International consensus on mitotane treatment in pediatric patients with adrenal cortical tumors: indications, therapy, and management of adverse effects.

OBJECTIVE: Mitotane is an important cornerstone in the treatment of pediatric adrenal cortical tumors (pACC), but experience with the drug in the pediatric age group is still limited and current practice is not guided by robust evidence. Therefore, we have compiled international consensus statements from pACC experts on mitotane indications, therapy, and management of adverse effects. METHODS: A Delphi method with 3 rounds of questionnaires within the pACC expert consortium of the international network groups European Network for the Study of Adrenal Tumors pediatric working group (ENSAT-PACT) and International Consortium of pediatric adrenocortical tumors (ICPACT) was used to create 21 final consensus statements. RESULTS: We divided the statements into 4 groups: environment, indications, therapy, and adverse effects. We reached a clear consensus for mitotane treatment for advanced pACC with stages III and IV and with incomplete resection/tumor spillage. For stage II patients, mitotane is not generally indicated. The timing of initiating mitotane therapy depends on the clinical condition of the patient and the setting of the planned therapy. We recommend a starting dose of 50 mg/kg/d (1500 mg/m²/d) which can be increased up to 4000 mg/m2/d. Blood levels should range between 14 and 20 mg/L. Duration of mitotane treatment depends on the clinical risk profile and tolerability. Mitotane treatment causes adrenal insufficiency in virtually all patients requiring glucocorticoid replacement shortly after beginning. As the spectrum of adverse effects of mitotane is wide-ranging and can be life-threatening, frequent clinical and neurological examinations (every 2-4 weeks), along with evaluation and assessment of laboratory values, are required. CONCLUSIONS: The Delphi method enabled us to propose an expert consensus statement, which may guide clinicians, further adapted by local norms and the individual patient setting. In order to generate evidence, well-constructed studies should be the focus of future efforts.

Management aspects of congenital adrenal hyperplasia during adolescence and transition to adult care.

The adolescent period is characterised by fundamental hormonal changes, which affect sex steroid production, cortisol metabolism and insulin sensitivity. These physiological changes have a significant impact on patients with congenital adrenal hyperplasia (CAH). An essential treatment aim across the lifespan in patients with CAH is to replace glucocorticoids sufficiently to avoid excess adrenal androgen production but equally to avoid cardiometabolic risks associated with excess glucocorticoid intake. The changes to the hormonal milieu at puberty, combined with poor adherence to medical therapy, often result in unsatisfactory control exacerbating androgen excess and increasing the risk of metabolic complications due to steroid over-replacement. With the physical and cognitive maturation of the adolescent with CAH, fertility issues and sexual function become a new focus of patient care in the paediatric clinic. This requires close surveillance for gonadal dysfunction, such as irregular periods/hirsutism or genital surgery-associated symptoms in girls and central hypogonadism or testicular adrenal rest tumours in boys. To ensure good health outcomes across the lifespan, the transition process from paediatric to adult care of patients with CAH must be planned carefully and early from the beginning of adolescence, spanning over many years into young adulthood. Its key aims are to empower the young person through education with full disclosure of their medical history, to ensure appropriate follow-up with experienced physicians and facilitate access to multispecialist teams addressing the complex needs of patients with CAH.

The broad phenotypic spectrum of 17α-hydroxylase/17,20-lyase (CYP17A1) deficiency: a case series.

CONTEXT: 17α-Hydroxylase/17,20-lyase deficiency (17OHD) caused by mutations in the CYP17A1 gene is a rare form of congenital adrenal hyperplasia typically characterised by cortisol deficiency, mineralocorticoid excess and sex steroid deficiency. OBJECTIVE: To examine the phenotypic spectrum of 17OHD by clinical and biochemical assessment and corresponding in silico and in vitro functional analysis. DESIGN: Case series. PATIENTS AND RESULTS: We assessed eight patients with 17OHD, including four with extreme 17OHD phenotypes: two siblings presented with failure to thrive in early infancy and two with isolated sex steroid deficiency and normal cortisol reserve. Diagnosis was established by mass spectrometry-based urinary steroid profiling and confirmed by genetic CYP17A1 analysis, revealing homozygous and compound heterozygous sequence variants. We found novel (p.Gly111Val, p.Ala398Glu, p.Ile371Thr) and previously described sequence variants (p.Pro409Leu, p.Arg347His, p.Gly436Arg, p.Phe53/54del, p.Tyr60IlefsLys88X). In vitro functional studies employing an overexpression system in HEK293 cells showed that 17,20-lyase activity was invariably decreased while mutant 17α-hydroxylase activity retained up to 14% of WT activity in the two patients with intact cortisol reserve. A ratio of urinary corticosterone over cortisol metabolites reflective of 17α-hydroxylase activity correlated well with clinical phenotype severity. CONCLUSION: Our findings illustrate the broad phenotypic spectrum of 17OHD. Isolated sex steroid deficiency with normal stimulated cortisol has not been reported before. Attenuation of 17α-hydroxylase activity is readily detected by urinary steroid profiling and predicts phenotype severity. SIGNIFICANCE STATEMENT: Here we report, supported by careful phenotyping, genotyping and functional analysis, a prismatic case series of patients with congenital adrenal hyperplasia due to 17α-hydroxylase (CYP17A1) deficiency (17OHD). These range in severity from the abolition of function, presenting in early infancy, and unusually mild with isolated sex steroid deficiency but normal ACTH-stimulated cortisol in adult patients. These findings will guide improved diagnostic detection of CYP17A1 deficiency.

Steroid Metabolome Analysis in Disorders of Adrenal Steroid Biosynthesis and Metabolism.

Steroid biosynthesis and metabolism are reflected by the serum steroid metabolome and, in even more detail, by the 24-hour urine steroid metabolome, which can provide unique insights into alterations of steroid flow and output indicative of underlying conditions. Mass spectrometry-based steroid metabolome profiling has allowed for the identification of unique multisteroid signatures associated with disorders of steroid biosynthesis and metabolism that can be used for personalized approaches to diagnosis, differential diagnosis, and prognostic prediction. Additionally, steroid metabolome analysis has been used successfully as a discovery tool, for the identification of novel steroidogenic disorders and pathways as well as revealing insights into the pathophysiology of adrenal disease. Increased availability and technological advances in mass spectrometry-based methodologies have refocused attention on steroid metabolome profiling and facilitated the development of high-throughput steroid profiling methods soon to reach clinical practice. Furthermore, steroid metabolomics, the combination of mass spectrometry-based steroid analysis with machine learning-based approaches, has facilitated the development of powerful customized diagnostic approaches. In this review, we provide a comprehensive up-to-date overview of the utility of steroid metabolome analysis for the diagnosis and management of inborn disorders of steroidogenesis and autonomous adrenal steroid excess in the context of adrenal tumors.

Causes, patterns and severity of androgen excess in 487 consecutively recruited pre- and post-pubertal children.

Objective Androgen excess in childhood is a common presentation and may signify sinister underlying pathology. Data describing its patterns and severity are scarce, limiting the information available for clinical decision processes. Here, we examined the differential diagnostic value of serum DHEAS, androstenedione (A4) and testosterone in childhood androgen excess. Design Retrospective review of all children undergoing serum androgen measurement at a single center over 5 years. Methods Serum A4 and testosterone were measured by tandem mass spectrometry and DHEAS by immunoassay. Patients with at least one increased androgen underwent phenotyping by clinical notes review. Results In 487 children with simultaneous DHEAS, A4 and testosterone measurements, we identified 199 with androgen excess (140 pre- and 59 post-pubertal). Premature adrenarche (PA) was the most common pre-pubertal diagnosis (61%), characterized by DHEAS excess in 85%, while A4 and testosterone were only increased in 26 and 9% respectively. PCOS was diagnosed in 40% of post-pubertal subjects, presenting equally frequent with isolated excess of DHEAS (29%) or testosterone (25%) or increases in both A4 and testosterone (25%). CAH patients (6%) predominantly had A4 excess (86%); testosterone and DHEAS were increased in 50 and 33% respectively. Concentrations increased above the two-fold upper limit of normal were mostly observed in PA for serum DHEAS (>20-fold in the single case of adrenocortical carcinoma) and in CAH for serum androstenedione. Conclusions Patterns and severity of childhood androgen excess provide pointers to the underlying diagnosis and can be used to guide further investigations.

Human DHEA sulfation requires direct interaction between PAPS synthase 2 and DHEA sulfotransferase SULT2A1.

The high-energy sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), generated by human PAPS synthase isoforms PAPSS1 and PAPSS2, is required for all human sulfation pathways. Sulfotransferase SULT2A1 uses PAPS for sulfation of the androgen precursor dehydroepiandrosterone (DHEA), thereby reducing downstream activation of DHEA to active androgens. Human PAPSS2 mutations manifest with undetectable DHEA sulfate, androgen excess, and metabolic disease, suggesting that ubiquitous PAPSS1 cannot compensate for deficient PAPSS2 in supporting DHEA sulfation. In knockdown studies in human adrenocortical NCI-H295R1 cells, we found that PAPSS2, but not PAPSS1, is required for efficient DHEA sulfation. Specific APS kinase activity, the rate-limiting step in PAPS biosynthesis, did not differ between PAPSS1 and PAPSS2. Co-expression of cytoplasmic SULT2A1 with a cytoplasmic PAPSS2 variant supported DHEA sulfation more efficiently than co-expression with nuclear PAPSS2 or nuclear/cytosolic PAPSS1. Proximity ligation assays revealed protein-protein interactions between SULT2A1 and PAPSS2 and, to a lesser extent, PAPSS1. Molecular docking studies showed a putative binding site for SULT2A1 within the PAPSS2 APS kinase domain. Energy-dependent scoring of docking solutions identified the interaction as specific for the PAPSS2 and SULT2A1 isoforms. These findings elucidate the mechanistic basis for the selective requirement for PAPSS2 in human DHEA sulfation.

Causes, Patterns, and Severity of Androgen Excess in 1205 Consecutively Recruited Women.

Context: Androgen excess in women is predominantly due to underlying polycystic ovary syndrome (PCOS). However, there is a lack of clarity regarding patterns and severity of androgen excess that should be considered predictive of non-PCOS pathology. Objective: We examined the diagnostic utility of simultaneous measurement of serum dehydroepiandrosterone sulfate (DHEAS), androstenedione (A4), and testosterone (T) to delineate biochemical signatures and cutoffs predictive of non-PCOS disorders in women with androgen excess. Design: Retrospective review of all women undergoing serum androgen measurement at a large tertiary referral center between 2012 and 2016. Serum A4 and T were measured by tandem mass spectrometry and DHEAS by immunoassay. Patients with at least one increased serum androgen underwent phenotyping by clinical notes review. Results: In 1205 women, DHEAS, A4, and T were measured simultaneously. PCOS was the most common diagnosis in premenopausal (89%) and postmenopausal women (29%). A4 was increased in all adrenocortical carcinoma (ACC) cases (n = 15) and T in all ovarian hyperthecosis (OHT) cases (n = 7); all but one case of congenital adrenal hyperplasia (CAH; n = 18) were identified by increased levels of A4 and/or T. In premenopausal women, CAH was a prevalent cause of severe A4 (59%) and T (43%) excess; severe DHEAS excess was predominantly due to PCOS (80%). In postmenopausal women, all cases of severe DHEAS and A4 excess were caused by ACC and severe T excess equally by ACC and OHT. Conclusions: Pattern and severity of androgen excess are important predictors of non-PCOS pathology and may be used to guide further investigations as appropriate.

Evidence for Increased 5α-Reductase Activity During Early Childhood in Daughters of Women With Polycystic Ovary Syndrome.

CONTEXT: Polycystic ovary syndrome (PCOS) is a heritable, complex genetic disease. Animal models suggest that androgen exposure at critical developmental stages contributes to disease pathogenesis. We hypothesized that genetic variation resulting in increased androgen production produces the phenotypic features of PCOS by programming during critical developmental periods. Although we have not found evidence for increased in utero androgen levels in cord blood in the daughters of women with PCOS (PCOS-d), target tissue androgen production may be amplified by increased 5α-reductase activity analogous to findings in adult affected women. It is possible to noninvasively test this hypothesis by examining urinary steroid metabolites. OBJECTIVE: We performed this study to investigate whether PCOS-d have altered androgen metabolism during early childhood. DESIGN, SETTING, AND PARTICIPANTS: Twenty-one PCOS-d, 1-3 years old, and 36 control girls of comparable age were studied at an academic medical center. MAIN OUTCOME MEASURES: Urinary steroid metabolites were measured by gas chromatography/mass spectrometry. Twenty-four hour steroid excretion rates and precursor to product ratios suggestive of 5α-reductase and 11ß-hydroxysteroid dehydrogenase activities were calculated. RESULTS: Age did not differ but weight for length Z-scores were higher in PCOS-d compared to control girls (P = .02). PCOS-d had increased 5α-tetrahydrocortisol:tetrahydrocortisol ratios (P = .04), suggesting increased global 5α-reductase activity. There was no evidence for differences in 11ß-hydroxysteroid dehydrogenase activity. Steroid metabolite excretion was not correlated with weight. CONCLUSIONS: Our findings suggest that differences in androgen metabolism are present in early childhood in PCOS-d. Increased 5α-reductase activity could contribute to the development of PCOS by amplifying target tissue androgen action.

The Regulation of Steroid Action by Sulfation and Desulfation.

Steroid sulfation and desulfation are fundamental pathways vital for a functional vertebrate endocrine system. After biosynthesis, hydrophobic steroids are sulfated to expedite circulatory transit. Target cells express transmembrane organic anion-transporting polypeptides that facilitate cellular uptake of sulfated steroids. Once intracellular, sulfatases hydrolyze these steroid sulfate esters to their unconjugated, and usually active, forms. Because most steroids can be sulfated, including cholesterol, pregnenolone, dehydroepiandrosterone, and estrone, understanding the function, tissue distribution, and regulation of sulfation and desulfation processes provides significant insights into normal endocrine function. Not surprisingly, dysregulation of these pathways is associated with numerous pathologies, including steroid-dependent cancers, polycystic ovary syndrome, and X-linked ichthyosis. Here we provide a comprehensive examination of our current knowledge of endocrine-related sulfation and desulfation pathways. We describe the interplay between sulfatases and sulfotransferases, showing how their expression and regulation influences steroid action. Furthermore, we address the role that organic anion-transporting polypeptides play in regulating intracellular steroid concentrations and how their expression patterns influence many pathologies, especially cancer. Finally, the recent advances in pharmacologically targeting steroidogenic pathways will be examined.

Role of ALADIN in human adrenocortical cells for oxidative stress response and steroidogenesis.

Triple A syndrome is caused by mutations in AAAS encoding the protein ALADIN. We investigated the role of ALADIN in the human adrenocortical cell line NCI-H295R1 by either over-expression or down-regulation of ALADIN. Our findings indicate that AAAS knock-down induces a down-regulation of genes coding for type II microsomal cytochrome P450 hydroxylases CYP17A1 and CYP21A2 and their electron donor enzyme cytochrome P450 oxidoreductase, thereby decreasing biosynthesis of precursor metabolites required for glucocorticoid and androgen production. Furthermore we demonstrate that ALADIN deficiency leads to increased susceptibility to oxidative stress and alteration in redox homeostasis after paraquat treatment. Finally, we show significantly impaired nuclear import of DNA ligase 1, aprataxin and ferritin heavy chain 1 in ALADIN knock-down cells. We conclude that down-regulating ALADIN results in decreased oxidative stress response leading to alteration in steroidogenesis, highlighting our knock-down cell model as an important in-vitro tool for studying the adrenal phenotype in triple A syndrome.

PAPSS2 deficiency causes androgen excess via impaired DHEA sulfation--in vitro and in vivo studies in a family harboring two novel PAPSS2 mutations.

CONTEXT: PAPSS2 (PAPS synthase 2) provides the universal sulfate donor PAPS (3'-phospho-adenosine-5'-phosphosulfate) to all human sulfotransferases, including SULT2A1, responsible for sulfation of the crucial androgen precursor dehydroepiandrosterone (DHEA). Impaired DHEA sulfation is thought to increase the conversion of DHEA toward active androgens, a proposition supported by the previous report of a girl with inactivating PAPSS2 mutations who presented with low serum DHEA sulfate and androgen excess, clinically manifesting with premature pubarche and early-onset polycystic ovary syndrome. PATIENTS AND METHODS: We investigated a family harboring two novel PAPSS2 mutations, including two compound heterozygous brothers presenting with disproportionate short stature, low serum DHEA sulfate, but normal serum androgens. Patients and parents underwent a DHEA challenge test comprising frequent blood sampling and urine collection before and after 100 mg DHEA orally, with subsequent analysis of DHEA sulfation and androgen metabolism by mass spectrometry. The functional impact of the mutations was investigated in silico and in vitro. RESULTS: We identified a novel PAPSS2 frameshift mutation, c.1371del, p.W462Cfs*3, resulting in complete disruption, and a novel missense mutation, c.809G>A, p.G270D, causing partial disruption of DHEA sulfation. Both patients and their mother, who was heterozygous for p.W462Cfs*3, showed increased 5α-reductase activity at baseline and significantly increased production of active androgens after DHEA intake. The mother had a history of oligomenorrhea and chronic anovulation that required clomiphene for ovulation induction. CONCLUSIONS: We provide direct in vivo evidence for the significant functional impact of mutant PAPSS2 on DHEA sulfation and androgen activation. Heterozygosity for PAPSS2 mutations can be associated with a phenotype resembling polycystic ovary syndrome.

Prenatal diagnosis of congenital adrenal hyperplasia caused by P450 oxidoreductase deficiency.

CONTEXT: Mutations in the electron donor enzyme P450 oxidoreductase (POR) result in congenital adrenal hyperplasia with apparent combined 17α-hydroxylase/17,20 lyase and 21-hydroxylase deficiencies, also termed P450 oxidoreductase deficiency (PORD). Major clinical features present in PORD are disordered sex development in affected individuals of both sexes, glucocorticoid deficiency, and multiple skeletal malformations. OBJECTIVE: The objective of the study was to establish a noninvasive approach to prenatal diagnosis of PORD including assessment of malformation severity to facilitate optimized prenatal diagnosis and timely treatment. DESIGN: We analyzed 20 pregnancies with children homozygous or compound heterozygous for disease-causing POR mutations and 1 pregnancy with a child carrying a heterozygous POR mutation by recording clinical and biochemical presentations and fetal ultrasound findings. In 4 of the pregnancies (3 homozygous and 1 heterozygous for disease-causing POR mutations), prenatal analysis of steroid metabolite excretion in maternal urine was carried out by gas chromatography/mass spectrometry during gestational weeks 11-23. RESULTS: Pregnancy complications in our cohort included maternal virilization (6 of 20) with onset in the second trimester. Seven pregnant women presented with low unconjugated estriol at prenatal screening (triple or quadruple antenatal screening test). Overt dysmorphic features were noted in 19 of the 20 babies at birth but observed in only 5 by prenatal ultrasound. These 5 had the most severe malformation phenotypes and poor outcome, whereas the other babies showed normal development. Steroid profiling of maternal urine revealed significantly increased steroids of fetal origin, namely the pregnenolone metabolite epiallopregnanediol and the androgen metabolite androsterone, with concomitant low values for estriol. Diagnostic steroid ratios conclusively indicated PORD as early as gestational week 12. In the heterozygous pregnancy, steroid ratios were only slightly elevated and estriol excretion was normal. CONCLUSION: Prenatal diagnosis in PORD is readily established via urinary steroid metabolite analysis of maternal urine. Visible malformations at prenatal ultrasound predict a severe malformation phenotype.

Pubertal presentation in seven patients with congenital adrenal hyperplasia due to P450 oxidoreductase deficiency.

CONTEXT: P450 oxidoreductase (POR) is a crucial electron donor to all microsomal P450 cytochrome (CYP) enzymes including 17α-hydroxylase (CYP17A1), 21-hydroxylase (CYP21A2) and P450 aromatase. Mutant POR causes congenital adrenal hyperplasia with combined glucocorticoid and sex steroid deficiency. P450 oxidoreductase deficiency (ORD) commonly presents neonatally, with disordered sex development in both sexes, skeletal malformations, and glucocorticoid deficiency. OBJECTIVE: The aim of the study was to describe the clinical and biochemical characteristics of ORD during puberty. DESIGN: Clinical, biochemical, and genetic assessment of seven ORD patients (five females, two males) presenting during puberty was conducted. RESULTS: Predominant findings in females were incomplete pubertal development (four of five) and large ovarian cysts (five of five) prone to spontaneous rupture, in some only resolving after combined treatment with estrogen/progestin, GnRH superagonists, and glucocorticoids. Pubertal development in the two boys was more mildly affected, with some spontaneous progression. Urinary steroid profiling revealed combined CYP17A1 and CYP21A2 deficiencies indicative of ORD in all patients; all but one failed to mount an appropriate cortisol response to ACTH stimulation indicative of adrenal insufficiency. Diagnosis of ORD was confirmed by direct sequencing, demonstrating disease-causing POR mutations. CONCLUSION: Delayed and disordered puberty can be the first sign leading to a diagnosis of ORD. Appropriate testosterone production during puberty in affected boys but manifest primary hypogonadism in girls with ORD may indicate that testicular steroidogenesis is less dependent on POR than adrenal and ovarian steroidogenesis. Ovarian cysts in pubertal girls may be driven not only by high gonadotropins but possibly also by impaired CYP51A1-mediated production of meiosis-activating sterols due to mutant POR.