ]. DHT is formed by conversion of testosterone by the enzyme 5- α-reductase (5AR) [2,3 ]. In recognizing the central role of DHT in the growth of the prostate, interference with androgen metabolism, e.g. by 5AR inhibition, became one of the cornerstones of medical management in men with BPH. The role of DHT accumulation as a causative factor in the aetiology of BPH, however, remains speculative. Previously reported increased levels of androgens in BPH tissue and in prostate cancer were not confirmed by later studies. Much of the debate came from the finding that substantial methodological differences and inconsistencies were found between research groups reporting on intraprostatic androgen levels, and that earlier studies from the 1970s and 1980s were hampered by methodological flaws. Validated tools for appropriate tissue handling and for tissue androgen steroid level assessment are now available but no consensus has been reached about which method is best. In the present review (Part I), we provide an overview of the methods currently used for androgen assessment, the conditions for appropriate tissue processing, and reported levels of intraprostatic androgens (DHT and testosterone) in normal adult prostate, in untreated BPH and in prostate cancer. In Part II, we highlight the levels of androgens in medically treated BPH and prostate cancer. METHODS FOR ANDROGEN ANALYSIS Androgen concentrations can be measured using various techniques, all of which have , doi: /j x x

2 INTRAPROSTATIC ANDROGEN ASSESSMENT IN UNTREATED BPH AND PROSTATE CANCER TABLE 1 Advantages and disadvantages of methods for testosterone and DHT measurement Method Advantage Disadvantage IA Fast, easy, relatively inexpensive Low specificity and sensitivity owing to cross reactivity (in low concentrations: not better than a guess ) Low sample volume No unequivocal identification IA with extraction and/or chromatography Good specificity and sensitivity Relatively highly skilled technicians needed Limited throughput and relatively high turn-around time Relatively expensive, relatively large sample volume ID-GC-MS or ID-LC-MS(/MS) High specificity, accuracy and sensitivity Highly skilled technicians needed Low sample volume (ID-LC-MS/MS) Limited throughput and relatively high turnaround time. Detect more analytes High sample volume and lengthy sample preparation (ID-GC-MS) Relatively expensive advantages and disadvantages (Table 1 ). Generally, two types of techniques for measuring androgens exist: immunoassay (IA) and mass spectrometry (MS). IA METHODS The IA is the most widely used assay for determination of testosterone concentrations. IAs are based on the principle that a labelled analogue of the hormone competes with the hormone in the sample for a limited number of binding sites, usually provided by antibodies. In these so-called competitive hormone assays, the proportion of labelled analogue bound to the binding sites is inversely proportional to the concentration of androgen in the sample. Depending on the characteristics of the label, IA is known as either radioactive IA (RIA) or chemiluminescence IA. Some IAs are fully automated and, consequently, it is possible to process many samples in a short amount of time, which makes it relatively inexpensive. The disadvantage of IA is its low specificity owing to cross-reactivity with related steroid hormones. These disadvantages can be overcome by performing extraction and/or chromatography before the IA. During extraction, testosterone is released from (testosterone-binding) proteins, whereas in chromatography, testosterone is separated from related steroid hormones. The free-testosterone thus released, enters the competition reaction with the labelled analogue. Despite an increased specificity, extraction and/or chromatography before IA lead to a prolonged processing time and increased costs. Although automated DHT assays are not available, the advantages and disadvantages of DHT measurement are the same as for testosterone measurement [4 ]. MS METHODS A mass spectrometer detects molecules according to their mass. A combination of two serially linked mass spectrometers ( MS/ MS or tandemms ) may be used to enhance specificity. Testosterone travelling through the first MS is selected based on its molecular mass. The testosterone molecules are subsequently fragmented and their specific fragmentation pattern is detected by the second mass spectrometer. MS(/MS) detection of testosterone is typically preceded by chromatography either gas chromatography (GC) or liquid chromatography (LC) and requires some form of sample preparation, such as extraction. In the case of LC, MS/MS is highly preferred over a one-detector system in order to compensate for the more rudimentary separation as compared with GC. The use of an isotope-labelled internal standard to correct for losses during sample preparation is indicated by the ID (isotope dilution) prefix. At the moment, ID-GC-MS is the reference method for measuring testosterone concentrations (Table 1 ) [5,6 ]. Its main advantage is its high specificity and accuracy. Disadvantages are the need for large sample volumes and meticulous sample preparation. As a consequence, ID-GC-MS is not preferentially used in clinical practice. ID-LC-MS/MS, however, could be more attractive to use in clinical settings, as it tolerates smaller sample volumes without necessarily losing specificity and accuracy, while the process of sample evaluation appears to be less laborious than with ID-GC-MS. There are pitfalls that hamper the specificity of the ID-LC-MS/MS method [7 ]. Rigorous chromatography is needed to separate the isomers of testosterone and DHT. Derivatization strategies sometimes need to be employed to increase the performance of used assays [8 11 ]. Even though derivatization might improve the fragmentation pattern of DHT, transitions should still be chosen carefully. Kalhorn et al. [8 ] measured a loss of an acetonitrileadduct as the transition for the oxime derivative of DHT. This transition is non-specific and might hamper the specificity of the DHT measurements. Also, standardization of measurements using ID-LC-MS/MS or ID-GC-MS needs attention [12 ]. Thorough analytical validation of MS methods should include calibration against a reference method to ensure accuracy [13 ]. In a comparative study of 10 different IAs and ID-GC-MS, it was shown that IAs deviated % compared with ID-GC-MS in the low ( <8 ng/dl) testosterone ranges [14 ]. None of the IAs tested was sufficiently reliable for investigation of sera in those with low testosterone concentrations. Triggered by this study, Herold and Fitzgerald [15 ],

3 VAN DER SLUIS ET AL. created a random number generator that measured female testosterone concentrations, and showed that guessing was nearly as good as most IAs, and even superior to some. The Endocrine Society and other endorsing organizations (including the AUA), stated that direct IA methods jeopardize the health of those patients whose medical care relies upon accurate measurement of testosterone levels [16 ]. INTRAPROSTATIC ANDROGEN MEASUREMENT TISSUE PROCESSING In recent years, serum measurement of testosterone has become a routine procedure in most clinical laboratories. Whereas serum androgen concentrations can be determined relatively easily, paraffin-embedded tissue is less accessible for androgen measurement. The chemical properties (e.g. the hydrophobic character) of most steroid hormones may lead to loss of androgens during embedding and hence lead to erroneous results. Consequently, an off-the-shelf assay for tissue androgen assessment is not routinely available. Moreover, the process of tissue homogenization, resuspension, and extraction of steroid hormones is timeconsuming, and early enzymatic decay of androgens calls for fast tissue storage and appropriate tissue handling techniques. Walsh et al. [17 ] determined the influence of enzymatic decay on DHT content in prostatic tissue. In a cadaver study ( n = 7), they showed that DHT levels were reasonably low (five- to -sixfold lower) compared with those in freshly obtained prostate specimens. Subsequent in vitro studies of fresh prostate tissue incubated at 37 C showed that more than half of the DHT content is lost within 2 h of tissue harvesting. The extent of this enzymatic decay in prostate tissue after disconnection from the blood circulation is not quantified in studies with larger numbers of cases. So, for proper assessment of tissue androgen concentrations utmost care must be taken to preserve the in vivo androgen concentration. Because the conversion of testosterone to DHT mainly takes place intracellularly, tissue should be disintegrated to destroy cell membranes and subsequently be homogenized. Dismembranated tissue can be obtained by exposure to ultrasound waves, physical tissue grinding or exposure to very low temperatures. After tissue homogenization, soluble androgens are extracted using chemical compounds [18,19 ]. ANDROGEN LEVELS IN NORMAL PROSTATE AND BPH TISSUE Until the 1980s, it was widely believed that prostatic concentrations of DHT might be increased in men with BPH [20 26 ]. In the reporting of physiological levels of androgens in the normal prostate and in BPH, data were initially obtained from autopsy studies and from studies assessing surgically removed prostates, respectively. Overall, intraprostatic androgen levels fell within wide ranges, between 0.7 and 2.1 for DHT in normal prostatic tissue [17,24 ], and between 1.0 and 8.15 for DHT in BPH tissue (Table 2 [17 48 ] ). In paraffin-embedded tissue obtained from four healthy volunteers undergoing prostate needle biopsy, concentrations of 9.3 for DHT and 1.8 for testosterone were reported [45 ]. Contrary to previous assumptions, it was shown that, if freshly obtained, testosterone and DHT levels in normal prostatic tissue obtained from 21 men at the time of radical prostatectomy or cystoprostatectomy were not statistically different from corresponding androgen levels in BPH tissue removed from 20 patients at open surgical procedures (Table 2 [17 ] ). Controversy also remains about the androgen concentrations in the different regions of the prostate. In 1977, Hammond et al. [ 20 ] found similar levels of DHT in the peripheral prostatic zone and BPH tissue. Di Silverio et al. [35,36 ] reported that, by contrast to this, DHT levels were highest in the periurethral zone (7.32 ), the region responsible for urinary obstruction, and lowest in the subcapsular region (4.22 [P < 0.01 ] ). Again however, DHT levels in three men < 45 years old were reported not to differ significantly from those of 18 patients > 45 years old with established BPH [17 ]. ANDROGEN LEVELS IN PROSTATE CANCER There is still no consensus on the true prostatic steroid concentrations in men within different stages and grades of prostate cancer. In a study by Geller et al. [49 ], 21 patients with previously untreated stage D prostate cancer underwent palliative TURP. In the resected tissue, a mean value of 3.9 DHT was reported, significantly less ( P < 0.05) as compared with those with BPH (i.e. 5.1 ). Nishiyama et al. [18 ] reported no difference in DHT concentrations between prostate cancer and untreated BPH tissue. Conflicting outcomes were reported by Heracek et al. [50 ] who compared intraprostatic concentrations of testosterone and DHT in 57 men who underwent transvesical prostatectomy for BPH with those in 121 men undergoing radical prostatectomy for prostate cancer. DHT levels in radical prostatectomy specimens (2.55 ) were significantly higher ( P < 0.01) than those in BPH tissue (1.86 ). Similar results were found for intraprostatic testosterone (1.34 vs 0.99 ng/g tissue, P < 0.05). In line with this, Ji et al. [ 51 ] reported a 42% higher DHT level in eight prostate tumours compared with their paired benign tissue samples (6.98 ng/g tissue vs 4.96 [ P < 0.05 ] ). Nishiyama et al. [44 ] investigated androgen levels in prostate cancer tissue of different Gleason scores in 47 patients with clinically localized prostate cancer. Prostatic tissue was obtained by prostate needle biopsy, and after snap-freezing and homogenization of tissue, testosterone and DHT levels were measured using the LC-MS technique. The mean concentration of intraprostatic DHT in prostate cancer was In those with a biopsy Gleason score 6, mean tissue DHT (5.94 ) was significantly higher than those with a Gleason score 7 10 (4.29 ; P = 0.025) [44 ]. These findings were not confirmed in a later study from the same group [52 ]. Geller et al. [53 ] showed that men with prostate cancer and a DHT level > 2.5 had a significantly longer disease-free survival than those with DHT levels <2.0 (P < 0.001). Only one study has so far reported on the concentration of androgens within prostate cancer lymph node (LN) metastases. LNs from 22 patients undergoing pelvic LN dissection for prostate cancer were assessed

4 INTRAPROSTATIC ANDROGEN ASSESSMENT IN UNTREATED BPH AND PROSTATE CANCER TABLE 2 Testosterone and DHT concentrations in normal prostatic tissue, BPH tissue and prostate cancer tissue Normal tissue DHT, Normal tissue testosterone, BPH tissue DHT, ng/g tissue BPH tissue testosterone, Prostate cancer tissue DHT, ng/g tissue Authors [ref] n Method of determination Tissue acquisition Krieg et al. [22] 7/14/7 RIA autopsy/op Belis et al. [25] 4/42/13 RIA OP/TURP/PB Walsh et al. [17] 21/20 RIA RP/RC/OP /5 RIA autopsy 0.7 ND 1.0 ND Hammond et al. [20] 18/10 RIA autopsy Meikle et al. [21] 5/12 RIA autopsy/turp 1.3 ND 4.0 ND Siiteri et al. [23] 15/10 RIA autopsy/op Geller et al. [24] 6/8 RIA autopsy/op 2.1 ND 5.6 ND Page et al. [45] 4 RIA PB Habib et al. [26] 10/10 RIA TURP ND Heracek et al. [50] 57/121 RIA OP Nishiyama et al. [18] 34/69 LC-MS PB 5.61 ND 5.19 ND Ji et al. [51] 8/8 RIA RP 4.96 ND 6.98 ND Geller et al.* [49] 25/23 RIA TURP 5.1 ND 3.9 ND Geller et al. [27] 6 RIA TURP 3.9 ND Geller et al. [28] 25 RIA TURP 6.0 ND McConnell et al. [32] 20 RIA TURP Norman et al. [33] 10 RIA TURP Span et al. [37] 7 RIA TURP Mizokami et al. [40] 15 LC-MS/MS TURP 2.48 ND Wurzel et al. [47] 21 RIA TURP Rittmaster et al. [48] 118 LC-MS/GC-MS TURP/RP Shibata et al. [38] 27 LC-MS/MS OP/TURP/RC Salerno et al. [29] 19 GC-MS OP Forti et al. [31] 19 GC-MS OP Hill et al. [34] 15 RIA OP Di Silverio et al. [35] 15 RIA OP Monti et al. [36] 15 RIA OP Marks et al. [19] 15 RIA PB Marks et al. [19] 20 RIA PB Marks et al. [46] 19 RIA PB Marks et al. [46] 21 RIA PB Mohler et al. [41] 30 RIA RP Titus et al. [42] 18 LC-MS RP Andriole et al. [39] 19 GC-MS RP Gleave et al. [43] 14 GC-MS RP Geller et al. [54]* 20 RIA RP 4.80 ND 22 RIA 2.23 ND Nishiyama et al. [44] 47 LC-MS PB 5.42 ND Nishiyama et al. [52] 28 LC-MS PB 5.70 ND Prostate cancer tissue testosterone, *Similar study group; Similar study group; Tissue levels are medians; Measurement in lymph node metastasis. Tissue testosterone and DHT levels are mean values except those marked with which are median values. Testosterone: 1 nmol/l = 28.8 ng/dl; 1 nmol/l = 29.0 ng/dl. DHT: 1 ng = nmol; 1 ng = nmol. PB, prostate biopsy; RP/RC, radical prostatectomy/radical cystoprostatectomy; OP, open prostatectomy; ND, no data. for androgen levels using RIA. Prostate tumour tissue from 20 different patients with prostate cancer was collected simultaneously during surgery. A mean DHT concentration of 2.23 was found in LN metastases, compared with 4.80 ng/g tissue in primary cancer ( P < 0.001). Apparently, in LN metastases of prostate cancer, DHT levels are found to be >50% lower than in primary prostate cancer [54 ]. DISCUSSION Prostatic diseases are highly prevalent in the aging male population owing to an increased life expectancy and PSA-based

5 VAN DER SLUIS ET AL. screening for prostate cancer. During a man s lifetime, the prostate is continuously affected by androgens, with DHT being the principle androgen within the prostate gland. Historically, accumulation of tissue androgens, DHT in particular, was assumed to play a causative role in the aetiology of BPH and in the initiation and progression of prostate cancer [20 26 ]. Consequently, the androgenic pathway has become an important target for treatment. Reproducible data on the true intraprostatic levels of androgens within the normal adult prostate are currently lacking ( Table 2 ). Published data on intraprostatic androgen levels differ widely because of patient selection, methods of tissue retrieval, methods of tissue processing, and assays of androgen determination used. Older studies mostly do not describe coefficient of variation, sd or statistical tests. A standardized technique to process prostatic tissue with the purpose of androgen level determination is needed. In the coming years, further confirmation of intraprostatic androgen levels can be expected when using techniques with high specificity, accuracy and sensitivity, e.g. ID-LC-MS/MS. Cadaver studies have evaluated androgen levels in the normal prostate, but most of these studies are hampered by substantial methodological flaws attributable to androgen decay in the time between tissue harvesting and androgen level measurements. Mean tissue testosterone levels in normal prostatic tissue ranged between 0.26 and 1.8 and mean tissue DHT levels ranged between 0.7 and 9.3 ( Table 2 ). As BPH tissue is much easier to obtain and process, research efforts have focused on intraprostatic androgen levels in BPH tissue obtained at the time of TURP. In these studies, no consensus was reached on the absolute concentrations of androgens. Values found in untreated BPH tissue ranged from to 2.0 for testosterone, and from 1.0 to 8.15 for DHT. These figures indicate that when prostatic tissue is harvested appropriately, testosterone and DHT content of the normal prostate is similar to that in BPH tissue. Also, recent findings fail to show any major difference in the steroid composition between the histologically distinct regions of the prostate. More detailed measurement in epithelial and stromal prostatic compartments might provide further understanding of the processes that lead to the initiation and progression of BPH and prostate cancer. In general, levels of steroids have been studied in greater detail in BPH than in prostate cancer. To date, there has been no consensus on testosterone and DHT concentrations in men diagnosed with prostate cancer. Similarly to BPH, the reported testosterone and DHT levels differ widely between study groups ( Table 2 ). The measured concentrations of tissue DHT in (untreated) prostate cancer varied from 0.66 to Intriguingly, reported values of intraprostatic androgens for BPH and prostate cancer have a similar distribution, both with respect to testosterone and to DHT levels. Further confirmation with more robust techniques is required to elucidate differences in androgen concentrations between tumour grade and/or histological site. In conclusion, the current literature lacks conclusive data on intraprostatic androgen levels, DHT in particular, as the most important mediator of AR-regulated processes. To date, no difference has been shown between DHT concentrations in normal adult prostate and BPH tissue, nor is there a proven difference in androgen level between histologically distinct regions of the prostate. Also, recent literature does not reveal major differences in steroid composition between BPH and prostate cancer. The role of intraprostatic DHT in the pathogenesis of BPH and in the initiation and progression of prostate cancer thus remains to be elucidated. Increased knowledge of the mechanisms of androgenic steroid pathways in prostatic diseases, with a special interest in intraprostatic androgen level determination, may lead to more optimized and more personalized forms of treatment, and probably new therapeutic targets as well. Recent advances in the methods for the assessment of androgens, such as ID-LC-MS/ MS, will allow resolving some, if not all, of these issues. CONFLICT OF INTEREST None declared. REFERENCES 1 Farnsworth WE, Brown JR. Metabolism of testosterone by the human prostate. JAMA 1963 ; 183 : Bruchovsky N, Wilson JD. The intranuclear binding of testosterone and 5-alpha-androstan-17-beta-ol-3-one by rat prostate. J Biol Chem 1968 ; 243 : Vis AN, Schr ö der FH. Key targets of hormonal treatment of prostate cancer. Part 1: the androgen receptor and steroidogenic pathways. 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