Diurnal 11-ketotestosterone and 17-hydroxyprogesterone saliva profiles in paediatric classical congenital adrenal hyperplasia.

OBJECTIVES: The most suitable biochemical markers for therapy adjustment in patients with congenital adrenal hyperplasia are controversial. 11-Oxygenated androgens are a promising new approach. The objective of this study was to investigate the diurnal rhythm of 11-ketotestosterone in children and adolescents in saliva and to correlate it with salivary 17-hydroxyprogesterone. METHODS: Fifty-one samples of steroid day-profiles from 17 patients were additionally analysed for 11-ketotestosterone, retrospectively. All patients were treated in our university outpatient clinic for paediatric endocrinology between 2020 and 2022. Steroid day-profiles of 17 patients could be examined. The cohort showed a balanced sex ratio. The median age was 13 years. The measurements for 17-hydroxyprogesterone were carried out during routine care by immunoassay. The measurements of 11-ketotestosterone were performed from frozen saliva samples using an implemented in-house protocol for liquid chromatography - tandem mass spectrometry (LC-MS/MS). The most important outcome were the absolute values for 11-ketotestosterone, their diurnal rhythmicity and the correlation with 17-hydroxyprogesterone. RESULTS: Both steroids show a circadian diurnal rhythm. 17-hydroxyprogesterone and 11-ketotestosterone correlate significantly. 11-Ketotestosterone showed a positive correlation with BMI at all times of the day. CONCLUSIONS: 11-Ketotestosterone shows circadian rhythmicity in our cohort and correlates with 17-hydroxyprogesterone. These findings serve as an important basis for prospective research into 11-oxygenated androgens as therapeutic markers in paediatrics. However, 11-ketotestosterone appears to be very dependent on BMI.

Gonadal androgens are associated with decreased type I interferon production by plasmacytoid dendritic cells and increased IgG titres to BNT162b2 following co-vaccination with live attenuated influenza vaccine in adolescents.

mRNA vaccine technologies introduced following the SARS-CoV-2 pandemic have highlighted the need to better understand the interaction of adjuvants and the early innate immune response. Type I interferon (IFN-I) is an integral part of this early innate response that primes several components of the adaptive immune response. Women are widely reported to respond better than men to tri- and quadrivalent influenza vaccines. Plasmacytoid dendritic cells (pDCs) are the primary cell type responsible for IFN-I production, and female pDCs produce more IFN-I than male pDCs since the upstream pattern recognition receptor Toll-like receptor 7 (TLR7) is encoded by X chromosome and is biallelically expressed by up to 30% of female immune cells. Additionally, the TLR7 promoter contains several putative androgen response elements, and androgens have been reported to suppress pDC IFN-I in vitro. Unexpectedly, therefore, we recently observed that male adolescents mount stronger antibody responses to the Pfizer BNT162b2 mRNA vaccine than female adolescents after controlling for natural SARS-CoV-2 infection. We here examined pDC behaviour in this same cohort to determine the impact of IFN-I on anti-spike and anti-receptor-binding domain IgG titres to BNT162b2. Through flow cytometry and least absolute shrinkage and selection operator (LASSO) modelling, we determined that serum-free testosterone was associated with reduced pDC IFN-I, but contrary to the well-described immunosuppressive role for androgens, the most bioactive androgen dihydrotestosterone was associated with increased IgG titres to BNT162b2. Also unexpectedly, we observed that co-vaccination with live attenuated influenza vaccine boosted the magnitude of IgG responses to BNT162b2. Together, these data support a model where systemic IFN-I increases vaccine-mediated immune responses, yet for vaccines with intracellular stages, modulation of the local IFN-I response may alter antigen longevity and consequently improve vaccine-driven immunity.

Report from the HarmoSter study: different LC-MS/MS androstenedione, DHEAS and testosterone methods compare well; however, unifying calibration is a double-edged sword.

OBJECTIVES: Current LC-MS/MS applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for DHEAS. We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification. METHODS: Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued. RESULTS: Intra-laboratory CVs ranged between 4.2-13.2 % for androstenedione, 1.6-10.8 % for DHEAS, and 4.3-8.7 % and 2.6-7.1 % for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20 %. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6 % for androstenedione (p<0.001), 7.2 vs. 4.9 % for DHEAS (p<0.001), 6.4 vs. 7.6 % for female testosterone (p<0.001) and 6.8 and 7.4 % for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5 % and -4.9 to 18.7 % for androstenedione, -10.9 to 4.8 % and -3.4 to 3.5 % for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6 % for testosterone in females, and -7.0 to 8.5 % and -7.5 to 11.8 % for testosterone in males, respectively. CONCLUSIONS: Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability.

Discriminatory Value of Steroid Hormones on Polycystic Ovary Syndrome and Clustering of Hyperandrogenism and Metabolic Factors.

OBJECTIVE: We determined (1) if 11-oxygenated androgens better identify polycystic ovary syndrome (PCOS) diagnosis in women with obesity compared to total or free testosterone (T) and free androgen index; (2) how biochemical hyperandrogenism and metabolic factors cluster in a cohort of women with infertility and obesity. METHODS: Women with obesity and PCOS comprised the study group (N = 132). Ovulatory women with obesity and idiopathic, tubal or male factor infertility were the control group (N = 83). Steroid hormones were measured by means of liquid chromatography tandem mass spectrometry. Receiver operating characteristic curves and principal component analysis were used. RESULTS: Women with obesity and PCOS had higher 11-ketotestosterone (11 KT) (1.22 nmol/L [0.84; 1.65] vs 1.05 [0.78; 1.35], P = .04) compared to controls, but not 11ß-hydroxyandrostenedione 4.30 [2.87; 5.92] vs 4.06 [3.22; 5.73], P = .44). 11-ketotestosterone (area under the curve: 0.59) did not better discriminate PCOS in women with obesity compared to: total T (0.84), free T (0.91), and free androgen index (0.85). We identified 4 principal components (PCs) in the PCOS group (72.1% explained variance): (1) insulin resistance status; (2) blood pressure; (3) obesity; (4) androgen status and 4 PCs in the control group (68.7% explained variance) with variables representing metabolism being dispersed in component 2, 3, and 4. CONCLUSIONS: Eleven-oxygenated androgens do not aid in the diagnosis of PCOS in women with obesity. Insulin resistance is the strongest PC in the PCOS group. There is no major dominant characteristic that defines obese non-PCOS women.

Transposable Elements Are Co-opted as Oncogenic Regulatory Elements by Lineage-Specific Transcription Factors in Prostate Cancer.

Transposable elements hold regulatory functions that impact cell fate determination by controlling gene expression. However, little is known about the transcriptional machinery engaged at transposable elements in pluripotent and mature versus oncogenic cell states. Through positional analysis over repetitive DNA sequences of H3K27ac chromatin immunoprecipitation sequencing data from 32 normal cell states, we report pluripotent/stem and mature cell state-specific "regulatory transposable elements." Pluripotent/stem elements are binding sites for pluripotency factors (e.g., NANOG, SOX2, OCT4). Mature cell elements are docking sites for lineage-specific transcription factors, including AR and FOXA1 in prostate epithelium. Expanding the analysis to prostate tumors, we identify a subset of regulatory transposable elements shared with pluripotent/stem cells, including Tigger3a. Using chromatin editing technology, we show how such elements promote prostate cancer growth by regulating AR transcriptional activity. Collectively, our results suggest that oncogenesis arises from lineage-specific transcription factors hijacking pluripotent/stem cell regulatory transposable elements. SIGNIFICANCE: We show that oncogenesis relies on co-opting transposable elements from pluripotent stem cells as regulatory elements altering the recruitment of lineage-specific transcription factors. We further discover how co-option is dependent on active chromatin states with important implications for developing treatment options against drivers of oncogenesis across the repetitive DNA. This article is featured in Selected Articles from This Issue, p. 2293.

Tissue Glucocorticoid Metabolism in Adrenal Insufficiency: A Prospective Study of Dual-release Hydrocortisone Therapy.

BACKGROUND: Patients with adrenal insufficiency (AI) require life-long glucocorticoid (GC) replacement therapy. Within tissues, cortisol (F) availability is under the control of the isozymes of 11ß-hydroxysteroid dehydrogenase (11ß-HSD). We hypothesize that corticosteroid metabolism is altered in patients with AI because of the nonphysiological pattern of current immediate release hydrocortisone (IR-HC) replacement therapy. The use of a once-daily dual-release hydrocortisone (DR-HC) preparation, (Plenadren®), offers a more physiological cortisol profile and may alter corticosteroid metabolism in vivo. STUDY DESIGN AND METHODS: Prospective crossover study assessing the impact of 12 weeks of DR-HC on systemic GC metabolism (urinary steroid metabolome profiling), cortisol activation in the liver (cortisone acetate challenge test), and subcutaneous adipose tissue (microdialysis, biopsy for gene expression analysis) in 51 patients with AI (primary and secondary) in comparison to IR-HC treatment and age- and BMI-matched controls. RESULTS: Patients with AI receiving IR-HC had a higher median 24-hour urinary excretion of cortisol compared with healthy controls (72.1 µg/24 hours [IQR 43.6-124.2] vs 51.9 µg/24 hours [35.5-72.3], P = .02), with lower global activity of 11ß-HSD2 and higher 5-alpha reductase activity. Following the switch from IR-HC to DR-HC therapy, there was a significant reduction in urinary cortisol and total GC metabolite excretion, which was most significant in the evening. There was an increase in 11ß-HSD2 activity. Hepatic 11ß-HSD1 activity was not significantly altered after switching to DR-HC, but there was a significant reduction in the expression and activity of 11ß-HSD1 in subcutaneous adipose tissue. CONCLUSION: Using comprehensive in vivo techniques, we have demonstrated abnormalities in corticosteroid metabolism in patients with primary and secondary AI receiving IR-HC. This dysregulation of pre-receptor glucocorticoid metabolism results in enhanced glucocorticoid activation in adipose tissue, which was ameliorated by treatment with DR-HC.

Serum cortisol assay performance following the 1 mg overnight dexamethasone suppression test.

BACKGROUND: The 1 mg overnight dexamethasone suppression test (ONDST) is recommended for the differential diagnosis of Cushing's syndrome and the investigation of adrenal incidentalomas. Despite documented variation in serum cortisol immunoassay performance, little has been published regarding its effect on the ONDST. AIMS: Assess the performance of three immunoassay platforms (Roche Elecsys II, Abbott Alinity & Siemens Centaur) when compared to a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. METHODS: Samples (n = 77) sent to the laboratory as part of an ONDST were retrieved prior to disposal, anonymized, and analysed on all platforms. Samples with factors impacting immunoassay analysis quality were excluded. Results were statistically compared to an LC-MS/MS method that previously demonstrated excellent comparability to a candidate reference method. RESULTS: The Roche gen II showed a mean bias of -2.4 nmol/L and a Passing-Bablok fit of y = -0.9 + 0.97x. This was not affected by sex. The Abbott showed a mean bias -18.8 nmol/L, and a fit of y = -11.3 + 0.88x. This bias was -20.7 nmol/L in females versus -17.2 nmol/L in males. The Siemens had a mean bias of 2.3 nmol/L and a fit of y = 1.4 + 1.07x. This bias was 5.7 nmol/L in males versus -1.0 nmol/L in females. CONCLUSIONS: Clinicians should be aware of the method-dependent variation that exists within serum cortisol analysis during the ONDSTs. Roche and Siemens aligned more closely with LC-MS/MS while the Abbot may cause a reduction in ONDST sensitivity. This data supports assay-specific cut-offs for the ONDST.

Chronic hypoxia favours adoption to a castration-resistant cell state in prostate cancer.

Predicting and treating recurrence in intermediate-risk prostate cancer patients remains a challenge despite having identified genomic instability [1] and hypoxia [2, 3] as risk factors. This underlies challenges in assigning the functional impact of these risk factors to mechanisms promoting prostate cancer progression. Here we show chronic hypoxia (CH), as observed in prostate tumours [4], leads to the adoption of an androgen-independent state in prostate cancer cells. Specifically, CH results in prostate cancer cells adopting transcriptional and metabolic alterations typical of castration-resistant prostate cancer cells. These changes include the increased expression of transmembrane transporters for the methionine cycle and related pathways leading to increased abundance of metabolites and expression of enzymes related to glycolysis. Targeting of the Glucose Transporter 1 (GLUT1) identified a dependency on glycolysis in androgen-independent cells. Overall, we identified a therapeutically targetable weakness in chronic hypoxia and androgen-independent prostate cancer. These findings may offer additional strategies for treatment development against hypoxic prostate cancer.

Classic and 11-oxygenated androgens in serum and saliva across adulthood: a cross-sectional study analyzing the impact of age, body mass index, and diurnal and menstrual cycle variation.

OBJECTIVE: 11-oxygenated androgens significantly contribute to the circulating androgen pool. Understanding the physiological variation of 11-oxygenated androgens and their determinants is essential for clinical interpretation, for example, in androgen excess conditions. We quantified classic and 11-oxygenated androgens in serum and saliva across the adult age and body mass index (BMI) range, also analyzing diurnal and menstrual cycle-dependent variation. DESIGN: Cross-sectional. Morning serum samples were collected from 290 healthy volunteers (125 men, 22-95 years; 165 women, 21-91 years). Morning saliva samples were collected by a sub-group (51 women and 32 men). Diurnal saliva profiles were collected by 13 men. Twelve women collected diurnal saliva profiles and morning saliva samples on 7 consecutive days during both follicular and luteal menstrual cycle phases. METHODS: Serum and salivary steroids were quantified by liquid chromatography-tandem mass spectrometry profiling assays. RESULTS: Serum classic androgens decreased with age-adjusted BMI, for example, %change kg/m2 for 5α-dihydrotestosterone: men -5.54% (95% confidence interval (CI) -8.10 to -2.98) and women -1.62% (95%CI -3.16 to -0.08). By contrast, 11-oxygenated androgens increased with BMI, for example, %change kg/m2 for 11-ketotestosterone: men 3.05% (95%CI 0.08-6.03) and women 1.68% (95%CI -0.44 to 3.79). Conversely, classic androgens decreased with age in both men and women, while 11-oxygenated androgens did not. Salivary androgens showed a diurnal pattern in men and in the follicular phase in women; in the luteal phase, only 11-oxygenated androgens showed diurnal variation. CONCLUSIONS: Classic androgens decrease while active 11-oxygenated androgens increase with increasing BMI, pointing toward the importance of adipose tissue mass for the activation of 11-oxygenated androgens. Classic but not 11-oxygenated androgens decline with age.

11-Oxygenated androgens are not secreted by the human ovary: in-vivo data from four different cases of hyperandrogenism.

Objective: Differentiation of an adrenal from an ovarian source of hyperandrogenemia can be challenging. Recent studies have highlighted the importance of 11-oxygenated C19 steroids to the androgen pool in humans. The aim of this study was to confirm the origin of 11-oxygenated androgens in females and to explore their potential use in the diagnostics of hyperandrogenic disorders. Methods: We measured testosterone and its precursors (dehydroepiandrosterone-sulfate and androstenedione) and 11-oxygenated androgens (11ß-hydroxyandrostenedione (11-OHA4) and 11-ketotestosterone (11-KT)) in the periphery, adrenal and ovarian veins in four different cases of hyperandrogenism in females (polycystic ovary syndrome (PCOS), primary bilateral macronodular adrenal hyperplasia, Sertoli-Leydig cell tumor and ovarian steroid cell tumor). Results: Two patients demonstrate excessive testosterone secretion in neoplastic ovarian tumors which was not paralleled by a significant secretion of 11-oxygenated androgens as determined by adrenal and ovarian vein sampling. In androgen-secreting bilateral adrenal macronodular hyperplasia, steroid profiles were characterized by elevated 11-KT and 11-OHA4 concentrations in adrenal veins and the periphery. In the patient with PCOS, peripheral 11-KT concentrations were slightly elevated in comparison to the other patients, but the 11-KT and 11-OHA4 concentrations were comparable in ovarian veins and in the periphery. Conclusion: This study confirms that 11-OHA4 and 11-KT are not biosynthesized by the ovary. We propose that the testosterone/11-KT ratio as well as 11-OHA4 could help identify predominant adrenal androgen excess and distinguish neoplastic and non-neoplastic ovarian androgen source. Significance statement: This study confirms that 11ß-hydroxyandrostenedione (11-OHA4) and 11-ketotestosterone (11-KT) are not biosynthesized by the human ovary. We propose that the testosterone/11-KT ratio as well as 11-OHA4 could help to identify predominant adrenal androgen excess and distinguish neoplastic and non-neoplastic ovarian androgen source.

11-Oxygenated C19 steroids are the predominant androgens responsible for hyperandrogenemia in Cushing's disease.

Background: Symptoms of hyperandrogenism are common in patients with Cushing's disease (CD), yet they are not sufficiently explained by androgen concentrations. In this study, we analyzed the contribution of 11-oxygenated C19 steroids (11oxC19) to hyperandrogenemia in female patients with CD. Methods: We assessed saliva day profiles in females with CD pre (n = 23) and post (n = 13) successful transsphenoidal surgery, 26 female controls, 5 females with CD treated with metyrapone and 5 treated with osilodrostat for cortisol, cortisone, androstenedione (A4), 11-hydroxyandrostenedione (11OHA4), testosterone (TS), 11-ketotestosterone (11KT), as well as metabolites of classic and 11-oxygenated androgens in 24-h urine. In addition, morning baseline levels of gonadotropins and estradiol, sex hormone-binding globulin, cortisol and dehydroepiandrosterone sulfate (DHEAS) in serum and adrenocorticotrophic hormone in plasma in patients and controls were investigated. Results: Treatment-naïve females with CD showed a significantly elevated area under the curve of 11OHA4 and 11KT in saliva throughout the day compared to controls (11OHA4 mean rank difference (mrd) 18.13, P = 0.0002; 11KT mrd 17.42; P = 0.0005), whereas A4, TS and DHEAS were comparable to controls. Gonadotropin concentrations were normal in all patients with CD. After transsphenoidal surgery, 11oxC19 and their metabolites dropped significantly in saliva (11OHA4 P < 0.0001; 11KT P = 0.0010) and urine (11-oxo-androsterone P = 0.0011; 11-hydroxy-androsterone P < 0.0001), treatment with osilodrostat and metyrapone efficaciously blocked 11oxC19 synthesis. Conclusion: Hyperandrogenemia in CD is predominantly caused by excess of 11oxC19 steroids.

Multi-steroid profiling by UHPLC-MS/MS with post-column infusion of ammonium fluoride.

BACKGROUND: Multi-steroid profiling is a powerful analytical tool that simultaneously quantifies steroids from different biosynthetic pathways. Here we present an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the profiling of 23 steroids using post-column infusion of ammonium fluoride. METHODS: Following liquid-liquid extraction, steroids were chromatographically separated over 5 min using a Phenomenex Luna Omega C18 column and a water (0.1 % formic acid) methanol gradient. Quantification was performed on a Waters Acquity UHPLC and Xevo® TQ-XS mass spectrometer. Ammonium fluoride (6 mmol/L, post-column infusion) and formic acid (0.1 % (vol/vol), mobile phase additive) were compared as additives to aid ionisation. RESULTS: Post-column infusion of ammonium fluoride enhanced ionisation in a steroid structure-dependent fashion compared to formic acid (122-140 % for 3ßOH-Δ5 steroids and 477-1274 % for 3-keto-Δ4 steroids). Therefore, we analytically validated post-column infusion of ammonium fluoride. Lower limits of quantification ranged from 0.3 to 3 nmol/L; All analytes were quantifiable with acceptable accuracy (bias range -14 % to 11.9 % for 21/23, -21 % to 11.9 % for all analytes). Average recovery ranged from 91.6 % to 113.6 % and average matrix effects from -29.9 % to 19.9 %. Imprecision ranged from 2.3 % to 23 % for all analytes and was < 15 % for 18/23 analytes. The serum multi-steroid profile of 10 healthy men and 10 healthy women was measured. CONCLUSIONS: UHPLC-MS/MS with post-column infusion of ammonium fluoride enables comprehensive multi-steroid profiling through enhanced ionisation particularly benefiting the detection of 3-keto-Δ4 steroids.

Report from the HarmoSter study: impact of calibration on comparability of LC-MS/MS measurement of circulating cortisol, 17OH-progesterone and aldosterone.

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for measuring circulating steroids. However, assays display technical heterogeneity. So far, reproducibility of corticosteroid LC-MS/MS measurements has received scant attention. The aim of the study was to compare LC-MS/MS measurements of cortisol, 17OH-progesterone and aldosterone from nine European centers and assess performance according to external quality assessment (EQA) materials and calibration. METHODS: Seventy-eight patient samples, EQA materials and two commercial calibration sets were measured twice by laboratory-specific procedures. Results were obtained by in-house (CAL1) and external calibrations (CAL2 and CAL3). We evaluated intra and inter-laboratory imprecision, correlation and agreement in patient samples, and trueness, bias and commutability in EQA materials. RESULTS: Using CAL1, intra-laboratory CVs ranged between 2.8-7.4%, 4.4-18.0% and 5.2-22.2%, for cortisol, 17OH-progesterone and aldosterone, respectively. Trueness and bias in EQA materials were mostly acceptable, however, inappropriate commutability and target value assignment were highlighted in some cases. CAL2 showed suboptimal accuracy. Median inter-laboratory CVs for cortisol, 17OH-progesterone and aldosterone were 4.9, 11.8 and 13.8% with CAL1 and 3.6, 10.3 and 8.6% with CAL3 (all p<0.001), respectively. Using CAL1, median bias vs. all laboratory-medians ranged from -6.6 to 6.9%, -17.2 to 7.8% and -12.0 to 16.8% for cortisol, 17OH-progesterone and aldosterone, respectively. Regression lines significantly deviated from the best fit for most laboratories. Using CAL3 improved cortisol and 17OH-progesterone between-method bias and correlation. CONCLUSIONS: Intra-laboratory imprecision and performance with EQA materials were variable. Inter-laboratory performance was mostly within specifications. Although residual variability persists, adopting common traceable calibrators and RMP-determined EQA materials is beneficial for standardization of LC-MS/MS steroid measurements.

Mutations in Noncoding Cis-Regulatory Elements Reveal Cancer Driver Cistromes in Luminal Breast Cancer.

Whole-genome sequencing of primary breast tumors enabled the identification of cancer driver genes and noncoding cancer driver plexuses from somatic mutations. However, differentiating driver from passenger events among noncoding genetic variants remains a challenge. Herein, we reveal cancer-driver cis-regulatory elements linked to transcription factors previously shown to be involved in development of luminal breast cancers by defining a tumor-enriched catalogue of approximately 100,000 unique cis-regulatory elements from 26 primary luminal estrogen receptor (ER)+ progesterone receptor (PR)+ breast tumors. Integrating this catalog with somatic mutations from 350 publicly available breast tumor whole genomes, we uncovered cancer driver cistromes, defined as the sum of binding sites for a transcription factor, for ten transcription factors in luminal breast cancer such as FOXA1 and ER, nine of which are essential for growth in breast cancer with four exclusive to the luminal subtype. Collectively, we present a strategy to find cancer driver cistromes relying on quantifying the enrichment of noncoding mutations over cis-regulatory elements concatenated into a functional unit. IMPLICATIONS: Mapping the accessible chromatin of luminal breast cancer led to discovery of an accumulation of mutations within cistromes of transcription factors essential to luminal breast cancer. This demonstrates coopting of regulatory networks to drive cancer and provides a framework to derive insight into the noncoding space of cancer.

Reorganization of the 3D Genome Pinpoints Noncoding Drivers of Primary Prostate Tumors.

Prostate cancer is a heterogeneous disease whose progression is linked to genome instability. However, the impact of this instability on the noncoding genome and its three-dimensional organization to aid progression is unclear. Using primary benign and tumor tissue, we find a high concordance in higher-order three-dimensional genome organization. This concordance argues for constraints to the topology of prostate tumor genomes. Nonetheless, we identified changes in focal chromatin interactions, typical of loops bridging noncoding cis-regulatory elements, and showed how structural variants can induce these changes to guide cis-regulatory element hijacking. Such events resulted in opposing differential expression of genes found at antipodes of rearrangements. Collectively, these results argue that changes to focal chromatin interactions, as opposed to higher-order genome organization, allow for aberrant gene regulation and are repeatedly mediated by structural variants in primary prostate cancer. SIGNIFICANCE: This work showcases how the noncoding genome can be hijacked by focal insults to its three-dimensional organization that contribute to prostate cancer oncogenesis.

Salivary Profiles of 11-oxygenated Androgens Follow a Diurnal Rhythm in Patients With Congenital Adrenal Hyperplasia.

CONTEXT: Several studies have highlighted the importance of the 11-oxygenated 19-carbon (11oxC19) adrenal-derived steroids as potential biomarkers for monitoring patients with 21-hydroxylase deficiency (21OHD). OBJECTIVE: To analyze circadian rhythmicity of 11oxC19 steroids in saliva profiles and evaluate their relevance as potential monitoring parameters in 21OHD. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional single-center study including 59 patients with classic 21OHD (men = 30; women = 29) and 49 body mass index- and age-matched controls (men = 19; women = 30). OUTCOME MEASURES: Salivary concentrations of the following steroids were analyzed by liquid chromatography-tandem mass spectrometry: 17-hydroxyprogesterone (17OHP), androstenedione (A4), testosterone (T), 11ß-hydroxyandrostenedione (11OHA4), and 11-ketotestosterone (11KT). RESULTS: Similar to the previously described rhythmicity of 17OHP, 11OHA4 and 11KT concentrations followed a distinct diurnal rhythm in both patients and controls with highest concentrations in the early morning and declining throughout the day (11-OHA4: mean reduction of hormone concentrations between timepoint 1 and 5 (Δ mean) in male patients = 66%; male controls Δ mean = 83%; female patients Δ mean = 47%; female controls Δ mean = 86%; 11KT: male patients Δ mean = 57%; male controls Δ mean = 63%; female patients Δ mean = 50%; female controls Δ mean = 76%). Significant correlations between the area under the curve for 17OHP and 11KT (rpmale = 0.773<0.0001; rpfemale = 0.737<0.0001), and 11OHA4 (rpmale = 0.6330.0002; rpfemale = 0.5640.0014) were observed in patients but not present or reduced in controls. CONCLUSIONS: Adrenal 11oxC19 androgens are secreted following a diurnal pattern. This should be considered when evaluating their utility for monitoring treatment control.

Steroid Ligands, the Forgotten Triggers of Nuclear Receptor Action; Implications for Acquired Resistance to Endocrine Therapy.

PURPOSE: There is strong epidemiologic evidence indicating that estrogens may not be the sole steroid drivers of breast cancer. We hypothesize that abundant adrenal androgenic steroid precursors, acting via the androgen receptor (AR), promote an endocrine-resistant breast cancer phenotype. EXPERIMENTAL DESIGN: AR was evaluated in a primary breast cancer tissue microarray (n = 844). Androstenedione (4AD) levels were evaluated in serum samples (n = 42) from hormone receptor-positive, postmenopausal breast cancer. Levels of androgens, progesterone, and estradiol were quantified using LC/MS-MS in serum from age- and grade-matched recurrent and nonrecurrent patients (n = 6) before and after aromatase inhibitor (AI) therapy (>12 months). AR and estrogen receptor (ER) signaling pathway activities were analyzed in two independent AI-treated cohorts. RESULTS: AR protein expression was associated with favorable progression-free survival in the total population (Wilcoxon, P < 0.001). Pretherapy serum samples from breast cancer patients showed decreasing levels of 4AD with age only in the nonrecurrent group (P < 0.05). LC/MS-MS analysis of an AI-sensitive and AI-resistant cohort demonstrated the ability to detect altered levels of steroids in serum of patients before and after AI therapy. Transcriptional analysis showed an increased ratio of AR:ER signaling pathway activities in patients failing AI therapy (t test P < 0.05); furthermore, 4AD mediated gene changes associated with acquired AI resistance. CONCLUSIONS: This study highlights the importance of examining the therapeutic consequences of the steroid microenvironment and demonstrable receptor activation using indicative gene expression signatures.

11-oxygenated androgens and their relation to hypothalamus-pituitary-gonadal-axis disturbances in adults with congenital adrenal hyperplasia.

CONTEXT: Hypothalamus-pituitary-gonadal (HPG)-axis disturbances are a common phenomenon in patients with classic congenital adrenal hyperplasia (CAH). 11-oxygenated androgens have been suggested to play a role in this context. DESIGN: Cross-sectional single center study including 89 patients (N = 42 men, N = 55 women) with classic CAH. MAIN OUTCOME MEASURES: Differences in steroid markers in men with hypogonadism and women with secondary amenorrhea with a special focus on 11-ketotestosterone (11KT) and 11ß-hydroxyandrostenedione (11OHA4). RESULTS: Hypogonadotropic hypogonadism was present in 23 % of men and 61 % of those women currently not on contraceptives suffered from irregular menstrual cycles or amenorrhea. Testicular adrenal rest tumor (TART) was documented in 28 % of men. 11KT (3.5x) and 11OHA4 (5.7x) among other adrenal steroids were significantly elevated in men with hypogonadism and in women with amenorrhea in comparison to those with a regular cycle (11KT: 5.2x; 11OHA4: 3.7x). 11-oxygenated androgens were not higher in men with TART than in those without. There was a negative association of 11KT and 11OHA4 with FSH but not with LH in men. As expected, all steroids were strongly correlated with each other and cases of disproportionally elevated 11-oxygenated androgens that could explain for HPG-disturbances or TART in otherwise controlled patients were rare and also found in eugonadal individuals. CONCLUSIONS: In CAH, 11-oxygenated androgens are elevated in women with menstrual disturbances and in men with hypogonadotropic hypogonadism. Due to the close correlation of 11-oxygenated androgens with other adrenal steroids it remains to be shown if their measurement is superior to conventional markers of androgen control.

Intracrine Testosterone Activation in Human Pancreatic ß-Cells Stimulates Insulin Secretion.

Testosterone (T) affects ß-cell function in men and women. T is a prohormone that undergoes intracrine conversion in target tissues to the potent androgen dihydrotestosterone (DHT) via the enzyme 5α-reductase (5α-R) or to the active estrogen 17ß-estradiol (E2) via the aromatase enzyme. Using male and female human pancreas sections, we show that the 5α-R type 1 isoform (SRD5A1) and aromatase are expressed in male and female ß-cells. We show that cultured male and female human islets exposed to T produce DHT and downstream metabolites. In these islets, exposure to the 5α-R inhibitors finasteride and dutasteride inhibited T conversion into DHT. We did not detect T conversion into E2 from female islets. However, we detected T conversion into E2 in islets from two out of four male donors. In these donors, exposure to the aromatase inhibitor anastrozole inhibited E2 production. Notably, in cultured male and female islets, T enhanced glucose-stimulated insulin secretion (GSIS). In these islets, exposure to 5α-R inhibitors or the aromatase inhibitor both inhibited T enhancement of GSIS. In conclusion, male and female human islets convert T into DHT and E2 via the intracrine activities of SRD5A1 and aromatase. This process is necessary for T enhancement of GSIS.