Adrenocortical tumors and pheochromocytoma/paraganglioma initially mistaken as neuroblastoma — experiences from the GPOH-MET registry

In children and adolescents, neuroblastoma (NBL), pheochromocytoma (PCC), and adrenocortical tumors (ACT) can arise from the adrenal gland. It may be difficult to distinguish between these three entities including associated extra-adrenal tumors (paraganglioma, PGL). Precise discrimination, however, is of crucial importance for management. Biopsy in ACT or PCC is potentially harmful and should be avoided whenever possible. We herein report data on 10 children and adolescents with ACT and five with PCC/PGL, previously mistaken as NBL. Two patients with adrenocortical carcinoma died due to disease progression. Two (2/9, missing data in one patient) patients with a final diagnosis of ACT clearly presented with obvious clinical signs and symptoms of steroid hormone excess, while seven patients did not. Blood analyses indicated increased levels of steroid hormones in one additional patient; however, urinary steroid metabolome analysis was not performed in any patient. Two (2/10) patients underwent tumor biopsy, and in two others tumor rupture occurred intraoperatively. In 6/10 patients, ACT diagnosis was only established by a reference pediatric pathology laboratory. Four (4/5) patients with a final diagnosis of PCC/PGL presented with clinical signs and symptoms of catecholamine excess. Urine tests indicated possible catecholamine excess in two patients, while no testing was carried out in three patients. Measurements of plasma metanephrines were not performed in any patient. None of the five patients with PCC/PGL received adrenergic blockers before surgery. In four patients, PCC/PGL diagnosis was established by a local pathologist, and in one patient diagnosis was revised to PGL by a pediatric reference pathologist. Genetic testing, performed in three out of five patients with PCC/PGL, indicated pathogenic variants of PCC/PGL susceptibility genes. The differential diagnosis of adrenal neoplasias and associated extra-adrenal tumors in children and adolescents may be challenging, necessitating interdisciplinary and multidisciplinary efforts. In ambiguous and/or hormonally inactive cases through comprehensive biochemical testing, microscopical complete tumor resection by an experienced surgeon is vital to preventing poor outcome in children and adolescents with ACT and/or PCC/PGL. Finally, specimens need to be assessed by an experienced pediatric pathologist to establish diagnosis

Sunitinib Inhibits Cell Proliferation and Alters Steroidogenesis by Down-Regulation of HSD3B2 in Adrenocortical Carcinoma Cells

The multi-tyrosine kinase inhibitor sunitinib is used in the treatment of several solid tumors. Animal experiments pointed to an adrenotoxic effect of sunitinib. Therefore, we evaluated the expression of key targets of sunitinib in human adrenocortical carcinoma (ACC) tumor samples and investigated its in vitro effects in ACC cell lines. We carried out immunohistochemistry for vascular endothelial growth factor (VEGF) and its receptor (VEGF-R2) in 157 ACC samples and nine normal adrenal glands. VEGF and VEGF-R2 protein were expressed in 72 and 99% of ACC samples, respectively. Using NCI-H295 and SW13 ACC cell lines, we investigated the effects of sunitinib on cell proliferation. Sunitinib reduced dose-dependently cell viability of both NCI-H295 and SW13 cells (SW13: 0.1 μM 96 ± 7%, 1 μM 90 ± 9%*, 5 μM 62 ± 6%*, controls 100 ± 9%; *p < 0.05). To determine sunitinib effects on steroidogenesis, we measured steroid hormones in cell culture supernatant by gas chromatography–mass spectrometry. We observed a pronounced decrease of cortisol secretion (1 μM 90.1 ± 1.5%*, 5 μM 57.2 ± 0.3%*, controls 100 ± 2.4%) and a concomitant increase in the DHEA/4-androstenedione and 17-hydroxypregnenolone/17-hydroxyprogesterone ratios, indicating specific inhibition of 3β-hydroxysteroid dehydrogenase (HSD3B2). In yeast microsomes transformed with HSD3B2, no direct inhibition of HSD3B2 by sunitinib was detected. Sunitinib induced down-regulation of HSD3B2 mRNA and protein in ACC cell lines (mRNA: 1 μM 44 ± 16%*; 5 μM 22 ± 2%*; 10 μM 19 ± 4%*; protein: 1 μM 82 ± 8%; 5 μM 63 ± 8%*; 10 μM 55 ± 9%*). CYP11B1 was down-regulated at mRNA but not at protein level and CYP11A1 remained unchanged. In conclusion, target molecules of sunitinib are expressed in the vast majority of ACC samples. Sunitinib exhibits anti-proliferative effects in vitro, and appears to specifically block adrenal steroidogenesis by down-regulation of HSD3B2, rendering it a promising option for treatment of ACC

17α-hydroxylase deficiency diagnosed in early infancy caused by a novel mutation of the CYP17A1 Gene

Mutations of the CYP17A1 gene cause 17α-hydroxylase deficiency (17OHD) resulting in 46,XY disorder of sex development, hypertension, hypokalemia and absent pubertal development. It is a rare, autosomal recessive form of congenital adrenal hyperplasia (CAH).We report on a neonate with prenatally determined 46,XY karyotype. At 20 weeks of gestation, lack of development of male external genitalia was noticed. A phenotypically female child was born at 41 weeks of gestation.Postnatal ultrasound revealed testes in both labia majora, an absence of uterus and normal adrenal glands. Steroid hormone analysis in serum revealed low basal levels of cortisol, testosterone and androstenedione in the presence of massively elevated corticosterone at the age of 2 weeks. The urinary steroid profile from spot urine showed excessive excretion of 17-desoxysteroids, decreased glucocorticoid metabolites and absent C19 steroids, thus proving 17OHD. Molecular analysis identified a novel mutation of the CYP17A1 gene: c.896T>A (p.I299N) in exon 5. Substitution with hydrocortisone was started. The child is raised as a girl and is developing well so far.Herein, we report the unusually early diagnosis of a newborn with the rare CAH form of 17OHD allowing an early start of treatment

Ensuring quality in 17OHP mass spectrometry measurement:an international study assessing isomeric steroid interference

Objectives: Interference from isomeric steroids is a potential cause of disparity between mass spectrometry-based 17-hydroxyprogesterone (17OHP) results. We aimed to assess the proficiency of mass spectrometry laboratories to report 17OHP in the presence of known isomeric steroids. Methods:A series of five samples were prepared using a previously demonstrated commutable approach. These samples included a control (spiked to 15.0 nmol/L 17OHP) and four challenge samples further enriched with equimolar concentrations of 17OHP isomers (11α-hydroxyprogesterone, 11β-hydroxyprogesterone, 16α-hydroxyprogesterone or 21-hydroxyprogesterone). These samples were distributed to 38 participating laboratories that reported serum 17OHP results using mass spectrometry in two external quality assurance programs. The result for each challenge sample was compared to the control sample submitted by each participant. Results: Twenty-six laboratories (68 % of distribution) across three continents returned results. Twenty-five laboratories used liquid chromatography-tandem mass spectrometry (LC-MS/MS), and one used gas chromatography-tandem mass spectrometry to measure 17OHP. The all-method median of the control sample was 14.3 nmol/L, ranging from 12.4 to 17.6 nmol/L. One laboratory had results that approached the lower limit of tolerance (minus 17.7 % of the control sample), suggesting the isomeric steroid caused an irregular result. Conclusions: Most participating laboratories demonstrated their ability to reliably measure 17OHP in the presence of the four clinically relevant isomeric steroids. The performance of the 12 (32 %) laboratories that did not engage in this activity remains unclear. We recommend that all laboratories offering LC-MS/MS analysis of 17OHP in serum, plasma, or dried bloodspots determine that the isomeric steroids are appropriately separated.</p

Ensuring quality in 17OHP mass spectrometry measurement:an international study assessing isomeric steroid interference

Objectives: Interference from isomeric steroids is a potential cause of disparity between mass spectrometry-based 17-hydroxyprogesterone (17OHP) results. We aimed to assess the proficiency of mass spectrometry laboratories to report 17OHP in the presence of known isomeric steroids. Methods:A series of five samples were prepared using a previously demonstrated commutable approach. These samples included a control (spiked to 15.0 nmol/L 17OHP) and four challenge samples further enriched with equimolar concentrations of 17OHP isomers (11α-hydroxyprogesterone, 11β-hydroxyprogesterone, 16α-hydroxyprogesterone or 21-hydroxyprogesterone). These samples were distributed to 38 participating laboratories that reported serum 17OHP results using mass spectrometry in two external quality assurance programs. The result for each challenge sample was compared to the control sample submitted by each participant. Results: Twenty-six laboratories (68 % of distribution) across three continents returned results. Twenty-five laboratories used liquid chromatography-tandem mass spectrometry (LC-MS/MS), and one used gas chromatography-tandem mass spectrometry to measure 17OHP. The all-method median of the control sample was 14.3 nmol/L, ranging from 12.4 to 17.6 nmol/L. One laboratory had results that approached the lower limit of tolerance (minus 17.7 % of the control sample), suggesting the isomeric steroid caused an irregular result. Conclusions: Most participating laboratories demonstrated their ability to reliably measure 17OHP in the presence of the four clinically relevant isomeric steroids. The performance of the 12 (32 %) laboratories that did not engage in this activity remains unclear. We recommend that all laboratories offering LC-MS/MS analysis of 17OHP in serum, plasma, or dried bloodspots determine that the isomeric steroids are appropriately separated.</p

Delineating endogenous Cushing's syndrome by GC-MS urinary steroid metabotyping

BACKGROUND Diagnosing Cushing's syndrome (CS) is highly complex. As the diagnostic potential of urinary steroid metabolome analysis by gas chromatography-mass spectrometry (GC-MS) in combination with systems biology has not yet been fully exploited, we studied a large cohort of patients with CS. METHODS We quantified daily urinary excretion rates of 36 steroid hormone metabolites. Applying cluster analysis, we investigated a control group and 168 patients: 44 with Cushing's disease (CD) (70% female), 18 with unilateral cortisol-producing adrenal adenoma (83% female), 13 with primary bilateral macronodular adrenal hyperplasia (PBMAH) (77% female), and 93 ruled-out CS (73% female). FINDINGS Cluster-Analysis delineated five urinary steroid metabotypes in CS. Metabotypes 1, 2 and 3 revealing average levels of cortisol and adrenal androgen metabolites included patients with exclusion of CS or and healthy controls. Metabotype 4 reflecting moderately elevated cortisol metabolites but decreased DHEA metabolites characterized the patients with unilateral adrenal CS and PBMAH. Metabotype 5 showing strong increases both in cortisol and DHEA metabolites, as well as overloaded enzymes of cortisol inactivation, was characteristic of CD patients. 11-oxygenated androgens were elevated in all patients with CS. The biomarkers THS, F, THF/THE, and (An + Et)/(11β-OH-An + 11β-OH-Et) correctly classified 97% of patients with CS and 95% of those without CS. An inverse relationship between 11-deoxygenated and 11-oxygenated androgens was typical for the ACTH independent (adrenal) forms of CS with an accuracy of 95%. INTERPRETATION GC-MS based urinary steroid metabotyping allows excellent identification of patients with endogenous CS and differentiation of its subtypes. FUNDING The study was funded by the Else Kröner-Fresenius-Stiftung and the Eva-Luise-und-Horst-Köhler-Stiftung

Characterization of the micro-environment of the testis that shapes the phenotype and function of testicular macrophages

Tissue-specific macrophages are important for the activation of innate immune responses and general organ homeostasis. Testicular macrophages (TM) reside in the testicular interstitial space and comprise the largest leukocyte population in the testis and are assumed to play a role in maintaining testicular immune privilege. Numerous studies have indicated that the interstitial fluid (IF) surrounding the TM has immunosuppressive properties, which may influence the TM phenotype. However, the identity of the immunosuppressive molecules present in the IF is poorly characterized. In this thesis it is shown that in the rat, IF shifts the M1 phenotype of granulocyte macrophage-colony stimulating factor induced bone marrow derived macrophages towards the M2 phenotype. M2 macrophages polarized by IF mimic the properties of TM such as increased expression of CD163, high secretion of IL-10 and low secretion of TNF-alpha. In addition, IF-polarized macrophages display immunoregulatory functions by inducing the expansion of immunosuppressive regulatory T cells. This thesis provides evidence that PGE2, PGI2, testosterone and corticosterone are important immunoregulatory molecules in the IF, playing a relevant role in determining the phenotype of TM. Except corticosterone, all of these factors are able to inhibit the NF-kB signaling pathway to suppress the production of pro-inflammatory cytokines and thus maintain an immunosuppressive microenvironment of the testis. Corticosterone was found to be the principal immunosuppressive molecule in the IF. Its receptor, the glucocorticoid receptor, was found to be present in TM immunohistochemically. In addition, TM locally produce small amounts of corticosterone, which suppress the expression of inflammatory genes and render TM refractory to inflammatory stimuli. Taken together, these results suggest that testicular corticosterone shapes the immunosuppressive function and phenotype of TM. This steroid hormone may therefore play also an important role in the establishment and maintenance of the immune privilege of the testis.Gewebsspezifische Makrophagen haben eine wichtige Funktion bei der Aktivierung angeborener Immunantworten und der Organhomeostase. Testikuläre Makrophagen (TM) befinden sich im Interstitium des Hodens und stellen die größte Leukozytenpopulation in der männlichen Gonade dar. Es wird angenommen, dass sie eine wichtige Funktion in der Aufrechterhaltung des Immunprivilegs des Hodens ausüben. Studien haben gezeigt, dass die interstitielle Flüssigkeit (IF), wleche die TM umgibt, immunsuppressive Eigenschaften aufweist, die den Phänotyp der TM beeinflussen könnten. Allerdings konnten immunsuppressive Moleküle in der IF bislang kaum charakterisiert werden. In der vorliegenden Arbeit wird für die Ratte als Modell gezeigt, dass die IF den durch Granulozyten- Makrophagen-Kolonie-stimulierenden Faktor (GM-CSF) induzierten M1 Phänotyp von Makrophagen, die aus dem Knochenmark isoliert wurden, in Richtung des M2 Phänotyps verschieben kann. IF-polarisierte M2-Makrophagen zeigen damit charakteristische Eigenschaften von TM, wie z. Bsp. erhöhte Expression von CD163, hohe Level von sezerniertem IL-10 bei geringer TNF-alpha Sekretion. Darüber hinaus zeigen IF-polarisierte Makrophagen immunoregulatorische Funktionen, indem sie die Expansion von immunsuppressiven regulatorischen T-Zellen induzieren. In dieser Studie werden erstmals auch Ergebnisse vorgestellt, die zeigen, dass PGE2, PGI2, Testosteron und Corticosteron wichtige immunregulatorische Moleküle in der IF darstellen und eine wesentliche Rolle bei der Bestimmung des TM-Phänotyps spielen. Mit Ausnahme von Corticosteron sind die genannten Faktoren in der Lage, den NF-kB-Signalweg zu hemmen, und damit die Produktion von entzündungshemmenden Zytokinen zu unterdrücken. Bei Corticosteron war der NFkB Signalweg bei der Immunsuppression nicht blockiert. Corticosteron konnte als wichtigster immunsuppressiver Faktor in der IF identifiziert werden. Dessen Rezeptor, der Glucocorticoidrezeptor, konnte in TM mittels Immunhistochemie gefunden werden. TM produzieren lokal moderate Mengen an Corticosteron, die die Expression inflammatorischer Gene unterdrücken und TM unempfindlich gegenüber entzündlichen Stimuli machen können. Zusammengenommen zeigen diese Ergebnisse, dass testikuläres Corticosteron maßgeblich für die immunsuppressive Funktion und den spezifischen Phänotyp der TM verantwortlich ist. Damit könnte das Steroidhormon auch eine wichtige Rolle bei der Etablierung und Aufrechterhaltung des Immunprivilegs im Hoden spielen

Cooperation of cancer drivers with regulatory germline variants shapes clinical outcomes

Pediatric malignancies including Ewing sarcoma (EwS) feature a paucity of somatic alterations except for pathognomonic driver-mutations that cannot explain overt variations in clinical outcome. Here, we demonstrate in EwS how cooperation of dominant oncogenes and regulatory germline variants determine tumor growth, patient survival and drug response. Binding of the oncogenic EWSR1-FLI1 fusion transcription factor to a polymorphic enhancerlike DNA element controls expression of the transcription factor MYBL2 mediating these phenotypes. Whole-genome and RNA sequencing reveals that variability at this locus is inherited via the germline and is associated with variable inter-tumoral MYBL2 expression. High MYBL2 levels sensitize EwS cells for inhibition of its upstream activating kinase CDK2 in vitro and in vivo, suggesting MYBL2 as a putative biomarker for anti-CDK2-therapy. Collectively, we establish cooperation of somatic mutations and regulatory germline variants as a major determinant of tumor progression and highlight the importance of integrating the regulatory genome in precision medicine